| 1. |
Escobar-Páramo P,
Giudicelli C,
Parsot C,
Denamur E,
( 2003 ) The evolutionary history of Shigella and enteroinvasive Escherichia coli revised. PMID : 14562958 : DOI : 10.1007/s00239-003-2460-3 Abstract >>
In Shigella and enteroinvasive Escherichia coli (EIEC), the etiologic agents of shigellosis in humans, the determinants responsible for entry of bacteria into and dissemination within epithelial cells are encoded by a virulence plasmid. To understand the evolution of the association between the virulence plasmid and the chromosome, we performed a phylogenetic analysis using the sequences of four chromosomal genes (trpA, trpB, pabB, and putP) and three virulence plasmid genes (ipaB, ipaD, and icsA) of a collection of 51 Shigella and EIEC strains. The phylogenetic tree derived from chromosomal genes showed a typical "star" phylogeny, indicating a fast diversification of Shigella and EIEC groups. Phylogenetic groups obtained from the chromosomal and plasmidic genes were similar, suggesting that the virulence plasmid and the chromosome share similar evolutionary histories. The few incongruences between the trees could be attributed to exchanges of fragments of different plasmids and not to the transfer of an entire plasmid. This indicates that the virulence plasmid was not transferred between the different Shigella and EIEC groups. These data support a model of evolution in which the acquisition of the virulence plasmid in an ancestral E. coli strain preceded the diversification by radiation of all Shigella and EIEC groups, which led to highly diversified but highly specialized pathogenic groups.
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2. |
Leyton DL,
Sloan J,
Hill RE,
Doughty S,
Hartland EL,
( 2003 ) Transfer region of pO113 from enterohemorrhagic Escherichia coli: similarity with R64 and identification of a novel plasmid-encoded autotransporter, EpeA. PMID : 14573650 : DOI : 10.1128/iai.71.11.6307-6319.2003 PMC : PMC219559 Abstract >>
Enterohemorrhagic Escherichia coli (EHEC) is a prominent, food-borne cause of diarrhea, bloody diarrhea, and the hemolytic uremic syndrome in industrialized countries. Most strains of EHEC carry the locus for enterocyte effacement (LEE) pathogenicity island, but a proportion of isolates from patients with severe disease do not carry LEE and very little is known about virulence factors in these organisms. LEE-negative strains of EHEC typically express Shiga toxin 2 and carry a large plasmid that encodes the production of EHEC hemolysin. In this study, we determined the nucleotide sequence of the transfer region of pO113, the large hemolysin plasmid from LEE-negative EHEC O113:H21 (EH41). This 63.9-kb region showed a high degree of similarity with the transfer region of R64, and pO113 was capable of self-transmission at low frequencies. Unlike R64 and the related dot/icm system of Legionella pneumophila, however, pO113 was unable to mobilize RSF1010. In addition, the pO113 transfer region encoded a novel high-molecular-weight serine protease autotransporter of Enterobacteriaceae (SPATE) protein, termed EpeA. Like other SPATEs, EpeA exhibited protease activity and mucinase activity, but expression was not associated with a cytopathic effect on epithelial cells. Analysis of a second high-molecular-weight secreted protein revealed that pO113 also encodes EspP, a cytopathic SPATE identified previously in EHEC O157:H7. The nucleotide sequences encoding the predicted beta-domains of espP and epeA were identical and also shared significant homology with a third SPATE protein, EspI. Both espP and epeA were detected in several LEE-negative clinical isolates of EHEC and thus may contribute to the pathogenesis of this subset of EHEC.
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3. |
Loudon JA,
Loughlin RE,
( 1992 ) Mutagenesis and regulation of the cysJ promoter of Escherichia coli K-12. PMID : 1452024 : DOI : 10.1016/0378-1119(92)90027-m Abstract >>
The cysJ promoter of Escherichia coli K-12, which is positively controlled by the CysB regulatory protein, was localized through the formation of a fusion of cysJ, the gene encoding NADPH-cytochrome c reductase with lacZ. The position of the transcription start point was determined and the orientation of transcription was shown to be counterclockwise on the E. coli K-12 map. Oligodeoxyribonucleotide-directed mutagenesis of the inferred -10 and -35 regions indicated that both sites could be altered to produce promoter 'down' mutations. When the -10 region was made to agree with the -10 consensus sequence, there was increased function under conditions of repression (that is, in the presence of cysteine). Upstream deletions, as well as mutations in a region proposed to be involved in binding of the CysB regulatory protein, identified sequences important for promoter activity from -90 to -78 and from -71 to -66. By comparison of the sequences of four cys promoters, a possible CysB-binding site was found which included the region shown to be required for the positive regulation of the cysJ promoter.
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4. |
Ostanin K,
Harms EH,
Stevis PE,
Kuciel R,
Zhou MM,
Van Etten RL,
( 1992 ) Overexpression, site-directed mutagenesis, and mechanism of Escherichia coli acid phosphatase. PMID : 1429631 : Abstract >>
Site-directed mutagenesis was used to examine the catalytic importance of 2 histidine and 4 arginine residues in Escherichia coli periplasmic acid phosphatase (EcAP). The residues that were selected as targets for mutagenesis were those that were also conserved in a number of high molecular weight acid phosphatases from eukaryotic organisms, including human prostatic and lysosomal acid phosphatases. Both wild type EcAP and mutant proteins were overproduced in E. coli using an expression system based on the T7 RNA polymerase promoter, and the proteins were purified to homogeneity. Examination of the purified mutant proteins by circular dichroism and proton NMR spectroscopy revealed no significant conformational changes. The replacement of Arg16 and His17 residues that were localized in a conserved N-terminal RHGXRXP motif resulted in the complete elimination of EcAP enzymatic activity. Critical roles for Arg20, Arg92, and His303 were also established because the corresponding mutant proteins exhibited residual activities that were not higher than 0.4% of that of wild type enzyme. In contrast, the replacement of Arg63 did not cause a significant alteration of the kinetic parameters. The results are in agreement with a previously postulated distant relationship between acid phosphatases, phosphoglycerate mutases, and fructose-2,6-bisphosphatase. These and earlier results are also consistent with the conclusion that 2 histidine residues participate in the catalytic mechanism of acid phosphatases, with His17 playing the role of a nucleophilic acceptor of the phospho group, whereas His303 may act as a proton donor to the alcohol or phenol.
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5. |
Nelson K,
Selander RK,
( 1992 ) Evolutionary genetics of the proline permease gene (putP) and the control region of the proline utilization operon in populations of Salmonella and Escherichia coli. PMID : 1400239 : DOI : 10.1128/jb.174.21.6886-6895.1992 PMC : PMC207367 Abstract >>
Virtually complete sequences (1,467 bp) of the proline permease gene (putP) and complete sequences (416 to 422 bp) of the control region of the proline utilization operon were determined for 16 strains of Salmonella, representing all eight subspecies, and 13 strains of Escherichia coli recovered from natural populations. Strains of Salmonella and E. coli differed, on average, at 16.3% of putP nucleotide sites and 17.5% of control region sites; the average difference between strains was much larger for Salmonella strains (4.6% of putP sites and 3.4% of control region sites) than for E. coli (2.4 and 0.9%, respectively). There was no difference in the distribution of polymorphic amino acid positions between the membrane-spanning and loop regions of the permease molecule, and rates of synonymous nucleotide substitution were virtually the same for the two domains. Statistical analysis yielded evidence of three probable cases of intragenic recombination, including the acquisition of a large segment of putP by strains of Salmonella subspecies VII from an unidentified source, the exchange of a 21-bp segment between two strains of E. coli, and the acquisition by one strain of E. coli of a cluster of 14 unique polymorphic control region sites from an unknown donor. An evolutionary tree for the putP and control region sequences was generally concordant with a tree for the gapA gene and a tree based on multilocus enzyme electrophoresis, thus providing evidence that for neither gene nor for enzyme genes in general has recombination occurred at rates sufficiently high or over regions sufficiently large to completely obscure phylogenetic relationships dependent on mutational divergence. It is suggested that the recombination rate varies among genes in relation to functional type, being highest for genes encoding cell surface and other proteins for which there is an adaptive advantage in structural diversity.
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6. |
Casalino M,
Latella MC,
Prosseda G,
Colonna B,
( 2003 ) CadC is the preferential target of a convergent evolution driving enteroinvasive Escherichia coli toward a lysine decarboxylase-defective phenotype. PMID : 14500464 : DOI : 10.1128/iai.71.10.5472-5479.2003 PMC : PMC201042 Abstract >>
Enteroinvasive E. coli (EIEC), like Shigella, is the etiological agent of bacillary dysentery, a particularly severe syndrome in children in developing countries. All EIEC strains share with Shigella the inability to synthesize lysine decarboxylase (the LDC phenotype). The lack of this function is considered a pathoadaptive mutation whose emergence was necessary to obtain the full expression of invasiveness. Cadaverine, the product of lysine decarboxylation, is a small polyamine which interferes mainly with the inflammatory process induced by dysenteric bacteria. Genes coding for lysine decarboxylase and its transporter constitute a single operon (cadBA) and are expressed at low pH under the positive control of CadC. This regulator is an inner membrane protein that is able to sense pH variation and to respond by transcriptionally activating the cadBA genes. In this study we show that, unlike in Shigella, mutations affecting the cad locus in the EIEC strains we have analyzed are not followed by a novel gene arrangement and that the LCD(-) phenotype is dependent mainly on inactivation of the cadC gene. Introduction of a functional CadC restores cadaverine expression in all EIEC strains harboring either an IS2 element or a defective cadC promoter. Comparative analysis between the cad regions of S. flexneri and EIEC suggests that the LDC(-) phenotype has been attained by different strategies within the E. coli species.
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7. |
Salomón RA,
Farías RN,
( 1992 ) Microcin 25, a novel antimicrobial peptide produced by Escherichia coli. PMID : 1429464 : DOI : 10.1128/jb.174.22.7428-7435.1992 PMC : PMC207439 Abstract >>
Microcin 25, a peptide antibiotic excreted by an Escherichia coli strain isolated from human feces, was purified to homogeneity and characterized. Composition analysis and data from gel filtration indicated that microcin 25 may contain 20 amino acid residues. It has a blocked amino-terminal end. Microcin synthesis and immunity are plasmid determined, and the antibiotic was produced in minimal medium when the cultures entered the stationary phase of growth. The peptide appears to interfere with cell division, since susceptible cells filamented when exposed to it. This response does not seem to be mediated by the SOS system.
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8. |
Bonacorsi S,
Clermont O,
Houdouin V,
Cordevant C,
Brahimi N,
Marecat A,
Tinsley C,
Nassif X,
Lange M,
Bingen E,
( 2003 ) Molecular analysis and experimental virulence of French and North American Escherichia coli neonatal meningitis isolates: identification of a new virulent clone. PMID : 12792866 : DOI : 10.1086/375347 Abstract >>
Phylogenetic relationships, virulence factors, alone and in specific combinations, and virulence in a rat meningitis model were examined among 132 isolates of Escherichia coli neonatal meningitis from France and North America. Isolates belonging to phylogenetic groups A (n=11), D (n=20), and B2 (n=99) had similar high prevalence rates of the siderophores aerobactin and yersiniabactin and the K1 capsule (>/=70%) yet induced different level of experimental bacteremia. Ectochromosomal DNA-like domains involved in blood-brain barrier passage (PAI III(536) [sfa/foc and iroN; 34%]; GimA [ibeA and ptnC; 38%]; PAI II(J96) [hly, cnf1, and hra; 10%]) were restricted to B2 isolates. Among group B2 isolates, representatives of the O45:K1 clonal group (n=30), which lacked these domains, were as able as the archetypal O18:K1 strain C5 to cause meningitis. Molecular epidemiology combined with experimental virulence assays demonstrate that known virulence factors are insufficient to fully explain the pathophysiology of ECNM and to allow for rational search for new virulence factors.
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9. |
Sharp PM,
Kelleher JE,
Daniel AS,
Cowan GM,
Murray NE,
( 1992 ) Roles of selection and recombination in the evolution of type I restriction-modification systems in enterobacteria. PMID : 1409708 : DOI : 10.1073/pnas.89.20.9836 PMC : PMC50228 Abstract >>
Restriction-modification systems can protect bacteria against viral infection. Sequences of the hsdM gene, encoding one of the three subunits of type I restriction-modification systems, have been determined for four strains of enterobacteria. Comparison with the known sequences of EcoK and EcoR124 indicates that all are homologous, though they fall into three families (exemplified by EcoK, EcoA, and EcoR124), the first two of which are apparently allelic. The extent of amino acid sequence identity between EcoK and EcoA is so low that the genes encoding them might be better termed pseudoalleles; this almost certainly reflects genetic exchange among highly divergent species. Within the EcoK family the ratio of intra- to interspecific divergence is very high. The extent of divergence between the genes from Escherichia coli K-12 and Salmonella typhimurium LT2 is similar to that for other genes with the same level of codon usage bias. In contrast, intraspecific divergence (between E. coli strains B and K-12) is extremely high and may reflect the action of frequency-dependent selection mediated by bacteriophages. There is also evidence of lateral transfer of a short sequence between E. coli and S. typhimurium.
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10. |
Shao J,
Li M,
Jia Q,
Lu Y,
Wang PG,
( 2003 ) Sequence of Escherichia coli O128 antigen biosynthesis cluster and functional identification of an alpha-1,2-fucosyltransferase. PMID : 14550554 : DOI : 10.1016/s0014-5793(03)00980-3 Abstract >>
O128 is one of the most common atypical enteropathogenic Escherichia coli isolated from diarrhea patients worldwide. The primary structure of E. coli O128 repeat units has previously been determined as -->3)-beta-D-GalNAc-(1-->4)-alpha-D-Gal-(1-->3)-beta-D-GalNAc-(1-->6)-[alpha-L-Fuc-(1-->2)]-beta-D-Gal-(1--> pentasaccharide. Here we report the complete sequencing of E. coli O128 antigen biosynthesis gene cluster and its flanking regions. Comparative sequence analysis revealed the expected O128 antigen process genes, GDP-fucose biosynthesis genes and four potential glycosyltransferase genes responsible for the assembly of E. coli O128 antigen repeats. WbsJ was shown to encode an alpha-1,2-fucosyltransferase by enzymatic assays and nuclear magnetic resonance spectroscopy analysis.
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11. |
Casarégola S,
Jacq A,
Laoudj D,
McGurk G,
Margarson S,
Tempête M,
Norris V,
Holland IB,
( 1992 ) Cloning and analysis of the entire Escherichia coli ams gene. ams is identical to hmp1 and encodes a 114 kDa protein that migrates as a 180 kDa protein. PMID : 1447789 : DOI : 10.1016/0022-2836(92)90489-7 Abstract >>
We have used an antibody to a previously identified 180 kDa (Hmp1) protein in Escherichia coli to clone the corresponding gene, which encodes a polypeptide of 114 kDa that has a mobility equivalent to 180 kDa in SDS/PAGE. We have demonstrated that the 180 kDa polypeptide is the primary gene product and not due to aggregation with other molecules. Moreover, our data indicate that the highly charged C-terminal region of the protein is responsible for its anomalous behaviour when analysed by SDS/PAGE. The hmp1 gene is in fact identical to ams (abnormal mRNA stability), also designated rne (RnaseE), and reported to have an ORF of 91 kDa. This discrepancy with the data in this paper can be ascribed to the omission of two bases in the previously reported sequence, generating an apparent stop codon. We previously demonstrated that the 180 kDa Hmp1/Ams protein cross reacted with both a polyclonal antibody and a monoclonal antibody raised against a yeast heavy chain myosin. However, we could detect no homology with myosin genes in the ams/hmp1 sequence. From the DNA sequence data, we identified a putative nucleotide binding site and a transmembrane domain in the N-terminal half of the molecule. In the C-terminal half, which appears to constitute a separate domain dominated by proline and charged amino acids, we also identified a region homologous to the highly conserved 70 kDa snRNP protein, involved in RNA splicing in eukaryotes. This feature would be consistent with reports that ams encodes RNaseE, an enzyme required for the processing of several stable RNAs in E. coli.
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12. |
Bakker D,
Willemsen PT,
Willems RH,
Huisman TT,
Mooi FR,
Oudega B,
Stegehuis F,
de Graaf FK,
( 1992 ) Identification of minor fimbrial subunits involved in biosynthesis of K88 fimbriae. PMID : 1400188 : DOI : 10.1128/jb.174.20.6350-6358.1992 PMC : PMC207580 Abstract >>
The nucleotide sequences of the genes faeF, faeH, faeI, and faeJ encoding K88 minor fimbrial subunits were determined. Analysis of the primary structure of the gene products revealed that all four proteins are synthesized with an amino-terminal signal sequence. The molecular masses of the mature FaeF, FaeH, FaeI, and FaeJ proteins were calculated to be 15,161, 25,461, 24,804, and 25,093 Da, respectively. FaeH, FaeI, and FaeJ showed significant homology with FaeG, the major fimbrial subunit of K88 fimbriae. Mutations in the respective genes were constructed. Analysis of the mutants showed that the minor fimbrial subunits FaeF and FaeH play an essential role in the biogenesis but not in the adhesive properties of the K88 fimbriae. Mutations in faeI or faeJ had no significant effect on K88 production or adhesive capacity. Specific antisera against FaeF and FaeH were raised by immunization with hybrid Cro-LacZ-FaeF and Cro-LacZ-FaeH proteins. Immunoblotting and immunoelectron microscopy revealed that FaeF and FaeH are located in or along the K88 fimbrial structure.
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13. |
Salazar L,
Lopéz J,
Andrés I,
Ortiz JM,
Rodríguez JC,
( 1992 ) Characterization and nucleotide sequence of the oriT-traM-finP region of the IncFVII plasmid pSU233. PMID : 1406590 : DOI : 10.1007/bf00538704 Abstract >>
By hybridizing the IncFVII haemolytic plasmid pSU233 with a probe containing the origin of transfer of the IncFII plasmid R1, we isolated a 1.9 kb BglII fragment containing at least the origin of transfer (oriT), and the genes traM and finP. Functional complementation analysis of deletion derivatives was used to map the origin of transfer. We also determined the nucleotide sequence of traM and finP. Comparison with similar regions of several plasmids, also belonging to the Rep-FIIA family, revelaed that pSU233 resembles the F plasmid by very close. The homology is not evenly distributed along this region, but clustered into homologous regions (TraZb-oriT, TraMb-oriT and traM separated by non-homologous regions (TraYb-oriT, finP). This organization resembles that reported for the replication region and also suggests evolution by exchange of modules. In addition, the nucleotide sequence of finP is different from those previously described for other IncF plasmids and constitutes a new allele, which we have denominated allele VI.
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14. |
Shaikh N,
Tarr PI,
( 2003 ) Escherichia coli O157:H7 Shiga toxin-encoding bacteriophages: integrations, excisions, truncations, and evolutionary implications. PMID : 12775697 : DOI : 10.1128/jb.185.12.3596-3605.2003 PMC : PMC156235 Abstract >>
As it descended from Escherichia coli O55:H7, Shiga toxin (Stx)-producing E. coli (STEC) O157:H7 is believed to have acquired, in sequence, a bacteriophage encoding Stx2 and another encoding Stx1. Between these events, sorbitol-fermenting E. coli O157:H(-) presumably diverged from this clade. We employed PCR and sequence analyses to investigate sites of bacteriophage integration into the chromosome, using evolutionarily informative STEC to trace the sequence of acquisition of elements encoding Stx. Contrary to expectations from the two currently sequenced strains, truncated bacteriophages occupy yehV in almost all E. coli O157:H7 strains that lack stx(1) (stx(1)-negative strains). Two truncated variants were determined to contain either GTT or TGACTGTT sequence, in lieu of 20,214 or 18,895 bp, respectively, of the bacteriophage central region. A single-nucleotide polymorphism in the latter variant suggests that recombination in that element extended beyond the inserted octamer. An stx(2) bacteriophage usually occupies wrbA in stx(1)(+)/stx(2)(+) E. coli O157:H7, but wrbA is unexpectedly unoccupied in most stx(1)-negative/stx(2)(+) E. coli O157:H7 strains, the presumed progenitors of stx(1)(+)/stx(2)(+) E. coli O157:H7. Trimethoprim-sulfamethoxazole promotes the excision of all, and ciprofloxacin and fosfomycin significantly promote the excision of a subset of complete and truncated stx bacteriophages from the E. coli O157:H7 strains tested; bile salts usually attenuate excision. These data demonstrate the unexpected diversity of the chromosomal architecture of E. coli O157:H7 (with novel truncated bacteriophages and multiple stx(2) bacteriophage insertion sites), suggest that stx(1) acquisition might be a multistep process, and compel the consideration of multiple exogenous factors, including antibiotics and bile, when chromosome stability is examined.
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15. |
Motallebi-Veshareh M,
Balzer D,
Lanka E,
Jagura-Burdzy G,
Thomas CM,
( 1992 ) Conjugative transfer functions of broad-host-range plasmid RK2 are coregulated with vegetative replication. PMID : 1376390 : DOI : 10.1111/j.1365-2958.1992.tb01541.x Abstract >>
The kilB locus (which is unclonable in the absence of korB) of broad-host-range plasmid RK2 (60 kb) lies between the trfA operon (co-ordinates 16.4 to 18.2 kb), which encodes a protein essential for vegetative replication, and the Tra2 block of conjugative transfer genes (co-ordinates 20.0 to 27.0 kb). Promoter probe studies indicated that kilB is transcribed clockwise from a region containing closely spaced divergent promoters, one of which is the trfA promoter. The repression of both promoters by korB suggested that kilB may also play a role in stable maintenance of RK2. We have sequenced the region containing kilB and analysed it by deletion and insertion mutagenesis. Loss of the KilB+ phenotype does not result in decreased stability of mini RK2 plasmids. However insertion in ORFI (kilBI) of the region analysed results in a Tra- phenotype in plasmids which are otherwise competent for transfer, demonstrating that this locus is essential for transfer and is probably the first gene of the Tra2 region. From the kilBI DNA sequence KilBI is predicted to be 34995 Da, in line with M(r) = 36,000 observed by sodium dodecyl sulphate/polyacrylamide gel electrophoresis, and contains a type I ATP-binding motif. The purified product was used to raise antibody which allowed the level of KilBI produced from RK2 to be estimated at approximately 2000 molecules per bacterium. Protein sequence comparisons showed the highest homology score with VirB11, which is essential for the transfer of the Agrobacterium tumefaciens Ti plasmid DNA from bacteria to plant cells. The sequence similarity of both KilBI and VirB11 to a family of protein export functions suggested that KilBI may be involved in assembly of the surface-associated Tra functions. The data presented in this paper provide the first demonstration of coregulation of genes required for vegetative replication and conjugative transfer on a bacterial plasmid.
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16. |
Wilson KA,
Kalkum M,
Ottesen J,
Yuzenkova J,
Chait BT,
Landick R,
Muir T,
Severinov K,
Darst SA,
( 2003 ) Structure of microcin J25, a peptide inhibitor of bacterial RNA polymerase, is a lassoed tail. PMID : 14531691 : DOI : 10.1021/ja036756q Abstract >>
Microcin J25 (MccJ25) is a 21-amino acid peptide inhibitor active against the DNA-dependent RNA polymerase of Gram negative bacteria. Previously, the structure of MccJ25 was reported to be a head-to-tail circle, cyclo(-G(1)GAGHVPEYF(10)VGIGTPISFY(20)G-). On the basis of biochemical studies, mass spectrometry, and NMR, we show that this structure is incorrect, and that the peptide has an extraordinary structural fold. MccJ25 contains an internal lactam linkage between the alpha-amino group of Gly1 and the gamma-carboxyl of Glu8. The tail (Tyr9-Gly21) passes through the ring (Gly1-Glu8), with Phe19 and Tyr20 straddling each side of the ring, sterically trapping the tail in a noncovalent interaction we call a lassoed tail.
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17. |
Kholodii G,
Mindlin S,
Petrova M,
Minakhina S,
( 2003 ) Tn5060 from the Siberian permafrost is most closely related to the ancestor of Tn21 prior to integron acquisition. PMID : 14553919 : DOI : 10.1016/S0378-1097(03)00559-7 Abstract >>
A Tn21-related mercury resistance transposon, Tn5060, has been isolated from Pseudomonas sp. strain A19-1 from a 8,000-10,000-year-old Siberian permafrost sample, and sequenced. Like Tn21, the element transposes to different plasmids at a frequency of 10(-2)-10(-3) per target plasmid transfer. Comparison of the complete Tn5060 DNA sequence (8,667 bp) with that of Tn21 (19,672 bp) shows that Tn5060 does not contain integron In2 and deviates from Tn21 in four nucleotide positions. These and other comparative data demonstrate that Tn5060 is the most closely related of the characterized mercury resistances to the as yet hypothetical immediate ancestor of Tn21, TnX.
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18. |
Waters VL,
Strack B,
Pansegrau W,
Lanka E,
Guiney DG,
( 1992 ) Mutational analysis of essential IncP alpha plasmid transfer genes traF and traG and involvement of traF in phage sensitivity. PMID : 1400217 : DOI : 10.1128/jb.174.20.6666-6673.1992 PMC : PMC207648 Abstract >>
Although the broad-host-range IncP plasmids can vegetatively replicate in diverse gram-negative bacteria, the development of shuttle vector systems has established that the host range for IncP plasmid conjugative transfer is greater than the range of bacteria that sustain IncP replicons. Towards understanding IncP plasmid conjugation and the connection between IncP conjugation and Agrobacterium tumefaciens T-DNA transfer to plants, two sets of mutants were generated in the larger transfer region (Tra1) of the IncP alpha plasmid RK2. Mutagenesis strategies were chosen to minimize transcriptional polar effects. Mutant Tra1 clones were mapped, sequenced, and processed to reconstruct 49.5-kb Tra2-containing plasmid derivatives in order to assay for transfer activity and IncP plasmid-specific phage sensitivity. Focusing on the activities of the gene products of traF and traG in Escherichia coli, we found that mutations in traF abolished transfer activity and rendered the host cells phage resistant and mutations in traG abolished transfer activity but had no effect on phage sensitivity. Complementation of these mutant derivatives with corresponding trans-acting clones carrying traF or traG restored transfer activity and, in the case of the traF mutant, the phage sensitivity of the host cell. We conclude that in E. coli, both TraF and TraG are essential for IncP plasmid transfer and that TraF is necessary (but not sufficient) for donor-specific phage sensitivity, and sequencing data suggest that both TraF and TraG are membrane spanning.
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19. |
Raha M,
Kawagishi I,
Müller V,
Kihara M,
Macnab RM,
( 1992 ) Escherichia coli produces a cytoplasmic alpha-amylase, AmyA. PMID : 1400215 : DOI : 10.1128/jb.174.20.6644-6652.1992 PMC : PMC207642 Abstract >>
In the gap between two closely linked flagellar gene clusters on the Escherichia coli and Salmonella typhimurium chromosomes (at about 42 to 43 min on the E. coli map), we found an open reading frame whose sequence suggested that it encoded an alpha-amylase; the deduced amino acid sequences in the two species were 87% identical. The strongest similarities to other alpha-amylases were to the excreted liquefying alpha-amylases of bacilli, with > 40% amino acid identity; the N-terminal sequence of the mature bacillar protein (after signal peptide cleavage) aligned with the N-terminal sequence of the E. coli or S. typhimurium protein (without assuming signal peptide cleavage). Minicell experiments identified the product of the E. coli gene as a 56-kDa protein, in agreement with the size predicted from the sequence. The protein was retained by spheroplasts rather than being released with the periplasmic fraction; cells transformed with plasmids containing the gene did not digest extracellular starch unless they were lysed; and the protein, when overproduced, was found in the soluble fraction. We conclude that the protein is cytoplasmic, as predicted by its sequence. The purified protein rapidly digested amylose, starch, amylopectin, and maltodextrins of size G6 or larger; it also digested glycogen, but much more slowly. It was specific for the alpha-anomeric linkage, being unable to digest cellulose. The principal products of starch digestion included maltotriose and maltotetraose as well as maltose, verifying that the protein was an alpha-amylase rather than a beta-amylase. The newly discovered gene has been named amyA. The natural physiological role of the AmyA protein is not yet evident.
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20. |
Jordi BJ,
Dagberg B,
de Haan LA,
Hamers AM,
van der Zeijst BA,
Gaastra W,
Uhlin BE,
( 1992 ) The positive regulator CfaD overcomes the repression mediated by histone-like protein H-NS (H1) in the CFA/I fimbrial operon of Escherichia coli. PMID : 1378396 : PMC : PMC556738 Abstract >>
CFA/I fimbriae of enterotoxigenic Escherichia coli are expressed at 37 degrees C and not at 20 degrees C. Expression of CFA/I fimbriae requires two DNA regions (regions 1 and 2) which are separated by 40 kb on the wild type plasmid. Region 2 encodes a protein (CfaD) which activates the promoter in region 1. We investigated whether the histone-like protein H-NS (H1) of E.coli is involved in the temperature regulated expression of CFA/I fimbriae. As demonstrated recently with other temperature regulated genes, a mutation in the gene coding for this nucleoid-associated H-NS (H1) protein resulted in derepression of CFA/I expression. CFA/I fimbriae were now expressed both at 20 degrees C and 37 degrees C. More strinkingly, the positive regulator CfaD was not needed for CFA/I expression in an H-NS- strain. This indicates that CfaD diminishes an inhibitory effect of the H-NS nucleoid-associated protein. We also showed that in the H-NS- strain the CfaD protein still has a positive effect on the transcription of CFA/I.
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21. |
Rosengren KJ,
Clark RJ,
Daly NL,
Göransson U,
Jones A,
Craik DJ,
( 2003 ) Microcin J25 has a threaded sidechain-to-backbone ring structure and not a head-to-tail cyclized backbone. PMID : 14531690 : DOI : 10.1021/ja0367703 Abstract >>
Microcin J25 is a 21 amino acid bacterial peptide that has potent antibacterial activity against Gram-negative bacteria, resulting from its interaction with RNA polymerase. The peptide was previously proposed to have a head-to-tail cyclized peptide backbone and a tight globular structure (Blond, A., P?duzzi, J., Goulard, C., Chiuchiolo, M. J., Barth?l?my, M., Prigent, Y., Salom?n, R. A., Far?as, R. N., Moreno, F. & Rebuffat, S. Eur. J. Biochem. 1999, 259, 747-755). It exhibits remarkable thermal stability for a peptide of its size lacking disulfide bonds and in part this was previously proposed to derive from its macrocyclic structure. We show here that in fact the peptide does not have a head-to-tail cyclic structure but rather a side chain to backbone cyclization between Glu8 and the N-terminus. This creates an embedded ring that is threaded by the C-terminal tail of the molecule, forming a noose-like feature. The three-dimensional structure deduced from NMR data suggests that slippage of the noose is prevented by two aromatic residues flanking the embedded ring. Unthreading does not occur even when the molecule is enzymatically digested with thermolysin. The new structural interpretation fully accounts for previously reported NMR and biophysical data and is consistent with the remarkable stability of this potent antimicrobial peptide.
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22. |
Bayro MJ,
Mukhopadhyay J,
Swapna GV,
Huang JY,
Ma LC,
Sineva E,
Dawson PE,
Montelione GT,
Ebright RH,
( 2003 ) Structure of antibacterial peptide microcin J25: a 21-residue lariat protoknot. PMID : 14531661 : DOI : 10.1021/ja036677e Abstract >>
The antibacterial peptide microcin J25 (MccJ25) inhibits bacterial transcription by binding within, and obstructing, the nucleotide-uptake channel of bacterial RNA polymerase. Published covalent and three-dimensional structures indicate that MccJ25 is a 21-residue cycle. Here, we show that the published covalent and three-dimensional structures are incorrect, and that MccJ25 in fact is a 21-residue "lariat protoknot", consisting of an 8-residue cyclic segment followed by a 13-residue linear segment that loops back and threads through the cyclic segment. MccJ25 is the first example of a lariat protoknot involving a backbone-side chain amide linkage.
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23. |
Bockmann J,
Heuel H,
Lengeler JW,
( 1992 ) Characterization of a chromosomally encoded, non-PTS metabolic pathway for sucrose utilization in Escherichia coli EC3132. PMID : 1435727 : DOI : 10.1007/bf00286177 Abstract >>
A wild-type isolate, EC3132, of Escherichia coli, that is able to grow on sucrose was isolated and its csc genes (mnemonic for chromosomally coded sucrose genes) transferred to strains of E. coli K12. EC3132 and all sucrose-positive exconjugants and transductants invariably showed a D-serine deaminase (Dsd)-negative phenotype. The csc locus maps adjacent to dsdA, the structural gene for the D-serine deaminase, and contains an inducible regulon, controlled by a sucrose-specific repressor CscR, together with structural genes for a sucrose hydrolase (invertase) CscA, for a D-fructokinase CscK, and for a transport system CscB. Based on DNA sequencing studies, this last codes for a hydrophobic protein of 415 amino acids. CscB is closely related to the beta-galactoside transport system LacY (31.2% identical residues) and a raffinose transport system RafB (32.3% identical residues) of the enteric bacteria, both of the proton symport type. A two-dimensional model common to the three transport proteins, which is based on the integrated consensus sequence, will be discussed.
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24. |
van der Woude MW,
Braaten BA,
Low DA,
( 1992 ) Evidence for global regulatory control of pilus expression in Escherichia coli by Lrp and DNA methylation: model building based on analysis of pap. PMID : 1357527 : DOI : 10.1111/j.1365-2958.1992.tb01418.x Abstract >>
Pyelonephritis-associated pilus (Pap) expression is regulated by a phase variation control mechanism involving PapB, Papl, catabolite activator protein (CAP), leucine-responsive regulatory protein (Lrp) and deoxyadenosine methylase (Dam). Lrp and Papl bind to a specific non-methylated pap regulatory DNA region containing the sequence 'GATC' and facilitate the formation of an active transcriptional complex. Evidence indicates that binding of Lrp and Papl to this region inhibits methylation of the GATC site by Dam. However, if this GATC site is first methylated by Dam, binding of Lrp and Papl is inhibited. These events lead to the formation of two different pap methylation states characteristic of active (ON) and inactive (OFF) pap transcription states. The fae (K88), daa (F1845) and sfa (S) pilus operons share conserved 'GATC-box' domains with pap and may be subject to a similar regulatory control mechanism involving Lrp and DNA methylation.
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25. |
Csitkovits VC,
Zechner EL,
( 2003 ) Extent of single-stranded DNA required for efficient TraI helicase activity in vitro. PMID : 14506243 : DOI : 10.1074/jbc.M310025200 Abstract >>
The IncF plasmid protein TraI functions during bacterial conjugation as a site- and strand-specific DNA transesterase and a highly processive 5' to 3' DNA helicase. The N-terminal DNA transesterase domain of TraI localizes the protein to nic and cleaves this site within the plasmid transfer origin. In the cell the C-terminal DNA helicase domain of TraI is essential for driving the 5' to 3' unwinding of plasmid DNA from nic to provide the strand destined for transfer. In vitro, however, purified TraI protein cannot enter and unwind nicked plasmid DNA and instead requires a 5' tail of single-stranded DNA at the duplex junction. In this study we evaluate the extent of single-stranded DNA adjacent to the duplex that is required for efficient TraI-catalyzed DNA unwinding in vitro. A series of linear partial duplex DNA substrates containing a central stretch of single-stranded DNA of defined length was created and its structure verified. We found that substrates containing >or=27 nucleotides of single-stranded DNA 5' to the duplex were unwound efficiently by TraI, whereas substrates containing 20 or fewer nucleotides were not. These results imply that during conjugation localized unwinding of >20 nucleotides at nic is necessary to initiate unwinding of plasmid DNA strands.
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26. |
Ivanova A,
Renshaw M,
Guntaka RV,
Eisenstark A,
( 1992 ) DNA base sequence variability in katF (putative sigma factor) gene of Escherichia coli. PMID : 1437569 : DOI : 10.1093/nar/20.20.5479 PMC : PMC334364 Abstract >>
N/A
|
27. |
Chanal C,
Poupart MC,
Sirot D,
Labia R,
Sirot J,
Cluzel R,
( 1992 ) Nucleotide sequences of CAZ-2, CAZ-6, and CAZ-7 beta-lactamase genes. PMID : 1416873 : DOI : 10.1128/aac.36.9.1817 PMC : PMC192192 Abstract >>
CAZ-2, CAZ-6, and CAZ-7 are plasmid-mediated beta-lactamases that are markedly active against ceftazidime. The corresponding structural genes were amplified by the polymerase chain reaction. Nucleotide sequences were determined by direct sequencing of the amplified products. Analysis of the nucleotide and the deduced amino acid sequences showed that CAZ-2, CAZ-6, and CAZ-7 are derived from TEM-2 by three, four, and two amino acid substitutions, respectively. All these substitutions are located at positions 102, 162, 235, 236, and 237 (Sutcliffe numbering), which are known to extend the substrate range of beta-lactamases. These substitutions are Lys-102, Ser-162, and Ser-236 in CAZ-2; Lys-102, Ser-162, Thr-235, and Lys-237 in CAZ-6; and Lys-102 and His-162 in CAZ-7. These results indicate that the nucleotide sequence of CAZ-2 is identical to that of TEM-8. The nucleotide sequence of CAZ-7 possesses the two mutations described in TEM-16 by the oligotyping method. In contrast, the combination of mutations encountered in CAZ-6 has not yet been described, and this enzyme was designated TEM-24.
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28. |
Kim SR,
Komano T,
( 1992 ) Nucleotide sequence of the R721 shufflon. PMID : 1400257 : DOI : 10.1128/jb.174.21.7053-7058.1992 PMC : PMC207388 Abstract >>
The shufflon is a DNA region that undergoes complex rearrangement mediated by the product of a putative site-specific recombinase gene, rci. The DNA sequences of the shufflon region and the rci gene of IncI2 plasmid R721 were determined. The R721 shufflon consists of three invertible DNA segments that are homologous to the shufflon segments found in IncI1 plasmid R64. Structural analysis of open reading frames indicated that the R721 shufflon possibly functions as a biological switch for selecting one of the six pilV genes in which the N-terminal region is constant and the C-terminal region is variable. The R721 rci gene was shown to encode a basic protein of 374 amino acid residues.
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29. |
Marklund BI,
Tennent JM,
Garcia E,
Hamers A,
Båga M,
Lindberg F,
Gaastra W,
Normark S,
( 1992 ) Horizontal gene transfer of the Escherichia coli pap and prs pili operons as a mechanism for the development of tissue-specific adhesive properties. PMID : 1357526 : DOI : 10.1111/j.1365-2958.1992.tb01399.x Abstract >>
Escherichia coli strains bind to Gal alpha 1-4Gal-containing glycolipids via P pili-associated G-adhesins. Three functional classes of adhesins with different binding specificities are encoded by conserved G-alleles. We suggest that the Class I papG-allele of strain J96 is a novel acquisition possibly introduced via horizontal gene transfer into one of the two P pili gene clusters carried by this strain. Closely related strains in the ECOR collection of natural E. coli isolates carry either a Class II or a Class III G-adhesin. Data indicate that genetic exchanges involving either entire pap or prs gene clusters or individual pap/prs genes have occurred. We propose that the retention and spread of pap/prs DNA among E. coli is the result of selection pressure exerted by mammalian intestinal isoreceptors.
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30. |
Biskri L,
Mazel D,
( 2003 ) Erythromycin esterase gene ere(A) is located in a functional gene cassette in an unusual class 2 integron. PMID : 14506050 : DOI : 10.1128/aac.47.10.3326-3331.2003 PMC : PMC201170 Abstract >>
The gene ere(A) of the plasmid pIP1100 is larger than originally reported and is organized as an integron gene cassette. The ere(A) gene cassette carries its own promoter and is propagated by a class 2 integron with an insertion sequence element, IS1, inserted upstream of the intI2 gene. The mobility of the ere(A) cassette has been demonstrated.
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31. |
Janulaitis A,
Vaisvila R,
Timinskas A,
Klimasauskas S,
Butkus V,
( 1992 ) Cloning and sequence analysis of the genes coding for Eco57I type IV restriction-modification enzymes. PMID : 1334261 : DOI : 10.1093/nar/20.22.6051 PMC : PMC334472 Abstract >>
A 6.3 kb fragment of E.coli RFL57 DNA coding for the type IV restriction-modification system Eco57I was cloned and expressed in E.coli RR1. A 5775 bp region of the cloned fragment was sequenced which contains three open reading frames (ORF). The methylase gene is 1623 bp long, corresponding to a protein of 543 amino acids (62 kDa); the endonuclease gene is 2991 bp in length (997 amino acids, 117 kDa). The two genes are transcribed convergently from different strands with their 3'-ends separated by 69 bp. The third short open reading frame (186 bp, 62 amino acids) has been identified, that precedes and overlaps by 7 nucleotides the ORF encoding the methylase. Comparison of the deduced Eco57I endonuclease and methylase amino acid sequences revealed three regions of significant similarity. Two of them resemble the conserved sequence motifs characteristic of the DNA[adenine-N6] methylases. The third one shares similarity with corresponding regions of the PaeR7I, TaqI, CviBIII, PstI, BamHI and HincII methylases. Homologs of this sequence are also found within the sequences of the PaeR7I, PstI and BamHI restriction endonucleases. This is the first example of a family of cognate restriction endonucleases and methylases sharing homologous regions. Analysis of the structural relationship suggests that the type IV enzymes represent an intermediate in the evolutionary pathway between the type III and type II enzymes.
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32. |
Janulaitis A,
Petrusyte M,
Maneliene Z,
Klimasauskas S,
Butkus V,
( 1992 ) Purification and properties of the Eco57I restriction endonuclease and methylase--prototypes of a new class (type IV). PMID : 1334260 : DOI : 10.1093/nar/20.22.6043 PMC : PMC334471 Abstract >>
The Eco57I restriction endonuclease and methylase were purified to homogeneity from the E.coli RR1 strain carrying the eco57IRM genes on a recombinant plasmid. The molecular weight of the denaturated methylase is 63 kDa. The restriction endonuclease exists in a monomeric form with an apparent molecular weight of 104-108 kDa. R.Eco57I also possesses methylase activity. The methylation activities of both enzymes modify the outer A residue in the target sequence 5'CTGAAG yielding N6-methyladenine. M.Eco57I modifies both strands of the substrate while R.Eco57I modifies only one. Only the methylase enzyme is stimulated by Ca2+. The restriction endonuclease shows an absolute requirement for Mg2+ and is stimulated by AdoMet. ATP has no influence on either activity of the enzymes. The subunit structure and enzymatic properties of the Eco57I enzymes distinguish them from all other restriction-modification enzymes that have been described previously. Therefore, RM.Eco57I may be regarded as a representative of a novel class of restriction-modification systems, and we propose to classify it as type IV.
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33. |
Herzer PJ,
Inouye S,
Inouye M,
( 1992 ) Retron-Ec107 is inserted into the Escherichia coli genome by replacing a palindromic 34bp intergenic sequence. PMID : 1372675 : DOI : 10.1111/j.1365-2958.1992.tb01477.x Abstract >>
Some natural isolates of Escherichia coli have been shown to produce a unique branched RNA-linked single-stranded DNA called msDNA. These bacteria contain a retro-element called retron consisting of the msr-msd region and the gene for reverse transcriptase (RT). All three E. coli retrons characterized to date have been shown to be integrated into a prophage or to be associated with phage-related genes. In this report, we identified a new msDNA from an E. coli wild strain. Using the msDNA as a probe, the retron for the msDNA was cloned and its DNA sequence was determined. The retron was found to consist of a 1.3kb DNA fragment, making it the smallest retron isolated to date. The msDNA produced from the retron consists of a 107 base single-stranded DNA, which is considered to be branched out from the 18th G residue of a 75-base RNA molecule by a 2',5'-phosphodiester linkage. Thus, the msDNA and the retron were designated msDNA-Ec107 and retron-Ec107, respectively. Most significantly, retron-Ec107 was inserted into the E. coli genome by replacing a 34bp intergenic sequence between the pyrE and ttk genes located at 82 min on the E. coli chromosome. Interestingly, the retron contains palindromic structures at both ends and the E. coli 34bp intergenic sequence also contains a 10bp inverted repeat structure. These palindromic structures might have played a role in the integration of retron-Ec107 into the E. coli genome.
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34. |
Clark CA,
Heuzenroeder MW,
Manning PA,
( 1992 ) Colonization factor antigen CFA/IV (PCF8775) of human enterotoxigenic Escherichia coli: nucleotide sequence of the CS5 determinant. PMID : 1371766 : PMC : PMC257624 Abstract >>
Human enterotoxigenic Escherichia coli isolates expressing the colonization factor antigen CFA/IV (previously designated PCF8775) produce plasmid-encoded CS5 fimbriae. The nucleotide sequence of the region encoding the major CS5 fimbrial subunit was determined. The subunit is synthesized as a precursor of 203 amino acids (20.85 kDa) with a mature protein of 181 amino acids corresponding to a size of 18.6 kDa. The CS5 subunit shows homology to the corresponding component of porcine enterotoxigenic E. coli F41, particularly within the signal sequence and at the carboxy terminus.
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35. |
Juranka P,
Zhang F,
Kulpa J,
Endicott J,
Blight M,
Holland IB,
Ling V,
( 1992 ) Characterization of the hemolysin transporter, HlyB, using an epitope insertion. PMID : 1371277 : Abstract >>
The prokaryotic hlyB gene product is a member of a superfamily of ATP-binding transport proteins that include the eukaryotic multidrug-resistance P-glycoprotein, the yeast STE6, and the cystic fibrosis CFTR gene products (Juranka, P. F., Zastawny, R. L., and Ling, V. (1989) FASEB J. 3, 2583-2592). Previous genetic studies have indicated that HlyB is involved in the transport of the 107-kDa HlyA protein from Escherichia coli; however, the HlyB protein has not been purified for biochemical studies due to its low abundance. In this study, we have engineered a monoclonal antibody epitope into the C-terminal end of HlyB that did not destroy its function. This has allowed us to use immunological methods to identify and localize various molecular forms of the HlyB protein present in vivo.
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36. |
Sommerfelt H,
Grewal HM,
Svennerholm AM,
Gaastra W,
Flood PR,
Viboud G,
Bhan MK,
( 1992 ) Genetic relationship of putative colonization factor O166 to colonization factor antigen I and coli surface antigen 4 of enterotoxigenic Escherichia coli. PMID : 1354200 : PMC : PMC257392 Abstract >>
Plasmid DNA from two strains of enterotoxigenic Escherichia coli harboring genes encoding coli surface antigen 4 (CS4) and from seven Indian enterotoxigenic E. coli isolates cross-hybridized at low stringency but not at high stringency with two polynucleotide probes derived from the colonization factor antigen I (CFA/I) operon. Low-stringency Southern blot hybridization of PstI-digested plasmid DNA from the seven Indian isolates yielded characteristic restriction fragment patterns, distinct from those of CS4- and CFA/I-associated plasmid DNA. Two of the Indian strains were transformed with a recombinant plasmid harboring the cfaD gene, which encodes a positive regulator of CFA/I and CS4 genes. The cfaD transformants produced large amounts of putative colonization factor O166 (PCFO166) irrespective of whether the nutrient agar contained bile salts, a growth factor otherwise required for adequate PCFO166 expression. A considerable interstrain variation in the level of PCFO166 production could be explained by differences in the proportion of bacteria that were fimbriated, as visualized by electron microscopy. The N-terminal amino acid sequence of PCFO166 fimbrial protein showed a high degree of homology with the corresponding sequences of CFA/I and CS4.
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37. |
Donnenberg MS,
Girón JA,
Nataro JP,
Kaper JB,
( 1992 ) A plasmid-encoded type IV fimbrial gene of enteropathogenic Escherichia coli associated with localized adherence. PMID : 1362446 : DOI : 10.1111/j.1365-2958.1992.tb02210.x Abstract >>
Enteropathogenic Escherichia coli (EPEC) form adherent microcolonies on the surface of tissue culture cells in a pattern termed localized adherence. Localized adherence requires the presence of a large EPEC adherence factor (EAF) plasmid. Recently a bundle-forming pilus has been described in EPEC possessing the EAF plasmid. An analysis of 22 non-invasive EPEC TnphoA mutants revealed that seven have insertions in the EAF plasmid and are incapable of localized adherence. We report here the mapping of the TnphoA insertions in these mutants. The nucleotide sequence of the gene interrupted in these TnphoA mutants (bfpA) was determined and found to correspond to the N-terminal amino acid sequence of the major structural protein of the bundle-forming pilus. The bfpA gene bears sequence similarities to members of the type IV fimbrial gene family and encodes a potential site for processing by a prepilin peptidase. A plasmid containing bfpA as the only open reading frame directs the synthesis of a protein recognized by antiserum raised against the bundle-forming pilus. TnphoA mutants at this locus are unable to synthesize BfpA, but synthesis is restored by introduction of a plasmid containing the cloned gene. The minimum fragment of DNA required to restore localized adherence is considerably greater than that required to restore BfpA synthesis. BfpA expression, as assessed by alkaline phosphatase activity in bfpA::TnphoA mutants, is affected by temperature and growth medium. These studies describe an EPEC plasmid-encoded fimbrial gene, a candidate for the elusive EPEC adherence factor responsible for localized adherence.
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38. |
Sekizaki T,
Miyazaki S,
Ito H,
Asawa T,
Nonomura I,
( 1992 ) Isolation and characterization of type 1 fimbriae from a chicken pathogenic Escherichia coli serotype O78. PMID : 1362082 : DOI : 10.1292/jvms.54.1145 Abstract >>
Type 1 fimbriae from chicken pathogenic Escherichia coli strain PDI-386 (serotype O78) was purified and characterized. Because of the acid-induced autoagglutination (T. Sekizaki, Y. Nakasato, and I. Nonomura, J. Vet. Med. Sci. 54, 493-499, 1992), the fimbriae could be easily purified by repeating acid sedimentation, washing, and dissolving in buffer (pH 8.0). In electron microscopy, the purified fimbriae showed a filament of 8 nm in diameter and 10 microns in average length. The molecular mass of the protein subunit of the purified fimbriae estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was 19,000 daltons. The amino acid composition and its NH2-terminal sequence were similar to the previously described one of the Klebsiella pneumoniae type 1 fimbriae. Moreover, there was an immunological relatedness between them. These results indicated that a molecular diversity found between the fimbriae of E. coli and that of K. pneumoniae has already been existed among chicken pathogenic E. coli strains.
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39. |
Wang MX,
Church GM,
( 1992 ) A whole genome approach to in vivo DNA-protein interactions in E. coli. PMID : 1334233 : DOI : 10.1038/360606a0 Abstract >>
The increasingly rapid pace at which genomic DNA sequences are being determined has created a need for more efficient techniques to determine which parts of these sequences are bound in vivo by the proteins controlling processes such as gene expression, DNA replication and chromosomal mechanics. Here we describe a whole-genome approach to identify and characterize such DNA sequences. The method uses endogenous or artificially introduced methylases to methylate all genomic targets except those protected in vivo by protein or non-protein factors interfering with methylase action. These protected targets remain unmethylated in purified genomic DNA and are identified using methylation-sensitive restriction endonucleases. When the method was applied to the Escherichia coli genome, 0.1% of the endogenous adenine methyl-transferase (Dam methylase) targets were found to be unmethylated. Five foreign methylases were examined by transfection. Database-matched DNA sequences flanking the in vivo-protected Dam sites all fell in the non-coding regions of seven E. coli operons (mtl, cdd, flh, gut, car, psp and fep). In the first four operons these DNA sequences closely matched the consensus sequence that binds to the cyclic AMP-receptor protein. The in vivo protection at the Dam site upstream of the car operon was correlated with a downregulation of car expression, as expected of a feedback repressor-binding model.
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40. |
Makino S,
Tobe T,
Asakura H,
Watarai M,
Ikeda T,
Takeshi K,
Sasakawa C,
( 2003 ) Distribution of the secondary type III secretion system locus found in enterohemorrhagic Escherichia coli O157:H7 isolates among Shiga toxin-producing E. coli strains. PMID : 12791847 : DOI : 10.1128/jcm.41.6.2341-2347.2003 PMC : PMC156528 Abstract >>
The ability of the complete genome sequence of enterohemorrhagic Escherichia coli O157 led to the identification of a 17-kb chromosomal region which contained a type III secretion system gene cluster at min 64.5. This locus contains open reading frames whose amino acid sequences show high degrees of similarity with those of proteins that make up the type III secretion apparatus, which is encoded by the inv-spa-prg locus on a Salmonella SPI-1 pathogenicity island. This locus was designated ETT2 (E. coli type III secretion 2) and consisted of the epr, epa, and eiv genes. ETT2 was found in enteropathogenic E. coli strains and also in some non-O157 Shiga toxin-producing E. coli (STEC) strains, but most of them contained a truncated portion of ETT2. Most O157 isolates had a complete collection of toxin-encoding genes eae and hlyA and the ETT2 locus, while most O26 strains had toxin-encoding genes eae and hlyA genes but an incomplete ETT2 locus. Thus, an intact copy of ETT2 might mark a pathogenic distinction for particular STEC strains. Therefore, the presence of the ETT2 locus can be used for identification of truly pathogenic STEC strains and for molecular fingerprinting of the epidemic strains in humans and animals.
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41. |
Mabilat C,
Lourençao-Vital J,
Goussard S,
Courvalin P,
( 1992 ) A new example of physical linkage between Tn1 and Tn21: the antibiotic multiple-resistance region of plasmid pCFF04 encoding extended-spectrum beta-lactamase TEM-3. PMID : 1331747 : DOI : 10.1007/bf00286188 Abstract >>
The genetic environment of plasmid-borne blaTEM mutant genes, encoding nine distinct TEM-type extended-spectrum beta-lactamases, was studied in transconjugants from clinical isolates of enterobacteria. Colony hybridization with probes specific for tnpA and tnpR of Tn3, tnpA and tnpI of Tn21, aacA4, and IS15, and restriction endonuclease analysis of plasmid DNA indicated that the structural genes for the enzymes were always associated with intact or deleted variants of the Tn3 family. Four of the nine blaTEM variants, which account for 62% of 222 isolates in a molecular epidemiological study, were associated with replicons indistinguishable from the epidemic Inc7-M plasmid pCFF04 that carries the blaTEM-3 gene. This suggests that mutant genes were selected from the same prototype plasmid carrying penicillinase genes blaTEM-1 or -2. A 6.6 kb DNA fragment of pCFF04 containing blaTEM-3 was characterized by amplification mapping and sequencing. The results obtained indicated that blaTEM-3 was present on a copy of Tn1 interrupted at the start codon of the transposase by a DNA sequence reminiscent of the inverted repeats of class II transposons. This partial Tn1 copy was in turn, inserted into the transposase gene of a Tn21-like transposon containing an integron expressing an aacA4 gene. The presence of an integron can account for the various assortments of aminoglycoside resistance genes found associated with blaTEM-3.
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42. |
Bonnet R,
Recule C,
Baraduc R,
Chanal C,
Sirot D,
De Champs C,
Sirot J,
( 2003 ) Effect of D240G substitution in a novel ESBL CTX-M-27. PMID : 12775683 : DOI : 10.1093/jac/dkg256 Abstract >>
Escherichia coli clinical strain Gre-1 collected in 2000 from a French hospital harboured a novel CTX-M-encoding gene, designated blaCTX-M-27. CTX-M-27 differed from CTX-M-14 only by the substitution D240G and was the third CTX-M enzyme harbouring this mutation after CTX-M-15 and CTX-M-16. The Gly-240-harbouring enzyme CTX-M-27 conferred to E. coli higher MICs of ceftazidime (MIC, 8 versus 1 mg/L) than did the Asp-240-harbouring CTX-M-14 enzyme. Comparison of CTX-M-14 and CTX-M-27 showed that residue Gly-240 decreased Km for ceftazidime (205 versus 940 microM), but decreased hydrolytic activity against good substrates, such as cefotaxime (kcat, 113 versus 415 s-1), probably owing to the alteration of beta3 strand positioning during the catalytic process.
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43. |
van Biesen T,
Frost LS,
( 1992 ) Differential levels of fertility inhibition among F-like plasmids are related to the cellular concentration of finO mRNA. PMID : 1374147 : DOI : 10.1111/j.1365-2958.1992.tb01527.x Abstract >>
The FinOP system of F-like plasmids consists of an antisense RNA (FinP) and a 22 kDa protein (FinO) which act in concert to prevent the translation of TraJ, the positive regulator of the transfer operon. Earlier studies suggested that two different variants of finO were responsible for differential levels of fertility inhibition among F-like plasmids. We have shown that these variations are due to the presence of an additional open reading frame (orf286) upstream of the finO gene of conjugative plasmids that are highly repressed for transfer. When orf286 and finO are linked in cis, the level of FinO expression is increased because of a rise in the cellular concentration of finO mRNA. orf286 frameshift and deletion mutants also gave the same concentration of finO transcript, suggesting that the effect is due to mRNA stabilization. We suggest that the levels of fertility inhibition exhibited by F-like plasmids are a function of their cellular FinO concentration.
|
44. |
Parker MW,
Postma JP,
Pattus F,
Tucker AD,
Tsernoglou D,
( 1992 ) Refined structure of the pore-forming domain of colicin A at 2.4 A resolution. PMID : 1373773 : DOI : 10.1016/0022-2836(92)90550-4 Abstract >>
The E1 subgroup (E1, A, B, IA, IB, K and N) of anti-bacterial toxins called colicins is known to form voltage-dependent channels in lipid bilayers. The crystal structure of the pore-forming domain of colicin A from Escherichia coli has been refined to the diffraction limit of the crystals at 2.4 A resolution by means of molecular dynamics and restrained least-squares methods to a conventional R-factor of 0.18 for all data between 6.0 and 2.4 A resolution. The polypeptide chain of 204 amino acid residues consists of ten alpha-helices organized in a three-layer structure. The helices range in length from 9 to 23 residues with an average length of 125 residues. The packing arrangement of the helices has been analysed; the packing is different from that observed in four-helix bundle proteins. The sites of 83 water molecules have been located and refined. Analysis of the structure provides insights into the mechanism of formation of a voltage-gated channel by the protein. Although it is proposed that substantial tertiary structural changes occur during membrane insertion, the secondary structural elements remain conserved. This idea has been proposed recently for a number of other protein-membrane events and thus may have more general applicability.
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45. |
Blomberg P,
Nordström K,
Wagner EG,
( 1992 ) Replication control of plasmid R1: RepA synthesis is regulated by CopA RNA through inhibition of leader peptide translation. PMID : 1378398 : PMC : PMC556743 Abstract >>
The replication frequency of plasmid R1 is post-transcriptionally controlled by an antisense RNA, CopA, that binds to the leader region in the RepA mRNA, CopT, and ultimately inhibits the synthesis of the replication initiator protein RepA. We present results demonstrating that CopA controls RepA synthesis indirectly. A reading frame for a 24 amino acid leader peptide (Tap, translational activator peptide) is located in the region between the copA and repA genes. A translational fusion between the tap and lacZ genes was used to demonstrate that tap is translated and controlled by CopA. Stop codons (UAA, UAG and UGA) introduced at three different positions within the tap gene led to a severe decrease in repA expression. Specific suppression of the stop codons reversed the effect. This indicates that tap translation is required for RepA synthesis. Phylogenetic comparisons between IncFII-like plasmids, together with previous in vitro and in vivo results (Ohman and Wagner, 1989, 1991), suggest that a stable RNA stem-loop structure sequesters the repA ribosome binding site irrespective of CopA-CopT duplex formation. The results presented here show that ribosomes translating the tap reading frame have to terminate close to the start codon of repA to permit reinitiation (direct translational coupling), and that transient disruption of the inhibitory RNA stem-loop is insufficient for activation of repA translation. The possibility that direct translational coupling is required because of a suboptimal repA RBS cannot be excluded.(ABSTRACT TRUNCATED AT 250 WORDS)
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46. |
Pinner E,
Padan E,
Schuldiner S,
( 1992 ) Cloning, sequencing, and expression of the nhaB gene, encoding a Na+/H+ antiporter in Escherichia coli. PMID : 1317851 : Abstract >>
In Escherichia coli, expulsion of sodium ions is driven by proton flux via at least two distinct Na+/H+ antiporters, NhaA and NhaB. When the nhaA gene is deleted from the chromosome, the cell becomes sensitive to high salinity and alkaline pH (Padan, E., Maisler, N., Taglicht, D., Karpel, R., and Schuldiner, S. (1989) J. Biol. Chem. 264, 20297-20302). In the current work we cloned the nhaB gene by complementation of the delta nhaA strain. The gene codes for a membrane protein 504 amino acids long. Hydropathic analysis of the sequence indicates the presence of 12 putative transmembrane helices. NhaB has been specifically labeled with [35S]methionine; it is a membrane protein and displays an apparent M(r) of 47,000, slightly lower than that predicted from its amino acid sequence. Membranes from cells containing multiple dose of nhaB display enhanced Na+/H+ antiporter activity, as measured by the ability of Na+ to collapse a preformed pH gradient or by direct measurement of 22Na+ fluxes. In contrast to NhaA, whose activity increases with pH, NhaB is practically insensitive to pH. Limited homologies with Na+ transporters have been identified.
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47. |
Gibbs TW,
Gill DR,
Salmond GP,
( 1992 ) Localised mutagenesis of the fts YEX operon: conditionally lethal missense substitutions in the FtsE cell division protein of Escherichia coli are similar to those found in the cystic fibrosis transmembrane conductance regulator protein (CFTR) of human patients. PMID : 1379670 : DOI : 10.1007/bf00272353 Abstract >>
After localised mutagenesis of the 76 min region of the Escherichia coli chromosome, we isolated a number of conditionally lethal mutants. Some of these mutants had a filamentation temperature sensitive (fts) phenotype and were assigned to the cell division genes ftsE of ftsX, whereas others were defective in the heat shock regulator gene rpoH. Both missense and amber mutant alleles of these genes were produced. The missense mutant ftsE alleles were cloned and sequenced to determine whether or not the respective mutations mapped to the region of the gene encoding the putative nucleotide binding site. Surprisingly, most of these mutant FtsE proteins had missense substitutions in a different domain of the protein. This region of the FtsE protein is highly conserved in a large family of proteins involved in diverse transport processes in all living cells, from bacteria to man. One of the proteins in this large family of homologues is the human cystic fibrosis transmembrane conductance regulator (CFTR), and the FtsE substitutions were found to be in very closely linked, or identical, amino acid residues to those which are frequently altered in the CFTR of human patients. These results confirm the structural importance of this highly conserved region of FtsE and CFTR and add weight to the current structural model for the human protein.
|
48. |
Klann AG,
Hull RA,
Hull SI,
( 1992 ) Sequences of the genes encoding the minor tip components of Pap-3 pili of Escherichia coli. PMID : 1356886 : DOI : 10.1016/0378-1119(92)90071-v Abstract >>
We report the sequence of the papE, papF and papG genes from the O75:K5 uropathogenic P pili variant, Pap-3. Comparison of the deduced amino acid sequences with those of other P pili variants reveals regions of complete homology, as well as regions of variation. Analysis of the variations in the hydrophilic domains of these proteins will help elucidate the residues which determine binding specificity.
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49. |
Ziegelin G,
Pansegrau W,
Lurz R,
Lanka E,
( 1992 ) TraK protein of conjugative plasmid RP4 forms a specialized nucleoprotein complex with the transfer origin. PMID : 1324929 : Abstract >>
Conjugative transfer of the self-transmissible IncP plasmid RP4 requires the product of the RP4 traK gene. By using the phage T7 expression system, the traK gene product was efficiently overproduced and purified to near homogeneity. traK encodes a basic protein (pI = 10.7) of 14.6 kDa that, as shown by DNA fragment retention assay, interacts exclusively with its cognate transfer origin. The apparent equilibrium constant K(app) for the complex of TraK and oriT-DNA was estimated to be 4 nM. Footprinting experiments using DNase I or hydroxyl radicals indicate that several TraK molecules interact specifically with an intrinsically bent region of oriT, covering a range of almost 200 base pairs. The TraK target sequence maps in the leading region adjacent to the relaxation nick site and recognition sequences involved in relaxosome formation but does not overlap them. Specific interactions between TraK and the DNA occur only on one side of the double helix. Electron microscopy of TraK-oriT complexes demonstrates that binding of TraK to its recognition region apparently shrinks the length of the target DNA, suggesting that the nucleic acid becomes wrapped around a core of TraK molecules. Formation of this structure could be favored by the presence of the sequence-directed bend in the TraK recognition region.
|
50. |
Harel J,
Forget C,
Saint-Amand J,
Daigle F,
Dubreuil D,
Jacques M,
Fairbrother J,
( 1992 ) Molecular cloning of a determinant coding for fimbrial antigen F165(1), a Prs-like fimbrial antigen from porcine septicaemic Escherichia coli. PMID : 1355108 : DOI : 10.1099/00221287-138-7-1495 Abstract >>
The genetic determinant coding for F165(1) fimbriae was cloned from the chromosome of the porcine Escherichia coli wild-type strain 4787 (O115:K-:H51:F165). The fimbrial determinant was further subcloned into the BamHI site of pACYC184 and a restriction map was established. On Southern hybridization, identity between the chromosomally encoded prs-like determinant of strain 4787 and its cloned counterparts was demonstrated. The cloned F165(1) fimbriae and those of the wild-type strain possessed a major protein subunit of molecular mass 18.5 kDa. Strains expressing F165(1) fimbriae were detected using an F165-specific polyclonal antiserum and caused mannose-resistant haemagglutination and agglutination of Forssman latex beads. Antiserum against the cloned F165(1) fimbriae recognized a 18.5 kDa band in the parent strain 4787.
|
51. |
Garcia E,
Bergmans HE,
van der Zeijst BA,
Gaastra W,
( 1992 ) Nucleotide sequences of the major subunits of F9 and F12 fimbriae of uropathogenic Escherichia coli. PMID : 1360613 : Abstract >>
An Eco RV-Cla I fragment containing the gene encoding the F9 fimbrial subunit of the human uropathogenic Escherichia coli strain C1018 and a PstI-PstI fragment containing the F12 fimbrial subunit gene of the dog uropathogenic strain 1442 have been cloned and the nucleotide sequence of the fragments determined. The structural gene of the F9 fimbriae (FniA) codes for a protein of 165 amino acid residues with a signal peptide of 25 amino acids. The F12 fimbrial gene (FtwA) codes for a protein of 155 amino acids which is preceded by a single peptide of 21 amino acids. The amino acid sequences of the FniA and FtwA proteins deduced from the nucleotide sequence were compared with sequences of other known P-fimbrial subunit proteins. As expected, most differences between the various proteins were found in the hypervariable regions defined by van Die et al. (1987). The N-terminal sequence of the FtwA protein differs from the one published by Klemm et al. (1983). FtwA contains two deletions found in comparison to the other fimbrial subunits.
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52. |
Stokes HW,
Hall RM,
( 1992 ) The integron In1 in plasmid R46 includes two copies of the oxa2 gene cassette. PMID : 1334268 : Abstract >>
The sequence of the insert region of the integron In1 found in the IncN plasmid R46 was completed. The insert region is 2929 bases long and includes four gene cassettes, two of which are identical copies of the oxa2 gene cassette flanking an aadA1 cassette. The fourth cassette encodes an open reading frame orfD. From comparison of these data with published maps and sequences it is argued that the integrons found in the IncN plasmids pCU1 and R1767 and in the transposon Tn2410 are closely related to In1 from R46. Both site-specific gene insertion and recA-dependent recombination are likely to have contributed to the evolution of these integrons.
|
53. |
Becker EC,
Meyer RJ,
( 2003 ) Relaxed specificity of the R1162 nickase: a model for evolution of a system for conjugative mobilization of plasmids. PMID : 12775691 : DOI : 10.1128/jb.185.12.3538-3546.2003 PMC : PMC156234 Abstract >>
The primary DNA processing protein for conjugative mobilization of the plasmid R1162 is the transesterase MobA, which acts at a unique site on the plasmid, the origin of transfer (oriT). Both MobA and oriT are members of a large family of related elements that are widely distributed among bacteria. Each oriT consists of a highly conserved core and an adjacent region that is required for binding by its cognate MobA. The sequence of the adjacent region is important in determining the specificity of the interaction between the Mob protein and the oriT DNA. However, the R1162 MobA is active on the oriT of pSC101, another naturally occurring plasmid. We show here that MobA can recognize oriTs having different sequences in the adjacent region and, with varying frequencies, can cleave these oriTs at the correct position within the core. Along with the structure of the oriTs themselves, these characteristics suggest a model for the evolution of this group of transfer systems.
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54. |
Gamas P,
Craig NL,
( 1992 ) Purification and characterization of TnsC, a Tn7 transposition protein that binds ATP and DNA. PMID : 1317955 : DOI : 10.1093/nar/20.10.2525 PMC : PMC312388 Abstract >>
The bacterial transposon Tn7 encodes five transposition genes tnsABCDE. We report a simple and rapid procedure for the purification of TnsC protein. We show that purified TnsC is active in and required for Tn7 transposition in a cell-free recombination system. This finding demonstrates that TnsC participates directly in Tn7 transposition and explains the requirement for tnsC function in Tn7 transposition. We have found that TnsC binds adenine nucleotides and is thus a likely site of action of the essential ATP cofactor in Tn7 transposition. We also report that TnsC binds non-specifically to DNA in the presence of ATP or the generally non-hydrolyzable analogues AMP-PNP and ATP-gamma-S, and that TnsC displays little affinity for DNA in the presence of ADP. We speculate that TnsC plays a central role in the selection of target DNA during Tn7 transposition.
|
55. |
Bakker D,
Willemsen PT,
Simons LH,
van Zijderveld FG,
de Graaf FK,
( 1992 ) Characterization of the antigenic and adhesive properties of FaeG, the major subunit of K88 fimbriae. PMID : 1372075 : DOI : 10.1111/j.1365-2958.1992.tb02006.x Abstract >>
The two K88 serotypes, K88ab and K88ac, differ in terms of antigenic and adhesive properties. The structural determinants of the serotype-specific epitopes and the identify of the amino acid residues involved in fimbriae-receptor interaction were studied by the construction and analysis of K88 hybrid proteins in which various parts of the K88ab and K88ac fimbrial subunit FaeG were exchanged, and by in vitro mutagenesis of non-conserved amino acid residues. Using a set of monoclonal antibodies, several regions or amino acid residues involved in the formation of serotype-specific antigenic determinants were located. The haemagglutinating activity of the hybrid and mutant proteins revealed several amino acid residues involved in the formation of the receptor binding site. A clear correlation was found between the receptor binding site and the serotype-specific antigenic determinants.
|
56. |
Batchelor RA,
Alifano P,
Biffali E,
Hull SI,
Hull RA,
( 1992 ) Nucleotide sequences of the genes regulating O-polysaccharide antigen chain length (rol) from Escherichia coli and Salmonella typhimurium: protein homology and functional complementation. PMID : 1379582 : DOI : 10.1128/jb.174.16.5228-5236.1992 PMC : PMC206356 Abstract >>
In this article, we report on the nucleotide sequences of the rol genes of Escherichia coli O75 and Salmonella typhimurium LT2. The rol gene in E. coli was previously shown to encode a 36-kDa protein that regulates size distribution of the O-antigen moiety of lipopolysaccharide. The E. coli and S. typhimurium rol gene sequences consist of 978 and 984 nucleotides, respectively. The homology between the nucleotide sequences of these two genes was found to be 68.9%. Both the E. coli rol and S. typhimurium rol genes are transcribed counter to the histidine operon and code for deduced polypeptides of 325 and 327 amino acids, respectively. The S. typhimurium rol gene was previously identified to encode a protein of unknown function and to share a transcription termination region with his. The homology between these deduced polypeptide sequences was observed to be 72%. A complementation test was performed in which the S. typhimurium rol gene was placed in trans with an E. coli plasmid (pRAB3) which encodes the O75 rfb gene cluster and not rol. The protein expressed from the S. typhimurium rol gene was found to regulate the distribution of the O75 O polysaccharide on the lipopolysaccharide of the host strain, E. coli S phi 874. The mechanism of Rol action may be independent of O antigen subunit structure, and its presence may be conserved in members of the family Enterobacteriaceae and other gram-negative bacilli that express O polysaccharides on their surface membrane.
|
57. |
Jordi BJ,
Willshaw GA,
van der Zeijst BA,
Gaastra W,
( 1992 ) The complete nucleotide sequence of region 1 of the CFA/I fimbrial operon of human enterotoxigenic Escherichia coli. PMID : 1352712 : Abstract >>
The production of the plasmid-encoded fimbrial antigen CFA/I of enterotoxigenic Escherichia coli requires two DNA regions: CFA/I region 1 and CFA/I region 2. These two regions are separated by about 40 kb on the wildtype plasmid. CFA/I region 1 contains the structural genes, whereas CFA/I region 2 contains a positive regulator. The first two genes (cfaA and cfaB) and the cfaD' sequence of region 1 have already been described. Here the total nucleotide sequence of region 1 is presented. Two new genes in region 1 are described, named cfaC and cfaE. The GC content of the genes in region 1 is 33.6% which is substantially lower than normally found in E. coli genes (50%). The codon usage also differs from the standard codons used in E. coli.
|
58. |
Imberechts H,
De Greve H,
Schlicker C,
Bouchet H,
Pohl P,
Charlier G,
Bertschinger H,
Wild P,
Vandekerckhove J,
Van Damme J,
( 1992 ) Characterization of F107 fimbriae of Escherichia coli 107/86, which causes edema disease in pigs, and nucleotide sequence of the F107 major fimbrial subunit gene, fedA. PMID : 1348723 : PMC : PMC257102 Abstract >>
F107 fimbriae were isolated and purified from edema disease strain 107/86 of Escherichia coli. Plasmid pIH120 was constructed, which contains the gene cluster that codes for adhesive F107 fimbriae. The major fimbrial subunit gene, fedA, was sequenced. An open reading frame that codes for a protein with 170 amino acids, including a 21-amino-acid signal peptide, was found. The protein without the signal sequence has a calculated molecular mass of 15,099 Da. Construction of a nonsense mutation in the open reading frame of fedA abolished both fimbrial expression and the capacity to adhere to isolated porcine intestinal villi. In a screening of 28 reference edema disease strains and isolates from clinically ill piglets, fedA was detected in 24 cases (85.7%). In 20 (83.3%) of these 24 strains, fedA was found in association with Shiga-like toxin II variant genes, coding for the toxin that is characteristic for edema disease strains of E. coli. The fimbrial subunit gene was not detected in enterotoxigenic E. coli strains. Because of the capacity of E. coli HB101(pIH120) transformants to adhere to isolated porcine intestinal villi, the high prevalence of fedA in edema disease strains, and the high correlation with the Shiga-like toxin II variant toxin-encoding genes, we suggest that F107 fimbriae are an important virulence factor in edema disease strains of E. coli.
|
59. |
Prentki P,
( 1992 ) Nucleotide sequence of the classical lacZ deletion delta M15. PMID : 1339377 : DOI : 10.1016/0378-1119(92)90056-u Abstract >>
N/A
|
60. |
Allmeier H,
Cresnar B,
Greck M,
Schmitt R,
( 1992 ) Complete nucleotide sequence of Tn1721: gene organization and a novel gene product with features of a chemotaxis protein. PMID : 1312499 : DOI : 10.1016/0378-1119(92)90597-i Abstract >>
The complete 11,139-nucleotide sequence of transposon Tn1721 has been determined. It contains three 38-bp inverted repeats, and (in this order) a new orfI, a resolution site (res), genes encoding resolvase (tnpR), transposase (tnpA), tetracycline-resistance (TcR) repressor (tetR), TcR (tetA) and a truncated transposase gene (tnpA'). The modulator origin of Tn1721 from at least three separate sources is supported by the distinctive codon usages of orfI, tnpR/tnpA and tetR/tetA, and by sequence similarities with Tn501 (tnpR/tnpA) and RP1 (tetR/tetA). The ORFI-encoded 56-kDa polypeptide exhibits features of a methyl-accepting chemotaxis protein (MCP) with a conserved signal domain and a potential transmembrane domain; this polypeptide cross-reacts with anti-MCP antiserum. Like chemotaxis genes, orfI is transcribed from a sigma 28-like promoter. The overexpressed orfI gene product interferes with MCP-dependent chemotaxis suggesting that it completes for soluble transducer protein(s) in the cell. The potential selective advantage of this novel transposon-borne gene is discussed.
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61. |
Mendiola MV,
Jubete Y,
de la Cruz F,
( 1992 ) DNA sequence of IS91 and identification of the transposase gene. PMID : 1310503 : DOI : 10.1128/jb.174.4.1345-1351.1992 PMC : PMC206431 Abstract >>
IS91 is a 1,830-bp insertion sequence that inserts specifically at the sequence CAAG or GAAC of the target and does not duplicate any sequence upon insertion (23). By transposon mutagenesis, we have identified open reading frame 426 (ORF426; bp 454 to 1731) as the putative ORF for the transposase. It displays a cysteine-rich, potential metal-binding domain in its N-terminal region. Adjacent to ORF426, there is an ORF (ORF121) which precedes and terminally overlaps ORF426 by one amino acid. Tn1732 insertions in ORF121 do not affect the transposition frequency. IS91 has sequence similarities to IS801 from Pseudomonas syringae. Their putative transposases are 36% identical, including conservation of the cysteine-rich cluster. The information concerning IS801 insertion specificity and target duplication has been reevaluated in the light of our results.
|
62. |
Rahav-Manor O,
Carmel O,
Karpel R,
Taglicht D,
Glaser G,
Schuldiner S,
Padan E,
( 1992 ) NhaR, a protein homologous to a family of bacterial regulatory proteins (LysR), regulates nhaA, the sodium proton antiporter gene in Escherichia coli. PMID : 1316901 : Abstract >>
On the basis of protein homology, nhaR has previously been shown to belong to a large family of regulatory proteins, the LysR family (Henikoff, S., Haughn, G.W., Calvo, J.M., and Wallace, J.C. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 6602-6606). In this work we show that nhaR is a regulator of nhaA, a gene encoding a Na+/H+ antiporter in Escherichia coli. Multicopy plasmid bearing nhaR enhances the Na(+)-dependent induction of a chromosomal nhaA'-'lacZ fusion. Extracts derived from cells overexpressing nhaR exhibit specific DNA binding capacity to the upstream sequences of nhaA. Construction of an nhaR deletion mutant (OR100) shows that nhaR is required in addition to nhaA to tolerate the extreme conditions under which nhaA is indispensable. Whereas OR100 grows like the wild type at neutral pH even at high Na+ concentrations (700 mM), it becomes much more sensitive to Na+ (greater than 300 mM) at pH 8.5; furthermore, OR100 is more sensitive to Li+ (100 mM) than the wild type. Nevertheless, the phenotype of OR100, which is more resistant to Na+, Li+, and alkaline pH than a delta nhaA strain (NM81), implies that the regulation exerted by nhaR is not complete and that some expression of nhaA exists in OR100. Accordingly, the effect of nhaR in cells is dependent on the level of nhaA. OR200, a nhaA and nhaR deletion mutant, has the same phenotype as NM81. Multicopy plasmid bearing nhaR does not change the phenotype of either OR200 or NM81. On the other hand, multicopy nhaA renders the cells Li(+)- and and Na(+)-resistant even without nhaR.
|
63. |
Wiegand TW,
Reznikoff WS,
( 1992 ) Characterization of two hypertransposing Tn5 mutants. PMID : 1310499 : DOI : 10.1128/jb.174.4.1229-1239.1992 PMC : PMC206416 Abstract >>
Transposition of Tn5 in Escherichia coli is regulated by two transposon-encoded proteins: transposase (Tnp), promoting transposition preferentially in cis, and the trans-acting inhibitor (Inh). Two separate transposase mutants were isolated that replace glutamate with lysine at position 110 (EK110) and at position 345 (EK345). The EK transposase proteins increase the Tn5 transposition frequency 6- to 16-fold in cis and enhance the ability of transposase to act in trans. The purified mutant transposase proteins interact with transposon outside end DNA differently from the wild-type protein, resulting in the formation of a novel complex in gel retardation assays. During characterization of the transposase proteins in the absence of inhibitor, we found that wild-type transposase itself has a transposition-inhibiting function and that this inhibition is reduced for the mutant proteins. We present a model for the regulation of Tn5 transposition, which proposes the existence of two transposase species, one cis-activating and the other trans-inhibiting. The phenotype of the EK transposase mutants can be explained by a shift in the ratio of these two species.
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64. |
Sorsa LJ,
Dufke S,
Heesemann J,
Schubert S,
( 2003 ) Characterization of an iroBCDEN gene cluster on a transmissible plasmid of uropathogenic Escherichia coli: evidence for horizontal transfer of a chromosomal virulence factor. PMID : 12761110 : DOI : 10.1128/iai.71.6.3285-3293.2003 PMC : PMC155703 Abstract >>
The chromosomal iroBCDEN gene cluster first described for Salmonella enterica is involved in the uptake of catecholate-type siderophore compounds. An orthologous gene cluster has recently been detected in Escherichia coli strains which cause extraintestinal disease. This E. coli iroBCDEN gene cluster has an impact on virulence and has been reported to be located in a pathogenicity island on the chromosome. In this study we characterized an iro gene cluster of a uropathogenic E. coli isolate which is located on a transmissible plasmid related to the R64 plasmid of S. enterica. This cluster is highly homologous to the chromosomal iro cluster of E. coli. When introduced into an E. coli fepA cir fiu aroB mutant, IroN, but not IroBCDE, mediated the utilization of structurally related catecholate siderophores, including 2,3-dihydroxybenzoyl-L-serine, 2,3-dihydroxybenzoyl-D-ornithine, 2,3-dihydroxybenzoic acid, and enterochelin. This study supports the idea of an ongoing horizontal transfer of putative virulence factors and the mobilization of single virulence gene clusters, which lead to a modular assembly of virulence determinants such as pathogenicity islands.
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65. |
Parent R,
Roy PH,
( 1992 ) The chloramphenicol acetyltransferase gene of Tn2424: a new breed of cat. PMID : 1314803 : DOI : 10.1128/jb.174.9.2891-2897.1992 PMC : PMC205941 Abstract >>
We have sequenced the gene coding for the chloramphenicol acetyltransferase of Tn2424 of plasmid NR79. This gene codes for a protein of 23,500 Da, and the derived protein sequence is similar to those of the chromosomal chloramphenicol acetyltransferases of Agrobacterium tumefaciens and Pseudomonas aeruginosa and of unidentified open reading frames, which may encode chloramphenicol acetyltransferases, adjacent to the ermG macrolide-lincosamide-streptogramin resistance gene of Bacillus sphaericus and the vgb virginiamycin resistance gene of Staphylococcus aureus. Weaker similarity to the LacA (thiogalactoside acetyltransferase) and CysE (serine acetyltransferase) proteins of Escherichia coli and the NodL protein of Rhizobium leguminosarum is also observed. There is no significant similarity to any other chloramphenicol acetyltransferase genes, such as that of Tn9. The Tn2424 cat gene is part of a 4.5-kb region which also contains the aacA1a aminoglycoside-6'-N-acetyltransferase gene; Tn2424 is similar to Tn21 except for the presence of this region. Sequences flanking the cat gene are typical of those flanking other genes inserted into pVS1-derived "integrons" by a site-specific recombinational mechanism.
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66. |
Patzer SI,
Baquero MR,
Bravo D,
Moreno F,
Hantke K,
( 2003 ) The colicin G, H and X determinants encode microcins M and H47, which might utilize the catecholate siderophore receptors FepA, Cir, Fiu and IroN. PMID : 12949180 : DOI : 10.1099/mic.0.26396-0 Abstract >>
The colicin G producer Escherichia coli CA46, the colicin H producer E. coli CA58 and E. coli Nissle 1917 (DSM 6601) were shown to produce microcin H47 and the newly described microcin M. Both microcins were exported like colicin V by an RND-type export system, including TolC. The gene cluster encoding microcins H47 and M in strains CA46 and CA58 is nearly identical to that in strain DSM 6601, except that two additional genes are included. A Fur box identified in front of the microcin-encoding genes explained the observed iron regulation of microcin production. The catecholate siderophore receptors Fiu, Cir and FepA from E. coli and IroN, Cir and FepA from Salmonella were identified as receptors for microcins M, H47 and E492. IroN takes up the glucose-containing catecholate siderophore salmochelin, whose synthesis is encoded in the iro gene cluster found in Salmonella and certain, often uropathogenic, E. coli strains. A gene in this iro cluster, iroB, which encodes a putative glycosyltransferase, was also found in the microcin H47/M and microcin E492 gene clusters. These microcins could aid the producing strain in competing against enterobacteria that utilize catecholate siderophores.
|
67. |
Szumanski MB,
Boyle SM,
( 1992 ) Influence of cyclic AMP, agmatine, and a novel protein encoded by a flanking gene on speB (agmatine ureohydrolase) in Escherichia coli. PMID : 1310091 : DOI : 10.1128/jb.174.3.758-764.1992 PMC : PMC206152 Abstract >>
The speB gene of Escherichia coli encodes agmatine ureohydrolase (AUH), a putrescine biosynthetic enzyme. The speB gene is transcribed either from its own promoter or as a polycistronic message from the promoter of the speA gene encoding arginine decarboxylase. Two open reading frames (ORF1 and ORF2) are present on the strand complementary to speB; approximately 90% of ORF2 overlaps the speB coding region. Analysis of transcriptional and translational fusions of ORF1 or ORF2 to lacZ revealed that ORF1 encoded a novel protein while ORF2 was not transcribed. Deletion of ORF1 from a plasmid containing ORF1, ORF2, and speB reduced the activity of AUH by 83%. In contrast, the presence of plasmid-encoded ORF1 caused an 86% increase in chromosomally encoded AUH activity. ORF1 did not stimulate alkaline phosphatase expressed from a phi(speB-phoA) transcriptional fusion encoded on the same plasmid. Western analysis (immunoblot) of a phi(ORF1-lacZ) translational fusion revealed that ORF1 encodes a 25.3-kDa protein. Agmatine induced transcription of phi(speB-phoA) but not phi(speA-phoA) fusions. Consequently, agmatine affects selection between the monocistronic and the polycistronic modes of speB transcription. In contrast, cyclic AMP (cAMP) repressed AUH activity of chromosomally encoded AUH but had no effect on plasmid-borne speB nor phi(speB-phoA). It is concluded that ORF1 encodes a protein which is a posttranscriptional regulator of speB, agmatine induces speB independent of speA, and cAMP regulates speB indirectly.
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68. |
Mendiola MV,
de la Cruz F,
( 1992 ) IS91 transposase is related to the rolling-circle-type replication proteins of the pUB110 family of plasmids. PMID : 1321417 : DOI : 10.1093/nar/20.13.3521 PMC : PMC312521 Abstract >>
N/A
|
69. |
Collins CM,
Gutman DM,
( 1992 ) Insertional inactivation of an Escherichia coli urease gene by IS3411. PMID : 1310093 : DOI : 10.1128/jb.174.3.883-888.1992 PMC : PMC206166 Abstract >>
Ureolytic Escherichia coli are unusual clinical isolates that are found at various extraintestinal sites of infection, predominantly the urinary tract. The urease-positive phenotype is unstable in approximately 25% of these isolates, and urease-negative segregants are produced at a high frequency. We have studied the nature of the urease-positive-to-negative transition in one of these isolates, designated E. coli 1021. Southern hybridization experiments with genomic DNA extracted from seven independent E. coli 1021 urease-negative segregants revealed the presence of a 1.3-kb DNA insertion in the urease gene cluster. A DNA fragment containing the DNA insertion was cloned from one of the urease-negative segregants. This cloned DNA fragment was capable of mediating cointegrate formation with the conjugative plasmid pOX38, suggesting that the DNA insertion was a transposable element. The insert was identified as an IS3411 element in ureG by DNA sequence analysis. A 3-bp target duplication (CTG) flanking the insertion element was found. DNA spanning the insertion site was amplified from the other six urease-negative segregants by using the polymerase chain reaction. The DNA sequence of the amplified fragments indicated that an IS3411 element was found in an identical site in all urease-negative segregants examined. These data suggest that in E. coli 1021, IS3411 transposes at a high frequency into ureG at a CTG site, disrupting this gene and eliminating urease activity.
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70. |
Janka A,
Bielaszewska M,
Dobrindt U,
Greune L,
Schmidt MA,
Karch H,
( 2003 ) Cytolethal distending toxin gene cluster in enterohemorrhagic Escherichia coli O157:H- and O157:H7: characterization and evolutionary considerations. PMID : 12761152 : DOI : 10.1128/iai.71.6.3634-3638.2003 PMC : PMC155755 Abstract >>
We identified a cytolethal distending toxin (cdt) gene cluster in 87, 6, and 0% of sorbitol-fermenting (SF) enterohemorrhagic Escherichia coli (EHEC) O157:H(-), EHEC O157:H7, and E. coli O55:H7/H(-) strains, respectively. The toxin was expressed by the wild-type EHEC O157 strains and by a cdt-containing cosmid from a library of SF EHEC O157:H(-) strain 493/89. The cdt flanks in strain 493/89 were homologous to bacteriophages P2 and lambda. Our data demonstrate that cdt, encoding a potential virulence factor, is present in the EHEC O157 complex and suggest that cdt may have been acquired by phage transduction.
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71. |
Lügering A,
Benz I,
Knochenhauer S,
Ruffing M,
Schmidt MA,
( 2003 ) The Pix pilus adhesin of the uropathogenic Escherichia coli strain X2194 (O2 : K(-): H6) is related to Pap pili but exhibits a truncated regulatory region. PMID : 12777480 : DOI : 10.1099/mic.0.26266-0 Abstract >>
Adhesins provide a major advantage for uropathogenic Escherichia coli in establishing urinary tract infections (UTIs). A novel gene cluster responsible for the expression of a filamentous adhesin of the pyelonephritogenic E. coli strain X2194 has been identified, molecularly cloned, and characterized. The 'pix operon' contains eight open reading frames which exhibit significant sequence homology to corresponding genes in the pap operon encoding P pili, the prevalent E. coli adhesins in non-obstructive acute pyelonephritis in humans. Although a pixB gene corresponding to the PapB regulator was identified, a papI homologue could not be found in the pix operon. Instead, a fragment of the R6 gene of the highly uropathogenic E. coli strain CFT073 was identified upstream of pixB. The R6 gene is located in a pathogenicity island containing several pilus-encoding sequences and shows homology to a transposase of Chelatobacter heintzii. In a pixA-lacZ fusion system it was demonstrated that the expression of Pix pili is regulated at the transcriptional level by the R6 gene sequence. A significantly reduced transcription was observed by deleting this fragment and by lowering the growth temperature from 37 to 26 degrees C. In contrast to other filamentous adhesin systems, Pix pili are mainly expressed in the steady state growth phase and were not repressed by the addition of glucose.
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72. |
Mruk I,
Kaczorowski T,
( 2003 ) Genetic organization and molecular analysis of the EcoVIII restriction-modification system of Escherichia coli E1585-68 and its comparison with isospecific homologs. PMID : 12732532 : DOI : 10.1128/aem.69.5.2638-2650.2003 PMC : PMC154532 Abstract >>
The EcoVIII restriction-modification (R-M) system is carried by the Escherichia coli E1585-68 natural plasmid pEC156 (4,312 bp). The two genes were cloned and characterized. The G+C content of the EcoVIII R-M system is 36.1%, which is significantly lower than the average G+C content of either plasmid pEC156 (43.6%) or E. coli genomic DNA (50.8%). The difference suggests that there is a possibility that the EcoVIII R-M system was recently acquired by the genome. The 921-bp EcoVIII endonuclease (R. EcoVIII) gene (ecoVIIIR) encodes a 307-amino-acid protein with an M(r) of 35,554. The convergently oriented EcoVIII methyltransferase (M. EcoVIII) gene (ecoVIIIM) consists of 912 bp that code for a 304-amino-acid protein with an M(r) of 33,930. The exact positions of the start codon AUG were determined by protein microsequencing. Both enzymes recognize the specific palindromic sequence 5'-AAGCTT-3'. Preparations of EcoVIII R-M enzymes purified to homogeneity were characterized. R. EcoVIII acts as a dimer and cleaves a specific sequence between two adenine residues, leaving 4-nucleotide 5' protruding ends. M. EcoVIII functions as a monomer and modifies the first adenine residue at the 5' end of the specific sequence to N(6)-methyladenine. These enzymes are thus functionally identical to the corresponding enzymes of the HindIII (Haemophilus influenzae Rd) and LlaCI (Lactococcus lactis subsp. cremoris W15) R-M systems. This finding is reflected by the levels of homology of M. EcoVIII with M. HindIII and M. LlaCI at the amino acid sequence level (50 and 62%, respectively) and by the presence of nine sequence motifs conserved among m(6) N-adenine beta-class methyltransferases. The deduced amino acid sequence of R. EcoVIII shows weak homology with its two isoschizomers, R. HindIII (26%) and R. LlaCI (17%). A catalytic sequence motif characteristic of restriction endonucleases was found in the primary structure of R. EcoVIII (D(108)X(12)DXK(123)), as well as in the primary structures of R. LlaCI and R. HindIII. Polyclonal antibodies raised against R. EcoVIII did not react with R. HindIII, while anti-M. EcoVIII antibodies cross-reacted with M. LlaCI but not with M. HindIII. R. EcoVIII requires Mg(II) ions for phosphodiester bond cleavage. We found that the same ions are strong inhibitors of the M. EcoVIII enzyme. The biological implications of this finding are discussed.
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73. |
Burian J,
Ausió J,
Phipps B,
Moore S,
Dougan D,
Kay W,
( 2003 ) Hexamerization of RepA from the Escherichia coli plasmid pKL1. PMID : 12939157 : DOI : 10.1021/bi034341b Abstract >>
The Escherichia coli plasmid pKL1 is one of the smallest bacterial plasmids. It encodes a single, autoregulating structural gene, repA, responsible for replication and copy number control. The oligomerization of RepA was previously proposed as the basis of a strategy for pKL1 copy number control. To elucidate the oligomerization properties of RepA in solution, RepA was expressed in E. coli; purified by ion exchange and hydrophobic chromatography; and examined in solution by spectrapolarimetry, light scattering, sedimentation velocity, and equilibrium ultracentrifugation. RepA behaved as a concentration-dependent equilibrium of dimers and hexamers. Conformational parameters of the RepA hexameric complex were determined. These results support the proposed autogenous regulatory model whereby RepA hexamers negatively regulate repA expression thereby affecting the copy number control of pKL1. RepA of pKL1 is the first plasmid replication initiation protein documented to be in dimeric-hexameric forms.
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74. |
Yatsuyanagi J,
Saito S,
Miyajima Y,
Amano K,
Enomoto K,
( 2003 ) Characterization of atypical enteropathogenic Escherichia coli strains harboring the astA gene that were associated with a waterborne outbreak of diarrhea in Japan. PMID : 12734245 : DOI : 10.1128/jcm.41.5.2033-2039.2003 PMC : PMC154716 Abstract >>
The virulence traits of the Escherichia coli strain associated with a waterborne diarrhea outbreak were examined. Forty-one of 75 students (ages 12 to 15) in Akita Prefecture, Japan, showed clinical symptoms. Seven E. coli Ouk:K-:H45 isolates were isolated from the patients as the causative agent of this outbreak. One isolate (EC-3605) showed the presence of E. coli attaching-and-effacing (eaeA) and enteroaggregative E. coli heat-stable enterotoxin-1 (astA) genes and the absence of Shiga toxin (stx1 and stx2) genes. A polymorphic enteropathogenic E. coli (EPEC) adherence factor plasmid was detected in EC-3605 with a major structural gene deletion and a regulatory gene frameshift mutation, revealing that EC-3605 represents an atypical EPEC strain harboring the astA gene. The role that atypical EPEC strains harboring the astA gene play in human disease is unclear. Our results, along with those of others, present a possibility that these strains comprise a distinct category of diarrheagenic E. coli and that astA affects the age distribution of atypical-EPEC infection.
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75. |
Volokhov D,
Chizhikov V,
Chumakov K,
Rasooly A,
( 2003 ) Microarray analysis of erythromycin resistance determinants. PMID : 12969293 : Abstract >>
To develop a DNA microarray for analysis of genes encoding resistance determinants to erythromycin and the related macrolide, lincosamide and streptogramin B (MLS) compounds. We developed an oligonucleotide microarray containing seven oligonucleotide probes (oligoprobes) for each of the six genes (ermA, ermB, ermC, ereA, ereB and msrA/B) that account for more than 98% of MLS resistance in Staphylococcus aureus clinical isolates. The microarray was used to test reference and clinical S. aureus and Streptococcus pyrogenes strains. Target genes from clinical strains were amplified and fluorescently labelled using multiplex PCR target amplification. The microarray assay correctly identified the MLS resistance genes in the reference strains and clinical isolates of S. aureus, and the results were confirmed by direct DNA sequence analysis. Of 18 S. aureus clinical strains tested, 11 isolates carry MLS determinants. One gene (ermC) was found in all 11 clinical isolates tested, and two others, ermA and msrA/B, were found in five or more isolates. Indeed, eight (72%) of 11 clinical isolate strains contained two or three MLS resistance genes, in one of the three combinations (ermA with ermC, ermC with msrA/B, ermA with ermC and msrA/B). Oligonucleotide microarray can detect and identify the six MLS resistance determinants analysed in this study. Our results suggest that microarray-based detection of microbial antibiotic resistance genes might be a useful tool for identifying antibiotic resistance determinants in a wide range of bacterial strains, given the high homology among microbial MLS resistance genes.
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76. |
Nishi J,
Sheikh J,
Mizuguchi K,
Luisi B,
Burland V,
Boutin A,
Rose DJ,
Blattner FR,
Nataro JP,
( 2003 ) The export of coat protein from enteroaggregative Escherichia coli by a specific ATP-binding cassette transporter system. PMID : 12933818 : DOI : 10.1074/jbc.M306413200 Abstract >>
Enteroaggregative Escherichia coli (EAEC) is an emerging enteric pathogen characterized by aggregative adherence (AA) to cultured human mucosal epithelium cells. We have recently characterized a 10.2-kDa protein, called dispersin, which is exported from the bacteria and which promotes dispersal of EAEC across the intestinal mucosa. Here, we present evidence that dispersin is exported by a putative ABC transporter complex, which is encoded by a genetic locus of the EAEC virulence plasmid pAA2. We demonstrate that the locus comprises a cluster of five genes (designated aat-PABCD), including homologs of an inner-membrane permease (AatP), an ATP-binding cassette protein (AatC) and the outer membrane protein TolC (AatA). We show that, like TolC, AatA localizes to the outer membrane independently of its ABC partner. Dispersin appears to require the Aat complex for outer membrane translocation but not for secretion across the inner membrane. We also show that, like the dispersin gene, transcription of the aat cluster is dependent on AggR, a regulator of virulence genes in EAEC. We propose that the aat cluster encodes a specialized ABC transporter, which plays a role in the pathogenesis of EAEC by transporting dispersin out of the bacterial cell.
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77. |
Gobius KS,
Higgs GM,
Desmarchelier PM,
( 2003 ) Presence of activatable Shiga toxin genotype (stx(2d)) in Shiga toxigenic Escherichia coli from livestock sources. PMID : 12904389 : DOI : 10.1128/jcm.41.8.3777-3783.2003 PMC : PMC179786 Abstract >>
Stx2d is a recently described Shiga toxin whose cytotoxicity is activated 10- to 1000-fold by the elastase present in mouse or human intestinal mucus. We examined Shiga toxigenic Escherichia coli (STEC) strains isolated from food and livestock sources for the presence of activatable stx(2d). The stx(2) operons of STEC were first analyzed by PCR-restriction fragment length polymorphism (RFLP) analysis and categorized as stx(2), stx(2c vha), stx(2c vhb), or stx(2d EH250). Subsequently, the stx(2c vha) and stx(2c vhb) operons were screened for the absence of a PstI site in the stx(2A) subunit gene, a restriction site polymorphism which is a predictive indicator for the stx(2d) (activatable) genotype. Twelve STEC isolates carrying putative stx(2d) operons were identified, and nucleotide sequencing was used to confirm the identification of these operons as stx(2d). The complete nucleotide sequences of seven representative stx(2d) operons were determined. Shiga toxin expression in stx(2d) isolates was confirmed by immunoblotting. stx(2d) isolates were induced for the production of bacteriophages carrying stx. Two isolates were able to produce bacteriophages phi1662a and phi1720a carrying the stx(2d) operons. RFLP analysis of bacteriophage genomic DNA revealed that phi1662a and phi1720a were highly related to each other; however, the DNA sequences of these two stx(2d) operons were distinct. The STEC strains carrying these operons were isolated from retail ground beef. Surveillance for STEC strains expressing activatable Stx2d Shiga toxin among clinical cases may indicate the significance of this toxin subtype to human health.
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78. |
Bürk C,
Dietrich R,
Açar G,
Moravek M,
Bülte M,
Märtlbauer E,
( 2003 ) Identification and characterization of a new variant of Shiga toxin 1 in Escherichia coli ONT:H19 of bovine origin. PMID : 12734256 : DOI : 10.1128/jcm.41.5.2106-2112.2003 PMC : PMC154714 Abstract >>
A new variant of Shiga toxin 1 (Stx1), designated Stx1d, which deviates considerably more than any other known variant from Stx1 encoded by phage 933J, was identified in an Escherichia coli strain, ONT:H19, isolated from bovine feces. The complete stx(1) gene of this strain was amplified and sequenced. Nucleotide sequence homology with stx(1) from phage 933J was only 91%, resulting in the substitution of 20 amino acids in the A subunit and 7 amino acids in the B subunit of the protein. Cell culture supernatant of this strain, which was negative for stx(2) by PCR testing, was cytotoxic to Vero cells and gave positive results in two commercial enzyme-linked immunosorbent assays for Stx. PCR primers were constructed for the specific detection of the new variant. The findings of this study suggest that Stx1 is not as conserved as thought before and that there might be more variants which cannot be detected by commonly used PCR methods.
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79. |
Starcic Erjavec M,
Gaastra W,
van Putten J,
Zgur-Bertok D,
( 2003 ) Identification of the origin of replications and partial characterization of plasmid pRK100. PMID : 12932736 : Abstract >>
In search for the evolutionary origin of the conjugative F-like plasmid pRK100, the plasmid's functional replication regions were identified. Additionally targeted genetic analysis was used to investigate origins of other regions of the plasmid. Construction of minireplicons via ligation of Tn1725 with plasmid fragments and targeted cloning of putative replication regions, followed by sequence analysis indicated two functional replication regions, a F plasmid related RepFIB and a R1 plasmid related RepFIIA replication region. Partial nucleotide sequencing of regions of the plasmid revealed genes that encode a putative enterochelin iron uptake system previously associated with an Escherichia coli pathogenicity island, PAI III536, and the pColV-like aerobactin genes. In addition, a homologue of the R100 plasmid related rmoA gene was found that exhibits strong similarity to hha/ymoA encoding the Hha/YmoA class of modulators of gene expression. PCR and hybridization experiments further demonstrated that pRK100 harbors multiple IS2 and IS3 insertion sequences that may have facilitated in the acquisition of elements from other DNA molecules. These data together with the previous identification of a F-like tra region and a pColIa-like colicin Ia, indicate that pRK100 has a highly mosaic structure with elements derived from many different known large natural plasmids.
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80. |
Tóth I,
Hérault F,
Beutin L,
Oswald E,
( 2003 ) Production of cytolethal distending toxins by pathogenic Escherichia coli strains isolated from human and animal sources: establishment of the existence of a new cdt variant (Type IV). PMID : 12958258 : DOI : 10.1128/jcm.41.9.4285-4291.2003 PMC : PMC193864 Abstract >>
Three types of cytolethal distending toxin (CDT), namely, CDT-I, CDT-II, and CDT-III, have been described in Escherichia coli. Using primers designed for the detection of sequences common to the cdtB genes, we analyzed by PCR a set of 21 CDT-producing E. coli strains of intestinal and extraintestinal origins isolated from human and different animal species in several European countries and in the United States. On the basis of the existing differences in the cdtB genes, cdt-I-, cdt-II-, and cdt-III-specific primer pairs were designed and used for cdt typing. These new primers successfully differentiated all of the previously described cdt genes. Six strains proved to be cdt-I; eight strains proved to be cdt-III. However, none of the type I-, II-, and III-specific primers generated amplicons from six CDT(+) strains, suggesting the existence of a new cdt variant. Sequence analysis of the amplicons from two untypeable genes confirmed the existence of a new cdt variant that we called cdt-IV. Using the new specific primers, cdt-IV was detected in human, porcine, and poultry strains of intestinal and extraintestinal origins. To validate all sets of cdt specific primers, a group of 353 human E. coli strains isolated in Hungary was then investigated for the presence of cdt genes. This included 190 strains isolated from patients with urinary tract infections (UTI), 51 strains isolated from other (nonurinary) extraintestinal infections, and 112 intestinal strains isolated from healthy individuals. Of 190 UTI strains, 15 (7.9%) had cdt genes. Of 51 non-UTI extraintestinal strains 3 (5.9%) contained the cdt gene, and 1 (0.9%) of 112 healthy intestinal strains was PCR positive. Five strains proved to be cdt-I, and fourteen strains proved to be cdt-IV. The CDT-producing extraintestinal strains belonged to a wide variety of serogroups, including O2, O6, O75, and O170. In conclusion, we have developed a new PCR typing system for CDT able to detect a new CDT variant present in pathogenic E. coli strains obtained from animals and humans.
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81. |
Botelho BA,
Bando SY,
Trabulsi LR,
Moreira-Filho CA,
( 2003 ) Identification of EPEC and non-EPEC serotypes in the EPEC O serogroups by PCR-RFLP analysis of the fliC gene. PMID : 12732425 : Abstract >>
Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the flagellin gene (fliC) was performed in 233 strains of enteropathogenic Escherichia coli (EPEC) O serogroups for determining their flagellar antigen (H) status. The serological detection of flagellin is the basis for the H-codes typing system in E. coli. Thus, it is impossible to serotype nonmotile bacteria (i.e. to assign H-codes). Twenty-eight fliC restriction patterns were obtained for motile (H2, H4, H6, H7, H8, H9, H10, H11, H12, H18, H21, H27, H32, H34, H35, H40 and H51) and nonmotile serotypes (H(-)). Each motile serotype was characterized by one or two fliC specific restriction patterns. The only exception was serogroup O128ab, where a common restriction pattern was found for serotypes O128ab:H2 and O128ab:H35, even after digestion with RsaI, AluI and Sau3AI endonucleases. These two serotypes were, however, discriminated by single strand conformation polymorphism (SSCP) analysis of RsaI restriction fragments. Nonmotile strains showed fliC restriction patterns identical to some known H serotypes. The PCR-RFLP analysis of fliC gene proved to be a useful method for identifying the H variants in motile and nonmotile EPEC O serogroups.
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82. |
Vourli S,
Tzouvelekis LS,
Tzelepi E,
Lebessi E,
Legakis NJ,
Miriagou V,
( 2003 ) Characterization of In111, a class 1 integron that carries the extended-spectrum beta-lactamase gene blaIBC-1. PMID : 12900034 : DOI : 10.1016/S0378-1097(03)00510-X Abstract >>
A class 1 integron, In111, carried by a self-transferable plasmid from an Escherichia coli clinical strain was characterized. The variable region of In111 constituted an array of gene cassettes encoding the extended-spectrum beta-lactamase IBC-1, the aminoglycoside-modifying enzymes AAC(6')-Ib and ANT(3")-Ia, dihydrofolate reductase I and a putative polypeptide (SMR-2) sharing similarity with the Qac transporters. Transcription of the gene cassettes was driven by a hybrid-type P1 promoter located in a typical 5' conserved segment (CS). The 3'CS included sulI, qacEDelta1, orf5 and orf6. In111 was bounded on the right by an inversely oriented IRt. The 5'CS was preceded by an intact IS26 element followed by an aphA1 gene.
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83. |
Jelacic JK,
Damrow T,
Chen GS,
Jelacic S,
Bielaszewska M,
Ciol M,
Carvalho HM,
Melton-Celsa AR,
O'Brien AD,
Tarr PI,
( 2003 ) Shiga toxin-producing Escherichia coli in Montana: bacterial genotypes and clinical profiles. PMID : 12934188 : DOI : 10.1086/376999 Abstract >>
The diseases and virulence genes associated with Shiga toxin-producing Escherichia coli (STEC) are characterized incompletely. We analyzed, by polymerase chain reaction, 82 STEC isolates collected prospectively in Montana and profiled associated illnesses by patient chart review. All E. coli O157:H7 contained stx2-group genes, as well as eae, iha, espA, and ehxA; 84% contained stx1. Non-O157:H7 STEC less frequently contained stx1 (P=.046), stx2 (P<.001), iha (P<.001), eae, and espA (P=.039 for both), were isolated less often from patients treated in emergency departments (P=.022), and tended to be associated less frequently with bloody diarrhea (P=.061). There were no significant associations between stx genotype and bloody diarrhea, but isolates containing stx2c or stx(2d-activatable) were recovered more often from patients who underwent diagnostic or therapeutic procedures (P=.033). Non-O157:H7 STEC are more heterogeneous and cause bloody diarrhea less frequently than do E. coli O157:H7. Bloody diarrhea cannot be attributed simply to the stx genotype of the infecting organism.
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84. |
Betteridge T,
Yang J,
Pittard AJ,
Praszkier J,
( 2003 ) Interaction of the initiator protein of an IncB plasmid with its origin of DNA replication. PMID : 12644491 : DOI : 10.1128/jb.185.7.2210-2218.2003 PMC : PMC151506 Abstract >>
The replication initiator protein RepA of the IncB plasmid pMU720 was purified and used in DNase I protection assays in vitro. RepA protected a 68-bp region of the origin of replication of pMU720. This region, which lies immediately downstream of the DnaA box, contains four copies of the sequence motif 5'AANCNGCAA3'. Mutational analyses identified this sequence as the binding site specifically recognized by RepA (the RepA box). Binding of RepA to the RepA boxes was ordered and sequential, with the box closest to the DnaA binding site (box 1) occupied first and the most distant boxes (boxes 3 and 4) occupied last. However, only boxes 1, 2, and 4 were essential for origin activity, with box 3 playing a lesser role. Changing the spacing between box 1 and the other three boxes affected binding of RepA in vitro and origin activity in vivo, indicating that the RepA molecules bound to ori(B) interact with one another.
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85. |
Sablé S,
Duarte M,
Bravo D,
Lanneluc I,
Pons AM,
Cottenceau G,
Moreno F,
( 2003 ) Wild-type Escherichia coli producing microcins B17, D93, J25, and L; cloning of genes for microcin L production and immunity. PMID : 12897830 : DOI : 10.1139/w03-047 Abstract >>
For the first time, an Escherichia coli strain producing four microcins (Mcc), B17, D93, J25, and L, and showing immunity to Mcc V was isolated and characterized. Each of the gene clusters encoding the production of Mcc B17, D93, and L was cloned separately. The gene cluster for Mcc L was cloned within a 13.5-kb HindIII-SalI fragment, which includes the Mcc V immunity gene, cvi.
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86. |
Wang L,
Rothemund D,
Curd H,
Reeves PR,
( 2003 ) Species-wide variation in the Escherichia coli flagellin (H-antigen) gene. PMID : 12700273 : DOI : 10.1128/jb.185.9.2936-2943.2003 PMC : PMC154406 Abstract >>
Escherichia coli is a clonal species. The best-understood components of its clonal variation are the flagellar (H) and polysaccharide (O) antigens, both well documented since the mid-1930s because of their use in serotyping. Flagellin is the protein subunit of the flagellum that carries H-antigen specificity. We show that 43 of the 54 H-antigen specificities of E. coli map to the flagellin gene at fliC and sequenced all 43 forms and confirmed specificity of each by cloning and expression. This is, to our knowledge, the first time that all known forms of such a highly polymorphic gene have been fully sequenced and characterized for any species. The established distinction between a highly variable central region and more conserved flanking regions is upheld. The sequences fall into two groups, one of which may be derived from the fliC gene of the E. coli/Salmonella enterica common ancestor, the other perhaps obtained by lateral transfer since species divergence. Comparison of sequences revealed that both horizontal DNA transfer and fixation of mutations under diversifying selection pressure contributed to polymorphism in this locus.
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87. |
Fomenko DE,
Metlitskaya AZ,
Péduzzi J,
Goulard C,
Katrukha GS,
Gening LV,
Rebuffat S,
Khmel IA,
( 2003 ) Microcin C51 plasmid genes: possible source of horizontal gene transfer. PMID : 12936987 : DOI : 10.1128/aac.47.9.2868-2874.2003 PMC : PMC182647 Abstract >>
Microcin C51 (MccC51) is an antimicrobial nucleotide-heptapeptide produced by a natural Escherichia coli strain. A 5.7-kb fragment of the pC51 plasmid carrying the genes involved in MccC51 production, secretion, and self-immunity was sequenced, and the genes were characterized. The sequence of the MccC51 gene cluster is highly similar to that of the MccC7 gene. Recombinant plasmids carrying different combinations of the mcc genes involved in the MccC51 production or immunity were constructed to characterize their functional roles. The mccA, mccB, mccD, and mccE genes are involved in MccC51 production, while the mccC and mccE genes are responsible for immunity to MccC51. The mcc gene cluster is flanked by 44-bp direct repeats. Amino acid sequence comparisons allowed us to propose functions for each Mcc polypeptide in MccC51 biosynthesis. Plasmid pUHN containing the cloned mccA, mccB, mccC, and mccE genes, but lacking mccD, directed the synthesis of MccC51p, a substance chemically related to MccC51. MccC51p exhibited weak antibiotic activity against E. coli and was toxic to the producing cells. The immunity to exogenous MccC51 determined by the mccC and mccE genes did not overcome the toxic action of MccC51p on the producing cells. The G+C content of the MccC51 operon, markedly lower than that of the E. coli genome, and the presence of direct repeats suggest the possibility of horizontal transfer of this gene cluster.
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88. |
Honarvar S,
Choi BK,
Schifferli DM,
( 2003 ) Phase variation of the 987P-like CS18 fimbriae of human enterotoxigenic Escherichia coli is regulated by site-specific recombinases. PMID : 12657052 : DOI : 10.1046/j.1365-2958.2003.03419.x Abstract >>
The gene cluster of the CS18 (PCFO20) fimbriae of human enterotoxigenic Escherichia coli (ETEC) was found to include seven genes (fotA to fotG) that are similar to each of the seven structural and export proteins of the 987P fimbriae. However, no analogous gene to the fasH regulatory gene, which is located at the 3' end of the 987P gene cluster and encodes an AraC-like activator of transcription, could be detected. Surprisingly, two novel genes (fotS and fotT) encoding proteins similar to the site-specific recombinases of the type 1 fimbriae (FimB and FimE) were identified at the 5' end of the fot gene cluster. These genes were shown to be required for the catalysis of a 312 bp-inversion just upstream of fotA. The inversion determines CS18 fimbrial phase variation. FotS participates in inverting the 312 bp-segment in both the ON and OFF orientation, whereas FotT has a bias for the OFF oriented recombination. Similar regulators of fimbriation by phase variation were described in uropathogenic and commensal Enterobacteriaceae. In contrast, only AraC-like transcriptional activators were previously described as regulators of the intestinal colonization factors of human ETEC isolates. Thus, the CS18 and 987P gene clusters encode similar components for fimbrial biogenesis but different types of regulators for fimbriation. The combination of blocks of genes encoding similar structural products but different regulatory proteins underlines how modular DNA rearrangements can evolve by serving pathogen diversification. Acquisition of a phase variation module to regulate fimbrial genes is proposed to be beneficial for the adaptation and transmission of pathogens.
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89. |
Parreira VR,
Gyles CL,
( 2003 ) A novel pathogenicity island integrated adjacent to the thrW tRNA gene of avian pathogenic Escherichia coli encodes a vacuolating autotransporter toxin. PMID : 12933851 : DOI : 10.1128/iai.71.9.5087-5096.2003 PMC : PMC187369 Abstract >>
We report the complete nucleotide sequence and genetic organization of the Vat-encoding pathogenicity island (PAI) of avian pathogenic Escherichia coli strain Ec222. The 22,139-bp PAI is situated adjacent to the 3' terminus of the thrW tRNA gene, has a G+C content of 41.2%, and includes a bacteriophage SfII integrase gene, mobile genetic elements, two open reading frames with products exhibiting sequence similarity to known proteins, and several other open reading frames of unknown function. The PAI encodes an autotransporter protein, Vat (vacuolating autotransporter toxin), which induces the formation of intracellular vacuoles resulting in cytotoxic effects similar to those caused by the VacA toxin from Helicobacter pylori. The predicted 148.3-kDa protein product possesses the three domains that are typical of serine protease autotransporters of Enterobacteriaceae: an N-terminal signal sequence of 55 amino acids, a 111.8-kDa passenger domain containing a modified serine protease site (ATSGSG), and a C-terminal outer membrane translocator of 30.5 kDa. Vat has 75% protein homology with the hemagglutinin Tsh, an autotransporter of avian pathogenic E. coli. A vat deletion mutant of Ec222 showed no virulence in respiratory and cellulitis infection models of disease in broiler chickens. We conclude that the newly described PAI and Vat may be involved in the pathogenicity of avian septicemic E. coli strain Ec222 and other avian pathogenic E. coli strains.
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90. |
Merckel MC,
Tanskanen J,
Edelman S,
Westerlund-Wikström B,
Korhonen TK,
Goldman A,
( 2003 ) The structural basis of receptor-binding by Escherichia coli associated with diarrhea and septicemia. PMID : 12909017 : DOI : 10.1016/s0022-2836(03)00841-6 Abstract >>
GafD in Escherichia coli G (F17) fimbriae is associated with diarrheal disease, and the structure of the ligand-binding domain, GafD1-178, has been determined at 1.7A resolution in the presence of the receptor sugar N-acetyl-D-glucosamine. The overall fold is a beta-barrel jelly-roll fold. The ligand-binding site was identified and localized to the side of the molecule. Receptor binding is mediated by side-chain as well main-chain interactions. Ala43-Asn44, Ser116-Thr117 form the sugar acetamide specificity pocket, while Asp88 confers tight binding and Trp109 appears to position the ligand. There is a disulfide bond that rigidifies the acetamide specificity pocket. The three fimbrial lectins, GafD, FimH and PapG share similar beta-barrel folds but display different ligand-binding regions and disulfide-bond patterns. We suggest an evolutionary path for the evolution of the very diverse fimbrial lectins from a common ancestral fold.
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91. |
Paton AW,
Paton JC,
Heuzenroeder MW,
Goldwater PN,
Manning PA,
( 1992 ) Cloning and nucleotide sequence of a variant Shiga-like toxin II gene from Escherichia coli OX3:H21 isolated from a case of sudden infant death syndrome. PMID : 1291844 : Abstract >>
Escherichia coli OX3:H21 expressing a toxin related to Shiga-like toxin (SLT) was isolated from the small bowel contents of a case of Sudden Infant Death Syndrome (SIDS). This strain was lysogenic for a lambdoid bacteriophage, but this did not encode the toxin. Southern hybridization analysis of chromosomal DNA revealed that the SLT-related gene was located on a 4.6 kb PstI fragment, which was cloned into E. coli JM109 in both orientations, using the vector pUC19, to generate plasmids pJCP501 and pJCP502. JM109 cells harbouring the recombinant plasmid produced SLT, as judged by cytotoxicity for Vero cells. Nucleotide sequence analysis revealed that the SLT gene was related to, but distinct from, previously reported variants of Shiga-like toxin type II, produced by E. coli from both human and animal sources. The A subunit of the SLT gene from OX3:H21 exhibited 95.9% homology (at both the DNA and derived amino acid sequence level) to the A subunit of the most closely related SLT-II variant. The B subunit was less similar, exhibiting 88.6 and 88.8% homology to the related gene at the DNA and amino acid level, respectively.
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92. |
Batisson I,
Guimond MP,
Girard F,
An H,
Zhu C,
Oswald E,
Fairbrother JM,
Jacques M,
Harel J,
( 2003 ) Characterization of the novel factor paa involved in the early steps of the adhesion mechanism of attaching and effacing Escherichia coli. PMID : 12874331 : DOI : 10.1128/iai.71.8.4516-4525.2003 PMC : PMC166039 Abstract >>
Nonenterotoxigenic porcine Escherichia coli strains belonging to the serogroup O45 have been associated with postweaning diarrhea in swine and adhere to intestinal epithelial cells in a characteristic attaching and effacing (A/E) pattern. O45 porcine enteropathogenic E. coli (PEPEC) strain 86-1390 induces typical A/E lesions in a pig ileal explant model. Using TnphoA transposon insertion mutagenesis on strain 86-1390, we found a mutant that did not induce A/E lesions. The insertion was identified in a gene designated paa (porcine A/E-associated gene). Sequence analysis of paa revealed an open reading frame of 753 bp encoding a 27.6-kDa protein which displayed 100, 51.8, and 49% homology with Paa of enterohemorrhagic E. coli O157:H7 strains (EDL933 and Sakai), PEB3 of Campylobacter jejuni, and AcfC of Vibrio cholerae, respectively. Chromosomal localization studies indicated that the region containing paa was inserted between the yciD and yciE genes at about 28.3 min of the E. coli K-12 chromosome. The presence of paa and eae sequences in the porcine O45 strains is highly correlated with the A/E phenotype. However, the observation that three eae-positive but paa-negative PEPEC O45 strains were A/E negative provides further evidence for the importance of the paa gene in the A/E activity of O45 strains. As well, the complementation of the paa mutant restored the A/E activity of the 86-1390 strain, showing the involvement of Paa in PEPEC pathogenicity. These observations suggest that Paa contributes to the early stages of A/E E. coli virulence.
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93. |
Varsaki A,
Lucas M,
Afendra AS,
Drainas C,
de la Cruz F,
( 2003 ) Genetic and biochemical characterization of MbeA, the relaxase involved in plasmid ColE1 conjugative mobilization. PMID : 12675806 : DOI : 10.1046/j.1365-2958.2003.03441.x Abstract >>
MbeA is a 60 kDa protein encoded by plasmid ColE1. It plays a key role in conjugative mobilization. MbeA*, a slightly truncated version of MbeA, was purified for in vitro analysis. MbeA* catalysed DNA cleavage and strand-transfer reactions using oligonucleotides embracing the ColE1 nic site, which was mapped to 5'-(1469)CTGG/CTTA(1462)-3'. Thus MbeA is the relaxase for ColE1 conjugal mobilization, in spite of the fact that it lacks a three histidine motif considered the invariant signature of conjugative relaxases. Amino acid sequence comparisons suggest MbeA is nevertheless related to the common relaxase protein family. For instance, MbeA residue Y19 could correspond to the invariant tyrosine in Motif I, whereas H97, E104 and N106 may constitute the equivalent residues to the histidine triad in Motif III. This hypothesis was tested by site-directed mutagenesis. MbeA amino acid residues Y19, H97, E104 and N106 were changed to alanine. MbeA mutant N106A showed reduced oligonucleotide cleavage and strand-transfer activities, whereas mutation in the other three residues resulted in proteins without detectable activity, suggesting they are directly implicated in catalysis of DNA-cleavage and strand-transfer reactions. A double substitution of E104 and N106 by histidines, therefore reconstituting the canonical histidine triad, restored relaxase activities to 1% of wild type. Thus, MbeA is a variant of the common relaxase theme with a HEN signature motif, which has to be added to the canonical three histidine motif of previously reported relaxases.
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94. |
Vandemaele F,
Vandekerchove D,
Vereecken M,
Derijcke J,
Dho-Moulin M,
Goddeeris BM,
( N/A ) Sequence analysis demonstrates the conservation of fimH and variability of fimA throughout avian pathogenic Escherichia coli (APEC). PMID : 12657207 : DOI : 10.1051/vetres:2002062 Abstract >>
In this study we sequenced and analysed the fimH and fimA genes of 24 avian pathogenic Escherichia coli (APEC) isolates, in order to investigate their possible conserved nature. Additional parameters (serotype, presence of aerobactin receptor, expression of F1 pili and virulence for chickens) were investigated to look for correlations with the obtained sequences. The sequence analysis demonstrated that FimH is highly conserved among all investigated APEC strains (>99% homology), whereas the major subunit FimA is less conserved, presenting 6 variable regions distributed along the protein. A hydrophilicity analysis suggested several variable domains of FimA to be potential epitopes. We were able to classify the investigated strains into three main groups, on the basis of the amino-acid sequences of the variable regions. This grouping was consistent throughout all variable regions and was independent of serotype, leading to an improved classification of the F1 pili. No correlation was found between the fimH and fimA sequences and the following parameters: avian species, organ of isolation, serotype, presence of aerobactin receptor and virulence for chickens. This study elucidated the molecular structure and the degree of conservation of FimH and FimA among various avian pathogenic E. coli strains.
|
95. |
Wang M,
Tran JH,
Jacoby GA,
Zhang Y,
Wang F,
Hooper DC,
( 2003 ) Plasmid-mediated quinolone resistance in clinical isolates of Escherichia coli from Shanghai, China. PMID : 12821475 : DOI : 10.1128/aac.47.7.2242-2248.2003 PMC : PMC161834 Abstract >>
Although quinolone resistance usually results from chromosomal mutations, recent studies indicate that quinolone resistance can also be plasmid mediated. The gene responsible, qnr, is distinct from the known quinolone resistance genes and in previous studies seemed to be restricted to Klebsiella pneumoniae and Escherichia coli isolates from the University of Alabama in Birmingham, where this resistance was discovered. In Shanghai, the frequency of ciprofloxacin resistance in E. coli has exceeded 50% since 1993. Seventy-eight unique ciprofloxacin-resistant clinical isolates of E. coli from Shanghai hospitals were screened for the qnr gene by colony blotting and Southern hybridization of plasmid DNA. Conjugation experiments were done with azide-resistant E. coli J53 as a recipient with selection for plasmid-encoded antimicrobial resistance (chloramphenicol, gentamicin, or tetracycline) and azide counterselection. qnr genes were sequenced, and the structure of the plasmid DNA adjacent to qnr was analyzed by primer walking with a sequential series of outward-facing sequencing primers with plasmid DNA templates purified from transconjugants. Six (7.7%) of 78 strains gave a reproducible hybridization signal with a qnr gene probe on colony blots and yielded strong signals on plasmid DNA preparations. Quinolone resistance was transferred from all six probe-positive strains. Transconjugants had 16- to 250-fold increases in the MICs of ciprofloxacin relative to that of the recipient. All six strains contained qnr with a nucleotide sequence identical to that originally reported, except for a single nucleotide change (CTA-->CTG at position 537) encoding the same amino acid. qnr was located in complex In4 family class 1 integrons. Two completely sequenced integrons were designated In36 and In37. Transferable plasmid-mediated quinolone resistance associated with qnr is thus prevalent in quinolone-resistant clinical strains of E. coli from Shanghai and may contribute to the rapid increase in bacterial resistance to quinolones in China.
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96. |
Lim D,
( 1992 ) Structure and biosynthesis of unbranched multicopy single-stranded DNA by reverse transcriptase in a clinical Escherichia coli isolate. PMID : 1282191 : DOI : 10.1111/j.1365-2958.1992.tb01788.x Abstract >>
It has been shown that retrons, retro-elements in bacteria, produce a reverse transcriptase (RT) and multicopy single-stranded DNA (msDNA) whose 5' end is covalently linked to RNA (msdRNA) by a 2'-5' phosphodiester bond. Here, I show that a retron in clinical Escherichia coli strain 161 produces an msDNA unlinked to RNA. The msDNA produced by this retron is a 79-nucleotide-long single-stranded DNA with monophosphate on its 5' terminus. When the retron in strain 161 is cloned into E. coli K-12, the majority of msDNA produced in the clone is the same as the msDNA in the clinical strain. However, in the K-12 clone, about 10% of the msDNA produced is present as a DNA covalently linked to RNA. The DNA part of this RNA-DNA compound is an 83 nucleotides long with the same sequence as the unbranched msDNA, except for the presence of four additional nucleotides at the 5' side. From the analysis of the RNA-DNA compound and the results of in vitro synthesis, I show that the primary product of reverse transcription in this retron is an 83-nucleotide-long DNA covalently linked to RNA. This RNA-DNA compound is further processed to the final product, the 79-nucleotide-long msDNA with a terminal 5' monophosphate, by an endonucleolytic cleavage between the fourth and fifth positions of the DNA component of the RNA-DNA compound. The minimum region required for the production of such msDNA free of RNA contains only genes known to be required for the synthesis of branched msDNA-RNA compound in other retrons (msd, msr and ret). This suggests that either the RT has an endonuclease activity or that the msDNA-RNA compound is autocatalytically processed.
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97. |
Perelle S,
Dilasser F,
Grout J,
Fach P,
( 2003 ) Development of a 5'-nuclease PCR assay for detecting Shiga toxin-producing Escherichia coli O145 based on the identification of an 'O-island 29' homologue. PMID : 12631194 : Abstract >>
A DNA sequence, from Escherichia coli STEC O145, homologous to O-island 29 from STEC O157 is described, together with a real-time PCR assay for detecting it. PCR and sequencing were used to identify the 'O-island 29' homologous DNA sequence from STEC O145 (strain VTH34). The sequence divergence between the STEC O145 and O157 'O-island 29' allowed a STEC O145 5'-nuclease PCR assay to be developed. The characterization of a novel locus in STEC O145 has allowed a specific O145 serogroup 5'-nuclease PCR assay to be designed. These findings increase the number of serogroup PCR assays available as alternatives to classical O-serotyping of E. coli.
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98. |
Dobrindt U,
Agerer F,
Michaelis K,
Janka A,
Buchrieser C,
Samuelson M,
Svanborg C,
Gottschalk G,
Karch H,
Hacker J,
( 2003 ) Analysis of genome plasticity in pathogenic and commensal Escherichia coli isolates by use of DNA arrays. PMID : 12618447 : DOI : 10.1128/jb.185.6.1831-1840.2003 PMC : PMC150128 Abstract >>
Genomes of prokaryotes differ significantly in size and DNA composition. Escherichia coli is considered a model organism to analyze the processes involved in bacterial genome evolution, as the species comprises numerous pathogenic and commensal variants. Pathogenic and nonpathogenic E. coli strains differ in the presence and absence of additional DNA elements contributing to specific virulence traits and also in the presence and absence of additional genetic information. To analyze the genetic diversity of pathogenic and commensal E. coli isolates, a whole-genome approach was applied. Using DNA arrays, the presence of all translatable open reading frames (ORFs) of nonpathogenic E. coli K-12 strain MG1655 was investigated in 26 E. coli isolates, including various extraintestinal and intestinal pathogenic E. coli isolates, 3 pathogenicity island deletion mutants, and commensal and laboratory strains. Additionally, the presence of virulence-associated genes of E. coli was determined using a DNA "pathoarray" developed in our laboratory. The frequency and distributional pattern of genomic variations vary widely in different E. coli strains. Up to 10% of the E. coli K-12-specific ORFs were not detectable in the genomes of the different strains. DNA sequences described for extraintestinal or intestinal pathogenic E. coli are more frequently detectable in isolates of the same origin than in other pathotypes. Several genes coding for virulence or fitness factors are also present in commensal E. coli isolates. Based on these results, the conserved E. coli core genome is estimated to consist of at least 3,100 translatable ORFs. The absence of K-12-specific ORFs was detectable in all chromosomal regions. These data demonstrate the great genome heterogeneity and genetic diversity among E. coli strains and underline the fact that both the acquisition and deletion of DNA elements are important processes involved in the evolution of prokaryotes.
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99. |
Kim PD,
Banack T,
Lerman DM,
Tracy JC,
Camara JE,
Crooke E,
Oliver D,
Firshein W,
( 2003 ) Identification of a novel membrane-associated gene product that suppresses toxicity of a TrfA peptide from plasmid RK2 and its relationship to the DnaA host initiation protein. PMID : 12618445 : DOI : 10.1128/jb.185.6.1817-1824.2003 PMC : PMC150145 Abstract >>
The toxicity of a peptide derived from the amino-terminal portion of 33-kDa TrfA, one of the initiation proteins encoded by the broad-host-range plasmid RK2, was suppressed by a host protein related to DnaA, the initiation protein of Escherichia coli. The newly identified 28.4-kDa protein, termed a DnaA paralog (Dp) because it is similar to a region of DnaA but likely has a different function in initiation of plasmid RK2 replication, interacts physically with the 33-kDa TrfA initiation protein, including the initiation-active monomeric form. The Dp has a cellular distribution similar to that of the 33-kDa TrfA initiation protein, being found primarily in the inner membrane fraction, with lesser amounts detected in the outer membrane fraction and almost none in the soluble fraction of E. coli. Maintenance and inner membrane-associated replication of plasmid RK2 were enhanced in a Dp knockout strain and inhibited in strains containing extra copies of the Dp gene or in membrane extracts to which a tagged form of Dp was added. Recently, the Dp was independently shown to help prevent overinitiation in E. coli and was termed Hda (S. Kato and T. Katayama, EMBO J. 20:4253-4262, 2001).
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100. |
Yaron S,
White DG,
Matthews KR,
( 2003 ) Characterization of an Escherichia coli O157:H7 marR mutant. PMID : 12878386 : Abstract >>
One mechanism for generation of multiple antibiotic resistance in enteric bacteria involves the global regulatory system marRAB. The operon, when induced, encodes for resistance to structurally and functionally unrelated antibiotics. Exposure of Escherichia coli O157:H7 to increasing levels of chloramphenicol (CHL) resulted in survival of mutants that were resistant to the inducing agent and to tetracycline (TET), nalidixic acid (NAL), and ciprofloxacin (CIP). A mutant (RU122) that lacks MarR, the transcriptional repressor of the multiple antibiotic resistance (mar) operon, served as a genetic tool to study the role of MarR in growth of E. coli O157:H7. No significant difference (P>0.05) was observed in growth curves of the wild-type or the mutant under the conditions examined (rich and minimal media, acidic conditions, and temperatures from 24 to 42 C). A preconditioned mutant (indRU122; cultured for 17 h in Luria-Bertani (LB) containing chloramphenicol and then examined) exhibited greater growth under all treatments tested compared to the mutant. marCRAB of E. coli O157:H7 was sequenced and compared to other known mar sequences to determine divergence from other bacteria (Salmonella, Klebsiella, and Enterobacter).
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101. |
Buts L,
Bouckaert J,
De Genst E,
Loris R,
Oscarson S,
Lahmann M,
Messens J,
Brosens E,
Wyns L,
De Greve H,
( 2003 ) The fimbrial adhesin F17-G of enterotoxigenic Escherichia coli has an immunoglobulin-like lectin domain that binds N-acetylglucosamine. PMID : 12864853 : DOI : 10.1046/j.1365-2958.2003.03600.x Abstract >>
The F17-G adhesin at the tip of flexible F17 fimbriae of enterotoxigenic Escherichia coli mediates binding to N-acetyl-beta-D-glucosamine-presenting receptors on the microvilli of the intestinal epithelium of ruminants. We report the 1.7 A resolution crystal structure of the lectin domain of F17-G, both free and in complex with N-acetylglucosamine. The monosaccharide is bound on the side of the ellipsoid-shaped protein in a conserved site around which all natural variations of F17-G are clustered. A model is proposed for the interaction between F17-fimbriated E. coli and microvilli with enhanced affinity compared with the binding constant we determined for F17-G binding to N-acetylglucosamine (0.85 mM-1). Unexpectedly, the F17-G structure reveals that the lectin domains of the F17-G, PapGII and FimH fimbrial adhesins all share the immunoglobulin-like fold of the structural components (pilins) of their fimbriae, despite lack of any sequence identity. Fold comparisons with pilin and chaperone structures of the chaperone/usher pathway highlight the central role of the C-terminal beta-strand G of the immunoglobulin-like fold and provides new insights into pilus assembly, function and adhesion.
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102. |
Kita K,
Kawakami H,
Tanaka H,
( 2003 ) Evidence for horizontal transfer of the EcoT38I restriction-modification gene to chromosomal DNA by the P2 phage and diversity of defective P2 prophages in Escherichia coli TH38 strains. PMID : 12644501 : DOI : 10.1128/jb.185.7.2296-2305.2003 PMC : PMC151499 Abstract >>
A DNA fragment carrying the genes coding for a novel EcoT38I restriction endonuclease (R.EcoT38I) and EcoT38I methyltransferase (M.EcoT38I), which recognize G(A/G)GC(C/T)C, was cloned from the chromosomal DNA of Escherichia coli TH38. The endonuclease and methyltransferase genes were in a head-to-head orientation and were separated by a 330-nucleotide intergenic region. A third gene, the C.EcoT38I gene, was found in the intergenic region, partially overlapping the R.EcoT38I gene. The gene product, C.EcoT38I, acted as both a positive regulator of R.EcoT38I gene expression and a negative regulator of M.EcoT38I gene expression. M.EcoT38I purified from recombinant E. coli cells was shown to be a monomeric protein and to methylate the inner cytosines in the recognition sequence. R.EcoT38I was purified from E. coli HB101 expressing M.EcoT38I and formed a homodimer. The EcoT38I restriction (R)-modification (M) system (R-M system) was found to be inserted between the A and Q genes of defective bacteriophage P2, which was lysogenized in the chromosome at locI, one of the P2 phage attachment sites observed in both E. coli K-12 MG1655 and TH38 chromosomal DNAs. Ten strains of E. coli TH38 were examined for the presence of the EcoT38I R-M gene on the P2 prophage. Conventional PCR analysis and assaying of R activity demonstrated that all strains carried a single copy of the EcoT38I R-M gene and expressed R activity but that diversity of excision in the ogr, D, H, I, and J genes in the defective P2 prophage had arisen.
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103. |
Monteiro-Neto V,
Bando SY,
Moreira-Filho CA,
Girón JA,
( 2003 ) Characterization of an outer membrane protein associated with haemagglutination and adhesive properties of enteroaggregative Escherichia coli O111:H12. PMID : 12864813 : Abstract >>
Diarrhoeagenic Escherichia coli strains of serotype O111:H12 are characterized by their aggregative pattern of adherence on cultured epithelial cells and thus are considered enteroaggregative E. coli (EAEC). We have previously shown that these EAEC strains lack the genes encoding the aggregative fimbriae I and II described in other heterologous EAEC strains. In this paper, we show compelling data suggesting that a plasmid-encoded outer membrane 58 kDa protein termed aggregative protein 58 (Ap58) produced by EAEC O111:H12 strains, is associated with the adherence capabilities and haemagglutination of animal red blood cells. This conclusion is supported by several lines of evidence: (i) adherent O111:H12 strains are able to produce Ap58; (ii) non-adherent O111:H12 strains are unable to produce Ap58; (iii) antibodies raised against Ap58 inhibited adherence and haemagglutination of epithelial and bovine red blood cells, respectively; (iv) a non-adherent E. coli K-12 host strain containing the ap58 gene determinant on plasmid pVM15 displayed abundant adherence to cultured HEp-2 cells; and (v) the purified Ap58 bound specifically to HEp-2 and bovine red blood cells. Our findings indicate that the aggregative adherence in the O111:H12 strains may be also mediated by non-fimbrial adhesins. We believe our data contribute to the understanding of the adherence mechanisms of these organisms.
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104. |
Rumer L,
Jores J,
Kirsch P,
Cavignac Y,
Zehmke K,
Wieler LH,
( 2003 ) Dissemination of pheU- and pheV-located genomic islands among enteropathogenic (EPEC) and enterohemorrhagic (EHEC) E. coli and their possible role in the horizontal transfer of the locus of enterocyte effacement (LEE). PMID : 12635929 : DOI : 10.1078/1438-4221-00229 Abstract >>
We have recently shown that the locus of enterocyte effacement (LEE) of the bovine enterohemorrhagic E. coli RW1374 (O103:H2) resides within a large pathogenicity island (PAI), integrated in the vicinity of the phenylalanine tRNA gene pheV. Here we describe an additional, but LEE-negative genomic island in RW1374 in the vicinity of another phenylalanine tRNA gene, pheU, the sequence of which is identical to pheV. These two genomic islands revealed identity of the left, but a relative variability of their right end sequences. To investigate the mechanism of LEE-PAI distribution in E. coli, we analysed similar junctions in the pheU/pheV loci of additional EPEC and EHEC strains the LEE location of which had not been determined before. By hybridisation of NotI restriction fragments with probes specific for LEE, pheV locus, and pheU locus, the LEE was found linked to either one of these two loci. The results agreed well with recently published phylogenetic data and indicate that in the clones of diarrheagenic E. coli (Dec) Dec 11 and Dec 12, forming the phylogenetic cluster EPEC 2, and in the strains of the most typical serotypes of the Dec 8, belonging to the phylogenetic cluster EHEC 2, the LEE was linked with pheV and not with the pheU locus as previously assumed. Sequence comparison with other pheU- and pheV-located genomic islands from different E. coli pathotypes (uropathogenic E. coli, septicemic E. coli) as well as from Shigella indicated the same structural features at the junctions. These conserved structures suggested a common DNA cassette, serving as common vehicle for horizontal gene transfer of various PAls. In addition, the elements suggest an origin from a common pheU-located ancestor and integration into the chromosome through site-specific recombination. Our results indicate that pheU/pheV-located genomic islands played an important role in the evolution of several PAls in E. coli and related pathogens.
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105. |
Aubel D,
Darfeuille-Michaud A,
Martin C,
Joly B,
( 1992 ) Nucleotide sequence of the nfaA gene encoding the antigen 8786 adhesive factor of enterotoxigenic Escherichia coli. PMID : 1281130 : DOI : 10.1016/0378-1097(92)90169-o Abstract >>
The nucleotide sequence of a 714-bp DNA fragment containing the enterotoxic Escherichia coli (ETEC) adhesive factor 8786 structural gene, designated nfaA, revealed an open reading frame of 498 bp encoding a polypeptide of 166 amino acids. Primer-extension experiments showed that the nfaA gene is within a single transcription unit. No homology was found with the ETEC adhesive factors already sequenced. In contrast, a homology with Salmonella enteritidis fimbrin SEF 14 was observed.
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106. |
Schmitt L,
Benabdelhak H,
Blight MA,
Holland IB,
Stubbs MT,
( 2003 ) Crystal structure of the nucleotide-binding domain of the ABC-transporter haemolysin B: identification of a variable region within ABC helical domains. PMID : 12823972 : DOI : 10.1016/s0022-2836(03)00592-8 Abstract >>
The ABC-transporter haemolysin B is a central component of the secretion machinery that translocates the toxin, haemolysin A, in a Sec-independent fashion across both membranes of E. coli. Here, we report the X-ray crystal structure of the nucleotide-binding domain (NBD) of HlyB. The molecule shares the common overall architecture of ABC-transporter NBDs. However, the last three residues of the Walker A motif adopt a 3(10) helical conformation, stabilized by a bound anion. In consequence, this results in an unusual interaction between the Walker A lysine residue and the Walker B glutamate residue. As these residues are normally required to be available for ATP binding, for catalysis and for dimer formation of ABC domains, we suggest that this conformation may represent a latent monomeric form of the NBD. Surprisingly, comparison of available NBD structures revealed a structurally diverse region (SDR) of about 30 residues within the helical arm II domain, unique to each of the eight NBDs analyzed. As this region interacts with the transmembrane part of ABC-transporters, the SDR helps to explain the selectivity and/or targeting of different NBDs to their cognate transmembrane domains.
|
107. |
Steenbergen SM,
Vimr ER,
( 2003 ) Functional relationships of the sialyltransferases involved in expression of the polysialic acid capsules of Escherichia coli K1 and K92 and Neisseria meningitidis groups B or C. PMID : 12578835 : DOI : 10.1074/jbc.M208837200 Abstract >>
Polysialic acid (PSA) capsules are cell-associated homopolymers of alpha2,8-, alpha2,9-, or alternating alpha2,8/2,9-linked sialic acid residues that function as essential virulence factors in neuroinvasive diseases caused by certain strains of Escherichia coli and Neisseria meningitidis. PSA chains structurally identical to the bacterial alpha2,8-linked capsular polysaccharides are also synthesized by the mammalian central nervous system, where they regulate neuronal function in association with the neural cell adhesion molecule (NCAM). Despite the structural identity between bacterial and NCAM PSAs, the respective polysialyltransferases (polySTs) responsible for polymerizing sialyl residues from donor CMP-sialic acid are not homologous glycosyltransferases. To better define the mechanism of capsule biosynthesis, we established the functional interchangeability of bacterial polySTs by complementation of a polymerase-deficient E. coli K1 mutant with the polyST genes from groups B or C N. meningitidis and the control E. coli K92 polymerase gene. The biochemical and immunochemical results demonstrated that linkage specificity is dictated solely by the source of the polymerase structural gene. To determine the molecular basis for linkage specificity, we created chimeras of the K1 and K92 polySTs by overlap extension PCR. Exchanging the first 52 N-terminal amino acids of the K1 NeuS with the C terminus of the K92 homologue did not alter specificity of the resulting chimera, whereas exchanging the first 85 or reciprocally exchanging the first 100 residues did. These results demonstrated that linkage specificity is dependent on residues located between positions 53 and 85 from the N terminus. Site-directed mutagenesis of the K92 polyST N terminus indicated that no single residue alteration was sufficient to affect specificity, consistent with the proposed function of this domain in orienting the acceptor. The combined results provide the first evidence for residues critical to acceptor binding and elongation in polysialyltransferase.
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108. |
Kyaw CM,
De Araujo CR,
Lima MR,
Gondim EG,
Brígido MM,
Giugliano LG,
( 2003 ) Evidence for the presence of a type III secretion system in diffusely adhering Escherichia coli (DAEC). PMID : 12809805 : Abstract >>
Diffusely adhering Escherichia coli (E. coli) strains (DAEC) represent a potential cause of diarrhoea in infants, and the detection of type three secretion system (TTSS) genes in DAEC would substantiate their pathogenic nature. In this work, four isolates of DAEC, recovered from stools of diarrhoeic children, were analysed by PCR, in order to detect the presence of TTSS genes. Primers targeted to the escC, escJ, escN and escV, some of the most conserved TTSS genes in enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC and EHEC), were used in order to verify the occurrence of homologous genes in our DAEC isolates. By this approach, we were able to characterise DNA fragments corresponding to putative escJ and escN genes in all DAEC isolates. Furthermore, DNA fragments homologous to the escC and escV genes were also amplified from all isolates. Besides the similarity found among the DAEC esc homologues with EPEC and EHEC esc genes, the nucleotide sequence analysis of the flanking regions of the amplified DNA fragments suggests that the putative DAEC esc genes are organised in the same manner as observed in EPEC and in EHEC strains. The results described here provide strong evidence for the presence of a TTSS in the DAEC strains analysed, implicating a pathogenic nature of these isolates.
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109. |
Blank TE,
Lacher DW,
Scaletsky IC,
Zhong H,
Whittam TS,
Donnenberg MS,
( 2003 ) Enteropathogenic Escherichia coli O157 strains from Brazil. PMID : 12533292 : DOI : 10.3201/eid0901.020072 PMC : PMC2873750 Abstract >>
We describe two serogroup O157 Escherichia coli strains from Brazilian infants with diarrhea. A variety of assays indicate that these strains belong to the enteropathogenic, not the enterohemorrhagic, pathotype. These strains possess a novel bfpA allele encoding the type IV pilin characteristic of typical enteropathogenic E. coli strains. Our results emphasize the pitfalls of classifying pathogenic E. coli by serogroup.
|
110. |
Perreten V,
Boerlin P,
( 2003 ) A new sulfonamide resistance gene (sul3) in Escherichia coli is widespread in the pig population of Switzerland. PMID : 12604565 : DOI : 10.1128/aac.47.3.1169-1172.2003 PMC : PMC149312 Abstract >>
A new gene, sul3, which specifies a 263-amino-acid protein similar to a dihydropteroate synthase encoded by the 54-kb conjugative plasmid pVP440 from Escherichia coli was characterized. Expression of the cloned sul3 gene conferred resistance to sulfamethoxazole on E. coli. Two copies of the insertion element IS15Delta/26 flanked the region containing sul3. The sul3 gene was detected in one-third of the sulfonamide-resistant pathogenic E. coli isolates from pigs in Switzerland.
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111. |
Partridge SR,
Hall RM,
( 2003 ) In34, a complex In5 family class 1 integron containing orf513 and dfrA10. PMID : 12499211 : DOI : 10.1128/aac.47.1.342-349.2003 PMC : PMC149023 Abstract >>
A complex class 1 integron, In34, found in a conjugative plasmid from a multidrug-resistant Klebsiella pneumoniae strain isolated in 1997 at a hospital in Sydney, Australia, was shown to have a backbone related to that of In2, which belongs to the In5 family. In In34, the aadB gene cassette replaces the aadA1a cassette in In2, and two additional resistance genes, dfrA10 and aphA1, that are not part of a gene cassette are present. The aphA1 gene is in a Tn4352-like transposon that is located in the tniA gene. The dfrA10 gene lies adjacent to a 2,154-bp DNA segment, known as the common region, that contains an open reading frame predicting a product of 513 amino acids (Orf513). Orf513 is 66 and 55% identical to the products of two further open reading frames that, like the common region, are found adjacent to antibiotic resistance genes. A 27-bp conserved sequence was found at one end of each type of common region. The loss of dfrA10 due to homologous recombination between flanking direct repeats and incorporation of the excised circle by homologous recombination were demonstrated. Part of In34 is identical to the sequenced portion of In7, which is from a multidrug-resistant Escherichia coli strain that had been isolated 19 years earlier in the same hospital. In34 and In7 are in plasmids that contain the same six resistance genes conferring resistance to ampicillin, chloramphenicol, gentamicin, kanamycin, neomycin, tobramycin, trimethoprim, and sulfonamides, but the plasmid backbones appear to be unrelated, suggesting that translocation of a multiple-drug-resistance-determining region as well as horizontal transfer may have occurred.
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112. |
Essa AM,
Julian DJ,
Kidd SP,
Brown NL,
Hobman JL,
( 2003 ) Mercury resistance determinants related to Tn21, Tn1696, and Tn5053 in enterobacteria from the preantibiotic era. PMID : 12604550 : DOI : 10.1128/aac.47.3.1115-1119.2003 PMC : PMC149298 Abstract >>
Three mer transposons from the Murray collection of preantibiotic enterobacteria show >99% sequence identity to current isolates. Tn5073 is most closely related to Tn5036 and Tn1696, and Tn5074 is most closely related to Tn5053. Tn5075 is most closely related to Tn21 but lacks integron In2 and is flanked by insertion elements.
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113. |
Altboum Z,
Levine MM,
Galen JE,
Barry EM,
( 2003 ) Genetic characterization and immunogenicity of coli surface antigen 4 from enterotoxigenic Escherichia coli when it is expressed in a Shigella live-vector strain. PMID : 12595452 : DOI : 10.1128/iai.71.3.1352-1360.2003 PMC : PMC148885 Abstract >>
The genes that encode the enterotoxigenic Escherichia coli (ETEC) CS4 fimbriae, csaA, -B, -C, -E, and -D', were isolated from strain E11881A. The csa operon encodes a 17-kDa major fimbrial subunit (CsaB), a 40-kDa tip-associated protein (CsaE), a 27-kDa chaperone-like protein (CsaA), a 97-kDa usher-like protein (CsaC), and a deleted regulatory protein (CsaD'). The predicted amino acid sequences of the CS4 proteins are highly homologous to structural and assembly proteins of other ETEC fimbriae, including CS1 and CS2, and to CFA/I in particular. The csaA, -B, -C, -E operon was cloned on a stabilized plasmid downstream from an osomotically regulated ompC promoter. pGA2-CS4 directs production of CS4 fimbriae in both E. coli DH5alpha and Shigella flexneri 2a vaccine strain CVD 1204, as detected by Western blot analysis and bacterial agglutination with anti-CS4 immune sera. Electron-microscopic examination of Shigella expressing CS4 confirmed the presence of fimbriae on the bacterial surface. Guinea pigs immunized with CVD 1204(pGA2-CS4) showed serum and mucosal antibody responses to both the Shigella vector and the ETEC fimbria CS4. Among the seven most prevalent fimbrial antigens of human ETEC, CS4 is the last to be cloned and sequenced. These findings pave the way for CS4 to be included in multivalent ETEC vaccines, including an attenuated Shigella live-vector-based ETEC vaccine.
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114. |
Rodríguez E,
Laviña M,
( 2003 ) The proton channel is the minimal structure of ATP synthase necessary and sufficient for microcin h47 antibiotic action. PMID : 12499189 : DOI : 10.1128/aac.47.1.181-187.2003 PMC : PMC148971 Abstract >>
It had been previously determined that the presence of F(o)F(1) ATP synthase was required for microcin H47 antibiotic action. In this work, microcin-resistant atp mutants were genetically analyzed. Their mutations, originated by Tn5 insertion, in all cases were found to affect determinants for the F(o) portion of ATP synthase. To discern if microcin action required the presence of the entire complex or if the F(o) proton channel would suffice, recombinant plasmids carrying different segments of the atp operon were constructed and introduced into an atp deletion strain. The phenotypic analysis of the strains thus obtained clearly indicated that the presence of the F(o) proton channel was absolutely required for microcin H47 action, while the F(1) catalytic portion was found to be dispensable. Furthermore, when any of the three components of the proton channel was missing, total resistance to the antibiotic ensued. Complementation analysis between atp::Tn5 chromosomal mutations and recombinant atp plasmid constructions further supported the idea that the proton channel would be the minimal structure of the ATP synthase complex needed for microcin H47 antibiotic action.
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115. |
Rahn A,
Whitfield C,
( 2003 ) Transcriptional organization and regulation of the Escherichia coli K30 group 1 capsule biosynthesis (cps) gene cluster. PMID : 12581358 : DOI : 10.1046/j.1365-2958.2003.03354.x Abstract >>
Escherichia coli group 1 capsules are important virulence determinants, yet little is known about the transcriptional organization or regulation of their biosynthetic (cps) operons. Transcription of the prototype serotype K30 cluster is modulated by the JUMPStart-RfaH antitermination mechanism, with the cps promoter being localized to a region immediately upstream of the JUMPStart sequence. A putative stem-loop structure located within the K30 cps cluster separates conserved genes with products that are required for surface expression of capsule from serotype-specific genes encoding enzymes for polymer repeat-unit synthesis and polymerization. This putative stem-loop structure significantly reduces transcription in a termination-probe vector and may contribute to differential expression of the cps genes. Previous work indicated that increased amounts of group 1 capsular polysaccharide synthesis resulted from the overexpression of the Rcs (regulator of capsule synthesis) proteins. However, neither overexpression of the transcriptional activator RcsB nor an rcsB::aadA chromosomal insertion altered the level of transcription measured by cps::lacZ fusions. In the group 1 strains examined, an RcsAB box was found immediately upstream of galF, a gene involved in the production of sugar nucleotide precursors. Overexpression of RcsB was found to result in a threefold increase in transcription of a galF::lacZ chromosomal fusion. Moreover, overexpression of GalF gave rise to a two- to threefold increase in cell-free as well as cell-associated capsule, without affecting cps::lacZ activity. These results indicate that transcription of the E. coli group 1 capsule cluster itself is not regulated by the Rcs system and may, in fact, be constitutive. However, the Rcs system can potentially influence levels of capsular polysaccharide production by increasing galF transcription and influencing the available pool of biosynthetic precursors.
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116. |
Dubois V,
Arpin C,
Quentin C,
Texier-Maugein J,
Poirel L,
Nordmann P,
( 2003 ) Decreased susceptibility to cefepime in a clinical strain of Escherichia coli related to plasmid- and integron-encoded OXA-30 beta-lactamase. PMID : 12821505 : DOI : 10.1128/aac.47.7.2380-2381.2003 PMC : PMC161840 Abstract >>
N/A
|
117. |
Starcic M,
Zgur-Bertok D,
Jordi BJ,
Wösten MM,
Gaastra W,
van Putten JP,
( 2003 ) The cyclic AMP-cyclic AMP receptor protein complex regulates activity of the traJ promoter of the Escherichia coli conjugative plasmid pRK100. PMID : 12591879 : DOI : 10.1128/jb.185.5.1616-1623.2003 PMC : PMC148056 Abstract >>
The TraJ protein is a central activator of F-like plasmid conjugal transfer. In a search for regulators of traJ expression, we studied the possible regulatory role of the cyclic AMP (cAMP)-cAMP receptor protein (CRP) complex in traJ transcription using a traJ-lacZ reporter system. A comparison of the enzyme activities in the wild-type Escherichia coli strain MC4100 with those in cya and crp mutants indicated that disruption of the formation of the cAMP-CRP complex negatively influenced the activity of the traJ promoter of the F-like plasmid pRK100. The defect in the cya mutant was partially restored by addition of exogenous cAMP. Competitive reverse transcription-PCR performed with RNA isolated from the wild-type and mutant strains showed that the cAMP-CRP complex exerted its effect at the level of transcription. Electrophoretic mobility shift assays with purified CRP demonstrated that there was direct binding of CRP to the traJ promoter region. DNase I footprint experiments mapped the CRP binding site around position -67.5 upstream of the putative traJ promoter. Targeted mutagenesis of the traJ promoter region confirmed the location of the CRP binding site. Consistent with the demonstrated regulation of TraJ by the cAMP-CRP complex, mutants with defects in cya or crp exhibited reduced conjugal transfer from pRK100.
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118. |
Fratamico PM,
Briggs CE,
Needle D,
Chen CY,
DebRoy C,
( 2003 ) Sequence of the Escherichia coli O121 O-antigen gene cluster and detection of enterohemorrhagic E. coli O121 by PCR amplification of the wzx and wzy genes. PMID : 12843098 : DOI : 10.1128/jcm.41.7.3379-3383.2003 PMC : PMC165269 Abstract >>
The DNA sequence of the 15,155-bp O-antigen gene cluster of Escherichia coli O121 was determined, and 14 open reading frames were identified (all had the same transcriptional direction). Analyses of results indicated that the wzx (O-antigen flippase) and wzy (O-antigen polymerase) genes were E. coli O121 specific, so regions in these two genes were chosen for development of PCR assays. The PCR assays using DNA from 99 E. coli O121 strains, strains representative of non-O121 E. coli serogroups, and strains of other bacterial genera and PCR assays using DNA from seven enrichments of swine fecal samples naturally contaminated with E. coli O121 showed specificity for E. coli O121. Thus, the PCR assay can be employed to reliably identify E. coli O121 and to potentially detect the organism in food, fecal, and environmental samples.
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119. |
García A,
Marini RP,
Feng Y,
Vitsky A,
Knox KA,
Taylor NS,
Schauer DB,
Fox JG,
( 2002 ) A naturally occurring rabbit model of enterohemorrhagic Escherichia coli-induced disease. PMID : 12447748 : DOI : 10.1086/345371 Abstract >>
Enterohemorrhagic Escherichia coli (EHEC) causes hemorrhagic colitis and hemolytic-uremic syndrome (HUS) in humans. The exact mechanism by which EHEC induces disease remains unclear because of the lack of a natural animal model for the disease. An outbreak of bloody diarrhea and sudden death was investigated in a group of Dutch belted rabbits. Two of these rabbits harbored enteropathogenic E. coli O145:H(-), and 1 rabbit was coinfected with EHEC O153:H(-). A partial Shiga toxin 1 gene (stx1) fragment from E. coli O153:H(-) was confirmed by Southern blot and sequence analysis. Toxin production was demonstrated by a HeLa cell cytotoxicity assay. Histopathologic findings in all affected rabbits included erosive and necrotizing enterocolitis with adherent bacterial rods, proliferative glomerulonephritis, tubular necrosis, and fibrin thrombi within small vessels and capillaries. Our findings provide evidence for a naturally occurring animal model of EHEC-induced systemic disease that closely resembles human HUS.
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120. |
Waturangi DE,
Suwanto A,
Schwarz S,
Erdelen W,
( 2003 ) Identification of class 1 integrons-associated gene cassettes in Escherichia coli isolated from Varanus spp. in Indonesia. PMID : 12493806 : DOI : 10.1093/jac/dkf253 Abstract >>
N/A
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121. |
Karasawa T,
Ito H,
Tsukamoto T,
Yamasaki S,
Kurazono H,
Faruque SM,
Nair GB,
Nishibuchi M,
Takeda Y,
( 2002 ) Cloning and characterization of genes encoding homologues of the B subunit of cholera toxin and the Escherichia coli heat-labile enterotoxin from clinical isolates of Citrobacter freundii and E. coli. PMID : 12438400 : DOI : 10.1128/iai.70.12.7153-7155.2002 PMC : PMC133046 Abstract >>
We identified and characterized a gene encoding a homologue of the B subunits of cholera toxin (CTB) and heat-labile enterotoxin (LTB) of Escherichia coli from a clinical isolate of Citrobacter freundii that was found to produce a factor in the culture supernatant that cross-reacted with antibodies to CTB and LTB when assayed by enzyme-linked immunosorbent assay (ELISA). The gene encoding the ELISA-positive factor, cfxB, consisted of 375 nucleotides and was located downstream of an 852-nucleotide open reading frame, cfxA, with a 56-nucleotide intergenic space. The cfxB gene was predicted to encode a 125-amino-acid polypeptide, which had 73.8 and 72.8% identities with the amino acid sequences of LTB and CTB, respectively. However, the amino acid sequence of the deduced polypeptide CFXA had no homologies to those of the A subunits of CT or LT. DNA probes developed from the sequences of cfxA and cfxB were used to screen 67 C. freundii isolates and 152 E. coli isolates from diarrheal patients by colony blot hybridization. Two strains, C. freundii 48 and E. coli 176, reacted with both DNA probes under conditions of high stringency. We cloned homologues of the cfxA and cfxB genes from E. coli 176 and designated them ecxA and ecxB, respectively. The ecxA gene and the ecxB gene comprise 855 and 375 nucleotides, respectively, with a 50-nucleotide intergenic space, and encode a 285- and a 125-amino-acid residue polypeptides, respectively. The results of the present study may provide important clues to the origin and evolution of immunologically related factors sharing a common enterotoxin-like A and B subunit structures.
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122. |
Sijbrandi R,
Urbanus ML,
ten Hagen-Jongman CM,
Bernstein HD,
Oudega B,
Otto BR,
Luirink J,
( 2003 ) Signal recognition particle (SRP)-mediated targeting and Sec-dependent translocation of an extracellular Escherichia coli protein. PMID : 12466262 : DOI : 10.1074/jbc.M211630200 Abstract >>
Hemoglobin protease (Hbp) is a hemoglobin-degrading protein that is secreted by a human pathogenic Escherichia coli strain via the autotransporter mechanism. Little is known about the earliest steps in autotransporter secretion, i.e. the targeting to and translocation across the inner membrane. Here, we present evidence that Hbp interacts with the signal recognition particle (SRP) and the Sec-translocon early during biogenesis. Furthermore, Hbp requires a functional SRP targeting pathway and Sec-translocon for optimal translocation across the inner membrane. SecB is not required for targeting of Hbp but can compensate to some extent for the lack of SRP. Hbp is synthesized with an unusually long signal peptide that is remarkably conserved among a subset of autotransporters. We propose that these autotransporters preferentially use the co-translational SRP/Sec route to avoid adverse effects of the exposure of their mature domains in the cytoplasm.
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123. |
Dozois CM,
Daigle F,
Curtiss R,
( 2003 ) Identification of pathogen-specific and conserved genes expressed in vivo by an avian pathogenic Escherichia coli strain. PMID : 12506201 : DOI : 10.1073/pnas.232686799 PMC : PMC140941 Abstract >>
Escherichia coli is a diverse bacterial species that comprises commensal nonpathogenic strains such as E. coli K-12 and pathogenic strains that cause a variety of diseases in different host species. Avian pathogenic E. coli strain chi7122 (O78:K80:H9) was used in a chicken infection model to identify bacterial genes that are expressed in infected tissues. By using the cDNA selection method of selective capture of transcribed sequences and enrichment for the isolation of pathogen-specific (non-E. coli K-12) transcripts, pathogen-specific cDNAs were identified. Pathogen-specific transcripts corresponded to putative adhesins, lipopolysaccharide core synthesis, iron-responsive, plasmid- and phage-encoded genes, and genes of unknown function. Specific deletion of the aerobactin siderophore system and E. coli iro locus, which were identified by selective capture of transcribed sequences, demonstrated that these pathogen-specific systems contribute to the virulence of strain chi7122. Consecutive blocking to enrich for selection of pathogen-specific genes did not completely eliminate the presence of transcripts that corresponded to sequences also present in E. coli K-12. These E. coli conserved genes are likely to be highly expressed in vivo and contribute to growth or virulence. Overall, the approach we have used simultaneously provided a means to identify novel pathogen-specific genes expressed in vivo and insight regarding the global gene expression and physiology of a pathogenic E. coli strain in a natural animal host during the infectious process.
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124. |
Zhang WL,
Köhler B,
Oswald E,
Beutin L,
Karch H,
Morabito S,
Caprioli A,
Suerbaum S,
Schmidt H,
( 2002 ) Genetic diversity of intimin genes of attaching and effacing Escherichia coli strains. PMID : 12454140 : DOI : 10.1128/jcm.40.12.4486-4492.2002 PMC : PMC154638 Abstract >>
In this study, we determined the sequences of four intimin variant genes detected in attaching and effacing Escherichia coli isolates of human origin. Three of them were novel and were designated eae-eta (eta), eae-iota (iota), and eae-kappa (kappa). The fourth was identical to the recently described eae-zeta (zeta), isolated from a bovine E. coli O84:NM isolate. We compared these sequences with those of published intimin-alpha, intimin-beta, intimin-gamma1, intimin-gamma2, intimin- epsilon, and intimin-theta alleles. Sequence analysis of these 10 intimin alleles confirmed extensive genetic diversity within the intimin gene family in E. coli. The genetic diversity was more prominent in the 3' region (starting at bp 2,112), which encodes the binding domain of intimin. Phylogenetic analyses revealed four groups of closely related intimin genes: alpha and zeta; beta and kappa; gamma1 and gamma2/theta; and epsilon and eta. Calculation of homoplasy ratios of sequences of the 5' region of eae (positions 1 to 2,111) revealed evidence for intragenic recombination. Split decomposition analysis also indicates that recombination events have played a role in the evolutionary history of eae. In conclusion, we recommend an eae nomenclature system based on the Greek alphabet and provide an updated PCR scheme for amplification and typing of E. coli eae.
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125. |
Sun T,
Nukaga M,
Mayama K,
Braswell EH,
Knox JR,
( 2003 ) Comparison of beta-lactamases of classes A and D: 1.5-A crystallographic structure of the class D OXA-1 oxacillinase. PMID : 12493831 : DOI : 10.1110/ps.0224303 PMC : PMC2312410 Abstract >>
The crystallographic structure of the Escherichia coli OXA-1 beta-lactamase has been established at 1.5-A resolution and refined to R = 0.18. The 28.2-kD oxacillinase is a class D serine beta-lactamase that is especially active against the penicillin-type beta-lactams oxacillin and cloxacillin. In contrast to the structures of OXA-2, OXA-10, and OXA-13 belonging to other subclasses, the OXA-1 molecule is monomeric rather than dimeric and represents the subclass characterized by an enlarged Omega loop near the beta-lactam binding site. The 6-residue hydrophilic insertion in this loop cannot interact directly with substrates and, instead, projects into solvent. In this structure at pH 7.5, carboxylation of the conserved Lys 70 in the catalytic site is observed. One oxygen atom of the carboxylate group is hydrogen bonded to Ser 120 and Trp 160. The other oxygen atom is more exposed and hydrogen bonded to the Ogamma of the reactive Ser 67. In the overlay of the class D and class A binding sites, the carboxylate group is displaced ca. 2.6 A from the carboxylate group of Glu 166 of class A enzymes. However, each group is equidistant from the site of the water molecule expected to function in hydrolysis, and which could be activated by the carboxylate group of Lys 70. In this ligand-free OXA-1 structure, no water molecule is seen in this site, so the water molecule must enter after formation of the acyl-Ser 67 intermediate.
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126. |
Rabel C,
Grahn AM,
Lurz R,
Lanka E,
( 2003 ) The VirB4 family of proposed traffic nucleoside triphosphatases: common motifs in plasmid RP4 TrbE are essential for conjugation and phage adsorption. PMID : 12533481 : DOI : 10.1128/jb.185.3.1045-1058.2003 PMC : PMC142825 Abstract >>
Proteins of the VirB4 family are encoded by conjugative plasmids and by type IV secretion systems, which specify macromolecule export machineries related to conjugation systems. The central feature of VirB4 proteins is a nucleotide binding site. In this study, we asked whether members of the VirB4 protein family have similarities in their primary structures and whether these proteins hydrolyze nucleotides. A multiple-sequence alignment of 19 members of the VirB4 protein family revealed striking overall similarities. We defined four common motifs and one conserved domain. One member of this protein family, TrbE of plasmid RP4, was genetically characterized by site-directed mutagenesis. Most mutations in trbE resulted in complete loss of its activities, which eliminated pilus production, propagation of plasmid-specific phages, and DNA transfer ability in Escherichia coli. Biochemical studies of a soluble derivative of RP4 TrbE and of the full-length homologous protein R388 TrwK revealed that the purified forms of these members of the VirB4 protein family do not hydrolyze ATP or GTP and behave as monomers in solution.
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127. |
Briñas L,
Zarazaga M,
Sáenz Y,
Ruiz-Larrea F,
Torres C,
( 2002 ) Beta-lactamases in ampicillin-resistant Escherichia coli isolates from foods, humans, and healthy animals. PMID : 12234838 : DOI : 10.1128/aac.46.10.3156-3163.2002 PMC : PMC128764 Abstract >>
TEM-, SHV-, and OXA-type beta-lactamases were studied by PCR with 124 ampicillin-resistant (AMP(r)) Escherichia coli isolates recovered from foods of animal origin (n = 20) and feces of humans (n = 49) and healthy animals (n = 55). PCR showed that 103 isolates were positive for TEM and negative for SHV and OXA. Three E. coli isolates showed a positive reaction for OXA, and one showed a positive reaction for SHV. The remaining 17 E. coli isolates were negative for the three enzymes by PCR. Fifty-seven of the 103 bla(TEM) amplicons were sequenced. Different molecular variants of bla(TEM-1) were found in 52 isolates: bla(TEM-1a) (n = 9), bla(TEM-1b) (n = 36), bla(TEM-1c) (n = 6), and bla(TEM-1f) (n = 1). Four inhibitor-resistant TEM (IRT) beta-lactamase-encoding genes were also detected: bla(TEM-30c) (IRT-2), bla(TEM-34b) (IRT-6), bla(TEM-40b) (IRT-11), and bla(TEM-51a) (IRT-15). A new bla(TEM) gene, named bla(TEM-95b), which showed a mutation in amino acid 145 (P-->A) was detected. It was found in a food isolate of chicken origin (AMP(r), amoxicillin-clavulanic acid susceptible). The promoter region in 24 bla(TEM) amplicons was analyzed, and the weak P3 promoter was found in 23 of them (bla(TEM-1) in 20 amplicons and bla(TEM-51a), bla(TEM-30c), and bla(TEM-95b) in 1 amplicon each). The strong Pa/Pb promoter was found only in the bla(TEM-34b) gene. No extended-spectrum beta-lactamases were detected. Mutations at position -42 or -32 in the ampC gene promoter were demonstrated in 4 of 10 E. coli isolates for which the cefoxitin MIC was >/=16 micro g/ml. Different variants of bla(TEM-1) and IRT bla(TEM) genes were found among the AMP(r) E. coli isolates from foods and the feces of humans and healthy animals, and a new gene, bla(TEM-95b) (P3), was detected.
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128. |
Perelle S,
Dilasser F,
Grout J,
Fach P,
( 2002 ) Identification of the O-antigen biosynthesis genes of Escherichia coli O91 and development of a O91 PCR serotyping test. PMID : 12392520 : Abstract >>
The aims of the study were to characterize the O91 O-antigen gene cluster from Shiga toxin-producing Escherichia coli (STEC) O91 and to provide the basis for a specific PCR test for rapid detection of E. coli O91. The published primers complementary to JUMPstart and gnd gene, the conserved flanking sequences of O-antigen genes clusters in E. coli and related species were used to amplify the 10-kbp O91 O-antigen biosynthesis locus of STEC O91. A DNA library representative of this cluster allowed two O91 specific probes to be identified, and two specific PCR O91 serotyping tests to be successfully developed. These results confirm that the O-antigen gene cluster sequences of E. coli allow rapidly a specific O-antigen PCR assay to be designed. These findings increase the number of PCR-assays available to replace the classical O-serotyping among E. coli O-antigen.
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129. |
Lanz R,
Kuhnert P,
Boerlin P,
( 2003 ) Antimicrobial resistance and resistance gene determinants in clinical Escherichia coli from different animal species in Switzerland. PMID : 12441233 : DOI : 10.1016/s0378-1135(02)00263-8 Abstract >>
Antimicrobial susceptibility testing was performed on a total of 581 clinical Escherichia coli isolates from diarrhea and edema disease in pigs, from acute mastitis in dairy cattle, from urinary tract infections in dogs and cats, and from septicemia in laying hens collected in Switzerland between 1999 and 2001. Among the 16 antimicrobial agents tested, resistance was most frequent for sulfonamides, tetracycline, and streptomycin. Isolates from swine presented significantly more resistance than those from the other animal species. The distribution of the resistance determinants for sulfonamides, tetracycline, and streptomycin was assessed by hybridization and PCR in resistant isolates. Significant differences in the distribution of resistance determinants for tetracycline (tetA, tetB) and sulfonamides (sulII) were observed between the isolates from swine and those from the other species. Resistance to sulfonamides could not be explained by known resistance mechanisms in more than a quarter of the sulfonamide-resistant and sulfonamide-intermediate isolates from swine, dogs and cats. This finding suggests that one or several new resistance mechanisms for sulfonamides may be widespread among E. coli isolates from these animal species. The integrase gene (intI) from class I integrons was detected in a large proportion of resistant isolates in association with the sulI and aadA genes, thus demonstrating the importance of integrons in the epidemiology of resistance in clinical E. coli isolates from animals.
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130. |
Yuzenkova J,
Delgado M,
Nechaev S,
Savalia D,
Epshtein V,
Artsimovitch I,
Mooney RA,
Landick R,
Farias RN,
Salomon R,
Severinov K,
( 2002 ) Mutations of bacterial RNA polymerase leading to resistance to microcin j25. PMID : 12401787 : DOI : 10.1074/jbc.M209425200 Abstract >>
A mutation in the conserved segment of the rpoC gene, which codes for the largest RNA polymerase (RNAP) subunit, beta', was found to make Escherichia coli cells resistant to microcin J25 (MccJ25), a bactericidal 21-amino acid peptide active against Gram-negative bacteria (Delgado, M. A., Rintoul, M. R., Farias, R. N., and Salomon, R. A. (2001) J. Bacteriol. 183, 4543-4550). Here, we report that mutant RNAP prepared from MccJ25-resistant cells, but not the wild-type RNAP, is resistant to MccJ25 in vitro, thus establishing that RNAP is a true cellular target of MccJ25. We also report the isolation of additional rpoC mutations that lead to MccJ25 resistance in vivo and in vitro. The new mutations affect beta' amino acids in evolutionarily conserved segments G, G', and F and are exposed into the RNAP secondary channel, a narrow opening that connects the enzyme surface with the catalytic center. We also report that previously known rpoB (RNAP beta subunit) mutations that lead to streptolydigin resistance cause resistance to MccJ25. We hypothesize that MccJ25 inhibits transcription by binding in RNAP secondary channel and blocking substrate access to the catalytic center.
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131. |
van den Ent F,
Møller-Jensen J,
Amos LA,
Gerdes K,
Löwe J,
( 2002 ) F-actin-like filaments formed by plasmid segregation protein ParM. PMID : 12486014 : DOI : 10.1093/emboj/cdf672 PMC : PMC139093 Abstract >>
It was the general belief that DNA partitioning in prokaryotes is independent of a cytoskeletal structure, which in eukaryotic cells is indispensable for DNA segregation. Recently, however, immunofluorescence microscopy revealed highly dynamic, filamentous structures along the longitudinal axis of Escherichia coli formed by ParM, a plasmid-encoded protein required for accurate segregation of low-copy-number plasmid R1. We show here that ParM polymerizes into double helical protofilaments with a longitudinal repeat similar to filamentous actin (F-actin) and MreB filaments that maintain the cell shape of non-spherical bacteria. The crystal structure of ParM with and without ADP demonstrates that it is a member of the actin family of proteins and shows a domain movement of 25 degrees upon nucleotide binding. Furthermore, the crystal structure of ParM reveals major differences in the protofilament interface compared with F-actin, despite the similar arrangement of the subunits within the filaments. Thus, there is now evidence for cytoskeletal structures, formed by actin-like filaments that are involved in plasmid partitioning in E.coli.
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132. |
Tarr CL,
Large TM,
Moeller CL,
Lacher DW,
Tarr PI,
Acheson DW,
Whittam TS,
( 2002 ) Molecular characterization of a serotype O121:H19 clone, a distinct Shiga toxin-producing clone of pathogenic Escherichia coli. PMID : 12438362 : DOI : 10.1128/iai.70.12.6853-6859.2002 PMC : PMC133070 Abstract >>
Most illnesses caused by Shiga toxin-producing Escherichia coli (STEC) have been attributed to E. coli serotype O157:H7, but non-O157 STEC infections are now increasingly recognized as public health problems worldwide. The O121:H19 serotype is being isolated more frequently from clinical specimens and has been implicated in one waterborne outbreak. We used multilocus virulence gene profiling, a PCR-based assay, to characterize the virulence gene content of 24 isolates of serotype O121:H19 and nonmotile variants. We also performed multilocus enzyme electrophoresis and multilocus sequencing to establish the clonal relatedness of O121 isolates and to elucidate the relationship of O121 to common STEC clones. The 24 isolates were found to represent a single bacterial clone, as there was no allelic variation across 18 enzyme loci among the isolates. The complete nucleotide sequence of the intimin gene differed by four substitutions from that of the epsilon (Int- epsilon) allele of O103:H2 strain PMK5. The typical O121 virulence gene profile was similar to the profiles of enterohemorrhagic E. coli (EHEC) clones of E. coli: it included a Shiga toxin 2 gene (stx(2)), two genes on the EHEC plasmid (toxB and ehxA), and the gene encoding intimin (eae). Despite the similarities, putative virulence genes distributed on O islands-large chromosomal DNA segments present in the O157:H7 genome-were useful for discriminating among STEC serotypes and the O121:H19 clone had a composite profile that was distinct from the profiles of the other major EHEC clones of pathogenic E. coli. On the basis of sequencing analysis with 13 housekeeping genes, the O121:H19 clone did not fall into any of the four classical EHEC and enteropathogenic E. coli groups but instead was closely related to two eae-negative STEC strains.
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133. |
D'Souza JM,
Wang L,
Reeves P,
( 2002 ) Sequence of the Escherichia coli O26 O antigen gene cluster and identification of O26 specific genes. PMID : 12384293 : DOI : 10.1016/s0378-1119(02)00876-4 Abstract >>
Escherichia coli associated with outbreaks of gastroenteritis and hemolytic uremic syndrome include clones with O antigens O157 and O111. However, O26 has emerged as an O antigen present in pathogenic strains, particularly those implicated in cases of infantile gastroenteritis worldwide. The O26 O antigen gene cluster was sequenced. It was found to contain the genes expected for biosynthesis of nucleotide sugars L-rhamnose, N-acetyl-L-fucosamine and N-acetyl-glucosamine, as well genes for O unit flippase, O antigen polymerase and potential transferase genes. By polymerase chain reaction testing against representative strains for the 166 Escherichia coli O serogroups and some randomly selected Gram-negative bacteria, we identified three O antigen genes that are highly specific to O26. This work provides the basis for a sensitive test for the rapid detection of pathogenic clones with the O26 antigen, which has implications for public health, especially in the control of food-borne outbreaks.
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134. |
Shaver AC,
Dombrowski PG,
Sweeney JY,
Treis T,
Zappala RM,
Sniegowski PD,
( 2002 ) Fitness evolution and the rise of mutator alleles in experimental Escherichia coli populations. PMID : 12399371 : PMC : PMC1462288 Abstract >>
We studied the evolution of high mutation rates and the evolution of fitness in three experimental populations of Escherichia coli adapting to a glucose-limited environment. We identified the mutations responsible for the high mutation rates and show that their rate of substitution in all three populations was too rapid to be accounted for simply by genetic drift. In two of the populations, large gains in fitness relative to the ancestor occurred as the mutator alleles rose to fixation, strongly supporting the conclusion that mutator alleles fixed by hitchhiking with beneficial mutations at other loci. In one population, no significant gain in fitness relative to the ancestor occurred in the population as a whole while the mutator allele rose to fixation, but a substantial and significant gain in fitness occurred in the mutator subpopulation as the mutator neared fixation. The spread of the mutator allele from rarity to fixation took >1000 generations in each population. We show that simultaneous adaptive gains in both the mutator and wild-type subpopulations (clonal interference) retarded the mutator fixation in at least one of the populations. We found little evidence that the evolution of high mutation rates accelerated adaptation in these populations.
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135. |
Grozdanov L,
Zähringer U,
Blum-Oehler G,
Brade L,
Henne A,
Knirel YA,
Schombel U,
Schulze J,
Sonnenborn U,
Gottschalk G,
Hacker J,
Rietschel ET,
Dobrindt U,
( 2002 ) A single nucleotide exchange in the wzy gene is responsible for the semirough O6 lipopolysaccharide phenotype and serum sensitivity of Escherichia coli strain Nissle 1917. PMID : 12374825 : DOI : 10.1128/jb.184.21.5912-5925.2002 PMC : PMC135379 Abstract >>
Structural analysis of lipopolysaccharide (LPS) isolated from semirough, serum-sensitive Escherichia coli strain Nissle 1917 (DSM 6601, serotype O6:K5:H1) revealed that this strain's LPS contains a bisphosphorylated hexaacyl lipid A and a tetradecasaccharide consisting of one E. coli O6 antigen repeating unit attached to the R1-type core. Configuration of the GlcNAc glycosidic linkage between O-antigen oligosaccharide and core (beta) differs from that interlinking the repeating units in the E. coli O6 antigen polysaccharide (alpha). The wa(*) and wb(*) gene clusters of strain Nissle 1917, required for LPS core and O6 repeating unit biosyntheses, were subcloned and sequenced. The DNA sequence of the wa(*) determinant (11.8 kb) shows 97% identity to other R1 core type-specific wa(*) gene clusters. The DNA sequence of the wb(*) gene cluster (11 kb) exhibits no homology to known DNA sequences except manC and manB. Comparison of the genetic structures of the wb(*)(O6) (wb(*) from serotype O6) determinants of strain Nissle 1917 and of smooth and serum-resistant uropathogenic E. coli O6 strain 536 demonstrated that the putative open reading frame encoding the O-antigen polymerase Wzy of strain Nissle 1917 was truncated due to a point mutation. Complementation with a functional wzy copy of E. coli strain 536 confirmed that the semirough phenotype of strain Nissle 1917 is due to the nonfunctional wzy gene. Expression of a functional wzy gene in E. coli strain Nissle 1917 increased its ability to withstand antibacterial defense mechanisms of blood serum. These results underline the importance of LPS for serum resistance or sensitivity of E. coli.
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136. |
Doughty S,
Sloan J,
Bennett-Wood V,
Robertson M,
Robins-Browne RM,
Hartland EL,
( 2002 ) Identification of a novel fimbrial gene cluster related to long polar fimbriae in locus of enterocyte effacement-negative strains of enterohemorrhagic Escherichia coli. PMID : 12438351 : DOI : 10.1128/iai.70.12.6761-6769.2002 PMC : PMC133005 Abstract >>
Enterohemorrhagic Escherichia coli (EHEC) is a food-borne cause of bloody diarrhea and the hemolytic-uremic syndrome (HUS) in humans. Most strains of EHEC belong to a group of bacterial pathogens that cause distinctive lesions on the host intestine termed attaching-and-effacing (A/E) lesions. A/E strains of EHEC, including the predominant serotype, O157:H7, are responsible for the majority of HUS outbreaks worldwide. However, several serotypes of EHEC are not A/E pathogens because they lack the locus of enterocyte effacement (LEE) pathogenicity island. Nevertheless, such strains have been associated with sporadic cases and small outbreaks of hemorrhagic colitis and HUS. Of these LEE-negative organisms, O113:H21 is one of the most commonly isolated EHEC serotypes in many regions. Clinical isolates of LEE-negative EHEC typically express Shiga toxin 2 and carry an approximately 90-kb plasmid that encodes EHEC hemolysin, but in the absence of LEE, little is known about the way in which these pathogens colonize the host intestine. In this study we describe the identification of a novel fimbrial gene cluster related to long polar fimbriae in EHEC O113:H21. This chromosomal region comprises four open reading frames, lpfA to lfpD, and has the same location in the EHEC O113:H21 genome as O island 154 in the prototype EHEC O157:H7 strain, EDL933. In a survey of EHEC of other serotypes, homologues of lpfA(O113) were found in 26 of 28 LEE-negative and 8 of 11 non-O157:H7 LEE-positive EHEC strains. Deletion of the putative major fimbrial subunit gene, lpfA, from EHEC O113:H21 resulted in decreased adherence of this strain to epithelial cells, suggesting that lpf(O113) may function as an adhesin in LEE-negative isolates of EHEC.
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137. |
Sanchez S,
McCrackin Stevenson MA,
Hudson CR,
Maier M,
Buffington T,
Dam Q,
Maurer JJ,
( 2002 ) Characterization of multidrug-resistant Escherichia coli isolates associated with nosocomial infections in dogs. PMID : 12354850 : DOI : 10.1128/jcm.40.10.3586-3595.2002 PMC : PMC130861 Abstract >>
Multidrug-resistant opportunistic pathogens have become endemic to the veterinary hospital environment. Escherichia coli isolates resistant to 12 antibiotics were isolated from two dogs that were housed in the intensive care unit at The University of Georgia Veterinary Teaching Hospital within 48 h of each other. Review of 21 retrospective and prospective hospital-acquired E. coli infections revealed that the isolates had similar antibiotic resistance profiles, characterized by resistance to most cephalosporins, beta-lactams, and the beta-lactamase inhibitor clavulanic acid as well as resistance to tetracycline, spectinomycin, sulfonamides, chloramphenicol, and gentamicin. E. coli isolates with similar resistance profiles were also isolated from the environment in the intensive care unit and surgery wards. Multiple E. coli genetic types were endemic to the hospital environment, with the pulsed-field gel electrophoresis fingerprint identified among E. coli isolates from diseased animals and the hospital environment matching. The extended-spectrum cephalosporin resistance in these nosocomial E. coli isolates was attributed to the cephamycinase-encoding gene, bla(CMY2). Chloramphenicol resistance was due in part to the dissemination of the florfenicol resistance gene, flo, among these isolates. Resistance encoded by both genes was self-transmissible. Although bla(CMY2) and flo were common to the polyclonal, nosocomial E. coli isolates, there was considerable diversity in the genetic compositions of class 1 integrons, especially among isolates belonging to the same genetic type. Two or more integrons were generally present in these isolates. The gene cassettes present within each integron ranged in size from 0.6 to 2.4 kb, although a 1.7-kb gene cassette was the most prevalent. The 1.7-kb gene cassette contained spectinomycin resistance gene aadA5 and trimethoprim resistance gene dfrA17.
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138. |
Jahreis K,
Bentler L,
Bockmann J,
Hans S,
Meyer A,
Siepelmeyer J,
Lengeler JW,
( 2002 ) Adaptation of sucrose metabolism in the Escherichia coli wild-type strain EC3132. PMID : 12218016 : DOI : 10.1128/jb.184.19.5307-5316.2002 PMC : PMC135337 Abstract >>
Although Escherichia coli strain EC3132 possesses a chromosomally encoded sucrose metabolic pathway, its growth on low sucrose concentrations (5 mM) is unusually slow, with a doubling time of 20 h. In this report we describe the subcloning and further characterization of the corresponding csc genes and adjacent genes. The csc regulon comprises three genes for a sucrose permease, a fructokinase, and a sucrose hydrolase (genes cscB, cscK, and cscA, respectively). The genes are arranged in two operons and are negatively controlled at the transcriptional level by the repressor CscR. Furthermore, csc gene expression was found to be cyclic AMP-CrpA dependent. A comparison of the genomic sequences of the E. coli strains EC3132, K-12, and O157:H7 in addition to Salmonella enterica serovar Typhimurium LT2 revealed that the csc genes are located in a hot spot region for chromosomal rearrangements in enteric bacteria. The comparison further indicated that the csc genes might have been transferred relatively recently to the E. coli wild-type EC3132 at around the time when the different strains of the enteric bacteria diverged. We found evidence that a mobile genetic element, which used the gene argW for site-specific integration into the chromosome, was probably involved in this horizontal gene transfer and that the csc genes are still in the process of optimal adaptation to the new host. Selection for such adaptational mutants growing faster on low sucrose concentrations gave three different classes of mutants. One class comprised cscR(Con) mutations that expressed all csc genes constitutively. The second class constituted a cscKo operator mutation, which became inducible for csc gene expression at low sucrose concentrations. The third class was found to be a mutation in the sucrose permease that caused an increase in transport activity.
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139. |
Tian W,
Chua K,
Strober W,
Chu CC,
( 2002 ) ILG1 : a new integrase-like gene that is a marker of bacterial contamination by the laboratory Escherichia coli strain TOP10F'. PMID : 12393938 : PMC : PMC2040001 Abstract >>
Identification of differentially expressed genes between normal and diseased states is an area of intense current medical research that can lead to the discovery of new therapeutic targets. However, isolation of differentially expressed genes by subtraction often suffers from unreported contamination of the resulting subtraction library with clones containing DNA sequences not from the original RNA samples. Subtraction using cDNA representational difference analysis (RDA) was performed on human B cells from normal or common variable immunodeficiency patients. The material remaining after the subtraction was cloned and individual clones were sequenced. The sequence of one clone with similarity to integrases (ILG1, integrase-like gene-1) was used to obtain the full length cDNA sequence and as a probe for the presence of this sequence in RNA or genomic DNA samples. After five rounds of cDNA RDA, 23.3% of the clones from the resulting subtraction library contained Escherichia coli DNA. In addition, three clones contained the sequence of a new integrase, ILG1. The full length cDNA sequence of ILG1 exhibits prokaryotic, but not eukaryotic, features. At the DNA level, ILG1 is not similar to any known gene. At the protein level, ILG1 has 58% similarity to integrases from the cryptic P4 bacteriophage family (S clade). The catalytic domain of ILG1 contains the conserved features found in site-specific recombinases. The critical residues that form the catalytic active site pocket are conserved, including the highly conserved R-H-R-Y hallmark of these recombinases. Interestingly, ILG1 was not present in the original B cell populations. By probing genomic DNA, ILG1 could only be detected in the E. coli TOP10F' strain used in our laboratory for molecular cloning, but not in any of its precursor strains, including TOP10. Furthermore, bacteria cultured from the mouth of the laboratory worker who performed cDNA RDA were also positive for ILG1. In the course of our studies using cDNA RDA, we have isolated and identified ILG1, a likely active site-specific recombinase and new member of the bacteriophage P4 family of integrases. This family of integrases is implicated in the horizontal DNA transfer of pathogenic genes between bacterial species, such as those found in pathogenic strains of E. coli, Shigella, Yersinia, and Vibrio cholera. Using ILG1 as a marker of our laboratory E. coli strain TOP10F', our evidence suggests that contaminating bacterial DNA in our subtraction experiment is due to this laboratory bacterial strain, which colonized exposed surfaces of the laboratory worker. Thus, identification of differentially expressed genes between normal and diseased states could be dramatically improved by using extra precaution to prevent bacterial contamination of samples.
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140. |
La Ragione RM,
McLaren IM,
Foster G,
Cooley WA,
Woodward MJ,
( 2002 ) Phenotypic and genotypic characterization of avian Escherichia coli O86:K61 isolates possessing a gamma-like intimin. PMID : 12324341 : DOI : 10.1128/aem.68.10.4932-4942.2002 PMC : PMC126447 Abstract >>
Escherichia coli O86:K61 has long been associated with outbreaks of infantile diarrhea in humans and with diarrheal disease in many animal species. Studies in the late 1990s identified E. coli O86:K61 as the cause of mortality in a variety of wild birds, and in this study, 34 E. coli O86:K61 isolates were examined. All of the isolates were nonmotile, but most elaborated at least two morphologically distinct surface appendages that were confirmed to be type 1 and curli fimbriae. Thirty-three isolates were positive for the eaeA gene encoding a gamma type of intimin. No phenotypic or genotypic evidence was obtained for elaboration of Shiga-like toxins, but most isolates possessed the gene coding for the cytolethal distending toxin. Five isolates were selected for adherence assays performed with tissue explants and HEp-2 cells, and four of these strains produced attaching and effacing lesions on HEp-2 cells and invaded the cells, as determined by transmission electron microscopy. Two of the five isolates were inoculated orally into 1-day-old specific-pathogen-free chicks, and both of these isolates colonized, invaded, and persisted well in this model. Neither isolate produced attaching and effacing lesions in chicks, although some pathology was evident in the alimentary tract. No deaths were recorded in inoculated chicks. These findings are discussed in light of the possibility that wild birds are potential zoonotic reservoirs of attaching and effacing E. coli.
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141. |
Hargreaves D,
Santos-Sierra S,
Giraldo R,
Sabariegos-Jareño R,
de la Cueva-Méndez G,
Boelens R,
Díaz-Orejas R,
Rafferty JB,
( 2002 ) Structural and functional analysis of the kid toxin protein from E. coli plasmid R1. PMID : 12377128 : Abstract >>
We have determined the structure of Kid toxin protein from E. coli plasmid R1 involved in stable plasmid inheritance by postsegregational killing of plasmid-less daughter cells. Kid forms a two-component system with its antagonist, Kis antitoxin. Our 1.4 A crystal structure of Kid reveals a 2-fold symmetric dimer that closely resembles the DNA gyrase-inhibitory toxin protein CcdB from E. coli F plasmid despite the lack of any notable sequence similarity. Analysis of nontoxic mutants of Kid suggests a target interaction interface associated with toxicity that is in marked contrast to that proposed for CcdB. A possible region for interaction of Kid with the antitoxin is proposed that overlaps with the target binding site and may explain the mode of antitoxin action.
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142. |
Bernier C,
Gounon P,
Le Bouguénec C,
( 2002 ) Identification of an aggregative adhesion fimbria (AAF) type III-encoding operon in enteroaggregative Escherichia coli as a sensitive probe for detecting the AAF-encoding operon family. PMID : 12117939 : DOI : 10.1128/iai.70.8.4302-4311.2002 PMC : PMC128174 Abstract >>
Enteroaggregative Escherichia coli (EAEC) is recognized as an emerging cause of diarrhea in children and adults worldwide, and recent studies have implicated EAEC in persistent diarrhea in patients infected with human immunodeficiency virus (HIV). In this study, we identified aggregative adhesion fimbria type III (AAF-III) in isolate 55989, a typical EAEC strain. Analysis of the sequence of the plasmid-borne agg-3 gene cluster encoding AAF-III showed this cluster to be closely related to the agg and aaf operons and to the afa operons carried by diffusely adherent pathogenic E. coli. We investigated the adhesion properties of a collection of 25 EAEC strains isolated from HIV-infected patients presenting with persistent diarrhea. We found that a minority of strains (36%) carried sequences similar to those of the agg and aaf operons, which encode AAF-I and AAF-II, respectively. We developed PCR assays specific for the agg-3 operon. In our collection, the frequency of AAF-III strains was similar (12%) to that of AAF-I strains (16%) but higher than that of AAF-II isolates (0%). Differences between EAEC strains in terms of the virulence factors present render detection of these strains difficult with the available DNA probes. Based on comparison of the agg, aaf, and agg-3 operons, we defined an AAF probe internal to the adhesion gene clusters and demonstrated that it was efficient for the identification of EAEC strains. We investigated 32 EAEC isolates, of which only 34.4% were detected with the classical CVD432 probe (detecting pAA virulence plasmids) whereas 65.6% were detected with the AAF probe.
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143. |
Teel LD,
Melton-Celsa AR,
Schmitt CK,
O'Brien AD,
( 2002 ) One of two copies of the gene for the activatable shiga toxin type 2d in Escherichia coli O91:H21 strain B2F1 is associated with an inducible bacteriophage. PMID : 12117937 : DOI : 10.1128/iai.70.8.4282-4291.2002 PMC : PMC128153 Abstract >>
Shiga toxin (Stx) types 1 and 2 are encoded within intact or defective temperate bacteriophages in Stx-producing Escherichia coli (STEC), and expression of these toxins is linked to bacteriophage induction. Among Stx2 variants, only stx(2e) from one human STEC isolate has been reported to be carried within a toxin-converting phage. In this study, we examined the O91:H21 STEC isolate B2F1, which carries two functional alleles for the potent activatable Stx2 variant toxin, Stx2d, for the presence of Stx2d-converting bacteriophages. We first constructed mutants of B2F1 that produced one or the other Stx2d toxin and found that the mutant that produced only Stx2d1 made less toxin than the Stx2d2-producing mutant. Consistent with that result, the Stx2d1-producing mutant was attenuated in a streptomycin-treated mouse model of STEC infection. When the mutants were treated with mitomycin C to promote bacteriophage induction, Vero cell cytotoxicity was elevated only in extracts of the Stx2d1-producing mutant. Additionally, when mice were treated with ciprofloxacin, an antibiotic that induces the O157:H7 Stx2-converting phage, the animals were more susceptible to the Stx2d1-producing mutant. Moreover, an stx(2d1)-containing lysogen was isolated from plaques on strain DH5alpha that had been exposed to lysates of the mutant that produced Stx2d1 only, and supernatants from that lysogen transformed with a plasmid encoding RecA were cytotoxic when the lysogen was induced with mitomycin C. Finally, electron-microscopic examination of extracts from the Stx2d1-producing mutant showed hexagonal particles that resemble the prototypic Stx2-converting phage 933W. Together these observations provide strong evidence that expression of Stx2d1 is bacteriophage associated. We conclude that despite the sequence similarity of the stx(2d1)- and stx(2d2)-flanking regions in B2F1, Stx2d1 expression is repressed within the context of its toxin-converting phage while Stx2d2 expression is independent of phage induction.
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144. |
Caniça M,
Ferreira M,
Ferreira E,
Cabral L,
( 2002 ) Phenotype and molecular characterization of the first inhibitor-resistant TEM-derived beta-lactamase identified in Portugal. PMID : 12384395 : DOI : 10.1128/aac.46.11.3688-3689.2002 PMC : PMC128722 Abstract >>
N/A
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145. |
Perelle S,
Fach P,
Dilasser F,
Grout J,
( 2002 ) A PCR test for detecting Escherichia coli O157:H7 based on the identification of the small inserted locus (SILO157). PMID : 12147073 : Abstract >>
This paper provides identification of a DNA sequence derived from Shiga toxin-producing Escherichia coli (STEC) O157:H7 and information on its utilization for detecting STEC O157 by PCR. Random Amplified Polymorphic DNA and DNA library were used to identify in STEC O157:H7 (strain EDL 933) a 2634-bp Small Inserted Locus, designated SILO157. Analysis of 211 bacterial strains showed that the PCR assays amplifying the SILO157 region could be used to detect STEC O157 with a good specificity. Characterization of a novel locus in STEC O157 is attractive since the serotype O157:H7 of STEC is still by far the most important serotype associated with more serious diseases. This island encodes putative proteins and especially one that is predicted to be an outer membrane protein designated IHP1. Further investigations could now be developed to appreciate the role of the SILO157 in pathogenicity.
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146. |
Steiniger-White M,
Bhasin A,
Lovell S,
Rayment I,
Reznikoff WS,
( 2002 ) Evidence for "unseen" transposase--DNA contacts. PMID : 12367522 : DOI : 10.1016/s0022-2836(02)00877-x Abstract >>
In this study, evidence of novel, important interactions between a hyperactive Tn5 transposon recognition end sequence and hyperactive Tn5 transposase (Tnp) are presented. A hyperactive Tn5 end sequence, the mosaic end (ME), was isolated previously. The ME and a wild-type end sequence, the outside end (OE), differ at only three positions, yet transposition on the ME is tenfold higher than on the OE in vivo. Also, transposition on the ME is much more efficient than transposition on the OE in vitro. Here, we show that the decreased activity observed for the OE is caused by a defect in paired ends complex (PEC) formation resulting from the orientation of the A-T base-pair at position 4 of this end. Efficient PEC formation requires an interaction between the C5-methyl group (C5-Me) on the non-transferred strand thymine base at position 4 (T4) and Tnp. PEC formation on nicked substrates is much less affected by the orientation of the A-T base-pair at position 4, indicating that the C5-Me group is important only for steps preceding nicking. A recently determined co-crystal structure of Tn5 Tnp with the ME is discussed and a model explaining possible roles for the base-pair at position 4 is explored.
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147. |
Thanassi DG,
Stathopoulos C,
Dodson K,
Geiger D,
Hultgren SJ,
( 2002 ) Bacterial outer membrane ushers contain distinct targeting and assembly domains for pilus biogenesis. PMID : 12399496 : DOI : 10.1128/jb.184.22.6260-6269.2002 PMC : PMC151958 Abstract >>
Biogenesis of a superfamily of surface structures by gram-negative bacteria requires the chaperone/usher pathway, a terminal branch of the general secretory pathway. In this pathway a periplasmic chaperone works together with an outer membrane usher to direct substrate folding, assembly, and secretion to the cell surface. We analyzed the structure and function of the PapC usher required for P pilus biogenesis by uropathogenic Escherichia coli. Structural analysis indicated PapC folds as a beta-barrel with short extracellular loops and extensive periplasmic domains. Several periplasmic regions were localized, including two domains containing conserved cysteine pairs. Functional analysis of deletion mutants revealed that the PapC C terminus was not required for insertion of the usher into the outer membrane or for proper folding. The usher C terminus was not necessary for interaction with chaperone-subunit complexes in vitro but was required for pilus biogenesis in vivo. Interestingly, coexpression of PapC C-terminal truncation mutants with the chromosomal fim gene cluster coding for type 1 pili allowed P pilus biogenesis in vivo. These studies suggest that chaperone-subunit complexes target an N-terminal domain of the usher and that subunit assembly into pili depends on a subsequent function provided by the usher C terminus.
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148. |
Dobrindt U,
Blum-Oehler G,
Nagy G,
Schneider G,
Johann A,
Gottschalk G,
Hacker J,
( 2002 ) Genetic structure and distribution of four pathogenicity islands (PAI I(536) to PAI IV(536)) of uropathogenic Escherichia coli strain 536. PMID : 12379716 : DOI : 10.1128/iai.70.11.6365-6372.2002 PMC : PMC130402 Abstract >>
For the uropathogenic Escherichia coli strain 536 (O6:K15:H31), the DNA sequences of three pathogenicity islands (PAIs) (PAI I(536) to PAI III(536)) and their flanking regions (about 270 kb) were determined to further characterize the virulence potential of this strain. PAI I(536) to PAI III(536) exhibit features typical of PAIs, such as (i) association with tRNA-encoding genes; (ii) G+C content differing from that of the host genome; (iii) flanking repeat structures; (iv) a mosaic-like structure comprising a multitude of functional, truncated, and nonfunctional putative open reading frames (ORFs) with known or unknown functions; and (v) the presence of many fragments of mobile genetic elements. PAI I(536) to PAI III(536) range between 68 and 102 kb in size. Although these islands contain several ORFs and known virulence determinants described for PAIs of other extraintestinal pathogenic E. coli (ExPEC) isolates, they also consist of as-yet-unidentified ORFs encoding putative virulence factors. The genetic structure of PAI IV(536), which represents the core element of the so-called high-pathogenicity island encoding a siderophore system initially identified in pathogenic yersiniae, was further characterized by sample sequencing. For the first time, multiple PAI sequences (PAI I(536) to PAI IV(536)) in uropathogenic E. coli were studied and their presence in several wild-type E. coli isolates was extensively investigated. The results obtained suggest that these PAIs or at least large fragments thereof are detectable in other pathogenic E. coli isolates. These results support our view that the acquisition of large DNA regions, such as PAIs, by horizontal gene transfer is an important factor for the evolution of bacterial pathogens.
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149. |
Dai L,
Zimmerly S,
( 2002 ) The dispersal of five group II introns among natural populations of Escherichia coli. PMID : 12403467 : DOI : 10.1017/s1355838202023014 PMC : PMC1370338 Abstract >>
Group II introns are self-splicing RNAs that also act as retroelements in bacteria, mitochondria, and chloroplasts. Group II introns were identified in Escherichia coli in 1994, but have not been characterized since, and, instead, other bacterial group II introns have been studied for splicing and mobility properties. Despite their apparent intractability, at least five distinct group II introns exist naturally in E. coli strains. To illuminate their function and learn how the introns have dispersed in their natural host, we have investigated their distribution in the ECOR reference collection. Two introns were cloned and sequenced to complete their partial sequences. Unexpectedly, southern blots showed all ECOR strains to contain fragments and/or full-length copies of group II introns, with some strains containing up to 15 intron copies. One intron, E.c.14, has two natural homing sites in IS629 and IS911 elements, and the intron can be present in one, both, or neither homing site in a given strain. Nearly all strains that contain full-length introns also contain unfilled homing sites, suggesting either that mobility is highly inefficient or that most full-length copies are nonfunctional. The data indicate independent mobility of the introns, as well as mobility via the host DNA elements, and overall, the pattern of intron distribution resembles that of IS elements.
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150. |
Santapaola D,
Casalino M,
Petrucca A,
Presutti C,
Zagaglia C,
Berlutti F,
Colonna B,
Nicoletti M,
( 2002 ) Enteroinvasive Escherichia coli virulence-plasmid-carried apyrase (apy) and ospB genes are organized as a bicistronic operon and are subject to differential expression. PMID : 12177345 : DOI : 10.1099/00221287-148-8-2519 Abstract >>
In Shigella flexneri and enteroinvasive Escherichia coli (EIEC) the expression of the virulence-plasmid(pINV)-carried potential pathogenesis-associated apy gene, which encodes apyrase (ATP diphosphohydrolase), is regulated by the same regulators that govern the expression of virulence genes. To understand the transcriptional organization of the apy gene, the authors sequenced an 8023 bp PstI fragment of the pINV of EIEC strain HN280, which encompasses apy as well as its adjacent genes. The PstI fragment displays 99% identity with the corresponding fragment of pWR100, the pINV of S. flexneri strain M90T, and contains four genes. One of these genes, ospB, encodes a secreted protein of unknown activity and is located immediately upstream of apy. Analyses of sequence, Northern hybridization, RT-PCR and primer extension data and transcriptional fusions indicated that ospB and apy are co-transcribed as a 2 kb bicistronic, temperature-regulated mRNA from an upstream promoter that precedes ospB. The 2 kb mRNA is post-transcriptionally processed in the intercistronic ospB-apy region, leading to the considerable accumulation of a more stable 1 kb apy-specific mRNA (half-life of 2.2+/-0.3 min, versus 27+/-4 s for the 2 kb transcript). Upon temperature induction, peak expression of the ospB-apy operon occurs when bacteria enter into the late phases of bacterial growth, where the apy-specific transcript was found to be much more prevalent if compared to the ospB-apy transcript.
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151. |
Heroven AK,
Dersch P,
( 2002 ) Two different open reading frames named slyA in the E. coli sequence databases. PMID : 12088660 : Abstract >>
N/A
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152. |
Campbell JW,
Cronan JE,
( 2002 ) The enigmatic Escherichia coli fadE gene is yafH. PMID : 12057976 : DOI : 10.1128/jb.184.13.3759-3764.2002 PMC : PMC135136 Abstract >>
The identity of the gene encoding acyl coenzyme A dehydrogenase is a major remaining mystery of the Escherichia coli fatty acid degradation (fad) regulon. Our prior genome array analyses showed that transcription of the yafH gene is controlled by the FadR regulatory protein. We now report direct experimental proof that yafH and fadE are the same gene.
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153. |
Doi Y,
Shibata N,
Shibayama K,
Kamachi K,
Kurokawa H,
Yokoyama K,
Yagi T,
Arakawa Y,
( 2002 ) Characterization of a novel plasmid-mediated cephalosporinase (CMY-9) and its genetic environment in an Escherichia coli clinical isolate. PMID : 12121914 : DOI : 10.1128/aac.46.8.2427-2434.2002 PMC : PMC127380 Abstract >>
An Escherichia coli strain, HKYM68, which showed resistance to broad-spectrum cephalosporins was isolated from a sputum specimen in Japan. The high-level resistance of the strain to ceftazidime, cefpirome, and moxalactam was carried by a self-transferable plasmid. The beta-lactamase gene responsible for the resistance was cloned and sequenced. The deduced amino acid sequence of this gene product, CMY-9, had a single amino acid substitution (E85D), the residue reported to be part of the recognition site for the R1 side chain of beta-lactams, compared with the amino acid sequence of CMY-8 and also had 78% identity with the amino acid sequence of CepH, a chromosomal cephalosporinase of Aeromonas hydrophila. A sul1-type class 1 integron containing an aacA1-orfG gene cassette was identified upstream of bla(CMY-9) and ended with a truncated 3' conserved segment. The following 2.1 kb was almost identical to the common region of integrons In6 and In7 and the integron of pSAL-1, except that orf513 encoding a putative transposase was identified instead of orf341 due to addition of a single nucleotide. bla(CMY-9) was closely located downstream of the end of the common region. These observations are indicative of the exogenous derivation of bla(CMY-9) from some environmental microorganisms such as aeromonads.
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154. |
Atlung T,
Nielsen HV,
Hansen FG,
( 2002 ) Characterisation of the allelic variation in the rpoS gene in thirteen K12 and six other non-pathogenic Escherichia coli strains. PMID : 11810263 : DOI : 10.1007/s00438-001-0610-0 Abstract >>
The nucleotide sequence of rpoS, the gene for the stress sigma factor, was determined in 13 different K12 strains of Escherichia coli. The results indicate that the original K12 isolate carried an amber mutation at codon 33, which in 50% of the derivatives is mutated by a single base substitution to a coding triplet, in most cases to CAG encoding glutamine. The six non-K12 strains examined here had GAG, encoding glutamate, in position 33. The two most divergent strains had three and seven neutral substitutions in rpoS and carried insertions of 2100 and 2900 bp, respectively, just downstream of the gene. The genetic variations in rpoS were compared with the variation in RpoS-related phenotypes, by measuring catalase (KatE) activity, glycogen accumulation and acid phosphatase levels, and a katEp-gfp fusion was used to visualise katE gene transcription. The RpoS phenotypes of the six rpoS(33E) strains varied significantly more than that of the K12 rpoS(33Q) strains, especially with respect to acid phosphatase levels. This was due to the absence of the gene for the transcriptional activator AppY from four of the rpoS(33E) strains, while all the K12 derivatives carried this gene. When cloned into a LacI-controlled vector and compared in a rpoS::Tn 10 background, the RpoS(33Q) and RpoS(33E) variants showed the same activity.
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155. |
Taneike I,
Zhang HM,
Wakisaka-Saito N,
Yamamoto T,
( 2002 ) Enterohemolysin operon of Shiga toxin-producing Escherichia coli: a virulence function of inflammatory cytokine production from human monocytes. PMID : 12135770 : DOI : 10.1016/s0014-5793(02)03027-2 Abstract >>
Shiga toxin-producing Escherichia coli (STEC) is associated with hemolytic uremic syndrome (HUS). Although most clinical isolates of STEC produce hemolysin (called enterohemolysin), the precise role of enterohemolysin in the pathogenesis of STEC infections is unknown. Here we demonstrated that E. coli carrying the cloned enterohemolysin operon (hlyC, A, B, D genes) from an STEC human strain induced the production of interleukin-1beta (IL-1beta) through its mRNA expression but not tumor necrosis factor-alpha from human monocytes. No IL-1beta release was observed with an enterohemolysin (HlyA)-negative, isogenic E. coli strain carrying a mutation in the hlyA gene. The data suggest that enterohemolysin, a pore-forming toxin, induces the production of IL-1beta, which is one of serum risk markers for HUS.
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156. |
Sabaté M,
Navarro F,
Miró E,
Campoy S,
Mirelis B,
Barbé J,
Prats G,
( 2002 ) Novel complex sul1-type integron in Escherichia coli carrying bla(CTX-M-9). PMID : 12121950 : DOI : 10.1128/aac.46.8.2656-2661.2002 PMC : PMC127377 Abstract >>
For the present report, a novel complex class 1 integron, In60, was characterized. Part of this integron includes the bla(CTX-M-9) gene and its downstream nucleotide sequence, which shares 81% and 78% nucleotide identity with those of kluA-1 beta-lactamase and orf3 of K. ascorbata, respectively. Furthermore, a new insertion sequence, IS3000, has been found in In60. PCR analysis indicates that integron In60 is present in 33 of 34 nonclonal enterobacterial isolates carrying the putative beta-lactamase CTX-M-9.
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157. |
Park W,
Jeon CO,
Madsen EL,
( 2002 ) Interaction of NahR, a LysR-type transcriptional regulator, with the alpha subunit of RNA polymerase in the naphthalene degrading bacterium, Pseudomonas putida NCIB 9816-4. PMID : 12167532 : DOI : 10.1111/j.1574-6968.2002.tb11300.x Abstract >>
NahR, a LysR-type transcriptional regulator, is required for expression of naphthalene catabolic operons. However, detailed mechanisms of transcriptional activation by NahR are poorly understood. Many transcriptional activators make direct contact with RNA polymerase (RNAP) to initiate transcription. We investigated the hypothesis that direct contact between NahR and the alpha subunit of RNAP (alphaRNAP) may be involved in expression of the naphthalene catabolic operons in Pseudomonas putida NCIB 9816-4. Interactions between the NahR and alphaRNAP in P. putida NCIB 9816-4 were analyzed using the yeast two-hybrid system. The results obtained indicate that protein-protein interactions occur between alphaRNAP and the NahR. Gene activation by NahR is consistent with the general transcriptional mechanism of class I transcription factors, which function by contacting alphaRNAP.
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158. |
Bitinaite J,
Mitkaite G,
Dauksaite V,
Jakubauskas A,
Timinskas A,
Vaisvila R,
Lubys A,
Janulaitis A,
( 2002 ) Evolutionary relationship of Alw26I, Eco31I and Esp3I, restriction endonucleases that recognise overlapping sequences. PMID : 12172806 : DOI : 10.1007/s00438-002-0701-6 Abstract >>
Type II restriction endonucleases (ENases) have served as models for understanding the enzyme-based site-specific cleavage of DNA. Using the knowledge gained from the available crystal structures, a number of attempts have been made to alter the specificity of ENases by mutagenesis. The negative results of these experiments argue that the three-dimensional structure of DNA-ENase complexes does not provide enough information to enable us to understand the interactions between DNA and ENases in detail. This conclusion calls for alternative approaches to the study of structure-function relationships related to the specificity of ENases. Comparative analysis of ENases that manifest divergent substrate specificities, but at the same time are evolutionarily related to each other, may be helpful in this respect. The success of such studies depends to a great extent on the availability of related ENases that recognise partially overlapping nucleotide sequences (e.g. sets of enzymes that bind to recognition sites of increasing length). In this study we report the cloning and sequence analysis of genes for three Type IIS restriction-modification (RM) systems. The genes encoding the ENases Alw26I, Eco31I and Esp3I (whose recognition sequences are 5'-GTCTC-3', 5'-GGTCTC-3' and 5'-CGTCTC-3', respectively) and their accompanying methyltransferases (MTases) have been cloned and the deduced amino acid sequences of their products have been compared. In pairwise comparisons, the degree of sequence identity between Alw26I, Eco31I and Esp3I ENases is higher than that observed hitherto among ENases that recognise partially overlapping nucleotide sequences. The sequences of Alw26I, Eco31I and Esp3I also reveal identical mosaic patterns of sequence conservation, which supports the idea that they are evolutionarily related and suggests that they should show a high level of structural similarity. Thus these ENases represent very attractive models for the study of the molecular basis of variation in the specific recognition of DNA targets. The corresponding MTases are represented by proteins of unusual structural and functional organisation. Both M. Alw26I and M. Esp3I are represented by a single bifunctional protein, which is composed of an m(6)A-MTase domain fused to a m(5)C-MTase domain. In contrast, two separate genes encode the m(6)A-MTase and m(5)C-MTase in the Eco31I RM system. Among the known bacterial m(5)C-MTases, the m(5)C-MTases of M. Alw26I, M. Eco31I and M. Esp3I represent unique examples of the circular permutation of their putative target recognition domains together with the conserved motifs IX and X.
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159. |
Fukushima M,
Kakinuma K,
Kawaguchi R,
( 2002 ) Phylogenetic analysis of Salmonella, Shigella, and Escherichia coli strains on the basis of the gyrB gene sequence. PMID : 12149329 : DOI : 10.1128/jcm.40.8.2779-2785.2002 PMC : PMC120687 Abstract >>
Phylogenetic analysis of about 200 strains of Salmonella, Shigella, and Escherichia coli was carried out using the nucleotide sequence of the gene for DNA gyrase B (gyrB), which was determined by directly sequencing PCR fragments. The results establish a new phylogenetic tree for the classification of Salmonella, Shigella, and Escherichia coli in which Salmonella forms a cluster separate from but closely related to Shigella and E. coli. In comparison with 16S rRNA analysis, the gyrB sequences indicated a greater evolutionary divergence for the bacteria. Thus, in screening for the presence of bacteria, the gyrB gene might be a useful tool for differentiating between closely related species of bacteria such as Shigella spp. and E. coli. At present, 16S rRNA sequence analysis is an accurate and rapid method for identifying most unknown bacteria to the genus level because the highly conserved 16S rRNA region is easy to amplify; however, analysis of the more variable gyrB sequence region can identify unknown bacteria to the species level. In summary, we have shown that gyrB sequence analysis is a useful alternative to 16S rRNA analysis for constructing the phylogenetic relationships of bacteria, in particular for the classification of closely related bacterial species.
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160. |
Brown EW,
Kotewicz ML,
Cebula TA,
( 2002 ) Detection of recombination among Salmonella enterica strains using the incongruence length difference test. PMID : 12128032 : Abstract >>
Particular serovars of Salmonella enterica have emerged as significant foodborne pathogens in humans. At the chromosomal level, discrete regions in the Salmonella genome have been identified that are known to play important roles in the maintenance, survival, and virulence of S. enterica within the host. Interestingly, several of these loci appear to have been acquired by horizontal transfer of DNA among and between bacterial species. The profound importance of recombination in pathogen emergence is just now being realized, perhaps explaining the sudden interest in developing novel and facile ways for detecting putative horizontal transfer events in bacteria. The incongruence length difference (ILD) test offers one such means. ILD uses phylogeny to trace sequences that may have been acquired promiscuously by exchange of DNA during chromosome evolution. We show here that the ILD test readily detects recombinations that have taken place in several housekeeping genes in Salmonella as well as genes composing the type 1 pilin complex (14 min) and the inv-spa invasion gene complex (63 min). Moreover, the ILD test indicated that the mutS gene (64 min), whose product helps protect the bacterial genome from invasion by foreign DNA, appears to have undergone intragenic recombination within S. enterica subspecies I. ILD findings were supported using additional tests known to be independent of the ILD approach (e.g., split decomposition analysis and compatibility of sites). Taken together, these data affirm the application of the ILD test as one approach for identifying recombined sequences in the Salmonella chromosome. Furthermore, horizontally acquired sequences within mutS support a model whereby evolutionarily important recombinants of S. enterica are rescued from strains carrying defective mutS alleles via horizontal transfer.
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161. |
Sandt CH,
Hopper JE,
Hill CW,
( 2002 ) Activation of prophage eib genes for immunoglobulin-binding proteins by genes from the IbrAB genetic island of Escherichia coli ECOR-9. PMID : 12057959 : DOI : 10.1128/jb.184.13.3640-3648.2002 PMC : PMC135156 Abstract >>
Four distinct Escherichia coli immunoglobulin-binding (eib) genes, each of which encodes a surface-exposed protein that binds immunoglobulins in a nonimmune manner, are carried by separate prophages in E. coli reference (ECOR) strain ECOR-9. Each eib gene was transferred to test E. coli strains, both in the form of multicopy recombinant plasmids and as lysogenized prophage. The derived lysogens express little or no Eib protein, in sharp contrast to the parental lysogen, suggesting that ECOR-9 has an expression-enhancing activity that the derived lysogens lack. Supporting this hypothesis, we cloned from ECOR-9 overlapping genes, ibrA and ibrB (designation is derived from "immunoglobulin-binding regulator"), which together activated eib expression in the derived lysogens. The proteins encoded by ibrA and ibrB are very similar to uncharacterized proteins encoded by genes of Salmonella enterica serovar Typhi and E. coli O157:H7 (in a prophage-like element of the Sakai strain and in two O islands of strain EDL933). The genomic segment containing ibrA and ibrB has been designated the IbrAB island. It contains regions of homology to the Shiga toxin-converting prophage, Stx2, as well as genes homologous to phage antirepressor genes. The left boundary between the IbrAB island and the chromosomal framework is located near min 35.8 of the E. coli K-12 genome. Homology to IbrAB was found in certain other ECOR strains, including the other five eib-positive strains and most strains of the phylogenetic group B2. Sequencing of a 1.1-kb portion of ibrAB revealed that the other eib-positive strains diverge by
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162. |
Tauschek M,
Strugnell RA,
Robins-Browne RM,
( 2002 ) Characterization and evidence of mobilization of the LEE pathogenicity island of rabbit-specific strains of enteropathogenic Escherichia coli. PMID : 12067342 : DOI : 10.1046/j.1365-2958.2002.02968.x Abstract >>
We have characterized the LEE pathogenicity islands (PAIs) of two rabbit-specific strains of enteropathogenic E. coli (REPEC), 83/39 (serotype O15:H-) and 84/110-1 (O103:H2), and have compared them to homologous loci from the human enteropathogenic and enterohaemorrhagic E. coli strains, E2348/69 and EDL933, and another REPEC strain, RDEC-1. All five PAIs contain a 34 kb core region that is highly conserved in gene order and nucleotide sequence. However, the LEE of 83/39 is significantly larger (59 540 basepairs) than those of the human strains, which are less than 44 kb, and has inserted into pheU tRNA. The regions flanking the 34 kb core of 83/39 contain homologues of two putative virulence determinants, efa1/lifA and senA. The LEE of 84/110-1 is approximately 85 kb and is located at pheV tRNA. Its core is almost identical to those of 83/39 and RDEC-1, apart from a larger espF gene, but its flanking regions contain trcA, a putative virulence determinant of EPEC. All three REPEC LEE PAIs contain a gene for an integrase, Int-phe. The LEE PAI of 84/110-1 is also flanked by short direct repeats (representing the 3'-end of pheV tRNA), suggesting that it may be unstable. To investigate this possibility, we constructed a LEE::sacB derivative of 84/110-1 and showed that the PAI was capable of spontaneous deletion. We also showed that Int-phe can mediate site-specific integration of foreign DNA at the pheU tRNA locus of E. coli DH1. Together these results indicate possible mechanisms of mobilization and integration of the LEE PAI.
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163. |
Lee SH,
Kim JY,
Lee GS,
Cheon SH,
An YJ,
Jeong SH,
Lee KJ,
( 2002 ) Characterization of blaCMY-11, an AmpC-type plasmid-mediated beta-lactamase gene in a Korean clinical isolate of Escherichia coli. PMID : 11815567 : DOI : 10.1093/jac/49.2.269 Abstract >>
We report the description of a new plasmid-encoded AmpC-type beta-lactamase gene (bla(CMY-11)) from Escherichia coli K983802.1 that was isolated from a patient in South Korea suffering from a urinary tract infection. Antibiotic susceptibility testing, plasmid analysis, pI determination, transconjugation and Southern blot analysis were carried out to investigate the resistance mechanism to cefoxitin. PCR, sequencing and sequence analysis were used to identify and analyse the beta-lactamase gene (bla(CMY-11)) responsible for the cefoxitin resistance. CMY-11 and bla(CMY-11) are compared with other class C beta-lactamases and their genes to determine phylogenetic relationships. The cefoxitin-resistance phenotype of E. coli K983802.1 reflects the presence of a large plasmid [pYMG-2 (130 kb)]. A beta-lactamase with a pI value of 8.0 from a transconjugant of E. coli K983802.1 was identified by isoelectric focusing. A 1478 bp DNA fragment from pYMG-2 containing bla(CMY-11) was sequenced and an open reading frame coding for a 382 amino acid peptide (CMY-11) was found. Phylogenetic analysis clearly shows that bla(CMY-11) belongs to the group of ampC-related bla genes. It is likely that bla(CMY-11) evolved from bla(CMY-1) via bla(CMY-10).
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164. |
Jiang Y,
Pogliano J,
Helinski DR,
Konieczny I,
( 2002 ) ParE toxin encoded by the broad-host-range plasmid RK2 is an inhibitor of Escherichia coli gyrase. PMID : 12010492 : DOI : 10.1046/j.1365-2958.2002.02921.x Abstract >>
Broad-host-range plasmid RK2 encodes a post-segregational killing system, parDE, which contributes to the stable maintenance of this plasmid in Escherichia coli and many distantly related bacteria. The ParE protein is a toxin that inhibits cell growth, causes cell filamentation and eventually cell death. The ParD protein is a specific ParE antitoxin. In this work, the in vitro activities of these two proteins were examined. The ParE protein was found to inhibit DNA synthesis using an E. coli oriC supercoiled template and a replication-proficient E. coli extract. Moreover, ParE inhibited the early stages of both chromosomal and plasmid DNA replication, as measured by the DnaB helicase- and gyrase-dependent formation of FI*, a highly unwound form of supercoiled DNA. The presence of ParD prevented these inhibitory activities of ParE. We also observed that the addition of ParE to supercoiled DNA plus gyrase alone resulted in the formation of a cleavable gyrase-DNA complex that was converted to a linear DNA form upon addition of sodium dodecyl sulphate (SDS). Adding ParD before or after the addition of ParE prevented the formation of this cleavable complex. These results demonstrate that the target of ParE toxin activity in vitro is E. coli gyrase.
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165. |
Subbarayan PR,
Deutscher MP,
( 2001 ) Escherichia coli RNase M is a multiply altered form of RNase I. PMID : 11780627 : PMC : PMC1370210 Abstract >>
RNase M, an enzyme previously purified to homogeneity from Escherichia coli, was suggested to be the RNase responsible for mRNA degradation in this bacterium. Although related to the endoribonuclease, RNase I, its distinct properties led to the conclusion that RNase M was a second, low molecular mass, broad specificity endoribonuclease present in E. coli. However, based on sequence analysis, southern hybridization, and enzyme activity, we show that RNase M is, in fact, a multiply altered form of RNase I. In addition to three amino acid substitutions that confer the properties of RNase M on the mutated RNase I, the protein is synthesized from an rna gene that contains a UGA nonsense codon at position 5, apparently as a result of a low level of readthrough. We also suggest that RNase M is just one of several previously described endoribonuclease activities that are actually manifestations of RNase I.
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166. |
Reischl U,
Youssef MT,
Kilwinski J,
Lehn N,
Zhang WL,
Karch H,
Strockbine NA,
( 2002 ) Real-time fluorescence PCR assays for detection and characterization of Shiga toxin, intimin, and enterohemolysin genes from Shiga toxin-producing Escherichia coli. PMID : 12089277 : DOI : 10.1128/jcm.40.7.2555-2565.2002 PMC : PMC120605 Abstract >>
PCR assays have proved useful for detecting and characterizing Shiga toxin-producing Escherichia coli (STEC). Recent advances in PCR technology have facilitated the development of real-time fluorescence PCR assays with greatly reduced amplification times and improved methods for the detection of amplified target sequences. We developed and evaluated two such assays for the LightCycler instrument: one that simultaneously detects the genes for Shiga toxins 1 and 2 (stx(1) and stx(2)) and another that simultaneously detects the genes for intimin (eae) and enterohemolysin (E-hly). Amplification and sequence-specific detection of the two target genes were completed within 60 min. Findings from the testing of 431 STEC isolates of human and animal origin, 73 isolates of E. coli negative for stx genes, and 118 isolates of other bacterial species with the LightCycler PCR (LC-PCR) assays were compared with those obtained by conventional block cycler PCR analysis. The sensitivities and specificities of the LC-PCR assays were each 100% for the stx(1), eae, and E-hly genes and 96 and 100%, respectively, for the stx(2) gene. No stx(2) genes were detected from 10 stx(2f)-positive isolates because of significant nucleotide differences in their primer annealing regions. Melting curve analyses of the amplified Shiga toxin genes revealed sequence variation within each of the tested genes that correlated with described and novel gene variants. The performance characteristics of the LC-PCR assays, such as their speed, detection method, and the potential subtyping information available from melting curve analyses, make them attractive alternatives to block cycler PCR assays for detecting and characterizing STEC strains.
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167. |
Huang SH,
Chen YH,
Kong G,
Chen SH,
Besemer J,
Borodovsky M,
Jong A,
( 2001 ) A novel genetic island of meningitic Escherichia coli K1 containing the ibeA invasion gene (GimA): functional annotation and carbon-source-regulated invasion of human brain microvascular endothelial cells. PMID : 11793250 : DOI : 10.1007/s101420100039 Abstract >>
The IbeA (ibe10) gene is an invasion determinant contributing to E. coli K1 invasion of the blood-brain barrier. This gene has been cloned and characterized from the chromosome of an invasive cerebrospinal fluid isolate of E. coli K1, strain RS218 (018:K1: H7). In the present study, a genetic island of meningitic E. coli containing ibeA (GimA) has been identified. A 20.3-kb genomic DNA island unique to E. coli K1 strains has been cloned and sequenced from an RS218 E. coli K1 genomic DNA library. Fourteen new genes have been identified in addition to the ibeA. The DNA sequence analysis indicated that the ibeA gene cluster was localized to the 98 min region and consisted of four operons, ptnIPKC, cglDTEC, gcxKRCI and ibeRAT. The G+C content (46.2%) of unique regions of the island is substantially different from that (50.8%) of the rest of the E. coli chromosome. By computer-assisted analysis of the sequences with DNA and protein databases (GenBank and PROSITE databases), the functions of the gene products could be anticipated, and were assigned to the functional categories of proteins relating to carbon source metabolism and substrate transportation. Glucose was shown to enhance E. coli penetration of human brain microvascular endothelial cells and exogenous cAMP was able to block the stimulating effect of glucose, suggesting that catabolic regulation may play a role in control of E. coli K1 invasion gene expression. Our data suggest that this genetic island may contribute to E. coli invasion of the blood-brain barrier through a carbon-source-regulated process.
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168. |
Saladin M,
Cao VT,
Lambert T,
Donay JL,
Herrmann JL,
Ould-Hocine Z,
Verdet C,
Delisle F,
Philippon A,
Arlet G,
( 2002 ) Diversity of CTX-M beta-lactamases and their promoter regions from Enterobacteriaceae isolated in three Parisian hospitals. PMID : 12007800 : DOI : 10.1111/j.1574-6968.2002.tb11126.x Abstract >>
Nine clinical isolates of Enterobacteriaceae (six Escherichia coli and three Proteus mirabilis) isolated in three Parisian hospitals between 1989 and 2000 showed a particular extended-spectrum cephalosporin-resistance profile characterized by resistance to cefotaxime and aztreonam but not to ceftazidime. CTX-M-1, CTX-M-2, CTX-M-9, CTX-M-14 and two novel plasmid-mediated CTX-M beta-lactamases (CTX-M-20, and CTX-M-21) were identified by polymerase chain reaction and isoelectric focusing (pI>8) and were associated in eight cases with TEM-1 (pI=5.4) or TEM-2 (pI=5.6) beta-lactamases. We used internal ISEcp1 and IS26 forward primers and the CTX-M consensus reverse primer to characterize the CTX-M beta-lactamase promoter regions and showed their high degree of structure diversity. We found upstream of some bla(CTX-M) genes, a 266-bp sequence 100% identical to the sequence upstream of the Kluyvera ascorbata beta-lactamase gene, suggesting that this chromosomal enzyme is the progenitor of the CTX-M-2/5 cluster.
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169. |
Smeds A,
Hemmann K,
Jakava-Viljanen M,
Pelkonen S,
Imberechts H,
Palva A,
( 2001 ) Characterization of the adhesin of Escherichia coli F18 fimbriae. PMID : 11705982 : DOI : 10.1128/IAI.69.12.7941-7945.2001 PMC : PMC98896 Abstract >>
Previous research has suggested that the adhesin encoded by the F18 fimbrial operon in Escherichia coli is either the FedE or FedF protein. In this work, we show that anti-FedF antibodies, unlike anti-FedE serum, were able to inhibit E. coli adhesion to porcine enterocytes. Moreover, specific adhesion to enterocytes was shown with purified FedF-maltose binding protein.
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170. |
Sundin GW,
( 2002 ) Distinct recent lineages of the strA- strB streptomycin-resistance genes in clinical and environmental bacteria. PMID : 12029529 : DOI : 10.1007/s00284-001-0100-y Abstract >>
We report the linkage of the strA-strB streptomycin-resistance genes with Class 1 integron sequences on pSTR1, a 75-kb multiple antibiotic-resistance plasmid from Shigella flexneri. strA-strB had previously been detected only within Tn 5393, a Tn 3-family transposon, and on small nonconjugative broad-host-range plasmids such as RSF1010. The geographic range of Tn 5393 was also extended to Pseudomonas spp. isolated from apple trees in New Zealand and soil in the USA. Comparative sequence analyses indicated that strA-strB from Tn 5393 and nonconjugative plasmids constitute distinct recent lineages with strA-strB from pSTR1 intermediate between the other two. The carriage of strA-strB within an integron, a transposon, and on broad-host-range plasmids has facilitated the world-wide dissemination of this determinant among at least 21 bacterial genera.
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171. |
Tarr CL,
Whittam TS,
( 2002 ) Molecular evolution of the intimin gene in O111 clones of pathogenic Escherichia coli. PMID : 11751825 : DOI : 10.1128/jb.184.2.479-487.2002 PMC : PMC139570 Abstract >>
Intimin is an important virulence factor in two groups of enteric pathogens: enteropathogenic Escherichia coli (EPEC), which is a major cause of infant diarrhea in the developing world, and enterohemorrhagic E. coli (EHEC), which has caused large food-borne outbreaks of hemorrhagic colitis in the United States and other developed countries. Intimin is encoded on a 35-kb pathogenicity island called the locus of enterocyte effacement (LEE). At least five antigenic types have been described for the highly variable gene, and each type is generally characteristic of particular evolutionary lineages. We determined the nucleotide sequences of intimin and other LEE genes in two O111 clones that have not been amenable to typing. The sequences from both O111:H8 and O111:H9 differed from the Int-beta that is typical of other clones in the same evolutionary lineage. The sequence from the O111:H8 strains was a mosaic of divergent segments that alternately clustered with Int-alpha, Int-beta, or Int-gamma. The sequence from the O111:H9 clone consistently showed a close relationship with that from E2348/69, a distantly related strain that expresses Int-alpha. The results suggest that there have been multiple acquisitions of the LEE in the EHEC 2/EPEC 2 clonal lineage, with a recent turnover in either O111:H8 or its close relatives. Amino acid substitutions that alter residue charge occurred more frequently than would be expected under random substitution in the extracellular domains of intimin, suggesting that diversifying selection has promoted divergence in this region of the protein. An N-terminal domain that presumably functions in the periplasm may also be under positive selection.
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172. |
Srimanote P,
Paton AW,
Paton JC,
( 2002 ) Characterization of a novel type IV pilus locus encoded on the large plasmid of locus of enterocyte effacement-negative Shiga-toxigenic Escherichia coli strains that are virulent for humans. PMID : 12011003 : DOI : 10.1128/iai.70.6.3094-3100.2002 PMC : PMC128018 Abstract >>
The majority of Shiga-toxigenic Escherichia coli (STEC) strains isolated from humans with gastrointestinal disease carry large (approximately 90-kb) plasmids. We have been analyzing the megaplasmid (designated pO113) from an O113:H21 STEC strain (98NK2). This strain lacks the locus for enterocyte effacement (LEE) and yet was responsible for an outbreak of hemolytic uremic syndrome. In the present study, we demonstrate that pO113 carries a novel type IV pilus biosynthesis locus (pil) related to those of the IncI plasmids R721, R64, and ColIb9. The pO113 pil locus consists of 11 closely linked genes (pilL through pilV) with an additional separately transcribed upstream gene (pilI). It directs the expression of long thin pili on the 98NK2 surface and the hemagglutination of guinea pig erythrocytes. We also demonstrate that pO113 can be transferred by conjugation. However, the type IV pilus encoded by pO113 does not appear to be involved in the adherence of 98NK2 to HEp-2 or Hct-8 cells in vitro. Homologues of the pO113 pil locus were present in several other LEE-negative STEC strains but not in LEE-positive STEC strains belonging to serogroup O26, O111, or O157.
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173. |
Mindlin S,
Kholodii G,
Gorlenko Z,
Minakhina S,
Minakhin L,
Kalyaeva E,
Kopteva A,
Petrova M,
Yurieva O,
Nikiforov V,
( 2001 ) Mercury resistance transposons of gram-negative environmental bacteria and their classification. PMID : 11763242 : Abstract >>
A total of 29 mercury resistance transposons were isolated from mercury-resistant environmental strains of proteobacteria collected in different parts of Eurasia and the USA and tested for hybridization with probes specific for transposase genes of known mercury resistance transposons. 9 were related to Tn21 in this test, 12 were related to Tn5053, 4 to Tn5041 and 1 to Tn5044; three transposons were negative in this test. Restriction mapping and DNA sequencing revealed that 12 transposons were identical or nearly identical to their corresponding relatives while the rest showed varying divergence from their closest relatives. Most of these previously unknown transposons apparently arose as a result of homologous or site-specific recombination. One of these, Tn5046, was completely sequenced, and shown to be a chimera with the mer operon and the transposition module derived from the transposons related to Tn5041 and to Tn5044, respectively. Transposon Tn5070, showing no hybridization with the specific probes used in this study, was also completely sequenced. The transposition module of Tn5070 was most closely related to that of Tn3 while the mer operon was most closely related to that of plasmid pMERPH. The merR of Tn5070 is transcribed in the same direction as the mer structural genes, which is typical for mer operons of gram-positive bacteria. Our data suggest that environmental bacteria may harbor many not yet recognized mercury resistance transposons and warrant their further inventory.
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174. |
Tauschek M,
Gorrell RJ,
Strugnell RA,
Robins-Browne RM,
( 2002 ) Identification of a protein secretory pathway for the secretion of heat-labile enterotoxin by an enterotoxigenic strain of Escherichia coli. PMID : 12011463 : DOI : 10.1073/pnas.092152899 PMC : PMC124529 Abstract >>
Enterotoxigenic Escherichia coli (ETEC) is an enteric pathogen that causes cholera-like diarrhea in humans and animals. ETEC secretes a heat-labile enterotoxin (LT), which resembles cholera toxin, but the actual mechanism of LT secretion is presently unknown. We have identified a previously unrecognized type II protein secretion pathway in the prototypic human ETEC strain, H10407 (serotype O78:H11). The genes for this pathway are absent from E. coli K-12, although examination of the K-12 genome suggests that it probably once possessed them. The secretory pathway bears significant homology at the amino acid level to the type II protein secretory pathway required by Vibrio cholerae for the secretion of cholera toxin. With this in mind, we determined whether the homologous pathway of E. coli H10407 played a role in the secretion of LT. To this end, we inactivated the pathway by inserting a kanamycin-resistance gene into one of the genes (gspD) of the type II secretion pathway by homologous recombination. LT secretion by E. coli H10407 and the gspD mutant was assayed by enzyme immunoassay, and its biological activity was assessed by using Y-1 adrenal cells. This investigation showed that the protein secretory pathway is functional and necessary for the secretion of LT by ETEC. Our findings have revealed the mechanism for the secretion of LT by ETEC, which previously was unknown, and provide further evidence of close biological similarities of the LT and cholera toxin.
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175. |
Partridge SR,
Brown HJ,
Hall RM,
( 2002 ) Characterization and movement of the class 1 integron known as Tn2521 and Tn1405. PMID : 11959558 : DOI : 10.1128/aac.46.5.1288-1294.2002 PMC : PMC127177 Abstract >>
Two putative transposons, Tn2521 and Tn1405, carrying determinants for the PSE-4 beta-lactamase and for resistance to streptomycin, spectinomycin, and sulfonamides were previously isolated from the chromosome of Pseudomonas aeruginosa Dalgleish. Detailed mapping and determination of the complete sequence of Tn2521 revealed that it is a class 1 integron, here renamed In33, with a backbone structure identical to that of In4 from Tn1696. In33 contains two gene cassettes, blaP1 and aadA1, replacing the aacC1-orfE-aadA2-cmlA1 cassette array in In4. Although In33 does not include any transposition genes, movement of In33 (Tn2521) targeted to a single location in the IncP-1 plasmid R18-18 has been reported previously (M. I. Sinclair and B. W. Holloway, J. Bacteriol. 151:569-579, 1982). A 5-bp duplication of the target, which lies within the res site recognized by the ParA resolvase of R18-18, was present, indicating that the mechanism of movement was transposition. Together, these data indicate that class 1 integrons that are defective in self-transposition can move under appropriate circumstances. The Tn1405 isolate studied was found to represent only the cassette array of In33, which had replaced the cassette array in the recipient plasmid R388, probably by homologous recombination.
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176. |
D'Haeze W,
Verplancke C,
Mironov V,
Holsters M,
( 2002 ) pMH11, A tool for gene disruption and expression analysis in Azorhizobium caulinodans. PMID : 11982330 : DOI : 10.1006/plas.2002.1565 Abstract >>
Tools for mutagenesis and expression analyses are needed to study the role of bacterial genes. Here, we report the construction of pMH11, a small, mobilizable plasmid that replicates in Escherichia coli, but not in Azorhizobium caulinodans, a nodulating microsymbiont of Sesbania rostrata, and that contains a unique BamHI restriction site upstream of a promoterless lacZ gene. pMH11 and two derivatives with the multiple cloning site of pBluescript (KS(II)) are useful for mutagenesis by gene disruption and for expression analyses after selection for cointegration by kanamycin resistance. Weakly constitutive promoter activity from the vector allowed transcription of genes downstream of the integration site, so that no polar effects were caused by gene disruption.
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177. |
Schröder G,
Krause S,
Zechner EL,
Traxler B,
Yeo HJ,
Lurz R,
Waksman G,
Lanka E,
( 2002 ) TraG-like proteins of DNA transfer systems and of the Helicobacter pylori type IV secretion system: inner membrane gate for exported substrates? PMID : 11976307 : DOI : 10.1128/jb.184.10.2767-2779.2002 PMC : PMC135038 Abstract >>
TraG-like proteins are potential NTP hydrolases (NTPases) that are essential for DNA transfer in bacterial conjugation. They are thought to mediate interactions between the DNA-processing (Dtr) and the mating pair formation (Mpf) systems. TraG-like proteins also function as essential components of type IV secretion systems of several bacterial pathogens such as Helicobacter pylori. Here we present the biochemical characterization of three members of the family of TraG-like proteins, TraG (RP4), TraD (F), and HP0524 (H. pylori). These proteins were found to have a pronounced tendency to form oligomers and were shown to bind DNA without sequence specificity. Standard NTPase assays indicated that these TraG-like proteins do not possess postulated NTP-hydrolyzing activity. Surface plasmon resonance was used to demonstrate an interaction between TraG and relaxase TraI of RP4. Topology analysis of TraG revealed that TraG is a transmembrane protein with cytosolic N and C termini and a short periplasmic domain close to the N terminus. We predict that multimeric inner membrane protein TraG forms a pore. A model suggesting that the relaxosome binds to the TraG pore via TraG-DNA and TraG-TraI interactions is presented.
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178. |
Brinkkötter A,
Shakeri-Garakani A,
Lengeler JW,
( 2002 ) Two class II D-tagatose-bisphosphate aldolases from enteric bacteria. PMID : 11976750 : DOI : 10.1007/s00203-002-0406-6 Abstract >>
Escherichia coli, Salmonella enterica, Klebsiella pneumoniaeand Klebsiella oxytocawere found to contain two D-tagatose 1,6-bisphosphate (TagBP)-specific aldolases involved in catabolism of galactitol (genes gatY gatZ) and of N-acetyl-galactosamine and D-galactosamine (genes kbaY kbaZ,also called agaY agaZ). The two aldolases were closely related (> or = 53.8% identical amino acids) and could substitute for each other in vivo. The catalytic subunits GatY or KbaY alone were sufficient to show aldolase activity. Although substantially shorter than other aldolases (285 amino acids, instead of 358 and 349 amino acids), these subunits contained most or all of the residues that have been identified as essential in substrate/product recognition and catalysis for class II aldolases. In contrast to these, both aldolases required subunits GatZ or KbaZ (420 amino acids) for full activity and for good in vivo and in vitro stability. The Z subunits alone did not show any aldolase activity. Close relatives of these new TagBP aldolases were found in several gram-negative and gram-positive bacteria, e.g., Streptomyces coelicolor.
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179. |
Brown EW,
LeClerc JE,
Kotewicz ML,
Cebula TA,
( 2001 ) Three R's of bacterial evolution: how replication, repair, and recombination frame the origin of species. PMID : 11746762 : Abstract >>
The genetic diversity of bacteria results not only from errors in DNA replication and repair but from horizontal exchange and recombination of DNA sequences from similar and disparate species as well. New individuals carrying adaptive changes are thus being spawned constantly among the population at large. When new selection pressures appear, these are the individuals that survive, at the expense of the general population, to forge new populations. Depending on the severity and uniqueness of the selection pressure, this could lead to new speciation. It is becoming more and more evident that, as nucleotide sequences of numerous loci from many bacterial strains continue to amass, horizontal transfer has played a key role in configuring the Escherichia coli chromosome. Here, we examine views, both old and new, for the role of recombination in the evolution of bacterial chromosomes. We present novel phylogenetic evidence for horizontal transfer of three genes involved in DNA replication and repair (mutS, uvrD, and polA). These data reveal a prominent role for horizontal transfer in the evolution of genes known to play a key role in the fidelity of DNA replication and, thus, ultimate survival of the organism. Our data underscore that recombination plays both a diversifying and a homogenizing role in defining the structure of the E. coli genome.
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180. |
Otto BR,
van Dooren SJ,
Dozois CM,
Luirink J,
Oudega B,
( 2002 ) Escherichia coli hemoglobin protease autotransporter contributes to synergistic abscess formation and heme-dependent growth of Bacteroides fragilis. PMID : 11748157 : DOI : 10.1128/iai.70.1.5-10.2002 PMC : PMC127594 Abstract >>
Intra-abdominal infections (IAI) continue to be a serious clinical problem. Bacterial synergism is an important factor that influences the shift from contamination to IAI, leading to the development of lesions and abscess formation. Escherichia coli and Bacteroides fragilis are particularly abundant in IAI. The underlying molecular mechanisms of this pathogenic synergy are still unclear. The role of the hemoglobin protease (Hbp) autotransporter protein from E. coli in the synergy of IAI was investigated. Hbp is identical to Tsh, a temperature-sensitive hemagglutinin associated with avian pathogenic E. coli. Clinical isolates from miscellaneous extraintestinal infections were phenotypically and genotypically screened for Hbp. The presence of Hbp was significantly associated with E. coli isolated from IAI and other extraintestinal infections. In a murine infection model, Hbp was shown to contribute to the pathogenic synergy of abscess development. Mice immunized with Hbp were protected against mixed infections and did not develop abscess lesions. Furthermore, an E. coli wild-type strain that did not induce abscess formation in the synergy model was transformed with a plasmid encoding the hbp gene, and mixed infections with this strain lead to increased growth of B. fragilis and induction of abscess lesions. Growth-promoting studies showed that purified Hbp is able to deliver heme to B. fragilis strain BE1. In conclusion, results suggest the synergy of abscess formation by E. coli and B. fragilis can be partly explained by the capacity of B. fragilis to intercept Hbp and iron from heme to overcome the iron restrictions imposed by the host.
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181. |
Kim J,
Nietfeldt J,
Ju J,
Wise J,
Fegan N,
Desmarchelier P,
Benson AK,
( 2001 ) Ancestral divergence, genome diversification, and phylogeographic variation in subpopulations of sorbitol-negative, beta-glucuronidase-negative enterohemorrhagic Escherichia coli O157. PMID : 11698378 : DOI : 10.1128/JB.183.23.6885-6897.2001 PMC : PMC95530 Abstract >>
The O157:H7 lineage of enterohemorrhagic Escherichia coli is a geographically disseminated complex of highly related genotypes that share common ancestry. The common clone that is found worldwide carries several markers of events in its evolution, including markers for acquisition of virulence genes and loss of physiological characteristics, such as sorbitol fermentation ability and beta-glucuronidase production. Populations of variants that are distinct with respect to motility and the sorbitol and beta-glucuronidase markers appear to have diverged at several points along the inferred evolutionary pathway. In addition to these variants, distinct subpopulations of the contemporary non-sorbitol-fermenting, beta-glucuronidase-negative O157:H7 clone were recently detected among bovine and human clinical isolates in the United States by using high-resolution genome comparison. In order to determine if these recently described subpopulations were derived from a regional or ancestral divergence event, we used octamer-based genome scanning, marker sorting, and DNA sequence analysis to examine their phylogenetic relationship to populations of non-sorbitol-fermenting, beta-glucuronidase negative O157:H7 and O157:H- strains from Australia. The inferred phylogeny is consistent with the hypothesis that subpopulations on each continent resulted from geographic spread of an ancestral divergence event and subsequent expansion of distinct subpopulations. Marker sorting and DNA sequence analyses identified sets of monophyletic markers consistent with the pattern of divergence and demonstrated that phylogeographic variation occurred through emergence of regional subclones and concentration of regional polymorphisms among distinct subpopulations. DNA sequence analysis of representative polyphyletic markers showed that genome diversity accrued through random drift and bacteriophage-mediated events.
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182. |
Wang L,
Huskic S,
Cisterne A,
Rothemund D,
Reeves PR,
( 2002 ) The O-antigen gene cluster of Escherichia coli O55:H7 and identification of a new UDP-GlcNAc C4 epimerase gene. PMID : 11976290 : DOI : 10.1128/jb.184.10.2620-2625.2002 PMC : PMC135022 Abstract >>
Escherichia coli O55 is an important antigen which is often associated with enteropathogenic E. coli clones. We sequenced the genes responsible for its synthesis and identified genes for O-antigen polymerase, O-antigen flippase, four enzymes involved in GDP-colitose synthesis, and three glycosyltransferases, all by comparison with known genes. Upstream of the normal O-antigen region there is a gne gene, which encodes a UDP-GlcNAc epimerase for converting UDP-GlcNAc to UDP-GalNAc and is essential for O55 antigen synthesis. The O55 gne product has only 20 and 26% identity to the gne genes of Pseudomonas aeruginosa and E. coli O113, respectively. We also found evidence for the O55 gene cluster's having evolved from another gene cluster by gain and loss of genes. Only three of the GDP-colitose pathway genes are in the usual location, the other two being separated, although nearby. It is thought that the E. coli O157:H7 clone evolved from the O55:H7 clone in part by transfer of the O157 gene cluster into an O55 lineage. Comparison of genes flanking the O-antigen gene clusters of the O55:H7 and O157:H7 clones revealed one recombination site within the galF gene and located the other between the hisG and amn genes. Genes outside the recombination sites are 99.6 to 100% identical in the two clones, while most genes thought to have transferred with the O157 gene cluster are 95 to 98% identical.
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183. |
Lovell S,
Goryshin IY,
Reznikoff WR,
Rayment I,
( 2002 ) Two-metal active site binding of a Tn5 transposase synaptic complex. PMID : 11896402 : DOI : 10.1038/nsb778 Abstract >>
A synaptic complex of Tn5 transposase with an extended outside end DNA duplex was prepared and crystallized, and its crystal structure was determined in an effort to reveal the role of metal ions in catalysis. Two Mn2+ ions bound to the active site when a single nucleotide of donor DNA was added to the 3' end of the transferred strand. Marked conformational changes were observed in the DNA bases closest to the active site. The position of the metal ions and the conformational changes of the DNA provide insight into the mechanism of hairpin formation and cleavage, and is consistent with a two-metal model for catalysis.
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184. |
Seah JN,
Kwang J,
( 2001 ) Mapping of Escherichia coli H27-specific epitope from H-specific polypeptides. PMID : 11687451 : DOI : 10.1128/CDLI.8.6.1126-1130.2001 PMC : PMC96237 Abstract >>
A murine monoclonal antibody (MAb) reactive to H27 flagellin antigen was produced and characterized. Forty-nine partially purified native H-type flagellins were used to evaluate the specificity of the MAb. The fliC gene of H27 is 1,464 bp in length (487 amino acids [aa]; 50.88 kDa). The central variable region (CVR) of the H27 flagellin gene was defined by comparison with flagellin sequences derived from H8, H34, and H49. To study the distribution of antigenic epitopes, the CVR covering amino acid residues 70 to 457 (388 aa) was dissected into seven overlapping fragments. Fragments carrying the H-type-specific antigenic determinants were identified by H27-specific antiserum. Polyclonal antibodies raised against different H-type flagellin proteins were used to determine the cross-reactive determinants. Three fragments, spanning amino acid residues 240 to 380, which carried the potential H-specific determinants were used for MAb production. A MAb specific to H27 was produced, and the specific epitope was mapped to amino acid residues 330 to 340. In this study, we produced MAbs from predetermined H27-specific polypeptides and used whole flagellin in enzyme-linked immunosorbent assays to circumvent the interference of anti-glutathione S-transferase antibodies. These factors when combined could help to improve the identification of the desired MAb.
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185. |
Zhao S,
White DG,
McDermott PF,
Friedman S,
English L,
Ayers S,
Meng J,
Maurer JJ,
Holland R,
Walker RD,
( 2001 ) Identification and expression of cephamycinase bla(CMY) genes in Escherichia coli and Salmonella isolates from food animals and ground meat. PMID : 11709361 : DOI : 10.1128/AAC.45.12.3647-3650.2001 PMC : PMC90890 Abstract >>
Twenty-one Salmonella and 54 Escherichia coli isolates, recovered from food animals and retail ground meats, that exhibited decreased susceptibilities to ceftiofur and ceftriaxone were shown to possess a bla(CMY) gene. The bla(CMY-4) gene was identified in an E. coli isolate recovered from retail chicken and was further shown to be responsible for resistance to cephalothin, ampicillin, and amoxicillin-clavulanic acid and elevated MICs of ceftriaxone, cefoxitin, and ceftiofur.
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186. |
Pradel N,
Leroy-Setrin S,
Joly B,
Livrelli V,
( 2002 ) Genomic subtraction to identify and characterize sequences of Shiga toxin-producing Escherichia coli O91:H21. PMID : 11976103 : DOI : 10.1128/aem.68.5.2316-2325.2002 PMC : PMC127536 Abstract >>
To identify Shiga toxin-producing Escherichia coli genes associated with severe human disease, a genomic subtraction technique was used with hemolytic-uremic syndrome-associated O91:H21 strain CH014 and O6:H10 bovine strains. The method was adapted to the Shiga toxin-producing E. coli genome: three rounds of subtraction were used to isolate DNA fragments specific to strain CH014. The fragments were characterized by genetic support analysis, sequencing, and hybridization to the genome of a collection of Shiga toxin-producing E. coli strains. A total of 42 fragments were found, 19 of which correspond to previously identified unique DNA sequences in the enterohemorrhagic E. coli EDL933 reference strain, including 7 fragments corresponding to prophage sequences and others encoding candidate virulence factors, such a SepA homolog protein and a fimbrial usher protein. In addition, the subtraction procedure yielded plasmid-related sequences from Shigella flexneri and enteropathogenic and Shiga toxin-producing E. coli virulence plasmids. We found that lateral gene transfer is extensive in strain CH014, and we discuss the role of genomic mobile elements, especially bacteriophages, in the evolution and possible transfer of virulence determinants.
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187. |
Sandt CH,
Hill CW,
( 2001 ) Nonimmune binding of human immunoglobulin A (IgA) and IgG Fc by distinct sequence segments of the EibF cell surface protein of Escherichia coli. PMID : 11705900 : DOI : 10.1128/IAI.69.12.7293-7203.2001 PMC : PMC98814 Abstract >>
The eib genes of Escherichia coli encode surface-exposed proteins which bind immunoglobulins (Ig) such as the Fc fragment of human IgG (IgG Fc) in a nonimmune manner. The Eib proteins belong to a family which includes YadA of Yersinia, UspA2 of Moraxella, and DsrA of Haemophilus ducreyi. This family of surface-exposed proteins shares several features, such as the ability to impart resistance to human serum complement and a tendency to exist as stable multimers. Four genes, eibA, eibC, eibD and eibE, were previously identified and cloned from ECOR-9, a strain from the E. coli reference collection. EibC, -D, and -E bind human serum IgA in addition to IgG, but no IgA binding has been observed for EibA. Here, we report the cloning of a new eib gene, eibF, from a second strain of E. coli, ECOR-2. The product, EibF, has a relatively strong preference for IgA. Like the other eib genes, eibF attenuates serum sensitivity, occurs as a stable multimer, and is associated with a prophage. By subcloning portions of the eibA and eibF genes, we have identified distinct sequence segments sufficient to cause Ig binding, multimerization, and discrimination between IgA and IgG. The ability to multimerize is associated with a sequence close to the C terminus that is homologous to other family members such as YadA. Binding of IgG Fc is associated with a sequence that is highly conserved among all Eib proteins but otherwise unique. Binding of IgA is associated with a sequence of EibF that is not similar to any EibA sequence.
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188. |
Naumann TA,
Reznikoff WS,
( 2002 ) Tn5 transposase active site mutants. PMID : 11877443 : DOI : 10.1074/jbc.M200742200 Abstract >>
Tn5 transposase (Tnp) is a 53.3-kDa protein that is encoded by and facilitates movement of transposon Tn5. Tnp monomers contain a single active site that is responsible for catalyzing a series of four DNA breaking/joining reactions at one transposon end. Based on primary sequence homology and protein structural information, we designed and constructed a series of plasmids that encode for Tnps containing active site mutations. Following Tnp expression and purification, the active site mutants were tested for their ability to form protein-DNA complexes and perform each of the four catalytic steps in the transposition pathway in vitro. The results demonstrate that Asp-97, Asp-188, and Glu-326, visible in the active site of Tn5 crystal structures, are absolutely required for all catalytic steps. Mutations within a series of amino acid residues that are conserved in the IS4 family of transposases and retroviral integrases also impair Tnp catalytic activity. Mutations at either Tyr-319 or Arg-322 reduce both hairpin resolution and strand transfer activity within protein-DNA complexes. Mutations at Lys-333 reduce the ability of Tnps to form protein-DNA complexes, whereas mutations at the less strongly conserved Lys-330 have less of an effect on both synaptic complex formation and catalytic activity.
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189. |
Rintoul MR,
de Arcuri BF,
Salomón RA,
Farías RN,
Morero RD,
( 2001 ) The antibacterial action of microcin J25: evidence for disruption of cytoplasmic membrane energization in Salmonella newport. PMID : 11731133 : DOI : 10.1111/j.1574-6968.2001.tb10895.x Abstract >>
Microcin J25 (MccJ25) is a cyclic peptide of 21 unmodified amino acid residues produced by a fecal strain of Escherichia coli. It has previously been shown that the antibiotic activity of this peptide is mainly directed to Enterobacteriaceae, including several pathogenic E. coli, Salmonella and Shigella strains. In this paper we show that MccJ25 acts on the cytoplasmic membrane of Salmonella newport cells producing alteration of membrane permeability, and the subsequent gradient dissipation, that initiate the inhibition of process, such as oxygen consumption. These results, taken together with our in vitro observations [Rintoul et al. (2000) Biochim. Biophys. Acta 1509, 65-72], strongly suggest that the disruption of the cytoplasmic membrane gradient is closely related to the bactericidal activity of MccJ25 in S. newport.
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190. |
Ninomiya T,
Sugiura N,
Tawada A,
Sugimoto K,
Watanabe H,
Kimata K,
( 2002 ) Molecular cloning and characterization of chondroitin polymerase from Escherichia coli strain K4. PMID : 11943778 : DOI : 10.1074/jbc.M201719200 Abstract >>
Escherichia coli strain K4 produces the K4 antigen, a capsule polysaccharide consisting of a chondroitin backbone (GlcUA beta(1-3)-GalNAc beta(1-4))(n) to which beta-fructose is linked at position C-3 of the GlcUA residue. We molecularly cloned region 2 of the K4 capsular gene cluster essential for biosynthesis of the polysaccharide, and we further identified a gene encoding a bifunctional glycosyltransferase that polymerizes the chondroitin backbone. The enzyme, containing two conserved glycosyltransferase sites, showed 59 and 61% identity at the amino acid level to class 2 hyaluronan synthase and chondroitin synthase from Pasteurella multocida, respectively. The soluble enzyme expressed in a bacterial expression system transferred GalNAc and GlcUA residues alternately, and polymerized the chondroitin chain up to a molecular mass of 20 kDa when chondroitin sulfate hexasaccharide was used as an acceptor. The enzyme exhibited apparent K(m) values for UDP-GlcUA and UDP-GalNAc of 3.44 and 31.6 microm, respectively, and absolutely required acceptors of chondroitin sulfate polymers and oligosaccharides at least longer than a tetrasaccharide. In addition, chondroitin polymers and oligosaccharides and hyaluronan polymers and oligosaccharides served as acceptors for chondroitin polymerization, but dermatan sulfate and heparin did not. These results may lead to elucidation of the mechanism for chondroitin chain synthesis in both microorganisms and mammals.
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191. |
Keller R,
Ordoñez JG,
de Oliveira RR,
Trabulsi LR,
Baldwin TJ,
Knutton S,
( 2002 ) Afa, a diffuse adherence fibrillar adhesin associated with enteropathogenic Escherichia coli. PMID : 11953412 : DOI : 10.1128/iai.70.5.2681-2689.2002 PMC : PMC127892 Abstract >>
O55 is one of the most frequent enteropathogenic Escherichia coli (EPEC) O serogroups implicated in infantile diarrhea in developing countries. Multilocus enzyme electrophoresis analysis showed that this serogroup includes two major electrophoretic types (ET), designated ET1 and ET5. ET1 corresponds to typical EPEC, whilst ET5 comprises strains with different combinations of virulence genes, including those for localized adherence (LA) and diffuse adherence (DA). Here we report that ET5 DA strains possess a DA adhesin, designated EPEC Afa. An 11.6-kb chromosomal region including the DA adhesin operon from one O55:H(-) ET5 EPEC strain was sequenced and found to encode a protein with 98% identity to AfaE-1, an adhesin associated with uropathogenic E. coli. Although described as an afimbrial adhesin, we show that both AfaE-1 and EPEC Afa possess fine fibrillar structures. This is the first characterization and demonstration of an Afa adhesin associated with EPEC.
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192. |
McVey CE,
Walsh MA,
Dodson GG,
Wilson KS,
Brannigan JA,
( 2001 ) Crystal structures of penicillin acylase enzyme-substrate complexes: structural insights into the catalytic mechanism. PMID : 11601852 : DOI : 10.1006/jmbi.2001.5043 Abstract >>
The crystal structure of penicillin G acylase from Escherichia coli has been determined to a resolution of 1.3 A from a crystal form grown in the presence of ethylene glycol. To study aspects of the substrate specificity and catalytic mechanism of this key biotechnological enzyme, mutants were made to generate inactive protein useful for producing enzyme-substrate complexes. Owing to the intimate association of enzyme activity and precursor processing in this protein family (the Ntn hydrolases), most attempts to alter active-site residues lead to processing defects. Mutation of the invariant residue Arg B263 results in the accumulation of a protein precursor form. However, the mutation of Asn B241, a residue implicated in stabilisation of the tetrahedral intermediate during catalysis, inactivates the enzyme but does not prevent autocatalytic processing or the ability to bind substrates. The crystal structure of the Asn B241 Ala oxyanion hole mutant enzyme has been determined in its native form and in complex with penicillin G and penicillin G sulphoxide. We show that Asn B241 has an important role in maintaining the active site geometry and in productive substrate binding, hence the structure of the mutant protein is a poor model for the Michaelis complex. For this reason, we subsequently solved the structure of the wild-type protein in complex with the slowly processed substrate penicillin G sulphoxide. Analysis of this structure suggests that the reaction mechanism proceeds via direct nucleophilic attack of Ser B1 on the scissile amide and not as previously proposed via a tightly H-bonded water molecule acting as a "virtual" base.
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193. |
Paton AW,
Srimanote P,
Woodrow MC,
Paton JC,
( 2001 ) Characterization of Saa, a novel autoagglutinating adhesin produced by locus of enterocyte effacement-negative Shiga-toxigenic Escherichia coli strains that are virulent for humans. PMID : 11598075 : DOI : 10.1128/IAI.69.11.6999-7009.2001 PMC : PMC100080 Abstract >>
The capacity of Shiga toxigenic Escherichia coli (STEC) to adhere to the intestinal mucosa undoubtedly contributes to pathogenesis of human disease. The majority of STEC strains isolated from severe cases produce attaching and effacing lesions on the intestinal mucosa, a property mediated by the locus of enterocyte effacement (LEE) pathogenicity island. This element is not essential for pathogenesis, as some cases of severe disease, including hemolytic uremic syndrome (HUS), are caused by LEE-negative STEC strains, but the mechanism whereby these adhere to the intestinal mucosa is not understood. We have isolated a gene from the megaplasmid of a LEE-negative O113:H21 STEC strain (98NK2) responsible for an outbreak of HUS, which encodes an auto-agglutinating adhesin designated Saa (STEC autoagglutinating adhesin). Introduction of saa cloned in pBC results in a 9.7-fold increase in adherence of E. coli JM109 to HEp-2 cells and a semilocalized adherence pattern. Mutagenesis of saa in 98NK2, or curing the wild-type strain of its megaplasmid, resulted in a significant reduction in adherence. Homologues of saa were found in several unrelated LEE-negative STEC serotypes, including O48:H21 (strain 94CR) and O91:H21 (strain B2F1), which were also isolated from patients with HUS. Saa exhibits a low degree of similarity (25% amino acid [aa] identity) with YadA of Yersinia enterocolitica and Eib, a recently described phage-encoded immunoglobulin binding protein from E. coli. Saa produced by 98NK2 is 516 aa long and includes four copies of a 37-aa direct repeat sequence. Interestingly, Saa produced by other STEC strains ranges in size from 460 to 534 aa as a consequence of variation in the number of repeats and/or other insertions or deletions immediately proximal to the repeat domain.
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194. |
Pickens JC,
Merritt EA,
Ahn M,
Verlinde CL,
Hol WG,
Fan E,
( 2002 ) Anchor-based design of improved cholera toxin and E. coli heat-labile enterotoxin receptor binding antagonists that display multiple binding modes. PMID : 11880036 : Abstract >>
The action of cholera toxin and E. coli heat-labile enterotoxin can be inhibited by blocking their binding to the cell-surface receptor GM1. We have used anchor-based design to create 15 receptor binding inhibitors that contain the previously characterized inhibitor MNPG as a substructure. In ELISA assays, all 15 compounds exhibited increased potency relative to MNPG. Binding affinities for two compounds, each containing a morpholine ring linked to MNPG via a hydrophobic tail, were characterized by pulsed ultrafiltration (PUF) and isothermal titration calorimetry (ITC). Crystal structures for these compounds bound to toxin B pentamer revealed a conserved binding mode for the MNPG moiety, with multiple binding modes adopted by the attached morpholine derivatives. The observed binding interactions can be exploited in the design of improved toxin binding inhibitors.
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195. |
Springs SL,
Bass SE,
Bowman G,
Nodelman I,
Schutt CE,
McLendon GL,
( 2002 ) A multigeneration analysis of cytochrome b(562) redox variants: evolutionary strategies for modulating redox potential revealed using a library approach. PMID : 11914078 : DOI : 10.1021/bi012066s Abstract >>
The redox potential of cytochromes sets the energy yield possible in metabolism and is also a key determinant of the rate at which redox reactions proceed. Here, the heme protein, cytochrome b(562), is used to study the in vitro evolution of redox potential within a library of variants containing the same structural archetype, the four-helix bundle. Multisite variations in the active site of cytochrome b(562) were introduced. A library of variants containing random mutations in place of R98 and R106 was created, and the redox potentials of a statistical sampling of this library were measured. This procedure was carried out for both the low- and high-potential variants of a previously studied F61X/F65X, first-generation library [Springs, S. L., Bass, S. E., and McLendon, G. L. (2000) Biochemistry 39, 6075]. The second-generation library reported here has a range of redox potentials which is greater than 40% (160 mV) of the known accessible potential among cytochromes with identical axial ligands (but different folds) and exceeds the range exhibited phylogenetically by the cytochrome c' family which internally maintains the same axial ligation and fold. A statistical analysis of the libraries examined reveals that the redox potential of WT cyt b(562) is found at the high-potential extremum of the distribution, indicating that this protein apparently evolved to differentially stabilize the reduced protein. The 2.7 A crystal structure of F61I/F65Y/R106L (low-potential variant of the second-generation library) was solved and is compared to the wild-type structure and the 2.2 A resolution structure of the F61I/F65Y variant (low-potential variant of the first-generation library). The structures indicate that charge-dipole effects are responsible for shifting the redox equilibrium toward the oxidized state in both the F61I/F65Y and F61I/F65Y/R106L variants. Specifically, a new protein dipole is introduced into the heme microenvironment as a result of the F65Y mutation, two new internal water molecules (one in hydrogen-bonding distance of Y65) are found, and in the case of F61I/F65Y/R106L (DeltaE(m) = 158 mV vs NHE), increased solvent exposure of the heme as a result of the R106L substitution is identified.
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196. |
Zhang W,
Bielaszewska M,
Kuczius T,
Karch H,
( 2002 ) Identification, characterization, and distribution of a Shiga toxin 1 gene variant (stx(1c)) in Escherichia coli strains isolated from humans. PMID : 11923370 : DOI : 10.1128/jcm.40.4.1441-1446.2002 PMC : PMC140390 Abstract >>
By using sequence analysis of Shiga toxin 1 (Stx 1) genes from human and ovine Stx-producing Escherichia coli (STEC) strains, we identified an Stx1 variant in STEC of human origin that was identical to the Stx1 variant from ovine STEC, but demonstrated only 97.1 and 96.6% amino acid sequence identity in its A and B subunits, respectively, to the Stx1 encoded by bacteriophage 933J. We designated this variant "Stx1c" and developed stxB(1) restriction fragment length polymorphism and stx(1c)-specific PCR strategies to determine the frequency and distribution of stx(1c) among 212 STEC strains isolated from humans. stx(1c) was identified in 36 (17.0%) of 212 STEC strains, 19 of which originated from asymptomatic subjects and 16 of which were from patients with uncomplicated diarrhea. stx(1c) was most frequently (in 23 STEC strains [63.9%]) associated with stx(2d), but 12 (33.3%) of the 36 STEC strains possessed stx(1c) only. A single STEC strain possessed stx(1c) together with stx(2) and was isolated from a patient with hemolytic-uremic syndrome. All 36 stx(1c)-positive STEC strains were eae negative and belonged to 10 different serogroups, none of which was O157, O26, O103, O111, or O145. Stx1c was produced by all stx(1c)-containing STEC strains, but reacted weakly with a commercial immunoassay. We conclude that STEC strains harboring the stx(1c) variant account for a significant proportion of human STEC isolates. The procedures developed in this study now allow the determination of the frequency of STEC strains harboring stx(1c) among clinical STEC isolates and their association with human disease in prospective studies.
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197. |
Pai H,
Jacoby GA,
( 2001 ) Sequences of the NPS-1 and TLE-1 beta-lactamase genes. PMID : 11557499 : DOI : 10.1128/AAC.45.10.2947-2948.2001 PMC : PMC90761 Abstract >>
The NPS-1 and TLE-1 beta-lactamase genes were cloned and sequenced. NPS-1 differed from LCR-1 beta-lactamase in 8 of 260 amino acids. TLE-1 differed from TEM-1 by a single Asp(115)-->Gly substitution and has been renamed TEM-90.
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198. |
Jores J,
Rumer L,
Kiessling S,
Kaper JB,
Wieler LH,
( 2001 ) A novel locus of enterocyte effacement (LEE) pathogenicity island inserted at pheV in bovine Shiga toxin-producing Escherichia coli strain O103:H2. PMID : 11682182 : DOI : 10.1111/j.1574-6968.2001.tb10866.x Abstract >>
We describe a locus of enterocyte effacement (LEE) which is part of a new pathogenicity island (PAI) detected in the bovine Shiga toxin-producing Escherichia coli strain RW1374 (O103:H2). This PAI is at least 80 kb in size and inserted in the vicinity of the pheV tRNA gene at 67 min of the E. coli chromosome. Furthermore, the PAI differs from the previously described LEEs by unique flanking regions at both sides, which harbor one copy each of an insertion element in an inverted orientation that is 96% identical to insertion site (IS)629. In addition, a 5-kb PAI-specific sequence downstream of the LEE core region and adjacent to the E. coli K12 region is duplicated upstream of the LEE core region as well. The duplicated sequences are more than 80% identical to each other and consist partially of prophage sequences.
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199. |
Assfalg M,
Banci L,
Bertini I,
Ciofi-Baffoni S,
Barker PD,
( 2001 ) (15)N backbone dynamics of ferricytochrome b(562): comparison with the reduced protein and the R98C variant. PMID : 11669612 : DOI : 10.1021/bi0101300 Abstract >>
The backbone dynamics of ferricytochrome b(562), a four-helix bundle protein from Escherichia coli, have been studied by NMR spectroscopy. The consequences of the introduction of a c-type thioether linkage between the heme and protein and the reduction to the ferrous cytochrome have also been analyzed. (15)N relaxation rates R(1) and R(2) and (1)H-(15)N NOEs were measured at proton Larmor frequencies of 500 and 600 MHz for the oxidized and reduced protein as well as for the oxidized R98C variant. In the latter protein, an "artificial" thioether covalent bond has been introduced between the heme group and the protein frame [Arnesano, F., Banci, L., Bertini, I., Ciofi-Baffoni, S., de Lumley Woodyear, T., Johnson, C. M., and Barker, P. D. (2000) Biochemistry 39, 1499-1514]. The (15)N relaxation data were analyzed with the ModelFree protocol, and the mobility parameters on the picosecond to nanosecond time scale were compared for the three species. The three forms are rather rigid as a whole, with average generalized order parameters values of 0.87 +/- 0.08 (oxidized cytochrome b(562)), 0.84 +/- 0.07 (reduced cytochrome b(562)), and 0.85 +/- 0.07 (oxidized R98C cytochrome b(562)), indicating similar mobility for each system. Lower order parameters (S(2)) are found for residues belonging to loops 1 and 2. Higher mobility, as indicated by lower order parameters, is found for heme binding helices alpha 1 and alpha 4 in the R98C variant with respect to the wild-type protein. The analysis requires a relatively long rotational correlation time (tau(m) = 9.6 ns) whose value is accounted for on the basis of the anisotropy of the molecular shape and the high phosphate concentration needed to ensure the occurrence of monomer species. A parallel study of motions in the millisecond to microsecond time scale has also been performed on oxidized wild-type and R98C cytochrome b(562). In a CPMG experiment, decay rates were analyzed in the presence of spin-echo pulse trains of variable spacing. The dynamic behavior on this time scale is similar to that observed on the sub-nanosecond time scale, showing an increased mobility in the residues connected to the heme ligands in the R98C variant. It appears that the increased protein stability of the variant, established previously, is not correlated with an increase in rigidity.
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200. |
Tame JR,
Namba K,
Dodson EJ,
Roper DI,
( 2002 ) The crystal structure of HpcE, a bifunctional decarboxylase/isomerase with a multifunctional fold. PMID : 11863436 : DOI : 10.1021/bi015717t Abstract >>
The structure of the bifunctional enzyme HpcE (OPET decarboxylase/HHDD isomerase) from Escherichia coli shows that the protein consists of highly similar N and C terminal halves. Sequence matches suggest that this fold is widespread among different species, including man. Many of these homologues are uncharacterized but apparently connected with the metabolism of aromatic compounds. The domain shows similar topology to the C terminal domain of fumarylacetoacetate hydrolase (FAH), a functionally related enzyme, despite lacking significant overall sequence similarity. HpcE is known to catalyze two rather different reactions, and comparisons with FAH allow some tentative conclusions to be drawn about the active sites. Key mutations within the active site apparently allow enzymes with this fold to carry out a variety chemical processes.
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201. |
Russo TA,
Carlino UB,
Johnson JR,
( 2001 ) Identification of a new iron-regulated virulence gene, ireA, in an extraintestinal pathogenic isolate of Escherichia coli. PMID : 11553562 : DOI : 10.1128/IAI.69.10.6209-6216.2001 PMC : PMC98753 Abstract >>
Our laboratory is studying an extraintestinal pathogenic isolate of Escherichia coli (CP9) as a model pathogen. We have been using human urine, ascites, and blood ex vivo to identify genes with increased expression in these media relative to expression in Luria-Bertani (LB) broth. Such genes may represent new or unrecognized virulence traits. In this study, we report the identification of a new gene, ireA (iron-responsive element). This gene has an open reading frame of 2,049 nucleotides, and its peptide has a molecular mass of 75.3 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Its expression is increased a mean of 3.6-fold in human urine, 16.2-fold in human ascites, and 6.6-fold in human blood relative to expression in LB medium, and it is Fe repressible. IreA also exhibits peptide similarities (48 to 56%) to previously identified proteins that function as siderophore receptors, suggesting that IreA is involved in iron acquisition. PCR-based analysis of ireA's phylogenetic distribution detected ireA in none (0%) of 14 fecal isolates that represented probable commensal strains, but in 13 (26%) of 50 random urine and blood clinical isolates (P = 0.05) and in 5 (100%) of 5 representatives of the J96-like, clonal group of which CP9 is a member (P < 0.001). In a mouse urinary tract infection model, the presence of ireA contributed significantly to CP9's ability to colonize the bladder (P < 0.02), evidence that IreA is a urovirulence factor. Taken together, these findings demonstrate that ireA encodes a new virulence factor, which is likely involved in Fe acquisition.
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202. |
Wang L,
Qu W,
Reeves PR,
( 2001 ) Sequence analysis of four Shigella boydii O-antigen loci: implication for Escherichia coli and Shigella relationships. PMID : 11598067 : DOI : 10.1128/IAI.69.11.6923-6930.2001 PMC : PMC100072 Abstract >>
Shigella strains are in reality clones of Escherichia coli and are believed to have emerged relatively recently (G. M. Pupo, R. Lan, and P. R. Reeves, Proc. Natl. Acad. Sci. USA 97:10567-10572, 2000). There are 33 O-antigen forms in these Shigella clones, of which 12 are identical to O antigens of other E. coli strains. We sequenced O-antigen gene clusters from Shigella boydii serotypes 4, 5, 6, and 9 and also studied the O53- and O79-antigen gene clusters of E. coli, encoding O antigens identical to those of S. boydii serotype 4 and S. boydii serotype 5, respectively. In both cases the S. boydii and E. coli O-antigen gene clusters have the same genes and organization. The clusters of both S. boydii 6 and S. boydii 9 O antigens have atypical features, with a functional insertion sequence and a wzx gene located in the orientation opposite to that of all other genes in S. boydii serotype 9 and an rmlC gene located away from other rml genes in S. boydii serotype 6. Sequences of O-antigen gene clusters from another three Shigella clones have been published, and two of them also have abnormal structures, with either the entire cluster or one gene being located on a plasmid in Shigella sonnei or Shigella dysenteriae, respectively. It appears that a high proportion of clusters coding for O antigens specific to Shigella clones have atypical features, perhaps indicating recent formation of these gene clusters.
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203. |
Asakura H,
Makino S,
Kobori H,
Watarai M,
Shirahata T,
Ikeda T,
Takeshi K,
( 2001 ) Phylogenetic diversity and similarity of active sites of Shiga toxin (stx) in Shiga toxin-producing Escherichia coli (STEC) isolates from humans and animals. PMID : 11561972 : DOI : 10.1017/s0950268801005635 PMC : PMC2869726 Abstract >>
Nucleotide sequences of Shiga toxin (Stx) genes in STEC from various origins were determined and characterized by phylogenetic analysis based on Shiga toxin (Stx) with those deposited in GenBank. The phylogenetic trees placed Stx1 and Stx2 into two and five groups respectively, and indicated that Stx1 in sheep-origin STEC were placed into a different group from those in other STEC, and that Stx2 of deer-origin STEC also belonged to the unique group and appeared to be distantly related to human-origin STEC. On the other hand, Stx of STEC isolated from cattle, seagulls and flies were closely related to those of human-origin STEC. Such a diversity of Stx suggested that STEC might be widely disseminated in many animal species, and be dependent on their host species or their habitat. In addition, the active sites in both toxins were compared; the active sites in both subunits of Stx in all the animal-origin STEC were identical to those in human-origin STEC, suggesting that all the toxin of STEC from animals might be also cytotoxic, and therefore, such animal-origin STEC might have potential pathogenicity for humans.
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204. |
Schmidt H,
Zhang WL,
Hemmrich U,
Jelacic S,
Brunder W,
Tarr PI,
Dobrindt U,
Hacker J,
Karch H,
( 2001 ) Identification and characterization of a novel genomic island integrated at selC in locus of enterocyte effacement-negative, Shiga toxin-producing Escherichia coli. PMID : 11598060 : DOI : 10.1128/IAI.69.11.6863-6873.2001 PMC : PMC100065 Abstract >>
The selC tRNA gene is a common site for the insertion of pathogenicity islands in a variety of bacterial enteric pathogens. We demonstrate here that Escherichia coli that produces Shiga toxin 2d and does not harbor the locus of enterocyte effacement (LEE) contains, instead, a novel genomic island. In one representative strain (E. coli O91:H(-) strain 4797/97), this island is 33,014 bp long and, like LEE in E. coli O157:H7, is integrated 15 bp downstream of selC. This E. coli O91:H(-) island contains genes encoding a novel serine protease, termed EspI; an adherence-associated locus, similar to iha of E. coli O157:H7; an E. coli vitamin B12 receptor (BtuB); an AraC-type regulatory module; and four homologues of E. coli phosphotransferase proteins. The remaining sequence consists largely of complete and incomplete insertion sequences, prophage sequences, and an intact phage integrase gene that is located directly downstream of the chromosomal selC. Recombinant EspI demonstrates serine protease activity using pepsin A and human apolipoprotein A-I as substrates. We also detected Iha-reactive protein in outer membranes of a recombinant clone and 10 LEE-negative, Shiga toxin-producing E. coli (STEC) strains by immunoblot analysis. Using PCR analysis of various STEC, enteropathogenic E. coli, enterotoxigenic E. coli, enteroaggregative E. coli, uropathogenic E. coli, and enteroinvasive E. coli strains, we detected the iha homologue in 59 (62%) of 95 strains tested. In contrast, espI and btuB were present in only two (2%) and none of these strains, respectively. We conclude that the newly described island occurs exclusively in a subgroup of STEC strains that are eae negative and contain the variant stx(2d)gene.
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205. |
Rasko DA,
Phillips JA,
Li X,
Mobley HL,
( 2001 ) Identification of DNA sequences from a second pathogenicity island of uropathogenic Escherichia coli CFT073: probes specific for uropathogenic populations. PMID : 11574920 : DOI : 10.1086/323602 Abstract >>
Uropathogenic Escherichia coli is the leading cause of urinary tract infection and hospital visits in North America. Cystitis and acute pyelonephritis, infection of the bladder and kidney, respectively, are the two most common syndromes encountered in patients with urinary tract infection. We sequenced and annotated 71,684 bases of a previously unidentified pathogenicity-associated island (PAI) from E. coli strain CFT073. This PAI contained 89 open-reading frames encoding a pap operon, iron-regulated genes, mobile genetic elements, and a large proportion of unknown or unidentified open-reading frames. Dot blot analysis with 11 DNA sequences from this PAI demonstrated that 7 sequences were more prevalent among uropathogens: 2 probes were more prevalent among cystitis and pyelonephritis isolates, 2 among pyelonephritis isolates only, and 3 among cystitis isolates only than among fecal isolates. These data suggest that groups of uropathogens have genetic differences that may be responsible for the different clinical outcomes.
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206. |
Mruk I,
Sektas M,
Kaczorowski T,
( 2001 ) Characterization of pEC156, a ColE1-type plasmid from Escherichia coli E1585-68 that carries genes of the EcoVIII restriction-modification system. PMID : 11591138 : DOI : 10.1006/plas.2001.1534 Abstract >>
The complete 4312-bp sequence of the pEC156 plasmid from Escherichia coli E1585-68, which carries genes encoding the EcoVIII restriction-modification (R-M) system, an isoschizomer of HindIII from Haemophilus influenzae, has been determined. Two clustered and convergently oriented open reading frames, large enough to encode genes of the EcoVIII R-M system, were found. The transcriptional start points were mapped by the primer extension method. The relative molecular masses of the EcoVIII endonuclease and EcoVIII methyltransferase deduced from the nucleotide sequence are 35,554 and 33,910, respectively. Nucleotide sequence analysis of pEC156 suggests that this plasmid is a ColE1-type replicon. It consists of an origin of replication and two untranslated genes encoding RNA I and RNA II, both involved in the regulation of plasmid DNA replication. The replication region also contains the gene encoding a 64-aa Rom-like protein. Inactivation of the putative rom gene by insertion of a kanamycin-resistance cassette resulted in 4.5-fold increase in pEC156-derived plasmid copy number in E. coli cells. All of these elements (RNA I, RNA II, and rom) reveal a high level of similarity to ColE1 homologs. The replication of all ColE1-type plasmids is dependent on the activity of E. coli DNA polymerase I. It was shown that a pEC156 derivative (pIB8) carrying an antibiotic resistance gene indeed failed to replicate in an E. coli polA12(ts) mutant at 43 degrees C, and its copy number was reduced in the E. coli pcnB80 mutant. These results prove that pEC156 is a ColE1-type replicon.
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207. |
Robey M,
Benito A,
Hutson RH,
Pascual C,
Park SF,
Mackey BM,
( 2001 ) Variation in resistance to high hydrostatic pressure and rpoS heterogeneity in natural isolates of Escherichia coli O157:H7. PMID : 11571200 : DOI : 10.1128/aem.67.10.4901-4907.2001 PMC : PMC93247 Abstract >>
Several natural isolates of Escherichia coli O157:H7 have previously been shown to exhibit stationary-phase-dependent variation in their resistance to inactivation by high hydrostatic pressure. In this report we demonstrate that loss of the stationary-phase-inducible sigma factor RpoS resulted in decreased resistance to pressure in E. coli O157:H7 and in a commensal strain. Furthermore, variation in the RpoS activity of the natural isolates of O157:H7 correlated with the pressure resistance of those strains. Heterogeneity was noted in the rpoS alleles of the natural isolates that may explain the differences in RpoS activity. These results are consistent with a role for rpoS in mediating resistance to high hydrostatic pressure in E. coli O157:H7.
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208. |
Ishikawa S,
Matsumura Y,
Yoshizako F,
Tsuchido T,
( 2002 ) Characterization of a cationic surfactant-resistant mutant isolated spontaneously from Escherichia coli. PMID : 11849354 : Abstract >>
In order to investigate the mechanism of bacterial resistance to surfactants, a spontaneous mutant of Escherichia coli, OW66, resistant to a cationic surfactant cetyltrimethylammonium bromide (CTAB), was isolated and its physiological properties analysed. Strain OW66 grew in M9 medium containing CTAB at 45 micromol l(-1), whereas its parent strain, OW6, did not, even at 15 micromol l(-1). The mutant was also resistant to some other surfactants, antibiotics, heavy metals, organic solvents and oxidants examined. To determine the differences in physiology between strains OW66 and OW6, the compositions of their cell surface structures were analysed. In strain OW66, the relative content of OmpC in particular was higher than that of OmpF, whereas a reverse situation was seen in OW6 strain. The lipopolysaccharide (LPS) profile was different between these strains, and altered LPS in strain OW66 was suggested to be involved in the resistance to CTAB. A CTAB-resistant E. coli isolate possesses an altered outer membrane. Treatment with a relatively low concentration of CTAB was found to introduce multi-drug resistance into bacterial cells. This acquired resistance should be taken into account with the frequent use of surfactants in industries and various environments.
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209. |
Cid D,
Ruiz-Santa-Quiteria JA,
Marín I,
Sanz R,
Orden JA,
Amils R,
de la Fuente R,
( 2001 ) Association between intimin (eae) and EspB gene subtypes in attaching and effacing Escherichia coli strains isolated from diarrhoeic lambs and goat kids. PMID : 11496011 : DOI : 10.1099/00221287-147-8-2341 Abstract >>
Attaching and effacing Escherichia coli (AEEC) strains isolated from diarrhoeic lambs and goat kids were characterized for intimin (eae) and EspB (espB) gene subtypes by PCR and sequencing, and for genetic relatedness by PFGE. Fifty (23 ovine and 27 caprine) AEEC strains of 398 (246 ovine and 152 caprine) analysed were detected by colony blot hybridization. These strains were epidemiologically unrelated since they were isolated from different outbreaks of neonatal diarrhoea over a long period. Ovine AEEC strains belonged to serogroups O2, O4, O26, O80, O91 or were untypable, and caprine strains belonged to serogroups O3, O153 and O163. Two intimin subtypes were detected among the ovine and caprine strains studied. Most of the strains (43/50) had the beta type intimin gene, but seven ovine strains possessed a variant gamma type intimin gene (gamma(V)). Analysis of deduced amino acid sequences of the eae gene revealed that the sequences of beta intimin of ovine and caprine strains were virtually identical to those of beta intimin of rabbit EPEC, human EPEC clone 2 and swine AEEC, whereas the gamma(V) intimin present in seven ovine strains had 75-76% identity with gamma intimin of human EHEC clone 1 strains, and 96% of identity with intimin of the human EHEC strain 95NR1 of serotype O111:H-. A PCR test was developed to identify the three different espB gene subtypes, espB of human EPEC clone 1 (espBalpha), espB of human EHEC clone 1 (espBgamma) and espB of rabbit EPEC and human EPEC clone 2 (espBbeta). There was close correlation between the intimin beta type and the espBbeta gene subtype in the ovine and caprine AEEC strains. The seven ovine strains possessing the gamma(V) intimin gene possessed the espBalpha gene subtype. None of the strains studied possessed the espBgamma gene found in human O157:H7 EHEC strains. PFGE analysis of genomic DNA of selected strains showed a great diversity among strains. Cluster analysis of PFGE patterns showed greater divergence between strains with the gamma(V) intimin gene than between strains with the beta intimin gene. This study showed that most of the AEEC strains isolated from diarrhoeic lambs and goat kids possessed beta intimin and espB genes identical to those of rabbit EPEC, and they may be associated with enteric disease in small ruminants.
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210. |
Strader MB,
Smiley RD,
Stinnett LG,
VerBerkmoes NC,
Howell EE,
( 2001 ) Role of S65, Q67, I68, and Y69 residues in homotetrameric R67 dihydrofolate reductase. PMID : 11560482 : DOI : 10.1021/bi0110544 Abstract >>
R67 dihydrofolate reductase (DHFR) shares no sequence or structural homology with chromosomal DHFRs. This enzyme arose recently in response to the clinical use of the antibacterial drug trimethoprim. R67 DHFR is a homotetramer possessing a single active site pore. A high-resolution crystal structure shows the homotetramer possesses exact 222 symmetry [Narayana, N., et al. (1995) Nat. Struct. Biol. 2, 1018-1025]. This symmetry dictates four symmetry-related binding sites must exist for each substrate as well as each cofactor. Isothermal titration calorimetry studies, however, indicate only two molecules bind: either two dihydrofolate molecules, two NADPH molecules, or one substrate and one cofactor [Bradrick, T. D., et al. (1996) Biochemistry 35, 11414-11424]. The latter is the productive ternary complex. To evaluate the role of S65, Q67, I68, and Y69 residues, located near the center of the active site pore, site-directed mutagenesis was performed. One mutation in the gene creates four mutations per active site pore which typically result in large cumulative effects. Steady state kinetic data indicate the mutants have altered K(m) values for both cofactor and substrate. For example, the Y69F R67 DHFR displays an 8-fold increase in the K(m) for dihydrofolate and a 20-fold increase in the K(m) for NADPH. Residues involved in ligand binding in R67 DHFR display very little, if any, specificity, consistent with their possessing dual roles in binding. These results support a model where R67 DHFR utilizes an unusual "hot spot" binding surface capable of binding both ligands and indicate this enzyme has adopted a novel yet simple approach to catalysis.
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211. |
Chow JW,
Kak V,
You I,
Kao SJ,
Petrin J,
Clewell DB,
Lerner SA,
Miller GH,
Shaw KJ,
( 2001 ) Aminoglycoside resistance genes aph(2")-Ib and aac(6')-Im detected together in strains of both Escherichia coli and Enterococcus faecium. PMID : 11557456 : DOI : 10.1128/AAC.45.10.2691-2694.2001 PMC : PMC90718 Abstract >>
Escherichia coli SCH92111602 expresses an aminoglycoside resistance profile similar to that conferred by the aac(6')-Ie-aph(2")-Ia gene found in gram-positive cocci and was found to contain the aminoglycoside resistance genes aph(2")-Ib and aac(6')-Im (only 44 nucleotides apart). aph(2")-Ib had been reported previously in Enterococcus faecium SF11770. aac(6')-Im had not been detected previously in enterococci and was found to be present also 44 nucleotides downstream from aph(2")-Ib in E. faecium SF11770. aph(2")-Ib and aac(6')-Im are separate open reading frames, each with its own putative ribosome binding site, whereas aac(6')-Ie-aph(2")-Ia appears to be a fusion of two genes with just one start and one stop codon. The deduced AAC(6')-Im protein exhibits 56% identity and 80% similarity to the AAC(6')-Ie domain of the bifunctional enzyme AAC(6')-APH(2"). Our results document the existence of a member of the aph(2") family of genes in gram-negative bacteria and provide evidence suggesting the horizontal transfer of aph(2")-Ib and aac(6')-Im as a unit between gram-positive and gram-negative bacteria.
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212. |
Titheradge AJ,
King J,
Ryu J,
Murray NE,
( 2001 ) Families of restriction enzymes: an analysis prompted by molecular and genetic data for type ID restriction and modification systems. PMID : 11600708 : DOI : 10.1093/nar/29.20.4195 PMC : PMC60208 Abstract >>
Current genetic and molecular evidence places all the known type I restriction and modification systems of Escherichia coli and Salmonella enterica into one of four discrete families: type IA, IB, IC or ID. StySBLI is the founder member of the ID family. Similarities of coding sequences have identified restriction systems in E.coli and Klebsiella pneumoniae as probable members of the type ID family. We present complementation tests that confirm the allocation of EcoR9I and KpnAI to the ID family. An alignment of the amino acid sequences of the HsdS subunits of StySBLI and EcoR9I identify two variable regions, each predicted to be a target recognition domain (TRD). Consistent with two TRDs, StySBLI was shown to recognise a bipartite target sequence, but one in which the adenine residues that are the substrates for methylation are separated by only 6 bp. Implications of family relationships are discussed and evidence is presented that extends the family affiliations identified in enteric bacteria to a wide range of other genera.
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213. |
Lan R,
Lumb B,
Ryan D,
Reeves PR,
( 2001 ) Molecular evolution of large virulence plasmid in Shigella clones and enteroinvasive Escherichia coli. PMID : 11553574 : DOI : 10.1128/IAI.69.10.6303-6309.2001 PMC : PMC98765 Abstract >>
Three genes, ipgD, mxiC, and mxiA, all in the invasion region of the Shigella virulence plasmid, were sequenced from strains representing a range of Shigella serotypes and from two enteroinvasive Escherichia coli (EIEC) isolates. The plasmids can be classified into two relatively homogeneous sequence forms which are quite distinct. pINV A plasmids are found in Shigella flexneri strains F6 and F6A, S. boydii strains B1, B4, B9, B10, B14, and B15, S. dysenteriae strains D3, D4, D6, D8, D9, D10, and D13, and the two EIEC strains (M519 and M520). pINV B plasmids are present in S. flexneri strains F1A, F2A, F3A, F3C, F4A, and FY, two S. boydii strains (B11 and B12), and S. sonnei. The D1 pINV plasmid is a recombinant with ipgD gene more closely related to those of pINV A but with mxiA and mxiC genes more closely related to those of pINV B. The phylogenetic relationships of the plasmid and those of the chromosomal genes of Shigella strains are largely consistent. The cluster 1 and cluster 3 strains tested (G.M. Pupo, R. Lan, and P. R. Reeves, Proc. Natl. Acad. Sci. USA 97:10567-10572, 2000) have pINV A and pINV B plasmids, respectively. However, of the three cluster 2 strains (B9, B11, and B15), B9 and B15 have pINV A while B11 has a pINV B plasmid. Those Shigella (D8 and D10 and S. sonnei) and EIEC strains which do not group with the main body of Shigella strains based on chromosomal genes were found to have plasmids belonging to one or the other of the two types and must have acquired these by lateral transfer.
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214. |
Benz I,
Schmidt MA,
( 2001 ) Glycosylation with heptose residues mediated by the aah gene product is essential for adherence of the AIDA-I adhesin. PMID : 11442838 : DOI : 10.1046/j.1365-2958.2001.02487.x Abstract >>
The diffuse adherence of Escherichia coli strain 2787 (O126:H27) is mediated by the autotransporter adhesin AIDA-I (adhesin-involved-in-diffuse-adherence) encoded by the plasmid-borne aidA gene. AIDA-I exhibits an aberrant mobility in denaturing gel electrophoresis. Deletion of the open reading frame (ORF) A immediately upstream of aidA restores the predicted mobility of AIDA-I, but the adhesin is no longer functional. This indicates that the mature AIDA-I adhesin is post-translationally modified and the modification is essential for adherence function. Labelling with digoxigenin hydrazide shows AIDA-I to be glycosylated. Using carbohydrate composition analysis, AIDA-I contains exclusively heptose residues (ratio heptose:AIDA-I approximately 19:1). The deduced amino acid sequence of the cytoplasmic open reading frame (ORF) A gene product shows homologies to heptosyltransferases. In addition, the modification was completely abolished in an ADP-glycero-manno-heptopyranose mutant. Our results provide direct evidence for glycosylation of the AIDA-I adhesin by heptoses with the ORF A gene product as a specific (mono)heptosyltransferase generating the functional mature AIDA-I adhesin. Consequently, the ORF A gene has been denoted 'aah' (autotransporter-adhesin-heptosyltransferase). Glycosylation by heptoses represents a novel protein modification in eubacteria.
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215. |
Bertin Y,
Boukhors K,
Pradel N,
Livrelli V,
Martin C,
( 2001 ) Stx2 subtyping of Shiga toxin-producing Escherichia coli isolated from cattle in France: detection of a new Stx2 subtype and correlation with additional virulence factors. PMID : 11526129 : DOI : 10.1128/jcm.39.9.3060-3065.2001 PMC : PMC88297 Abstract >>
At least 11 Stx2 variants produced by Shiga toxin-producing Escherichia coli (STEC) isolated from patients and animals have been described. The Stx2 subtyping of STEC isolated from healthy cows positive for stx(2) (n = 104) or stx(2) and stx(1) (n = 63) was investigated. Stx2vh-b, Stx2 (renamed Stx2-EDL933), and Stx2vh-a were the subtypes mostly detected among the bovine isolates (39.5, 39, and 25.5%, respectively). Stx2e was not present, and subtypes included in the Stx2d group (Stx2d-OX3a, Stx2d-O111, and Stx2d-Ount) were found infrequently among the isolates examined (8.5%). A combination of two distinct Stx2 subtypes was observed among 23.5% of the strains. For the first time, a combination of three subtypes (Stx2-EDL933/Stx2vh-b/Stx2d and Stx2vh-a/Stx2vh-b/Stx2d) was detected (3.5% of the isolates). In addition, bovine STEC harboring stx(1) and one or two stx(2) genes appeared highly cytotoxic toward Vero cells. A new Stx2 subtype (Stx2-NV206), present among 14.5% of the isolates, showed high cytotoxicity for Vero cells. Two amino acid residues (Ser-291 and Glu-297) important for the activation of Stx2 by human intestinal mucus were conserved on the Stx2-NV206 A subunit. The gene encoding Ehx enterohemolysin was prominent among STEC harboring stx(2)-EDL933 alone (78%) or a combination of stx(2)-EDL933 and stx(2)vh-b (85%). In addition, Stx2-EDL933 and/or Stx2vh-b subtypes were highly associated with other putative virulence factors such as Stx1 and EspP extracellular serine protease, but not with EAST1 enterotoxin.
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216. |
Mansfield KG,
Lin KC,
Xia D,
Newman JV,
Schauer DB,
MacKey J,
Lackner AA,
Carville A,
( 2001 ) Enteropathogenic Escherichia coli and ulcerative colitis in cotton-top tamarins (Saguinus oedipus). PMID : 11517446 : DOI : 10.1086/322990 Abstract >>
The cotton-top tamarin (CTT; Saguinus oedipus) is an endangered New World primate that develops a highly prevalent idiopathic colitis resembling human ulcerative colitis. This study found that enteropathogenic Escherichia coli (EPEC) caused acute colitis in CTTs, which was associated with ulcerative colitis. EPEC clinical isolates revealed localized adherence patterns by HEp-2 assay and were devoid of Shiga-toxin production. Sequencing of the eae gene (GenBank accession no. AF319597) revealed 99.2% identity to sequences of human isolates (GenBank AF116899) and corresponded to the epsilon intimin gene subtype. Detection of intimin sequences by polymerase chain reaction on primary fecal cultures indicated widespread EPEC infection in the CTT colony. Prospective analysis revealed that animals with fecal cultures positive for intimin sequences had a higher frequency of active colitis (75.0% vs. 27.2%; P<.005, chi(2) test) and higher histological scores of colonic inflammation (0.875 vs. 0.455, respectively; P<.05, Mann-Whitney rank sum test).
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217. |
Taniguchi T,
Akeda Y,
Haba A,
Yasuda Y,
Yamamoto K,
Honda T,
Tochikubo K,
( 2001 ) Gene cluster for assembly of pilus colonization factor antigen III of enterotoxigenic Escherichia coli. PMID : 11500465 : DOI : 10.1128/iai.69.9.5864-5873.2001 PMC : PMC98705 Abstract >>
The assembly of pilus colonization factor antigen III (CFA/III) of enterotoxigenic Escherichia coli (ETEC) requires the processing of CFA/III major pilin (CofA) by a prepilin peptidase (CofP), similar to other type IV pilus formation systems. CofA is produced initially as a 26.5-kDa preform pilin (prepilin) and then processed to a 20.5-kDa mature pilin by CofP which is predicted to be localized in the inner membrane. In the present experiment, we determined the nucleotide sequence of the whole region for CFA/III formation and identified a cluster of 14 genes, including cofA and cofP. Several proteins encoded by cof genes were similar to previously described proteins, such as the toxin-coregulated pili of Vibrio cholerae and the bundle-forming pili of enteropathogenic E. coli. The G+C content of the cof gene cluster was 37%, which was significantly lower than the average for the E. coli genome (50%). The introduction of a recombinant plasmid containing the cof gene cluster into the E. coli K-12 strain conferred CFA/III biogenesis and the ability of adhesion to the human colon carcinoma cell line Caco-2. This is the first report of a complete nucleotide sequence of the type IV pili found in human ETEC, and our results provide a useful model for studying the molecular mechanism of CFA/III biogenesis and the role of CFA/III in ETEC infection.
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218. |
Okeke IN,
Borneman JA,
Shin S,
Mellies JL,
Quinn LE,
Kaper JB,
( 2001 ) Comparative sequence analysis of the plasmid-encoded regulator of enteropathogenic Escherichia coli Strains. PMID : 11500429 : DOI : 10.1128/iai.69.9.5553-5564.2001 PMC : PMC98669 Abstract >>
Enteropathogenic Escherichia coli (EPEC) strains that carry the EPEC adherence factor (EAF) plasmid were screened for the presence of different EAF sequences, including those of the plasmid-encoded regulator (per). Considerable variation in gene content of EAF plasmids from different strains was seen. However, bfpA, the gene encoding the structural subunit for the bundle-forming pilus, bundlin, and per genes were found in 96.8% of strains. Sequence analysis of the per operon and its promoter region from 15 representative strains revealed that it is highly conserved. Most of the variation occurs in the 5' two-thirds of the perA gene. In contrast, the C-terminal portion of the predicted PerA protein that contains the DNA-binding helix-turn-helix motif is 100% conserved in all strains that possess a full-length gene. In a minority of strains including the O119:H2 and canine isolates and in a subset of O128:H2 and O142:H6 strains, frameshift mutations in perA leading to premature truncation and consequent inactivation of the gene were identified. Cloned perA, -B, and -C genes from these strains, unlike those from strains with a functional operon, failed to activate the LEE1 operon and bfpA transcriptional fusions or to complement a per mutant in reference strain E2348/69. Furthermore, O119, O128, and canine strains that carry inactive per operons were deficient in virulence protein expression. The context in which the perABC operon occurs on the EAF plasmid varies. The sequence upstream of the per promoter region in EPEC reference strains E2348/69 and B171-8 was present in strains belonging to most serogroups. In a subset of O119:H2, O128:H2, and O142:H6 strains and in the canine isolate, this sequence was replaced by an IS1294-homologous sequence.
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219. |
Monday SR,
Whittam TS,
Feng PC,
( 2001 ) Genetic and evolutionary analysis of mutations in the gusA gene that cause the absence of beta-glucuronidase activity in Escherichia coli O157:H7. PMID : 11510000 : DOI : 10.1086/323154 Abstract >>
Escherichia coli serotype O157:H7 do not exhibit beta-glucuronidase (GUD) activity but carry the gusA gene (uidA) that encodes for GUD. In trans-complementation, the gusA gene cloned from the GUD-positive variant strain 493-89 effectively restored GUD activity in O157:H7 strain 35150. Comparison of gusA sequences from the GUD-negative 35150 strain to that of 493-89 revealed several base mutations, including a guanosine (G) dinucleotide insertion that caused a frameshift in the 35150 gusA gene and introduced a predicted premature termination codon. This explains the absence of GUD activity in O157:H7. A 35150 gusA construct from which the G-G insertion was deleted restored activity in GUD-negative O157:H7 transformants. The G-G insertion was present in all GUD-negative O157:H7 strains but was absent in their GUD-positive variants. The G-G insertion that produced the characteristic GUD-negative phenotype to O157:H7 strains appeared later than the other gusA mutations in the evolutionary emergence of O157:H7.
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220. |
Johnson JR,
O'Bryan TT,
Kuskowski M,
Maslow JN,
( 2001 ) Ongoing horizontal and vertical transmission of virulence genes and papA alleles among Escherichia coli blood isolates from patients with diverse-source bacteremia. PMID : 11500406 : DOI : 10.1128/iai.69.9.5363-5374.2001 PMC : PMC98646 Abstract >>
The phylogenetic distributions of multiple putative virulence factors (VFs) and papA (P fimbrial structural subunit) alleles among 182 Escherichia coli blood isolates from patients with diverse-source bacteremia were defined. Phylogenetic correspondence among these strains, the E. coli Reference (ECOR) collection, and other collections of extraintestinal pathogenic E. coli (ExPEC) was assessed. Although among the 182 bacteremia isolates phylogenetic group B2 predominated, exhibited the greatest concentration of individual VFs, and contained the largest number of familiar virulent clones, other phylogenetic groups exhibited greater concentrations of certain VFs than did group B2 and included several additional virulent clones. Certain of the newly detected VF genes, e.g., fyuA (yersiniabactin; 76%) and focG (F1C fimbriae; 25%), were as prevalent or more prevalent than their more familiar traditional counterparts, e.g., iut (aerobactin; 57%) and sfaS (S fimbriae; 14%), thus possibly offering additional useful targets for preventive interventions. Considerable diversity of VF profiles was observed at every level within the phylogenetic tree, including even within individual lineages. This suggested that many different pathways can lead to extraintestinal virulence in E. coli and that the evolution of ExPEC, which involves extensive horizontal transmission of VFs and continuous remodeling of pathogenicity-associated islands, is a highly active, ongoing process.
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221. |
Dodson KW,
Pinkner JS,
Rose T,
Magnusson G,
Hultgren SJ,
Waksman G,
( 2001 ) Structural basis of the interaction of the pyelonephritic E. coli adhesin to its human kidney receptor. PMID : 11440716 : DOI : 10.1016/s0092-8674(01)00388-9 DOI : 10.1016/s0092-8674(01)00388-9 Abstract >>
PapG is the adhesin at the tip of the P pilus that mediates attachment of uropathogenic Escherichia coli to the uroepithelium of the human kidney. The human specific allele of PapG binds to globoside (GbO4), which consists of the tetrasaccharide GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc linked to ceramide. Here, we present the crystal structures of a binary complex of the PapG receptor binding domain bound to GbO4 as well as the unbound form of the adhesin. The biological importance of each of the residues involved in binding was investigated by site-directed mutagenesis. These studies provide a molecular snapshot of a host-pathogen interaction that determines the tropism of uropathogenic E. coli for the human kidney and is critical to the pathogenesis of pyelonephritis.
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222. |
Karim A,
Poirel L,
Nagarajan S,
Nordmann P,
( 2001 ) Plasmid-mediated extended-spectrum beta-lactamase (CTX-M-3 like) from India and gene association with insertion sequence ISEcp1. PMID : 11470367 : DOI : 10.1111/j.1574-6968.2001.tb10762.x Abstract >>
Six non-clonally related enterobacterial isolates producing a same extended-spectrum beta-lactamase CTX-M-15 were isolated in 1999 from patients hospitalized in a New Delhi hospital. CTX-M-15 differed from CTX-M-3 by an asparagine to glycine substitution in position ABL238. Its gene was located on large plasmids varying in size. In each case, a same insertion sequence ISEcp1 was identified upstream of the 5' end of bla(CTX-M-15). Typical -35 and -10 promoter sequences of Enterobacteriaceae were identified in the 3' end of ISEcp1. The location of ISEcp1 upstream of plasmid-mediated CTX-M-type beta-lactamase genes may contribute to their spread or/and their expression.
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223. |
Harris SL,
Spears PA,
Havell EA,
Hamrick TS,
Horton JR,
Orndorff PE,
( 2001 ) Characterization of Escherichia coli type 1 pilus mutants with altered binding specificities. PMID : 11395476 : DOI : 10.1128/JB.183.13.4099-4102.2001 PMC : PMC95295 Abstract >>
PCR mutagenesis and a unique enrichment scheme were used to obtain two mutants, each with a single lesion in fimH, the chromosomal gene that encodes the adhesin protein (FimH) of Escherichia coli type 1 pili. These mutants were noteworthy in part because both were altered in the normal range of cell types bound by FimH. One mutation altered an amino acid at a site previously shown to be involved in temperature-dependent binding, and the other altered an amino acid lining the predicted FimH binding pocket.
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224. |
Delgado MA,
Rintoul MR,
Farías RN,
Salomón RA,
( 2001 ) Escherichia coli RNA polymerase is the target of the cyclopeptide antibiotic microcin J25. PMID : 11443089 : DOI : 10.1128/JB.183.15.4543-4550.2001 PMC : PMC95349 Abstract >>
Escherichia coli microcin J25 (MccJ25) is a plasmid-encoded, cyclic peptide antibiotic consisting of 21 unmodified amino acid residues. It is primarily active on gram-negative bacteria related to the producer strain, inducing cell filamentation in an SOS-independent way. A mutation causing resistance to MccJ25 was isolated. Genetic analysis indicated that it resided in the rpoC gene, encoding the beta' subunit of RNA polymerase, at 90 min on the E. coli genetic map. The mutation was genetically crossed on to a plasmid containing the wild-type rpoC gene. The presence of the recombinant plasmid conferred complete resistance to otherwise sensitive strains. Nucleotide sequencing of the plasmid-borne, mutant rpoC gene revealed a ACC (Thr)-to-ATC (Ile) change at codon 931, within homology block G, an evolutionarily conserved region in the large subunits of all RNA polymerases. MccJ25 decreased RNA synthesis both in vivo and in vitro. These results point to the RNA polymerase as the target of microcin action. We favor the possibility that the filamentous phenotype induced by MccJ25 results from impaired transcription of genes coding for cell division proteins. As far as we know, MccJ25 is the first peptide antibiotic shown to affect RNA polymerase.
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225. |
Buetow L,
Flatau G,
Chiu K,
Boquet P,
Ghosh P,
( 2001 ) Structure of the Rho-activating domain of Escherichia coli cytotoxic necrotizing factor 1. PMID : 11427886 : DOI : 10.1038/89610 Abstract >>
Certain uropathogenic and neonatal meningitis-causing strains of Escherichia coli express a 114 kDa protein toxin called cytotoxic necrotizing factor 1 (CNF1). The toxin causes alteration of the host cell actin cytoskeleton and promotes bacterial invasion of blood-brain barrier endothelial cells. CNF1 belongs to a unique group of large cytotoxins that cause constitutive activation of Rho guanosine triphosphatases (GTPases), which are key regulators of the actin cytoskeleton. This group also includes E. coli cytotoxic necrotizing factor 2 (CNF2, 114 kDa) and dermonecrotic toxins (DNT, 159 kDa) of Bordetella spp. with related sequences occurring in Yersinia spp. Here we show that the catalytic region of CNF1 exhibits a novel protein fold as determined by its 1.83 A resolution crystal structure. The structure reveals that CNF1 has a Cys-His-main chain oxygen catalytic triad reminiscent of enzymes belonging to the catalytic triad superfamily. The position of the catalytic Cys residue at the base of a deep pocket restricts access to potential substrates and helps explain the high specificity of this and related toxins.
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226. |
Bonnet R,
Dutour C,
Sampaio JL,
Chanal C,
Sirot D,
Labia R,
De Champs C,
Sirot J,
( 2001 ) Novel cefotaximase (CTX-M-16) with increased catalytic efficiency due to substitution Asp-240-->Gly. PMID : 11451684 : DOI : 10.1128/AAC.45.8.2269-2275.2001 PMC : PMC90641 Abstract >>
Three clinical strains (Escherichia coli Rio-6, E. coli Rio-7, and Enterobacter cloacae Rio-9) collected in 1996 and 1999 from hospitals in Rio de Janeiro (Brazil) were resistant to broad-spectrum cephalosporins and gave a positive double-disk synergy test. Two bla(CTX-M) genes encoding beta-lactamases of pl 7.9 and 8.2 were implicated in this resistance: the bla(CTX-M-9) gene observed in E. coli Rio-7 and E. cloacae Rio-9 and a novel CTX-M-encoding gene, designated bla(CTX-M-16), observed in E. coli strain Rio-6. The deduced amino acid sequence of CTX-M-16 differed from CTX-M-9 only by the substitution Asp-240-->Gly. The CTX-M-16-producing E. coli transformant exhibited the same level of resistance to cefotaxime (MIC, 16 microg/ml) but had a higher MIC of ceftazidime (MIC, 8 versus 1 microg/ml) than the CTX-M-9-producing transformant. Enzymatic studies revealed that CTX-M-16 had a 13-fold higher affinity for aztreonam and a 7.5-fold higher k(cat) for ceftazidime than CTX-M-9, thereby showing that the residue in position 240 can modulate the enzymatic properties of CTX-M enzymes. The two bla(CTX-M-9) genes and the bla(CTX-M-16) gene were located on different plasmids, suggesting the presence of mobile elements associated with CTX-M-encoding genes. CTX-M-2 and CTX-M-8 enzymes were found in Brazil in 1996, and two other CTX-M beta-lactamases, CTX-M-9 and CTX-M-16, were subsequently observed. These reports are evidence of the diversity of CTX-M-type extended-spectrum beta-lactamases in Brazil.
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227. |
Kawai S,
Mori S,
Mukai T,
Hashimoto W,
Murata K,
( 2001 ) Molecular characterization of Escherichia coli NAD kinase. PMID : 11488932 : DOI : 10.1046/j.1432-1327.2001.02358.x Abstract >>
NAD kinase was purified to homogeneity from Escherichia coli MG1655. The enzyme was a hexamer consisting of 30 kDa subunits and utilized ATP or other nucleoside triphosphates as phosphoryl donors for the phosphorylation of NAD, most efficiently at pH 7.5 and 60 degrees C. The enzyme could not use inorganic polyphosphates as phosphoryl donors and was designated as ATP-NAD kinase. The N-terminal amino-acid sequence of the purified enzyme was encoded by yfjB, which had been deposited as a gene of unknown function in the E. coli whole genomic DNA sequence database. yfjB was cloned and expressed in E. coli BL21(DE3)pLysS. The purified product (YfjB) showed NAD kinase activity, and was identical to ATP-NAD kinase purified from E. coli MG1655 in molecular structure and other enzymatic properties. The deduced amino-acid sequence of YfjB exhibited homology with that of Mycobacterium tuberculosis inorganic polyphosphate/ATP-NAD kinase [Kawai, S., Mori, S., Mukai, T., Suzuki, S., Hashimoto, W., Takeshi, Y. & Murata, K. (2000) Biochem. Biophys. Res. Commun. 276, 57-63], and those of many hypothetical proteins for which functions have not yet been revealed. The YfjB homologues were considered to be NAD kinases and alignment of their sequences revealed highly conserved regions, XXX-XGGDG-XL and DGXXX-TPTGSTAY, where X represents a hydrophobic amino-acid residue.
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228. |
Brunder W,
Khan AS,
Hacker J,
Karch H,
( 2001 ) Novel type of fimbriae encoded by the large plasmid of sorbitol-fermenting enterohemorrhagic Escherichia coli O157:H(-). PMID : 11401985 : DOI : 10.1128/IAI.69.7.4447-4457.2001 PMC : PMC98518 Abstract >>
Sorbitol-fermenting (SF) enterohemorrhagic Escherichia coli (EHEC) O157:H(-) have emerged as important causes of diarrheal diseases and the hemolytic-uremic syndrome in Germany. In this study, we characterized a 32-kb fragment of the plasmid of SF EHEC O157:H(-), pSFO157, which differs markedly from plasmid pO157 of classical non-sorbitol-fermenting EHEC O157:H7. We found a cluster of six genes, termed sfpA, sfpH, sfpC, sfpD, sfpJ, and sfpG, which mediate mannose-resistant hemagglutination and the expression of fimbriae. sfp genes are similar to the pap genes, encoding P-fimbriae of uropathogenic E. coli, but the sfp cluster lacks homologues of genes encoding subunits of a tip fibrillum as well as regulatory genes. The major pilin, SfpA, despite its similarity to PapA, does not cluster together with known PapA alleles in a phylogenetic tree but is structurally related to the PmpA pilin of Proteus mirabilis. The putative adhesin gene sfpG, responsible for the hemagglutination phenotype, shows significant homology neither to papG nor to other known sequences. Sfp fimbriae are 3 to 5 nm in diameter, in contrast to P-fimbriae, which are 7 nm in diameter. PCR analyses showed that the sfp gene cluster is a characteristic of SF EHEC O157:H(-) strains and is not present in other EHEC isolates, diarrheagenic E. coli, or other Enterobacteriaceae. The sfp gene cluster is flanked by two blocks of insertion sequences and an origin of plasmid replication, indicating that horizontal gene transfer may have contributed to the presence of Sfp fimbriae in SF EHEC O157:H(-).
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229. |
Wang L,
Briggs CE,
Rothemund D,
Fratamico P,
Luchansky JB,
Reeves PR,
( 2001 ) Sequence of the E. coli O104 antigen gene cluster and identification of O104 specific genes. PMID : 11404020 : DOI : 10.1016/s0378-1119(01)00471-1 Abstract >>
The Escherichia coli O104 polysaccharide is an important antigen, which contains sialic acid and is often associated with EHEC clones. Sialic acid is a component of many animal tissues, and its presence in bacterial polysaccharides may contribute to bacterial pathogenicity. We sequenced the genes responsible for O104 antigen synthesis and have found genes which from their sequences are identified as an O antigen polymerase gene, an O antigen flippase gene, three CMP-sialic acid synthesis genes, and three potential glycosyl transferase genes. The E. coli K9 group IB capsular antigen has the same structure as the O104 O antigen, and we find using gene by gene PCR that the K9 gene cluster is essentially the same as that for O104. It appears that the distinction between presence as group IB capsule or O antigen for this structure does not involve any difference in genes present in the O antigen gene cluster. By PCR testing against representative strains for the 166 E. coli O antigens and some randomly selected Gram-negative bacteria, we identified three O antigen genes which are highly specific to O104/K9. This work provides the basis for a sensitive test for rapid detection of O104 E. coli. This is important both for decisions on patient care as early treatment may reduce the risk of life-threatening complications and for a faster response in control of food borne outbreaks.
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230. |
Ramachandran V,
Hornitzky MA,
Bettelheim KA,
Walker MJ,
Djordjevic SP,
( 2001 ) The common ovine Shiga toxin 2-containing Escherichia coli serotypes and human isolates of the same serotypes possess a Stx2d toxin type. PMID : 11326016 : DOI : 10.1128/JCM.39.5.1932-1937.2001 PMC : PMC88051 Abstract >>
Shiga toxin 2 (Stx2) has been reported as the main Shiga toxin associated with human disease. In addition, the Stx2 toxin type can have a profound impact on the degree of tissue damage in animal models. We have characterized the stx(2) subtype of 168 Shiga toxin-producing Escherichia coli (STEC) isolates of which 146 were derived from ovine sources (principally feces and meat) and 22 were isolated from humans. The ovine STEC isolates were of serotypes that have been shown to occur commonly in the gastrointestinal tract of healthy sheep. The major stx(2) subtype in the ovine isolates was shown to be stx(2d-Ount) (119 of 146 [81.5%]) and was predominantly associated with serotypes O75:H(-)/H8/H40, O91:H(-), O123:H(-), O128:H2, and OR:H2. However, 17 of 18 (94.4%) ovine isolates of serotype O5:H(-) possessed a stx(2d-O111/OX3a) subtype. Furthermore, STEC isolates of serotypes commonly found in sheep and recovered from both clinical and nonclinical human infections also contained a stx(2d) (stx(2d-Ount/O111/OX3a)) subtype. These studies suggest that a specific stx(2) subtype(s) associates with serotype and may have important epidemiological implications for tracing sources of E. coli during outbreaks of STEC-associated diseases in humans.
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231. |
Boudeau J,
Barnich N,
Darfeuille-Michaud A,
( 2001 ) Type 1 pili-mediated adherence of Escherichia coli strain LF82 isolated from Crohn's disease is involved in bacterial invasion of intestinal epithelial cells. PMID : 11251843 : DOI : 10.1111/j.1365-2958.2001.02315.x Abstract >>
We previously characterized the invasive ability of Escherichia coli strain LF82, isolated from an ileal biopsy of a patient with Crohn's disease. In the present study, we performed TnphoA insertion mutagenesis to identify genes involved in LF82 invasion of intestinal epithelial cells. Most of the non-invasive mutants had an insertion mutation within the type 1 pili-encoding operon. Two non-invasive fim mutants, which harboured an insertion within the fimI and fimF genes, still adhered but had lost the ability to induce host cell membrane elongations at the sites of contact with the epithelial cells. Transcomplementation experiments with a fim operon cloned from E. coli K-12 restored both invasive ability and the ability to induce host cell membrane elongations. Expression of the cloned LF82 or K-12 fim operon into the non-invasive laboratory strain JM109 did not confer invasive properties. Thus, these findings showed that: (i) type 1 pili-mediated adherence is involved in LF82-induced perturbation of host cell signalling responsible for membrane elongations; (ii) native shafts are required for type 1 pilus-mediated induction of membrane elongations; (iii) this active phenomenon is a key step in the establishment of the invasive process; and (iv) type 1 pili alone are not sufficient to trigger bacterial internalization.
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232. |
Manning SD,
Zhang L,
Foxman B,
Spindler A,
Tallman P,
Marrs CF,
( 2001 ) Prevalence of known P-fimbrial G alleles in Escherichia coli and identification of a new adhesin class. PMID : 11329472 : DOI : 10.1128/CDLI.8.3.637-640.2001 PMC : PMC96115 Abstract >>
Screening a large Escherichia coli collection for P-fimbrial adhesin classes identified 20 unclassifiable strains. Cloning and sequencing of papG from an unclassifiable strain identified another G allele. The novel adhesin gene has 65% identity to the class I adhesin gene, 44% identity to the class II adhesin gene, and 43% identity to the class III adhesin gene.
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233. |
Allen NL,
Hilton AC,
Betts R,
Penn CW,
( 2001 ) Use of representational difference analysis to identify Escherichia coli O157-specific DNA sequences. PMID : 11313134 : DOI : 10.1111/j.1574-6968.2001.tb10603.x Abstract >>
Whereas several important virulence factors in Escherichia coli O157 have been identified, studies suggest they are not always essential and are probably insufficient to account for the severe clinical manifestation of E. coli O157 infection. Identification of putative virulence determinants is crucial to the understanding of bacterial pathogenesis and genomic comparison analysis may aid the characterisation of unidentified virulence attributes. In this study, representational difference analysis (RDA) was used for genomic comparison of E. coli O157 with the proposed ancestral strain, E. coli O55. Unique E. coli O157 gene sequences were isolated and one, termed RDA-1, taken forward for further analysis. Southern blotting with labelled RDA-1 as a probe showed it to be present in 77% of E. coli O157 isolates and absent in all non-E. coli O157 screened. Sequence flanking RDA-1 was obtained from a genomic clone identified by hybridisation, and contained an open reading frame predicted to encode a novel iron-regulated outer membrane protein.
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234. |
Lee SH,
Jeong SH,
Lee KJ,
( 2001 ) Evolution of TEM beta--lactamase genes identified by PCR with newly designed primers in Korean clinical isolates. PMID : 11298153 : Abstract >>
N/A
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235. |
Smajs D,
Weinstock GM,
( 2001 ) The iron- and temperature-regulated cjrBC genes of Shigella and enteroinvasive Escherichia coli strains code for colicin Js uptake. PMID : 11395459 : DOI : 10.1128/JB.183.13.3958-3966.2001 PMC : PMC95278 Abstract >>
A cosmid library of DNA from colicin Js-sensitive enteroinvasive Escherichia coli (EIEC) strain O164 was made in colicin Js-resistant strain E. coli VCS257, and colicin Js-sensitive clones were identified. Sensitivity to colicin Js was associated with the carriage of a three-gene operon upstream of and partially overlapping senB. The open reading frames were designated cjrABC (for colicin Js receptor), coding for proteins of 291, 258, and 753 amino acids, respectively. Tn7 insertions in any of them led to complete resistance to colicin Js. A near-consensus Fur box was found upstream of cjrA, suggesting regulation of the cjr operon by iron levels. CjrA protein was homologous to iron-regulated Pseudomonas aeruginosa protein PhuW, whose function is unknown; CjrB was homologous to the TonB protein from Pseudomonas putida; and CjrC was homologous to a putative outer membrane siderophore receptor from Campylobacter jejuni. Cloning experiments showed that the cjrB and cjrC genes are sufficient for colicin Js sensitivity. Uptake of colicin Js into sensitive bacteria was dependent on the ExbB protein but not on the E. coli K-12 TonB and TolA, -B, and -Q proteins. Sensitivity to colicin Js is positively regulated by temperature via the VirB protein and negatively controlled by the iron source through the Fur protein. Among EIEC strains, two types of colicin Js-sensitive phenotypes were identified that differed in sensitivity to colicin Js by 1 order of magnitude. The difference in sensitivity to colicin Js is not due to differences between the sequences of the CjrB and CjrC proteins.
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236. |
Culham DE,
Lu A,
Jishage M,
Krogfelt KA,
Ishihama A,
Wood JM,
( 2001 ) The osmotic stress response and virulence in pyelonephritis isolates of Escherichia coli: contributions of RpoS, ProP, ProU and other systems. PMID : 11390697 : DOI : 10.1099/00221287-147-6-1657 Abstract >>
Trehalose synthesis (RpoS-dependent) and betaine uptake mediated by transporters ProP and ProU contribute to the osmotolerance of Escherichia coli K-12. Pyelonephritis isolates CFT073 and HU734 were similar and diminished in osmotolerance, respectively, compared to E. coli K-12. The roles of RpoS, ProP and ProU in osmoregulation and urovirulence were assessed for these isolates. Strain HU734 expressed an RpoS variant which had low activity and a C-terminal extension. This bacterium accumulated very little trehalose and had poor stationary-phase thermotolerance. For E. coli CFT073, introduction of an rpoS deletion impaired trehalose accumulation, osmotolerance and stationary-phase thermotolerance. The rpoS defects accounted for the difference in osmotolerance between these strains in minimal medium of very high osmolality (1.4 mol kg(-1)) but not in medium of lower osmolality (0.4 mol kg(-1)). The slow growth of both pyelonephritis isolates in high-osmolality medium was stimulated by glycine betaine (GB) and deletion of proP and/or proU impaired GB uptake. An HU734 derivative lacking both proP and proU retained osmoprotective GB uptake activity that could be attributed to system BetU, which is not present in strain K-12 or CFT073. BetU transported GB (K(m), 22 microM) and proline betaine. High-osmolality human urine (0.92 mol kg(-1)) included membrane-permeant osmolyte urea (0.44 M) plus other constituents which contributed an osmolality of only approximately 0.4 mol kg(-1). Strains HU734 and CFT073 showed correspondingly low GB uptake activities after cultivation in this urine. Deletion of proP and proU slowed the growth of E. coli HU734 in this high-osmolality human urine (which contains betaines) but had little impact on its colonization of the murine urinary tract after transurethral inoculation. By contrast, deletion of rpoS, proP and proU had no effect on the very rapid growth of CFT073 in high-osmolality urine or on its experimental colonization of the murine urinary tract. RpoS-dependent gene expression is not essential for growth in human urine or colonization of the murine urinary tract. Additional osmoregulatory systems, some not present in E. coli K-12 (e.g. BetU), may facilitate growth of pyelonephritis isolates in human urine and colonization of mammalian urinary tracts. The contributions of systems ProP and ProU to urinary tract colonization cannot be definitively assessed until all such systems are identified.
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237. |
Dobrindt U,
Blum-Oehler G,
Hartsch T,
Gottschalk G,
Ron EZ,
Fünfstück R,
Hacker J,
( 2001 ) S-Fimbria-encoding determinant sfa(I) is located on pathogenicity island III(536) of uropathogenic Escherichia coli strain 536. PMID : 11401961 : DOI : 10.1128/IAI.69.7.4248-4256.2001 PMC : PMC98494 Abstract >>
The sfa(I) determinant encoding the S-fimbrial adhesin of uropathogenic Escherichia coli strains was found to be located on a pathogenicity island of uropathogenic E. coli strain 536. This pathogenicity island, designated PAI III(536), is located at 5.6 min of the E. coli chromosome and covers a region of at least 37 kb between the tRNA locus thrW and yagU. As far as it has been determined, PAI III(536) also contains genes which code for components of a putative enterochelin siderophore system of E. coli and Salmonella spp. as well as for colicin V immunity. Several intact or nonfunctional mobility genes of bacteriophages and insertion sequence elements such as transposases and integrases are present on PAI III(536). The presence of known PAI III(536) sequences has been investigated in several wild-type E. coli isolates. The results demonstrate that the determinants of the members of the S-family of fimbrial adhesins may be located on a common pathogenicity island which, in E. coli strain 536, replaces a 40-kb DNA region which represents an E. coli K-12-specific genomic island.
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238. |
Zhao S,
White DG,
Ge B,
Ayers S,
Friedman S,
English L,
Wagner D,
Gaines S,
Meng J,
( 2001 ) Identification and characterization of integron-mediated antibiotic resistance among Shiga toxin-producing Escherichia coli isolates. PMID : 11282605 : DOI : 10.1128/AEM.67.4.1558-1564.2001 PMC : PMC92769 Abstract >>
A total of 50 isolates of Shiga toxin-producing Escherichia coli (STEC), including 29 O157:H7 and 21 non-O157 STEC strains, were analyzed for antimicrobial susceptibilities and the presence of class 1 integrons. Seventy-eight (n = 39) percent of the isolates exhibited resistance to two or more antimicrobial classes. Multiple resistance to streptomycin, sulfamethoxazole, and tetracycline was most often observed. Class 1 integrons were identified among nine STEC isolates, including serotypes O157:H7, O111:H11, O111:H8, O111:NM, O103:H2, O45:H2, O26:H11, and O5:NM. The majority of the amplified integron fragments were 1 kb in size with the exception of one E. coli O111:H8 isolate which possessed a 2-kb amplicon. DNA sequence analysis revealed that the integrons identified within the O111:H11, O111:NM, O45:H2, and O26:H11 isolates contained the aadA gene encoding resistance to streptomycin and spectinomycin. Integrons identified among the O157:H7 and O103:H2 isolates also possessed a similar aadA gene. However, DNA sequencing revealed only 86 and 88% homology, respectively. The 2-kb integron of the E. coli O111:H8 isolate contained three genes, dfrXII, aadA2, and a gene of unknown function, orfF, which were 86, 100, and 100% homologous, respectively, to previously reported gene cassettes identified in integrons found in Citrobacter freundii and Klebsiella pneumoniae. Furthermore, integrons identified among the O157:H7 and O111:NM strains were transferable via conjugation to another strain of E. coli O157:H7 and to several strains of Hafnia alvei. To our knowledge, this is the first report of integrons and antibiotic resistance gene cassettes in STEC, in particular E. coli O157:H7.
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239. |
Jensen SO,
Reeves PR,
( 2001 ) Molecular evolution of the GDP-mannose pathway genes (manB and manC) in Salmonella enterica. PMID : 11238967 : DOI : 10.1099/00221287-147-3-599 Abstract >>
The evolutionary history of the GDP-mannose pathway in Salmonella enterica was studied via sequencing manB and manC genes from 13 representative strains for O antigens containing mannose and/or sugar derivatives of GDP-D-mannose. In addition, colanic acid (CA) manB and manC genes were sequenced from selected strains, as the basis for a detailed comparison. Interestingly, including the eight previously characterized O antigen gene clusters, 12 of the 21 S. enterica strains studied in total (each representing a different O antigen structure) possess a manB gene which displays DNA identity, ranging from 93 to 99%, to the CA manB gene of S. enterica LT2. Furthermore, the CA-like manB genes (as well as the CA manB and manC genes) display subspecies specificity, and the CA and CA-like manB genes (for individual strains) appear to be evolving in concert via gene conversion events. In comparison, the manC genes were generally not CA-like, a situation also apparent in Escherichia coli,and therefore most strongly reflected the evolutionary history of the S. enterica O antigen GDP-mannose pathway. It appears that, in relatively recent times, gene capture from a distant source has occurred infrequently, and that groups of manB and manC genes have been maintained and are continuing to evolve within S. enterica and more closely related species.
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240. |
Stockner T,
Plugariu C,
Koraimann G,
Högenauer G,
Bermel W,
Prytulla S,
Sterk H,
( 2001 ) Solution structure of the DNA-binding domain of TraM. PMID : 11258958 : DOI : 10.1021/bi002031c Abstract >>
The solution structure of the DNA-binding domain of the TraM protein, an essential component of the DNA transfer machinery of the conjugative resistance plasmid R1, is presented. The structure has been determined using homonuclear 2-dimensional NMR spectroscopy as well as 15N labeled heteronuclear 2- and 3-dimensional NMR spectroscopy. It turns out that the solution structure of the DNA binding domain of the TraM protein is globular and dominantly helical. The very first amino acids of the N-terminus are unstructured.
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241. |
Mansfield KG,
Lin KC,
Newman J,
Schauer D,
MacKey J,
Lackner AA,
Carville A,
( 2001 ) Identification of enteropathogenic Escherichia coli in simian immunodeficiency virus-infected infant and adult rhesus macaques. PMID : 11230413 : DOI : 10.1128/JCM.39.3.971-976.2001 PMC : PMC87859 Abstract >>
Enteropathogenic Escherichia coli (EPEC) was recognized as a common opportunistic pathogen of simian immunodeficiency virus-infected rhesus macaques (Macaca mulatta) with AIDS. Retrospective analysis revealed that 27 of 96 (28.1%) animals with AIDS had features of EPEC infection, and EPEC was the most frequent pathogen of the gastrointestinal tract identified morphologically. In 7.3% of animals dying with AIDS, EPEC represented the sole opportunistic agent of the gastrointestinal tract at death. In 20.8% of cases, it was seen in combination with one or more gastrointestinal pathogens, including Cryptosporidium parvum, Enterocytozoon bieneusi, Mycobacterium avium, Entamoeba histolytica, Balantidium coli, Strongyloides stercoralis, cytomegalovirus, and adenovirus. Clinically, infection was associated with persistent diarrhea and wasting and was more frequent in animals that died at under 1 year of age (P < 0.001, Fisher exact test). The organism was associated with the characteristic attaching and effacing lesion in colonic tissue sections and produced a focal adherence pattern on a HEp-2 assay but was negative for Shiga toxin production as assessed by PCR and a HeLa cell cytotoxicity assay. A 2.6-kb fragment encompassing the intimin gene was amplified and sequenced and revealed 99.2% identity to sequences obtained from human isolates (GenBank AF116899) corresponding to the epsilon intimin subtype. Further investigations with rhesus macaques may offer opportunities to study the impact of EPEC on AIDS pathogenesis and gastrointestinal dysfunction.
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242. |
Roche A,
McFadden J,
Owen P,
( 2001 ) Antigen 43, the major phase-variable protein of the Escherichia coli outer membrane, can exist as a family of proteins encoded by multiple alleles. PMID : 11160810 : DOI : 10.1099/00221287-147-1-161 Abstract >>
agn43 encodes a major phase-variable outer-membrane protein, antigen 43 (Ag43), involved in autoaggregation of Escherichia coli cells. The gene is present in single copy on the chromosome of E. coli K-12. In contrast, Southern hybridization and gene inactivation studies demonstrate that control producer strain E. coli ML308-225 possesses duplicate copies of agn43 (agn43A and agn43B). Construction and analyses of single and double knockout mutants clearly show that both alleles are capable of expressing antigen in a phase-variable manner, with observed differences in the ON<-->OFF switch frequencies appearing to favour expression of Ag43B under conditions of normal laboratory growth. Comparative analysis of agn43A and agn43B gene sequences revealed 98% identity at the nucleotide and predicted protein levels, with differences in the protein sequence of the surface-expressed alpha(43) subunit altering the surface probability of one of the predicted epitopes. Analysis of a panel of enteropathogenic E. coli strains by Southern hybridization using agn43-specific gene probes provided strong evidence for the presence of varying numbers of agn43 alleles within clinical isolates. Taken together, the results indicate the presence of a family of distinct Ag43 proteins encoded by multiple chromosomal alleles.
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243. |
Twining SS,
Goryshin IY,
Bhasin A,
Reznikoff WS,
( 2001 ) Functional characterization of arginine 30, lysine 40, and arginine 62 in Tn5 transposase. PMID : 11283001 : DOI : 10.1074/jbc.M010748200 Abstract >>
Three N-terminal basic residues of Tn5 transposase, which are associated with proteolytic cleavages by Escherichia coli proteinases, were mutated to glutamine residues with the goal of producing more stable transposase molecules. Mutation of either arginine 30 or arginine 62 to glutamine produced transposase molecules that were more stable toward E. coli proteinases than the parent hyperactive Tn5 transposase, however, they were inactive in vivo. In vitro analysis revealed these mutants were inactive, because both Arg(30) and Arg(62) are required for formation of the paired ends complexes when the transposon is attached to the donor backbone. These results suggest Arg(30) and Arg(62) play critical roles in DNA binding and/or synaptic complex formation. Mutation of lysine 40 to glutamine did not increase the overall stability of the transposase to E. coli proteinases. This mutant transposase was only about 1% as active as the parent hyperactive transposase in vivo; however, it retained nearly full activity in vitro. These results suggest that lysine 40 is important for a step in the transposition mechanism that is bypassed in the in vitro assay system, such as the removal of the transposase molecule from DNA following strand transfer.
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244. |
Harel J,
Daigle F,
Forget C,
Tessier MC,
Crost C,
Martin C,
( 2000 ) Phase variation of F165(1) (Prs-like) fimbriae from Escherichia coli causing septicaemia in animals. PMID : 11142399 : DOI : 10.1139/w00-109 Abstract >>
Escherichia coli O115:F165 strains are associated with septicaemia in young pigs and synthesize fimbriae involved in virulence, designated as F165(1). F165(1) fimbriae belong to the P fimbrial family and are encoded by the foo gene cluster. The foo regulatory region of strain 5131 possesses characteristics similar to that of members of the P regulatory family, including papI and papB homologues, and two GATC sites separated by 102 bp, targets of differential Dam methylation. In wild-type strains, the synthesis of F165(1) is repressed by leucine and the fimbriae undergo phase variation. Immunofluorescence staining showed that phase variation of F165(1) results in a majority of cells (98%) in the ON phase, in contrast with phase variation of other members of this regulatory family, for which the majority of the cells are in the OFF state. Using a translational fusion in strain 5131 between phoA and fooA, encoding for the major structural subunit of F165(1), it was shown that leucine inhibits the OFF to ON switch and modulates the basal transcription of the foo operon.
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245. |
Zhu C,
Agin TS,
Elliott SJ,
Johnson LA,
Thate TE,
Kaper JB,
Boedeker EC,
( 2001 ) Complete nucleotide sequence and analysis of the locus of enterocyte Effacement from rabbit diarrheagenic Escherichia coli RDEC-1. PMID : 11254564 : DOI : 10.1128/IAI.69.4.2107-2115.2001 PMC : PMC98136 Abstract >>
The pathogenicity island termed the locus of enterocyte effacement (LEE) is found in diverse attaching and effacing pathogens associated with diarrhea in humans and other animal species. To explore the relation of variation in LEE sequences to host specificity and genetic lineage, we determined the nucleotide sequence of the LEE region from a rabbit diarrheagenic Escherichia coli strain RDEC-1 (O15:H-) and compared it with those from human enteropathogenic E. coli (EPEC, O127:H6) and enterohemorrhagic E. coli (EHEC, O157:H7) strains. Differing from EPEC and EHEC LEEs, the RDEC-1 LEE is not inserted at selC and is flanked by an IS2 element and the lifA toxin gene. The RDEC-1 LEE contains a core region of 40 open reading frames, all of which are shared with the LEE of EPEC and EHEC. orf3 and the ERIC (enteric repetitive intergenic consensus) sequence present in the LEEs of EHEC and EPEC are absent from the RDEC-1 LEE. The predicted promoters of LEE1, LEE2, LEE3, tir, and LEE4 operons are highly conserved among the LEEs, although the upstream regions varied considerably for tir and the crucial LEE1 promoter, suggesting differences in regulation. Among the shared genes, high homology (>95% identity) between the RDEC-1 and the EPEC and EHEC LEEs at the predicted amino acid level was observed for the components of the type III secretion apparatus, the Ces chaperones, and the Ler regulator. In contrast, more divergence (66 to 88% identity) was observed in genes encoding proteins involved in host interaction, such as intimin (Eae) and the secreted proteins (Tir and Esps). A comparison of the highly variable genes from RDEC-1 with those from a number of attaching and effacing pathogens infecting different species and of different evolutionary lineages was performed. Although RDEC-1 diverges from some human-infecting EPEC and EHEC, most of the variation observed appeared to be due to evolutionary lineage rather than host specificity. Therefore, much of the observed hypervariability in genes involved in pathogenesis may not represent specific adaptation to different host species.
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246. |
Uhlich GA,
Keen JE,
Elder RO,
( 2001 ) Mutations in the csgD promoter associated with variations in curli expression in certain strains of Escherichia coli O157:H7. PMID : 11319125 : DOI : 10.1128/AEM.67.5.2367-2370.2001 PMC : PMC92880 Abstract >>
Single-base-pair csgD promoter mutations in human outbreak Escherichia coli O157:H7 strains ATCC 43894 and ATCC 43895 coincided with differential Congo red dye binding from curli fiber expression. Red phenotype csgD::lacZ promoter fusions had fourfold-greater expression than white promoter fusions. Cloning the red variant csgDEFG operon into white variants induced the red phenotype. Substrate utilization differed between red and white variants.
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247. |
Dartigalongue C,
Missiakas D,
Raina S,
( 2001 ) Characterization of the Escherichia coli sigma E regulon. PMID : 11274153 : DOI : 10.1074/jbc.M100464200 Abstract >>
Escherichia coli responds to the accumulation of misfolded proteins by inducing the transcription of heat shock genes. Efinal sigma(E) RNA polymerase controls one of the two heat shock regulons of E. coli. This regulon is activated upon accumulation of misfolded polypeptides in the double membrane envelope of E. coli. final sigma(E) (RpoE) is a member of the extracytoplasmic function subfamily of sigma factors. Here we asked how many genes are activated by Efinal sigma(E) RNA polymerase and what is the identity of these genes. Using two independent genetic approaches, 20 E. coli promoters were identified which activate reporter gene transcription in a final sigma(E)-dependent manner. In all cases examined, a canonical final sigma(E) binding site could be revealed upon mapping transcriptional start sites. 10 identified promoters activated the transcription of previously identified genes with four genes acting directly on the folding of E. coli envelope proteins (dsbC, fkpA, skp, and surA). The remaining promoters transcribed genes that are presumed to encode hitherto unknown extracytoplasmic functions and were named ecf (ecfA-ecfM). Two of these ecf genes were found to be essential for E. coli growth.
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248. |
Johnson JR,
Stell AL,
Kaster N,
Fasching C,
O'Bryan TT,
( 2001 ) Novel molecular variants of allele I of the Escherichia coli P fimbrial adhesin gene papG. PMID : 11254589 : DOI : 10.1128/IAI.69.4.2318-2327.2001 PMC : PMC98161 Abstract >>
P fimbriae of extraintestinal pathogenic Escherichia coli mediate digalactoside-specific adherence via the tip adhesin molecule PapG, which occurs in three known variants (I to III), which are encoded by the corresponding three alleles of papG. In the present study, newly discovered variants of papG allele I and the respective wild-type source strains were characterized. One of the new papG allele I variants conferred a unique agglutination phenotype that combined the phenotypes associated with papG alleles I, II, and III. Comparative hydrophilicity analysis of predicted PapG peptides revealed regions that might explain the observed phenotypic similarities and differences between the PapG variants. The new papG allele I variants occurred either as the sole papG allele or together with both papG alleles II and III, rather than with only papG allele III, as in archetypal strains J96 and CP9. They also occurred in the absence of the usual F13 papA allele. One of the new papG allele I variants occurred in a serogroup O6 strain that, according to random amplified polymorphic DNA analysis, was phylogenetically distant from the "J96-like" clonal group of E. coli O4:H5, which includes all previously identified examples of papG allele I. Cluster analysis of nucleotide and predicted peptide sequences suggested that papG allele I represents the earliest evolutionary branch from a common papG ancestor. These results demonstrate unexpected diversity within papG allele I and, together with previous findings, suggest that the J96-like clonal group of E. coli O4:H5 may represent the original source of papG within the species.
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249. |
Elwell C,
Chao K,
Patel K,
Dreyfus L,
( 2001 ) Escherichia coli CdtB mediates cytolethal distending toxin cell cycle arrest. PMID : 11292766 : DOI : 10.1128/IAI.69.5.3418-3422.2001 PMC : PMC98302 Abstract >>
We previously reported that the CdtB polypeptide of Escherichia coli cytolethal distending toxin (CDT) shares significant pattern-specific homology with mammalian type I DNases. In addition, the DNase-related residues of CdtB are required for cellular toxicity. Here we demonstrate that purified CdtB converts supercoiled plasmid DNA to relaxed and linear forms and promotes cell cycle arrest when combined with an E. coli extract containing CdtA and CdtC. CdtB alone had no effect on HeLa cells, however; introduction of the polypeptide into HeLa cells by electroporation resulted in cellular distension, chromatin fragmentation, and cell cycle arrest, all of which are consequences of CDT action. In contrast to these findings, purified CdtB(H154A) lacked both DNA-nicking and cell cycle arrest activities. These results suggest a functional relationship between DNase-related residues in CdtB and CDT biological activity.
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250. |
Goldstein C,
Lee MD,
Sanchez S,
Hudson C,
Phillips B,
Register B,
Grady M,
Liebert C,
Summers AO,
White DG,
Maurer JJ,
( 2001 ) Incidence of class 1 and 2 integrases in clinical and commensal bacteria from livestock, companion animals, and exotics. PMID : 11181350 : DOI : 10.1128/AAC.45.3.723-726.2001 PMC : PMC90363 Abstract >>
Many pathogenic and commensal organisms are multidrug resistant due to exposure to various antibiotics. Often, this antimicrobial resistance is encoded by integrons that occur on plasmids or that are integrated into the bacterial chromosome. Integrons are commonly associated with bacterial genera in the family Enterobacteriaceae. We determined that class 1 integrases were present in approximately 46% of the isolates from the family Enterobacteriaceae; class 2 integrases were present only among Escherichia coli and Salmonella isolates. Seven percent of veterinary isolates were positive for class 3 integrase by DNA-DNA hybridization but could not be confirmed to be positive by PCR. None of the veterinary isolates possessed the class 4 integrase gene. The distribution of these integrase genes was variable within the members of the family Enterobacteriaceae when some or all integrase classes were absent from a particular genus. There was also considerable variability in the distribution of these integrases within a species, depending on the animal host. Unlike the class 1 integrases, the other integrase class, intI2, appears to be more restricted in its distribution among the members of the family Enterobacteriaceae. There is also considerable variability in the distribution of the class 1 integrases within E. coli strains isolated from different food animals. The class 1 integrases are the most widely disseminated of the four classes among the members of the family Enterobacteriaceae from both the clinical and normal flora of animals. This is the first report to closely examine the distribution of class 2 integrases in members of the family Enterobacteriaceae isolated in the United States.
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251. |
Nagy G,
Dobrindt U,
Kupfer M,
Emödy L,
Karch H,
Hacker J,
( 2001 ) Expression of hemin receptor molecule ChuA is influenced by RfaH in uropathogenic Escherichia coli strain 536. PMID : 11179376 : DOI : 10.1128/IAI.69.3.1924-1928.2001 PMC : PMC98105 Abstract >>
The outer membrane protein ChuA responsible for hemin utilization has been recently identified in several pathogenic Escherichia coli strains. We report that the regulatory protein RfaH influences ChuA expression in the uropathogenic E. coli strain 536. In an rfaH mutant, the chuA transcript as well as the ChuA protein levels were significantly decreased in comparison with those in the wild-type strain. Within the chuA gene, a consensus motif known as the JUMPStart (just upstream of many polysaccharide associated gene starts) sequence was found, which is shared by RfaH-affected operons. Furthermore, the presence of two different subclasses of the chuA determinant and their distribution in E. coli pathogroups are described.
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252. |
Oliver A,
Pérez-Díaz JC,
Coque TM,
Baquero F,
Cantón R,
( 2001 ) Nucleotide sequence and characterization of a novel cefotaxime-hydrolyzing beta-lactamase (CTX-M-10) isolated in Spain. PMID : 11158766 : DOI : 10.1128/AAC.45.2.616-620.2001 PMC : PMC90338 Abstract >>
A cefotaxime-resistant, ceftazidime-susceptible Escherichia coli isolate was obtained from a patient with sepsis in 1997, from which a beta-lactamase with a pI of 8.1 was cloned. Cephaloridine and cefotaxime relative hydrolysis rates were 167 and 81, respectively (penicillin G rate = 100), whereas ceftazidime hydrolysis was not detected. The nucleotide sequence revealed a bla gene related to that coding for CTX-M-3. Despite 21 nucleotide substitutions, only 2 determined amino acid changes (Ala27Val and Arg38Gln). The amino acid sequence identity between this enzyme, designated CTX-M-10, and the chromosomal beta-lactamase of Kluyvera ascorbata was 81%.
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253. |
Huang SH,
Wan ZS,
Chen YH,
Jong AY,
Kim KS,
( 2001 ) Further characterization of Escherichia coli brain microvascular endothelial cell invasion gene ibeA by deletion, complementation, and protein expression. PMID : 11237832 : DOI : 10.1086/319290 Abstract >>
The ibeA gene (ibe10) previously identified by TnphoA mutagenesis is part of a 50-kDa full-length open-reading frame (ORF) encoded by a 1.37-kb DNA fragment. An isogenic in-frame deletion mutant of ibeA (ZD1) was constructed by chromosomal gene replacement with a suicide plasmid pCVD442 carrying a 2.1-kb DNA fragment with an ibeA deletion. Similar to the previously described TnphoA insertion mutant of ibeA, the isogenic ibeA deletion mutant ZD1 was significantly less invasive in human brain microvascular endothelial cells (BMECs) than the parent strain. The mutant ZD1 was fully complemented by the ibeA ORF. The ibeA gene was subcloned into pET28a(+) and was expressed as a recombinant protein with an N-terminal histidine tag. The recombinant IbeA protein had much greater activity (50 times) in blocking the invasion of BMECs by Escherichia coli K1 than did the partial protein fragment, which provides further evidence that ibeA is an important determinant for E. coli K1 invasion of BMECs.
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254. |
Mellies JL,
Navarro-Garcia F,
Okeke I,
Frederickson J,
Nataro JP,
Kaper JB,
( 2001 ) espC pathogenicity island of enteropathogenic Escherichia coli encodes an enterotoxin. PMID : 11119520 : DOI : 10.1128/IAI.69.1.315-324.2001 PMC : PMC97886 Abstract >>
At least five proteins are secreted extracellularly by enteropathogenic Escherichia coli (EPEC), a leading cause of infant diarrhea in developing countries. However only one, EspC, is known to be secreted independently of the type III secretion apparatus encoded by genes located within the 35.6-kb locus of enterocyte effacement pathogenicity island. EspC is a member of the autotransporter family of proteins, and the secreted portion of the molecule is 110 kDa. Here we determine that the espC gene is located within a second EPEC pathogenicity island at 60 min on the chromosome of E. coli. We also show that EspC is an enterotoxin, indicated by rises in short-circuit current and potential difference in rat jejunal tissue mounted in Ussing chambers. In addition, preincubation with antiserum against the homologous Pet enterotoxin of enteroaggregative E. coli eliminated EspC enterotoxin activity. Like the EAF plasmid, the espC pathogenicity island was found only in a subset of EPEC, suggesting that EspC may play a role as an accessory virulence factor in some but not all EPEC strains.
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255. |
Naas T,
Mikami Y,
Imai T,
Poirel L,
Nordmann P,
( 2001 ) Characterization of In53, a class 1 plasmid- and composite transposon-located integron of Escherichia coli which carries an unusual array of gene cassettes. PMID : 11114922 : DOI : 10.1128/JB.183.1.235-249.2001 PMC : PMC94871 Abstract >>
Further characterization of the genetic environment of the gene encoding the Escherichia coli extended-spectrum beta-lactamase, bla(VEB-1), revealed the presence of a plasmid-located class 1 integron, In53, which carried eight functional resistance gene cassettes in addition to bla(VEB-1). While the aadB and the arr-2 gene cassettes were identical to those previously described, the remaining cassettes were novel: (i) a novel nonenzymatic chloramphenicol resistance gene of the cmlA family, (ii) a qac allele encoding a member of the small multidrug resistance family of proteins, (iii) a cassette, aacA1b/orfG, which encodes a novel 6'-N-acetyltransferase, and (iv) a fused gene cassette, oxa10/aadA1, which is made of two cassettes previously described as single cassettes. In addition, oxa10 and aadA1 genes were expressed from their own promoter sequence present upstream of the oxa10 cassette. arr-2 coded for a protein that shared 54% amino acid identity with the rifampin ADP-ribosylating transferase encoded by the arr-1 gene from Mycobacterium smegmatis DSM43756. While in M. smegmatis, the main inactivated compound was 23-ribosyl-rifampin, the inactivated antibiotic recovered from E. coli culture was 23-O-ADP-ribosyl-rifampin. The integrase gene of In53 was interrupted by an IS26 insertion sequence, which was also present in the 3' conserved segment. Thus, In53 is a truncated integron located on a composite transposon, named Tn2000, bounded by two IS26 elements in opposite orientations. Target site duplication at both ends of the transposon indicated that the integron likely was inserted into the plasmid through a transpositional process. This is the first description of an integron located on a composite transposon.
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256. |
Brown EW,
LeClerc JE,
Li B,
Payne WL,
Cebula TA,
( 2001 ) Phylogenetic evidence for horizontal transfer of mutS alleles among naturally occurring Escherichia coli strains. PMID : 11160094 : DOI : 10.1128/JB.183.5.1631-1644.2001 PMC : PMC95048 Abstract >>
mutS mutators accelerate the bacterial mutation rate 100- to 1,000-fold and relax the barriers that normally restrict homeologous recombination. These mutators thus afford the opportunity for horizontal exchange of DNA between disparate strains. While much is known regarding the mutS phenotype, the evolutionary structure of the mutS(+) gene in Escherichia coli remains unclear. The physical proximity of mutS to an adjacent polymorphic region of the chromosome suggests that this gene itself may be subject to horizontal transfer and recombination events. To test this notion, a phylogenetic approach was employed that compared gene phylogeny to strain phylogeny, making it possible to identify E. coli strains in which mutS alleles have recombined. Comparison of mutS phylogeny against predicted E. coli "whole-chromosome" phylogenies (derived from multilocus enzyme electrophoresis and mdh sequences) revealed striking levels of phylogenetic discordance among mutS alleles and their respective strains. We interpret these incongruences as signatures of horizontal exchange among mutS alleles. Examination of additional sites surrounding mutS also revealed incongruous distributions compared to E. coli strain phylogeny. This suggests that other regional sequences are equally subject to horizontal transfer, supporting the hypothesis that the 61.5-min mutS-rpoS region is a recombinational hot spot within the E. coli chromosome. Furthermore, these data are consistent with a mechanism for stabilizing adaptive changes promoted by mutS mutators through rescue of defective mutS alleles with wild-type sequences.
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257. |
Peek AS,
Souza V,
Eguiarte LE,
Gaut BS,
( 2001 ) The interaction of protein structure, selection, and recombination on the evolution of the type-1 fimbrial major subunit (fimA) from Escherichia coli. PMID : 11244580 : Abstract >>
Fimbrial adhesins allow bacteria to interact with and attach to their environment. The bacteria possibly benefit from these interactions, but all external structures including adhesins also allow bacteria to be identified by other organisms. Thus adhesion molecules might be under multiple forms of selection including selection to constrain functional interactions or evolve novel epitopes to avoid recognition. We address these issues by studying genetic diversity in the Escherichia coli type-1 fimbrial major subunit, fimA. Overall, sequence diversity in fimA is high (pi = 0.07) relative to that in other E. coli genes. High diversity is a function of positive diversifying selection, as detected by d(N)/d(S) ratios higher than 1.0, and amino acid residuces subject to diversifying selection are nonrandomly clustered on the exterior surface of the peptide. In addition, McDonald and Kreitman tests suggest that there has been historical but not current directional selection at fimA between E. coli and Salmonella. Finally, some regions of the fimA peptide appear to be under strong structural constraint within E. coli, particularly the interior regions of the molecule that is involved in subunit to subunit interaction. Recombination also plays a major role contributing to E. coli fimA allelic variation and estimates of recombination (2N(e)c) and mutation (2N(e)mu) are about the same. Recombination may act to separate the diverse evolutionary forces in different regions of the fimA peptide.
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258. |
Gomis-Rüth FX,
Moncalián G,
Pérez-Luque R,
González A,
Cabezón E,
de la Cruz F,
Coll M,
( 2001 ) The bacterial conjugation protein TrwB resembles ring helicases and F1-ATPase. PMID : 11214325 : DOI : 10.1038/35054586 Abstract >>
The transfer of DNA across membranes and between cells is a central biological process; however, its molecular mechanism remains unknown. In prokaryotes, trans-membrane passage by bacterial conjugation, is the main route for horizontal gene transfer. It is the means for rapid acquisition of new genetic information, including antibiotic resistance by pathogens. Trans-kingdom gene transfer from bacteria to plants or fungi and even bacterial sporulation are special cases of conjugation. An integral membrane DNA-binding protein, called TrwB in the Escherichia coli R388 conjugative system, is essential for the conjugation process. This large multimeric protein is responsible for recruiting the relaxosome DNA-protein complex, and participates in the transfer of a single DNA strand during cell mating. Here we report the three-dimensional structure of a soluble variant of TrwB. The molecule consists of two domains: a nucleotide-binding domain of alpha/beta topology, reminiscent of RecA and DNA ring helicases, and an all-alpha domain. Six equivalent protein monomers associate to form an almost spherical quaternary structure that is strikingly similar to F1-ATPase. A central channel, 20 A in width, traverses the hexamer.
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259. |
Lalioui L,
Le Bouguénec C,
( 2001 ) afa-8 Gene cluster is carried by a pathogenicity island inserted into the tRNA(Phe) of human and bovine pathogenic Escherichia coli isolates. PMID : 11159989 : DOI : 10.1128/IAI.69.2.937-948.2001 PMC : PMC97973 Abstract >>
We recently described a new afimbrial adhesin, AfaE-VIII, produced by animal strains associated with diarrhea and septicemia and by human isolates associated with extraintestinal infections. Here, we report that the afa-8 operon, encoding AfaE-VIII adhesin, from the human blood isolate Escherichia coli AL862 is carried by a 61-kb genomic region with characteristics typical of a pathogenicity island (PAI), including a size larger than 10 kb, the presence of an integrase-encoding gene, the insertion into a tRNA locus (pheR), and the presence of a small direct repeat at each extremity. Moreover, the G+C content of the afa-8 operon (46.4%) is lower than that of the E. coli K-12/MG1655 chromosome (50.8%). Within this PAI, designated PAI I(AL862), we identified open reading frames able to code for products similar to proteins involved in sugar utilization. Four probes spanning these sequences hybridized with 74.3% of pathogenic afa-8-positive E. coli strains isolated from humans and animals, 25% of human pathogenic afa-8-negative E. coli strains, and only 8% of fecal strains (P = 0.05), indicating that these sequences are strongly associated with the afa-8 operon and that this genetic association may define a PAI widely distributed among human and animal afa-8-positive strains. One of the distinctive features of this study is that E. coli AL862 also carries another afa-8-containing PAI (PAI II(AL862)), which appeared to be similar in size and genetic organization to PAI I(AL862) and was inserted into the pheV gene. We investigated the insertion sites of afa-8-containing PAI in human and bovine pathogenic E. coli strains and found that this PAI preferentially inserted into the pheV gene.
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260. |
Azpiroz MF,
Rodríguez E,
Laviña M,
( 2001 ) The structure, function, and origin of the microcin H47 ATP-binding cassette exporter indicate its relatedness to that of colicin V. PMID : 11181394 : DOI : 10.1128/AAC.45.3.969-972.2001 PMC : PMC90407 Abstract >>
Microcin H47, a gene-encoded peptide antibiotic produced by a natural Escherichia coli strain, was shown to be secreted by a three-component ATP-binding cassette exporter which was revealed to be strongly related to that of colicin V. The results of sequence and gene fusion analyses, as well as heterologous complementation assays, are presented.
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261. |
Denamur E,
Lecointre G,
Darlu P,
Tenaillon O,
Acquaviva C,
Sayada C,
Sunjevaric I,
Rothstein R,
Elion J,
Taddei F,
Radman M,
Matic I,
( 2000 ) Evolutionary implications of the frequent horizontal transfer of mismatch repair genes. PMID : 11114328 : DOI : 10.1016/s0092-8674(00)00175-6 Abstract >>
Mutation and subsequent recombination events create genetic diversity, which is subjected to natural selection. Bacterial mismatch repair (MMR) deficient mutants, exhibiting high mutation and homologous recombination rates, are frequently found in natural populations. Therefore, we have explored the possibility that MMR deficiency emerging in nature has left some "imprint" in the sequence of bacterial genomes. Comparative molecular phylogeny of MMR genes from natural Escherichia coli isolates shows that, compared to housekeeping genes, individual functional MMR genes exhibit high sequence mosaicism derived from diverse phylogenetic lineages. This apparent horizontal gene transfer correlates with hyperrecombination phenotype of MMR-deficient mutators. The sequence mosaicism of MMR genes may be a hallmark of a mechanism of adaptive evolution that involves modulation of mutation and recombination rates by recurrent losses and reacquisitions of MMR gene functions.
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262. |
Klöss H,
Brinkkötter A,
( 2000 ) Pathways for the utilization of N-acetyl-galactosamine and galactosamine in Escherichia coli. PMID : 10931310 : DOI : 10.1046/j.1365-2958.2000.01969.x Abstract >>
Among enteric bacteria, the ability to grow on N-acetyl-galactosamine (GalNAc or Aga) and on D-galactosamine (GalN or Gam) differs. Thus, strains B, C and EC3132 of Escherichia coli are Aga+ Gam+ whereas E. coli K-12 is Aga- Gam-, similarly to Klebsiella pneumoniae KAY2026, Klebsiella oxytoca M5a1 and Salmonella typhimurium LT2. The former strains carry a complete aga/kba gene cluster at 70.5 min of their gene map. These genes encode an Aga-specific phosphotransferase system (PTS) or IIAga (agaVWE) and a GalN-specific PTS or IIGam (agaBCD). Both PTSs belong to the mannose-sorbose family, i.e. the IIB, IIC and IID domains are encoded by different genes, and they share a IIA domain (agaF). Furthermore, the genes encode an Aga6P-deacetylase (agaA), a GalN6P deaminase (agaI), a tagatose-bisphosphate aldolase comprising two different peptides (kbaYZ) and a putative isomerase (agaS), i.e. complete pathways for the transport and degradation of both amino sugars. The genes are organized in two adjacent operons (kbaZagaVWEFA and agaS kbaYagaBCDI) and controlled by a repressor AgaR. Its gene agaR is located upstream of kbaZ, and AgaR responds to GalNAc and GalN in the medium. All Aga- Gam- strains, however, carry a deletion covering genes agaW' EF 'A; consequently they lack active IIAga and IIGam PTSs, thus explaining their inability to grow on the two amino sugars. Remnants of a putative recombination site flank the deleted DNA in the various Aga- Gam- enteric bacteria. Derivatives with an Aga+ Gam- phenotype can be isolated from E. coli K-12. These retain the DeltaagaW' EF 'A deletion and carry suppressor mutations in the gat and nag genes for galactitol and N-acetyl-glucosamine metabolism, respectively, that allow growth on Aga but not on GalN.
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263. |
Liebert CA,
Watson AL,
Summers AO,
( 2000 ) The quality of merC, a module of the mer mosaic. PMID : 11116334 : Abstract >>
We examined a region of high variability in the mosaic mercury resistance (mer) operon of natural bacterial isolates from the primate intestinal microbiota. The region between the merP and merA genes of nine mer loci was sequenced and either the merC, the merF, or no gene was present. Two novel merC genes were identified. Overall nucleotide diversity, pi (per 100 sites), of the merC gene was greater (49.63) than adjacent merP (35.82) and merA (32.58) genes. However, the consequences of this variability for the predicted structure of the MerC protein are limited and putative functional elements (metal-binding ligands and transmembrane domains) are strongly conserved. Comparison of codon usage of the merTP, merC, and merA genes suggests that several merC genes are not coeval with their flanking sequences. Although evidence of homologous recombination within the very variable merC genes is not apparent, the flanking regions have higher homologies than merC, and recombination appears to be driving their overall sequence identities higher. The synonymous codon usage bias (EN(C)) values suggest greater variability in expression of the merC gene than in flanking genes in six different bacterial hosts. We propose a model for the evolution of MerC as a host-dependent, adventitious module of the mer operon.
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264. |
Yethon JA,
Whitfield C,
( 2001 ) Purification and characterization of WaaP from Escherichia coli, a lipopolysaccharide kinase essential for outer membrane stability. PMID : 11069912 : DOI : 10.1074/jbc.M008255200 Abstract >>
In Escherichia coli, Salmonella enterica, and Pseudomonas aeruginosa, the waaP (rfaP) gene product is required for the addition of phosphate to O-4 of the first heptose residue of the lipopolysaccharide (LPS) inner core region. This phosphate substitution is particularly important to the biology of these bacteria; it has previously been shown that WaaP is necessary for resistance to hydrophobic and polycationic antimicrobials in E. coli and that it is required for virulence in invasive strains of S. enterica. WaaP function is also known to be essential for the viability of P. aeruginosa. The predicted WaaP protein shows low levels of similarity (10-15% identity) to eukaryotic protein kinases, but its kinase activity has never been tested. Here we report the purification of WaaP and the reconstitution of its enzymatic activity in vitro. The purified enzyme catalyzes the incorporation of 33P from [gamma-33P]ATP into acceptor LPS purified from a defined E. coli waaP mutant. Enzymatic activity is dependent upon the presence of Mg2+ and is maximal from pH 8.0 to 9.0. The apparent Km (determined at saturating concentrations of the second substrate) is 0.13 mm for ATP and 76 microm for LPS. These data are the first proof that WaaP is indeed an LPS kinase. Further, site-directed mutagenesis of a predicted catalytic residue suggests that WaaP shares a common mechanism of action with eukaryotic protein kinases.
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265. |
Johnson JR,
Delavari P,
Stell AL,
Whittam TS,
Carlino U,
Russo TA,
( 2001 ) Molecular comparison of extraintestinal Escherichia coli isolates of the same electrophoretic lineages from humans and domestic animals. PMID : 11106542 : DOI : 10.1086/317662 Abstract >>
Molecular typing methods were used to characterize 38 Escherichia coli strains that originally were isolated from extraintestinal infections and represented 5 multilocus enzyme electrophoretic types (ETs) recovered from both humans and animals. Within each ET, the human and animal isolates did not consistently segregate by host group, according to individual virulence factors (VFs), composite VF-serotype profiles, or pulsed-field gel electrophoresis profiles. Several close matches with respect to VF-serotype profiles were identified between human and canine isolates from different locales. One canine and 2 human isolates of serogroup O6 closely resembled archetypal human pyelonephritis isolate 536 (O6:K15:H31), according to papA sequence and VF-serotype profile. These findings support the hypothesis that certain pathogenic lineages of E. coli cause disease in both humans and animals and that humans may acquire pathogenic E. coli from domestic pets.
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266. |
Chen CC,
Fang M,
Majumder A,
Wu HY,
( 2001 ) A 72-base pair AT-rich DNA sequence element functions as a bacterial gene silencer. PMID : 11121424 : DOI : 10.1074/jbc.M010501200 Abstract >>
We have previously demonstrated that sequential activation of the bacterial ilvIH-leuO-leuABCD gene cluster involves a promoter-relay mechanism. In the current study, we show that the final activation of the leuABCD operon is through a transcriptional derepression mechanism. The leuABCD operon is transcriptionally repressed by the presence of a 318-base pair AT-rich upstream element. LeuO is required for derepressing the repressed leuABCD operon. Deletion analysis of the repressive effect of the 318-bp element has led to the identification of a 72-bp AT-rich (78% A+T) DNA sequence element, AT4, which is capable of silencing a number of unrelated promoters in addition to the leuABCD promoter. AT4-mediated gene silencing is orientation-independent and occurs within a distance of 300 base pairs. Furthermore, an increased gene-silencing effect was observed with a tandemly repeated AT4 dimer. The possible mechanism of AT4-mediated gene silencing in bacteria is discussed.
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267. |
White DG,
Hudson C,
Maurer JJ,
Ayers S,
Zhao S,
Lee MD,
Bolton L,
Foley T,
Sherwood J,
( 2000 ) Characterization of chloramphenicol and florfenicol resistance in Escherichia coli associated with bovine diarrhea. PMID : 11101601 : PMC : PMC87642 Abstract >>
Florfenicol, a veterinary fluorinated analog of thiamphenicol, is approved for treatment of bovine respiratory pathogens in the United States. However, florfenicol resistance has recently emerged among veterinary Escherichia coli isolates incriminated in bovine diarrhea. The flo gene, which confers resistance to florfenicol and chloramphenicol, has previously been identified in Photobacterium piscicida and Salmonella enterica serovar Typhimurium DT104. The flo gene product is closely related to the CmlA protein identified in Pseudomonas aeruginosa. The cmlA gene confers nonenzymatic chloramphenicol resistance via an efflux mechanism. Forty-eight E. coli isolates recovered from calves with diarrhea, including 41 that were both chloramphenicol and florfenicol resistant, were assayed for the presence of both flo and cmlA genes. Forty-two of the 44 isolates for which florfenicol MICs were > or =16 microg/ml were positive via PCR for the flo gene. All E. coli isolates for which florfenicol MICs were < or =8 microg/ml were negative for the flo gene (n = 4) Twelve E. coli isolates were positive for cmlA, and chloramphenicol MICs for all 12 were > or =32 microg/ml. Additionally, eight isolates were positive for both flo and cmlA, and both florfenicol and chloramphenicol MICs for these isolates were > or =64 microg/ml. DNA sequence analysis of the E. coli flo gene demonstrated 98% identity to the published GenBank sequences of both serovar Typhimurium flo(St) and P. piscicida pp-flo. The flo gene was identified on high-molecular-weight plasmids of approximately 225 kb among the majority of florfenicol-resistant E. coli isolates. However, not all of the florfenicol-resistant E. coli isolates tested contained the large flo-positive plasmids. This suggests that several of the E. coli isolates may possess a chromosomal flo gene. The E. coli flo gene specifies nonenzymatic cross-resistance to both florfenicol and chloramphenicol, and its presence among bovine E. coli isolates of diverse genetic backgrounds indicates a distribution much wider than previously thought.
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268. |
Johnson JR,
Delavari P,
Kuskowski M,
Stell AL,
( 2001 ) Phylogenetic distribution of extraintestinal virulence-associated traits in Escherichia coli. PMID : 11106538 : DOI : 10.1086/317656 Abstract >>
The 72 member strains of the Escherichia coli Reference collection were assessed as to genotype for 31 putative extraintestinal virulence factor (VF) genes and DNA sequence for papA, the P fimbrial structural subunit gene. Although most VFs were concentrated in phylogenetic group B2 or jointly in groups B2 and D, others were concentrated primarily in group D, were broadly distributed (without group-specific associations), and/or occurred only outside of group B2. Statistical correlations among VFs suggested linkage on pathogenicity-associated islands or plasmids. Isolates from humans and nonhuman primates had more VFs than did isolates from other animals. Sequence diversity was minimal within each F type-specific papA allele group but was substantial among different papA allele groups. The distribution patterns of papA variants and other VFs suggested multiple horizontal transfer events. These findings provide new insights into the phylogenetic origins of extraintestinal VFs in E. coli.
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269. |
Handa N,
Kobayashi I,
Arnold DA,
( 2000 ) A novel, 11 nucleotide variant of chi, chi*: one of a class of sequences defining the Escherichia coli recombination hotspot chi. PMID : 10884344 : DOI : 10.1006/jmbi.2000.3861 Abstract >>
In wild-type Escherichia coli, recognition of the recombination hotspot, chi (5'-GCTGGTGG-3'), by the RecBCD enzyme is central to homologous recombination. However, in the recC* class of RecBCD mutants, stimulation of recombination by the canonical chi sequence is not detectable, but the levels of homologous recombination are nearly wild-type. In vivo studies demonstrate that a member of this class of mutants, the recC1004 allele, encodes an enzyme that responds to a novel variant of chi, termed chi* (5'-GCTGGTGCTCG-3'). Here, we establish that, in vitro, the chi* sequence is recognized more efficiently by the RecBC(1004)D enzyme than is the wild-type chi. This is manifest by both a greater modification of nuclease activity and a higher stimulation of RecA protein-mediated joint molecule formation at chi* than at chi. Sequencing of the recC1004 gene revealed that it contains a frameshift mutation, which results in a replacement of nine of the wild-type amino acid residues by eight in the mutant protein, and defines a locus that is important for the specificity of chi-recognition. In addition, we show that this novel, 11 nucleotide chi* sequence also regulates the wild-type RecBCD enzyme, supporting the notion that variants of the canonical chi constitute a class of sequences that regulate the recombination function of RecBCD enzyme.
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270. |
Bellin T,
Pulz M,
Matussek A,
Hempen HG,
Gunzer F,
( 2001 ) Rapid detection of enterohemorrhagic Escherichia coli by real-time PCR with fluorescent hybridization probes. PMID : 11136804 : DOI : 10.1128/JCM.39.1.370-374.2001 PMC : PMC87735 Abstract >>
In this report, we present a PCR protocol for rapid identification of enterohemorrhagic Escherichia coli on a LightCycler instrument. In a multiplex assay, the genes encoding Shiga toxin 1 and Shiga toxin 2 are detected in a single reaction capillary. A complete analysis of up to 32 samples takes about 45 min.
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271. |
Goryshin IY,
Reznikoff WS,
Davies DR,
( 2000 ) Three-dimensional structure of the Tn5 synaptic complex transposition intermediate. PMID : 10884228 : DOI : 10.1126/science.289.5476.77 Abstract >>
Genomic evolution has been profoundly influenced by DNA transposition, a process whereby defined DNA segments move freely about the genome. Transposition is mediated by transposases, and similar events are catalyzed by retroviral integrases such as human immunodeficiency virus-1 (HIV-1) integrase. Understanding how these proteins interact with DNA is central to understanding the molecular basis of transposition. We report the three-dimensional structure of prokaryotic Tn5 transposase complexed with Tn5 transposon end DNA determined to 2.3 angstrom resolution. The molecular assembly is dimeric, where each double-stranded DNA molecule is bound by both protein subunits, orienting the transposon ends into the active sites. This structure provides a molecular framework for understanding many aspects of transposition, including the binding of transposon end DNA by one subunit and cleavage by a second, cleavage of two strands of DNA by a single active site via a hairpin intermediate, and strand transfer into target DNA.
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272. |
Chang LL,
Chang YH,
Lee TM,
Chang CY,
( 2000 ) Two new gene cassettes, dfr17 (for trimethoprim resistance) and aadA4 (for spectinomycin/streptomycin resistance), inserted in an Escherichia coli class 1 integron. PMID : 10882694 : DOI : 10.1093/jac/46.1.87 Abstract >>
Two new gene cassettes, dfr17 and aadA4, inserted in a class 1 integron of Escherichia coli EC107, are described here. The dfr17 cassette encodes trimethoprim resistance and has 91% identity with the dfrVII dihydrofolate reductase gene. The aadA4 cassette confers resistance to spectinomycin and streptomycin and shows 94% identity with the aadA3 gene. The integron carrying the dfr17 and aadA4 cassettes was located on a conjugative plasmid, pEC1072.
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273. |
Blank TE,
Zhong H,
Bell AL,
Whittam TS,
Donnenberg MS,
( 2000 ) Molecular variation among type IV pilin (bfpA) genes from diverse enteropathogenic Escherichia coli strains. PMID : 11083828 : DOI : 10.1128/iai.68.12.7028-7038.2000 PMC : PMC97813 Abstract >>
Typical enteropathogenic Escherichia coli (EPEC) strains produce bundle-forming pili (BFP), type IVB fimbriae that have been implicated in EPEC virulence, antigenicity, autoaggregation, and localized adherence to epithelial cells (LA). BFP are polymers of bundlin, a pilin protein that is encoded by the bfpA gene found on a large EPEC plasmid. Striking sequence variation has previously been observed among type IV pilin genes of other gram-negative bacterial pathogens (e.g., Pseudomonas and Neisseria spp.). In contrast, the established sequences of bfpA genes from two distantly related prototype EPEC strains vary by only a single base pair. To determine whether bundlin sequences vary more extensively, we used PCR to amplify the bfpA genes from 19 EPEC strains chosen for their various serotypes and sites and years of isolation. Eight different bfpA alleles were identified by sequencing of the PCR products. These alleles can be classified into two major groups. The alpha group contains three alleles derived from strains carrying O55, O86, O111, O119, O127, or O128 somatic antigens. The beta group contains five alleles derived from strains carrying O55, O110, O128ab, O142, or nontypeable antigens. Sequence comparisons show that bundlin has highly conserved and variable regions, with most of the variation occurring in the C-terminal two-thirds of the protein. The results of multilocus enzyme electrophoresis support the hypothesis that bfpA sequences have spread horizontally across distantly related clonal lineages. Strains with divergent bundlin sequences express bundlin protein, produce BFP, and carry out autoaggregation and LA. However, four strains lack most or all of these phenotypes despite having an intact bfpA gene. These results have important implications for our understanding of bundlin structure, transmission of the bfp gene cluster among EPEC strains, and the role of bundlin variation in the evasion of host immune system responses.
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274. |
Dartigalongue C,
Nikaido H,
Raina S,
( 2000 ) Protein folding in the periplasm in the absence of primary oxidant DsbA: modulation of redox potential in periplasmic space via OmpL porin. PMID : 11080145 : DOI : 10.1093/emboj/19.22.5980 PMC : PMC305838 Abstract >>
Disulfide bond formation in Escherichia coli is a catalyzed reaction accomplished by DsbA. We found that null mutations in a new porin gene, ompL, allowed a total bypass of the DsbA requirement for protein oxidation. These mutations acted as extragenic null suppressors for dsbA, and restored normal folding of alkaline phosphatase and relieved sensitivity to dithiothreitol. ompL dsbA double mutants were completely like wild-type mutants in terms of motility and lack of mucoidy. This suppression was not dependent on DsbC and DsbG, since the oxidation status of these proteins was unaltered in ompL dsbA strains. Purified OmpL allowed diffusion of small solutes, including sugars, but the suppression was not dependent on the carbon sources used. Suppression by ompL null mutations required DsbB, leading us to propose a hypothesis that DsbB oxidizes yet unidentified, low-molecular-weight redox agents in the periplasm. These oxidized agents accumulate and substitute for DsbA if their leakage into the medium is prevented by the absence of OmpL, presumed to form a specific channel for their diffusion.
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275. |
Adams MD,
Phillips CM,
Neely MN,
Collett MS,
Cadavid L,
Riley MA,
( 2000 ) The newly characterized colicin Y provides evidence of positive selection in pore-former colicin diversification. PMID : 10878131 : DOI : 10.1099/00221287-146-7-1671 Abstract >>
Two evolutionary mechanisms have been proposed in the process of protein diversification of the large family of antimicrobial toxins of Escherichia coli, known as the colicins. Data from previous studies suggest that the relatively rare nuclease colicins appear to diversify primarily through the action of positive selection, whilst the more abundant pore-former colicins appear to diversify through the action of recombination. The complete DNA sequence of the newly characterized colicin plasmid, pCol-Let, isolated from a Yanomama Indian of South America, is presented here. This plasmid encodes a newly identified pore-former colicin, colicin Y. DNA and protein sequence comparisons of the colicin Y gene cluster and the encoded proteins with those of published pore-former colicins provide the first evidence that positive selection may also act to increase pore-former colicin diversity.
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276. |
Harris SL,
Spears PA,
Havell EA,
Hamrick TS,
( 2000 ) Genetic characterization of Escherichia coli type 1 pilus adhesin mutants and identification of a novel binding phenotype. PMID : 10869080 : DOI : 10.1128/jb.182.14.4012-4021.2000 PMC : PMC94587 Abstract >>
Five Escherichia coli type 1 pilus mutants that had point mutations in fimH, the gene encoding the type 1 pilus adhesin FimH, were characterized. FimH is a minor component of type 1 pili that is required for the pili to bind and agglutinate guinea pig erythrocytes in a mannose-inhibitable manner. Point mutations were located by DNA sequencing and deletion mapping. All mutations mapped within the signal sequence or in the first 28% of the predicted mature protein. All mutations were missense mutations except for one, a frameshift lesion that was predicted to cause the loss of approximately 60% of the mature FimH protein. Bacterial agglutination tests with polyclonal antiserum raised to a LacZ-FimH fusion protein failed to confirm that parental amounts of FimH cross-reacting material were expressed in four of the five mutants. The remaining mutant, a temperature-sensitive (ts) fimH mutant that agglutinated guinea pig erythrocytes after growth at 31 degrees C but not at 42 degrees C, reacted with antiserum at both temperatures in a manner similar to the parent. Consequently, this mutant was chosen for further study. Temperature shift experiments revealed that new FimH biosynthesis was required for the phenotypic change. Guinea pig erythrocyte and mouse macrophage binding experiments using the ts mutant grown at the restrictive and permissive temperatures revealed that whereas erythrocyte binding was reduced to a level comparable to that of a fimH insertion mutant at the restrictive temperature, mouse peritoneal macrophages were bound with parental efficiency at both the permissive and restrictive temperatures. Also, macrophage binding by the ts mutant was insensitive to mannose inhibition after growth at 42 degrees C but sensitive after growth at 31 degrees C. The ts mutant thus binds macrophages with one receptor specificity at 31 degrees C and another at 42 degrees C.
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277. |
Culham DE,
Wood JM,
( 2000 ) An Escherichia coli reference collection group B2- and uropathogen-associated polymorphism in the rpoS-mutS region of the E. coli chromosome. PMID : 11029456 : DOI : 10.1128/jb.182.21.6272-6276.2000 PMC : PMC94770 Abstract >>
Chromosomal DNAs of enterohemorrhagic, uropathogenic, and laboratory attenuated Escherichia coli strains differ in the rpoS-mutS region. Many uropathogens lack a deletion and an insertion characteristic of enterohemorrhagic strains. At the same chromosomal position, they harbor a 2.1-kb insertion of unknown origin with a base composition suggestive of horizontal gene transfer. Unlike virulence determinants associated with urinary tract infection and/or neonatal meningitis (pap or prs, sfa, kps, and hly), the 2.1-kb insertion is shared by all group B2 strains of the E. coli Reference Collection.
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278. |
Navarro F,
Miró E,
Vergés C,
Tarragó R,
Sabaté M,
( 2000 ) Cloning and sequence of the gene encoding a novel cefotaxime-hydrolyzing beta-lactamase (CTX-M-9) from Escherichia coli in Spain. PMID : 10858363 : DOI : 10.1128/aac.44.7.1970-1973.2000 PMC : PMC89994 Abstract >>
A new CTX-M-type beta-lactamase (CTX-M-9) has been cloned from a clinical cefotaxime-resistant Escherichia coli strain. Despite the close identity that exists between the CTX-M-9 and Toho-2 beta-lactamases (88%), the 35 amino acids located between residues Ala-185 and Ala-219 are totally different in both enzymes. Outside of this region there are only six amino acids substitutions between both proteins.
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279. |
Mihara H,
Kurihara T,
Lacourciere GM,
( 2000 ) Escherichia coli NifS-like proteins provide selenium in the pathway for the biosynthesis of selenophosphate. PMID : 10829016 : DOI : 10.1074/jbc.M000926200 Abstract >>
Selenophosphate synthetase (SPS), the selD gene product from Escherichia coli, catalyzes the biosynthesis of monoselenophosphate, AMP, and orthophosphate in a 1:1:1 ratio from selenide and ATP. Kinetic characterization revealed the K(m) value for selenide approached levels that are toxic to the cell. Our previous demonstration that a Se(0)-generating system consisting of l-selenocysteine and the Azotobacter vinelandii NifS protein can replace selenide for selenophosphate biosynthesis in vitro suggested a mechanism whereby cells can overcome selenide toxicity. Recently, three E. coli NifS-like proteins, CsdB, CSD, and IscS, have been overexpressed and characterized. All three enzymes act on selenocysteine and cysteine to produce Se(0) and S(0), respectively. In the present study, we demonstrate the ability of each E. coli NifS-like protein to function as a selenium delivery protein for the in vitro biosynthesis of selenophosphate by E. coli wild-type SPS. Significantly, the SPS (C17S) mutant, which is inactive in the standard in vitro assay with selenide as substrate, was found to exhibit detectable activity in the presence of CsdB, CSD, or IscS and l-selenocysteine. Taken together the ability of the NifS-like proteins to generate a selenium substrate for SPS and the activation of the SPS (C17S) mutant suggest a selenium delivery function for the proteins in vivo.
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280. |
Kormutakova R,
Klucar L,
Turna J,
( 2000 ) DNA sequence analysis of the tellurite-resistance determinant from clinical strain of Escherichia coli and identification of essential genes. PMID : 11016400 : Abstract >>
The tellurite-resistant Escherichia coli strain KL53 was found during testing of the group of clinical isolates for antibiotics and heavy metal ion resistance (Burian et al. 1990). Determinant of the tellurite resistance of the strain was located on the large conjugative plasmid pTE53 and cloned into pACYC184. Three different Ter clones harboring pLK2, pLK18 and pLK20 were isolated (Burian et al. 1998). The smallest functional Ter clone harboring pLK18 was chosen for further analysis. Plasmid pLK18 have been subcloned to obtain convenient DNA fragments for sequencing of tellurite-resistance determinant. Sequencing of this DNA fragments provided complete DNA sequence of the determinant, 5,250 bp in size. The sequence has been compared with nucleotide and protein databank (BLAST programs) and significant homology with the three known operons coding for tellurite resistance has been found (determinat on plasmid pR478 from Serratia marcescens, on plasmid pMER610 from Alcaligenes sp. and chromosomal tellurite resistance genes from Proteus mirabilis). We identified 5 ORFs coding for 5 genes named terB to terF. The clone harboring pLK18 was subjected to the transposition with Tn1737Km to disrupt determinant of the tellurite resistance. Plasmid DNA of several clones containing pLK18 with Tn1737Km was isolated to locate the target site of Tn1737Km. Analyses showed, the genes terB, terC, terD and terE are essential for conservation of the resistance whereas the gene terF is not important in this respect.
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281. |
Elwell CA,
Dreyfus LA,
( 2000 ) DNase I homologous residues in CdtB are critical for cytolethal distending toxin-mediated cell cycle arrest. PMID : 10972814 : DOI : 10.1046/j.1365-2958.2000.02070.x Abstract >>
Cytolethal distending toxins (CDTs) block cell division by arresting the eukaryotic cell cycle at G2/M. Although previously not recognized in standard BLAST searches, a position-specific iterated (PSI) BLAST search of the protein data bank using CDT polypeptides as query sequences indicated that CdtB bears significant position-specific homology to type I mammalian DNases. The PSIBLAST sequence alignment reveals that residues of DNase I involved in phosphodiester bond hydrolysis (His134 and His252) are conserved in CdtB as well as their respective hydrogen bond pairs (Glu78 and Asp212). CdtB also contains a pentapeptide motif found in all DNase I enzymes. Further, crude CDT preparations possess detectable DNase activity not associated with identical preparations from control cells. Five CdtB mutations in amino acids corresponding to DNase I active site residues were prepared and expressed together with wild-type CdtA and CdtC polypeptides. Mutation in four of the five DNase-specific active site residues resulted in CDT preparations that lacked DNase activity and failed to induce cellular distension or arrest division of HeLa cells. The fifth mutation, Glu86 (Glu78 in DNase I), retained the ability to induce a moderate level of cell cycle arrest and displayed reduced DNase activity relative to wild-type CDT. Together, these data suggest that the CDT holotoxin has intrinsic DNase activity that is associated with the CdtB polypeptide and that this DNase activity may be responsible for the CDT-induced cell cycle arrest.
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282. |
Gubbins MJ,
Ghetu AF,
( 2000 ) Crystal structure of the bacterial conjugation repressor finO. PMID : 10876242 : DOI : 10.1038/76790 Abstract >>
The conjugative transfer of F-like plasmids is repressed by FinO, an RNA binding protein. FinO interacts with the F-plasmid encoded traJ mRNA and its antisense RNA, FinP, stabilizing FinP against endonucleolytic degradation and facilitating sense-antisense RNA recognition. Here we present the 2.0 A resolution X-ray crystal structure of FinO, lacking its flexible N-terminal extension. FinO adopts a novel, elongated, largely helical conformation. An N-terminal region, previously shown to contact RNA, forms a positively charged alpha-helix (helix 1) that protrudes 45 A from the central core of FinO. A C-terminal region of FinO that is implicated in RNA interactions also extends out from the central body of the protein, adopting a helical conformation and packing against the base of the N-terminal helix. A highly positively charged patch on the surface of the FinO core may present another RNA binding surface. The results of an in vitro RNA duplexing assay demonstrate that the flexible N-terminal region of FinO plays a key role in FinP-traJ RNA recognition, and supports our proposal that this region and the N-terminus of helix 1 interact with and stabilize paired, complementary RNA loops in a kissing complex.
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283. |
Tarr PI,
Schoening LM,
Yea YL,
Ward TR,
Jelacic S,
Whittam TS,
( 2000 ) Acquisition of the rfb-gnd cluster in evolution of Escherichia coli O55 and O157. PMID : 11029441 : DOI : 10.1128/jb.182.21.6183-6191.2000 PMC : PMC94755 Abstract >>
The rfb region specifies the structure of lipopolysaccharide side chains that comprise the diverse gram-negative bacterial somatic (O) antigens. The rfb locus is adjacent to gnd, which is a polymorphic gene encoding 6-phosphogluconate dehydrogenase. To determine if rfb and gnd cotransfer, we sequenced gnd in five O55 and 13 O157 strains of Escherichia coli. E. coli O157:H7 has a gnd allele (allele A) that is only 82% identical to the gnd allele (allele D) of closely related E. coli O55:H7. In contrast, gnd alleles of E. coli O55 in distant lineages are >99.9% identical to gnd allele D. Though gnd alleles B and C in E. coli O157 that are distantly related to E. coli O157:H7 are more similar to allele A than to allele D, there are nucleotide differences at 4 to 6% of their sites. Alleles B and C can be found in E. coli O157 in different lineages, but we have found allele A only in E. coli O157 belonging to the DEC5 lineage. DNA 3' to the O55 gnd allele in diverse E. coli lineages has sequences homologous to tnpA of the Salmonella enterica serovar Typhimurium IS200 element, E. coli Rhs elements (including an H-rpt gene), and portions of the O111 and O157 rfb regions. We conclude that rfb and gnd cotransferred into E. coli O55 and O157 in widely separated lineages and that recombination was responsible for recent antigenic shifts in the emergence of pathogenic E. coli O55 and O157.
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284. |
Huang SH,
Stins MF,
Kim KS,
( 2000 ) Bacterial penetration across the blood-brain barrier during the development of neonatal meningitis. PMID : 11008113 : Abstract >>
Bacterial pathogens may breach the blood-brain barrier (BBB) and invade the central nervous system through paracellular and/or transcellular mechanisms. Transcellular penetration, e.g., transcytosis across the BBB has been demonstrated for Escherichia coli K1, group B streptococcus, Listeria monocytogenes, Citrobacter freundii and Streptococcus pneumonia strains. Genes contributing to invasion of brain microvascular endothelial cells include E. coli K1 genes ompA, ibeA, ibeB, and yijP. Understanding the mechanisms of bacterial penetration across the BBB may help develop novel approaches to preventing bacterial meningitis.
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285. |
Nataro JP,
Poteet-Smith CE,
Steiner TS,
( 2000 ) Enteroaggregative Escherichia coli expresses a novel flagellin that causes IL-8 release from intestinal epithelial cells. PMID : 10862792 : DOI : 10.1172/JCI8892 PMC : PMC378507 Abstract >>
Enteroaggregative Escherichia coli (EAEC) is an emerging cause of acute and persistent diarrhea worldwide. EAEC infections are associated with intestinal inflammation and growth impairment in infected children, even in the absence of diarrhea. We previously reported that prototype EAEC strains rapidly induce IL-8 production by Caco-2 intestinal epithelial cells, and that this effect is mediated by a soluble, heat-stable factor released by these bacteria in culture. We herein report the cloning, sequencing, and expression of this biologically active IL-8-releasing factor from EAEC, and its identification as a flagellin that is unique among known expressed proteins. Flagella purified from EAEC 042 and several other EAEC isolates potently release IL-8 from Caco-2 cells; an engineered aflagellar mutant of 042 does not release IL-8. Finally, cloned EAEC flagellin expressed in nonpathogenic E. coli as a polyhistidine-tagged fusion protein maintains its proinflammatory activity. These findings demonstrate a major new means by which EAEC may cause intestinal inflammation, persistent diarrhea, and growth impairment that characterize human infection with these organisms. Furthermore, they open new approaches for diagnosis and vaccine development. This novel pathogenic mechanism of EAEC extends an emerging paradigm of bacterial flagella as inflammatory stimuli.
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286. |
Liesegang A,
Tschäpe H,
Bielaszewska M,
Zhang WL,
( 2000 ) Molecular characteristics and epidemiological significance of Shiga toxin-producing Escherichia coli O26 strains. PMID : 10834966 : PMC : PMC86746 Abstract >>
Fifty-five Shiga toxin (Stx)-producing Escherichia coli (STEC) O26:H11 and O26:H(-) strains isolated from humans between 1965 and 1999 in Germany and the Czech Republic were investigated for their chromosomal and plasmid characteristics. All motile (n = 23) and nonmotile (n = 32) STEC O26 strains were shown to possess the identical flagellin subunit-encoding gene (fliC). We observed a striking recent shift of the stx genotype from stx(1) to stx(2) among the STEC O26 isolates. While stx(1) was the exclusive genotype identified in our collection until 1994, 94% of the isolates obtained after 1997 possessed stx(2) either alone (71%) or together with stx(1) (23%). Plasmid profiling demonstrated a remarkable heterogeneity with respect to plasmid sizes and combinations. Southern blot analysis of plasmid DNA with probes specific to potential accessory virulence genes revealed considerable additional variability in gene composition and arrangement. Pulsed-field gel electrophoresis (PFGE) differentiated 16 subgroups among the 55 STEC O26 strains. Using these techniques we demonstrate the emergence of a new clonal subgroup characterized by PFGE pattern A and a unique combination of virulence markers including stx(2) and a single, approximately 90-kb plasmid harboring the enterhemorrhagic E. coli hlyA and etp genes. The proportion of PFGE subgroup A strains among STEC O26 isolates rose from 30% in 1996 to more than 50% in 1999. Four clusters of infections with the clonal subgroup A were identified. We conclude that the STEC serogroup O26 is diverse and that pathogenic clonal subgroups can rapidly emerge during short intervals. The extensive genetic diversity of STEC O26 provides a basis for molecular subtyping of this important non-O157 STEC serogroup.
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287. |
Herbelin CJ,
Bumbaugh AC,
Reid SD,
( 2000 ) Parallel evolution of virulence in pathogenic Escherichia coli. PMID : 10894541 : DOI : 10.1038/35017546 Abstract >>
The mechanisms underlying the evolution and emergence of new bacterial pathogens are not well understood. To elucidate the evolution of pathogenic Escherichia coli strains, here we sequenced seven housekeeping genes to build a phylogenetic tree and trace the history of the acquisition of virulence genes. Compatibility analysis indicates that more than 70% of the informative sites agree with a single phylogeny, suggesting that recombination has not completely obscured the remnants of ancestral chromosomes. On the basis of the rate of synonymous substitution for E. coli and Salmonella enterica (4.7 x 10(-9) per site per year), the radiation of clones began about 9 million years ago and the highly virulent pathogen responsible for epidemics of food poisoning, E. coli O157:H7, separated from a common ancestor of E. coli K-12 as long as 4.5 million years ago. Phylogenetic analysis reveals that old lineages of E. coli have acquired the same virulence factors in parallel, including a pathogenicity island involved in intestinal adhesion, a plasmid-borne haemolysin, and phage-encoded Shiga toxins. Such parallel evolution indicates that natural selection has favoured an ordered acquisition of genes and the progressive build-up of molecular mechanisms that increase virulence.
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288. |
Knoechel DG,
Haynes CA,
Creagh AL,
Pfuetzner RA,
Frey EA,
Luo Y,
( 2000 ) Crystal structure of enteropathogenic Escherichia coli intimin-receptor complex. PMID : 10890451 : DOI : 10.1038/35016618 Abstract >>
Intimin and its translocated intimin receptor (Tir) are bacterial proteins that mediate adhesion between mammalian cells and attaching and effacing (A/E) pathogens. Enteropathogenic Escherichia coli (EPEC) causes significant paediatric morbidity and mortality world-wide. A related A/E pathogen, enterohaemorrhagic E. coli (EHEC; O157:H7) is one of the most important food-borne pathogens in North America, Europe and Japan. A unique and essential feature of A/E bacterial pathogens is the formation of actin-rich pedestals beneath the intimately adherent bacteria and localized destruction of the intestinal brush border. The bacterial outer membrane adhesin, intimin, is necessary for the production of the A/E lesion and diarrhoea. The A/E bacteria translocate their own receptor for intimin, Tir, into the membrane of mammalian cells using the type III secretion system. The translocated Tir triggers additional host signalling events and actin nucleation, which are essential for lesion formation. Here we describe the the crystal structures of an EPEC intimin carboxy-terminal fragment alone and in complex with the EPEC Tir intimin-binding domain, giving insight into the molecular mechanisms of adhesion of A/E pathogens.
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289. |
Bou G,
Oliver A,
Ojeda M,
Monzón C,
Martínez-Beltrán J,
( 2000 ) Molecular characterization of FOX-4, a new AmpC-type plasmid-mediated beta-lactamase from an Escherichia coli strain isolated in Spain. PMID : 10952615 : DOI : 10.1128/aac.44.9.2549-2553.2000 PMC : PMC90105 Abstract >>
A clinical strain of Escherichia coli (Ec GCE) displayed resistance to cefoxitin, cefotetan, cefotaxime, and ceftazidime. Susceptibility was not restored by the addition of clavulanic acid. Two beta-lactamases with apparent pIs of 5.4 and 6.4 were identified; the beta-lactamase with a pI of 6.4 was transferred by conjugation and associated with a 40-kb plasmid. Analysis of the nucleotide sequence showed a new ampC beta-lactamase gene that is closely related to those encoding the FOX-3, FOX-2, and FOX-1 beta-lactamases but whose product has four novel amino acid mutations, at positions 11 (M-->T), 43 (A-->E), 233 (V-->A), and 280 (Y-->H). This first cephamycinase from Spain was named FOX-4.
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290. |
Heincz MC,
McFall E,
( 1975 ) N-terminal amino acid sequences of D-serine deaminases of wild-type and operator-constitutive strains of Escherichia coli K-12. PMID : 1099073 : PMC : PMC235842 Abstract >>
The N-terminal amino acid sequences of the D-serine deaminases from strains of Escherichia coli K-12 that harbor wild-type and high-level constitutive catabolite-insensitive operator-initiator regions are identical: Met-Ser-GluNH2-Ser-Gly-Arg-His-Cys. This result indicates that the operator-initiator region is probably distinct from the D-serine deaminase structural gene.
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291. |
Unkmeir A,
Schmidt H,
( 2000 ) Structural analysis of phage-borne stx genes and their flanking sequences in shiga toxin-producing Escherichia coli and Shigella dysenteriae type 1 strains. PMID : 10948097 : DOI : 10.1128/iai.68.9.4856-4864.2000 PMC : PMC101682 Abstract >>
The stx-flanking regions of 49 Shiga toxin-producing Escherichia coli strains and nine Shigella dysenteriae serotype 1 strains containing either stx, stx(1), stx(2), or stx(2) variant genes, were examined. We analyzed these regions by PCR using a set of primers with one primer specific for the respective stx gene and a second primer complementary to sequences of Stx phages H-19B and 933W. We further characterized the amplification products by restriction endonuclease digestion and nucleotide sequencing. PCR products of stx(1)-containing E. coli strains of serogroups O157, O26, and 0103 showed the same lengths and similar restriction patterns. However, we failed to amplify the 3' stx-flanking region in stx(1)-harboring E. coli O111:H(-) strains. Stx2-producing E. coli strains revealed amplification products of different lengths and restriction patterns, suggesting greater heterogeneity than in stx(1)-positive strains. We also obtained specific PCR products for two Stx2c-producing and seven Stx2f-producing E. coli strains when they were subjected to PCR analysis. In nine S. dysenteriae type 1 strains, H-19B- and 933W-specific primers amplified only the 3' stx-flanking region. The results of our study demonstrate that the stx genes of all strains investigated are continuous with phage sequences. Whereas almost all strains except E. coli O111:H(-) strains were associated with a S-like gene, association with Q could not be demonstrated in nine S. dysenteriae type 1 strains and three E. coli strains. Furthermore, we showed that the organization of the stx-flanking regions is similar in all strains investigated, whereas fine-structure analysis showed subtle differences among the sequences examined. Our results support the hypothesis that stx genes in E. coli and S. dysenteriae are generally phage-borne.
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292. |
McVeigh A,
Fasano A,
Scott DA,
Jelacic S,
Moseley SL,
Robertson DC,
Savarino SJ,
( 2000 ) IS1414, an Escherichia coli insertion sequence with a heat-stable enterotoxin gene embedded in a transposase-like gene. PMID : 10992475 : DOI : 10.1128/iai.68.10.5710-5715.2000 PMC : PMC101527 Abstract >>
Enteroaggregative Escherichia coli (EAEC) heat-stable enterotoxin 1 (EAST1) was originally discovered in EAEC but has also been associated with enterotoxigenic E. coli (ETEC). Multiple genomic restriction fragments from each of three ETEC strains of human origin showed homology with an EAST1 gene probe. A single hybridizing fragment was detected on the plasmid of ETEC strain 27D that also encodes heat-stable enterotoxin Ib and colonization factor antigen I. We isolated and characterized this fragment, showing that it (i) carries an allele of astA nearly identical to that originally reported from EAEC 17-2 and (ii) expressed enterotoxic activity. Sequence analysis of the toxin coding region revealed that astA is completely embedded within a 1,209-bp open reading frame (ORF1), whose coding sequence is on the same strand but in the -1 reading frame in reference to the toxin gene. In vitro expression of the predicted M(r)- approximately 46,000 protein product of ORF1 was demonstrated. ORF1 is highly similar to transposase genes of IS285 from Yersinia pestis, IS1356 from Burkholderia cepacia, and ISRm3 from Rhizobium meliloti. It is bounded by 30-bp imperfect inverted repeat sequences and flanked by 8-bp direct repeats. Based on these structural features, pathognomonic of a regular insertion sequence, this element was designated IS1414. Preliminary experiments to show IS1414 translocation were unsuccessful. Overlapping genes of the type suggested by the IS1414 core region have heretofore not been described in bacteria. It seems to offer a most efficient mechanism for intragenomic and horizontal dissemination of EAST1.
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293. |
Pupo GM,
Lan R,
Reeves PR,
( 2000 ) Multiple independent origins of Shigella clones of Escherichia coli and convergent evolution of many of their characteristics. PMID : 10954745 : DOI : 10.1073/pnas.180094797 PMC : PMC27065 Abstract >>
The evolutionary relationships of 46 Shigella strains representing each of the serotypes belonging to the four traditional Shigella species (subgroups), Dysenteriae, Flexneri, Boydii, and Sonnei, were determined by sequencing of eight housekeeping genes in four regions of the chromosome. Analysis revealed a very similar evolutionary pattern for each region. Three clusters of strains were identified, each including strains from different subgroups. Cluster 1 contains the majority of Boydii and Dysenteriae strains (B1-4, B6, B8, B10, B14, and B18; and D3-7, D9, and D11-13) plus Flexneri 6 and 6A. Cluster 2 contains seven Boydii strains (B5, B7, B9, B11, B15, B16, and B17) and Dysenteriae 2. Cluster 3 contains one Boydii strain (B12) and the Flexneri serotypes 1-5 strains. Sonnei and three Dysenteriae strains (D1, D8, and D10) are outside of the three main clusters but, nonetheless, are clearly within Escherichia coli. Boydii 13 was found to be distantly related to E. coli. Shigella strains, like the other pathogenic forms of E. coli, do not have a single evolutionary origin, indicating convergent evolution of Shigella phenotypic properties. We estimate the three main Shigella clusters to have evolved within the last 35,000 to 270,000 years, suggesting that shigellosis was one of the early infectious diseases of humans.
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294. |
Herbelin CJ,
Chirillo SC,
Melnick KA,
Whittam TS,
( 2000 ) Gene conservation and loss in the mutS-rpoS genomic region of pathogenic Escherichia coli. PMID : 10986240 : DOI : 10.1128/jb.182.19.5381-5390.2000 PMC : PMC110980 Abstract >>
The extent and nature of DNA polymorphism in the mutS-rpoS region of the Escherichia coli genome were assessed in 21 strains of enteropathogenic E. coli (EPEC) and enterohemorrhagic E. coli (EHEC) and in 6 strains originally isolated from natural populations. The intervening region between mutS and rpoS was amplified by long-range PCR, and the resulting amplicons varied substantially in length (7.8 to 14.2 kb) among pathogenic groups. Restriction maps based on five enzymes and sequence analysis showed that strains of the EPEC 1, EPEC 2, and EHEC 2 groups have a long mutS-rpoS region composed of a approximately 6.0-kb DNA segment found in strain K-12 and a novel DNA segment (approximately 2.9 kb) located at the 3' end of rpoS. The novel segment contains three genes (yclC, pad1, and slyA) that occur in E. coli O157:H7 and related strains but are not found in K-12 or members of the ECOR group A. Phylogenetic analysis of the common sequences indicates that the long intergenic region is ancestral and at least two separate deletion events gave rise to the shorter regions characteristic of the E. coli O157:H7 and K-12 lineages.
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295. |
Rothemund D,
Wang L,
( 2000 ) Sequence diversity of the Escherichia coli H7 fliC genes: implication for a DNA-based typing scheme for E. coli O157:H7. PMID : 10790100 : PMC : PMC86588 Abstract >>
Flagellar (H) antigens are mostly encoded by genes at the fliC locus in E. coli. We have sequenced 11 H7 fliC genes from Escherichia coli strains that belong to seven O serotypes. These sequences, together with those of nine other H7 fliC genes (from strains of three different O serotypes) sequenced recently (S. D. Reid, R. K. Selander, and T. S. Whittam, J. Bacteriol. 181:153-160, 1999), include 10 different sequences. The differences between these 10 sequences range from 0.06 to 3.12%. By comparison with other E. coli flagellin genes, we have identified primer length sequences specific for H7 genes in general and others specific for H7 genes of O157 and O55 strains: the specificity was confirmed by PCR testing the type strains for all 53 E. coli H types. We have previously identified genes specific for the E. coli O157 antigen, and use of the combination of O157- and H7-specific primers allows the sensitive and rapid detection of O157:H7 E. coli strains, which cause the majority of hemorrhagic colitis cases.
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296. |
Brée A,
Fairbrother JM,
Dho-Moulin M,
Dozois CM,
( 2000 ) Relationship between the Tsh autotransporter and pathogenicity of avian Escherichia coli and localization and analysis of the Tsh genetic region. PMID : 10858231 : DOI : 10.1128/iai.68.7.4145-4154.2000 PMC : PMC101714 Abstract >>
The temperature-sensitive hemagglutinin Tsh is a member of the autotransporter group of proteins and was first identified in avian-pathogenic Escherichia coli (APEC) strain chi7122. The prevalence of tsh was investigated in 300 E. coli isolates of avian origin and characterized for virulence in a 1-day-old chick lethality test. Results indicate that among the tsh-positive APEC isolates, 90.6% belonged to the highest virulence class. Experimental inoculation of chickens with chi7122 and an isogenic tsh mutant demonstrated that Tsh may contribute to the development of lesions within the air sacs of birds but is not required for subsequent generalized infection manifesting as perihepatitis, pericarditis, and septicemia. Conjugation and hybridization experiments revealed that the tsh gene is located on a ColV-type plasmid in many of the APEC strains studied, including strain chi7122, near the colicin V genes in most of these strains. DNA sequences flanking the tsh gene of strain chi7122 include complete and partial insertion sequences and phage-related DNA sequences, some of which were also found on virulence plasmids and pathogenicity islands present in various E. coli pathotypes and other pathogenic members of the Enterobacteriaceae. These results demonstrate that the tsh gene is frequently located on the ColV virulence plasmid in APEC and suggest a possible role of Tsh in the pathogenicity of E. coli for chickens in the early stages of infection.
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297. |
Seah JN,
Kwang J,
( N/A ) Identification of H-specific determinants in flagellin of four Escherichia coli strains. PMID : 10985739 : Abstract >>
The H serogroup of Escherichia coli is determined by the flagellar antigen, flagellin. Sequence analysis of the flagellin gene, fliC, reveals a central variable region and the highly conserved N- and C-termini. This variable region has been shown to encode both H-specific and cross-reactive epitopes. Using polyclonal antibodies, we mapped the linear H-specific determinants in flagellin from four E. coli serotypes O157:H10, 0138:H14, O157:H42 and O157:H43. The specificity of all potential fragments was verified with 52 ECRC (Escherichia coli Reference Center) H-specific antisera. Our results indicated that: (a) a specific determinant of H10 flagellin (1263 bp long) maps to the region covering amino acid residues 305-331; (b) a specific determinant of H14 flagellin (1653 bp long) maps to the region covering amino acid residues 430-461; (c) a specific determinant of H42 flagellin (1281 bp long) maps to a region covering amino acid residues 171-201; and (d) a specific determinant of H43 flagellin (1506 bp long) maps to a region covering amino acid residues 200-260.
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298. |
Leflon-Guibout V,
Speldooren V,
Heym B,
Nicolas-Chanoine M,
( 2000 ) Epidemiological survey of amoxicillin-clavulanate resistance and corresponding molecular mechanisms in Escherichia coli isolates in France: new genetic features of bla(TEM) genes. PMID : 10991849 : DOI : 10.1128/aac.44.10.2709-2714.2000 PMC : PMC90140 Abstract >>
Amoxicillin-clavulanate resistance (MIC >16 microg/ml) and the corresponding molecular mechanisms were prospectively studied in Escherichia coli over a 3-year period (1996 to 1998) in 14 French hospitals. The overall frequency of resistant E. coli isolates remained stable at about 5% over this period. The highest frequency of resistant isolates (10 to 15%) was observed, independently of the year, among E. coli isolated from lower respiratory tract samples, and the isolation rate of resistant strains was significantly higher in surgical wards than in medical wards in 1998 (7.8 versus 2.8%). The two most frequent mechanisms of resistance for the 3 years were the hyperproduction of the chromosomal class C beta-lactamase (48, 38.4, and 39.7%) and the production of inhibitor-resistant TEM (IRT) enzymes (30.4, 37.2, and 41.2%). By using the single-strand conformational polymorphism-PCR technique and sequencing methods, we determined that 59 IRT enzymes corresponded to previously described IRT enzymes whereas 8 were new. Three of these new enzymes derived from TEM-1 by only one amino acid substitution (Ser130Gly, Arg244Gly, and Asn276Asp), whereas three others derived by two amino acid substitutions (Met69Leu and Arg244Ser, Met69Leu and Ile127Val, and Met69Val and Arg275Gln). The two remaining new IRTs showed three amino acid substitutions (Met69Val, Trp165Arg, and Asn276Asp and Met69Ile, Trp165Cys, and Arg275Gln). New genetic features were also found in bla(TEM) genes, namely, bla(TEM-1B) with either the promoters Pa and Pb, P4, or a promoter displaying a C-->G transversion at position 3 of the -35 consensus sequence and new bla(TEM) genes, notably one encoding TEM-1 but possessing the silent mutations originally described in bla(TEM-2) and then in some bla(TEM)-encoding IRT enzymes.
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299. |
Fang M,
Majumder A,
Tsai KJ,
Wu HY,
( 2000 ) ppGpp-dependent leuO expression in bacteria under stress. PMID : 11006083 : DOI : 10.1006/bbrc.2000.3440 Abstract >>
Despite the known potential transcription regulatory role of leuO gene product, LeuO, the condition when leuO expresses during bacterial growth cycle remains unclear. Mechanistically, leuO expression was shown to be part of promoter relay mechanism, however, the factor(s) responsible for the regulation of leuO expression is not known. Combining Northern and Western results, we demonstrate in the present communication that leuO expression is normally low and enhanced when bacteria are in transition from exponential growth to stationary phase. The stationary phase-associated leuO expression is ppGpp dependent and rpoS (sigma(s) factor) independent.
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300. |
Dychinco B,
Mazel D,
( 2000 ) Antibiotic resistance in the ECOR collection: integrons and identification of a novel aad gene. PMID : 10817710 : DOI : 10.1128/aac.44.6.1568-1574.2000 PMC : PMC89914 Abstract >>
The 72 Escherichia coli strains of the ECOR collection were examined for resistance to 10 different antimicrobial agents including ampicillin, tetracycline, mercury, trimethoprim, and sulfonamides. Eighteen strains were resistant to at least one of the antibiotics tested, and nearly 20% (14 of 72) were resistant to two or more. Several of the resistance determinants were shown to be carried on conjugative elements. The collection was screened for the presence of the three classes of integrons and for the sul1 gene, which is generally associated with class 1 integrons. The four strains found to carry a class 1 integron also had Tn21-encoded mercury resistance. One of the integrons encoded a novel streptomycin resistance gene, aadA7, with an attC site (or 59-base element) nearly identical to the attC site associated with the qacF gene cassette found in In40 (M.-C. Ploy, P. Courvalin, and T. Lambert, Antimicrob. Agents Chemother. 42:2557-2563, 1998). The conservation of associated attC sites among unrelated resistance cassettes is similar to arrangements found in the Vibrio cholerae superintegrons (D. Mazel, B. Dychinco, V. A. Webb, and J. Davies, Science 280:605-608, 1998) and supports the hypothesis that resistance cassettes are picked up from superintegron pools and independently assembled from unrelated genes and related attC sites.
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301. |
Cloeckaert A,
Baucheron S,
Flaujac G,
Schwarz S,
Kehrenberg C,
Martel JL,
Chaslus-Dancla E,
( 2000 ) Plasmid-mediated florfenicol resistance encoded by the floR gene in Escherichia coli isolated from cattle. PMID : 10991873 : DOI : 10.1128/aac.44.10.2858-2860.2000 PMC : PMC90164 Abstract >>
A florfenicol resistance gene almost identical to floR of Salmonella enterica serovar Typhimurium DT104 was detected on 110- to 125-kb plasmids in Escherichia coli isolates of animal origin. Analysis of the floR gene flanking regions of one of the plasmids showed that they were different from those encountered in S. enterica serovar Typhimurium DT104.
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302. |
Garcia MI,
Jouve M,
Nataro JP,
Gounon P,
Le Bouguénec C,
( 2000 ) Characterization of the AfaD-like family of invasins encoded by pathogenic Escherichia coli associated with intestinal and extra-intestinal infections. PMID : 10981717 : DOI : 10.1016/s0014-5793(00)01898-6 Abstract >>
The afimbrial adhesive sheath, encoded by the afa-3 gene cluster, is composed of two proteins with different roles in bacterium-HeLa cell interactions. AfaE is required for adhesion and AfaD for internalization. In this study, we found that the AfaD invasin was structurally and functionally conserved among human afa-expressing strains, independently of AfaE subtype and clinical origin of the Escherichia coli isolate. The AggB protein from enteroaggregative E. coli was also found to be an AfaD-related invasin. These data suggest that AfaD is the prototype of a family of invasins encoded by adhesion-associated operons in pathogenic E. coli.
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303. |
Kurihara T,
Mihara H,
( 2000 ) Kinetic and mutational studies of three NifS homologs from Escherichia coli: mechanistic difference between L-cysteine desulfurase and L-selenocysteine lyase reactions. PMID : 10739946 : DOI : 10.1093/oxfordjournals.jbchem.a022641 Abstract >>
We have purified three NifS homologs from Escherichia coli, CSD, CsdB, and IscS, that appear to be involved in iron-sulfur cluster formation and/or the biosynthesis of selenophosphate. All three homologs catalyze the elimination of Se and S from L-selenocysteine and L-cysteine, respectively, to form L-alanine. These pyridoxal 5'-phosphate enzymes were inactivated by abortive transamination, yielding pyruvate and a pyridoxamine 5'-phosphate form of the enzyme. The enzymes showed non-Michaelis-Menten behavior for L-selenocysteine and L-cysteine. When pyruvate was added, they showed Michaelis-Menten behavior for L-selenocysteine but not for L-cysteine. Pyruvate significantly enhanced the activity of CSD toward L-selenocysteine. Surprisingly, the enzyme activity toward L-cysteine was not increased as much by pyruvate, suggesting the presence of different rate-limiting steps or reaction mechanisms for L-cysteine desulfurization and the degradation of L-selenocysteine. We substituted Ala for each of Cys358 in CSD, Cys364 in CsdB, and Cys328 in IscS, residues that correspond to the catalytically essential Cys325 of Azotobacter vinelandii NifS. The enzyme activity toward L-cysteine was almost completely abolished by the mutations, whereas the activity toward L-selenocysteine was much less affected. This indicates that the reaction mechanism of L-cysteine desulfurization is different from that of L-selenocysteine decomposition, and that the conserved cysteine residues play a critical role only in L-cysteine desulfurization.
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304. |
Ling G,
Delavari P,
Fasching C,
Low DA,
O'Bryan TT,
Johnson JR,
( 2000 ) Evidence of commonality between canine and human extraintestinal pathogenic Escherichia coli strains that express papG allele III. PMID : 10816481 : DOI : 10.1128/iai.68.6.3327-3336.2000 PMC : PMC97593 Abstract >>
Although dogs have been proposed as carriers of extraintestinal pathogenic Escherichia coli (ExPEC) with infectious potential for humans, presumed host species-specific differences between canine and human ExPEC strains have cast doubt on this hypothesis. The recent discovery that allele III of papG (the P fimbrial adhesin gene) predominates among human cystitis isolates and confers an adherence phenotype resembling that of canine ExPEC prompted the present reevaluation of the canine-human ExPEC connection. Sixteen paired pap-positive urine and rectal E. coli isolates from dogs with urinary tract infection were studied. papG (adhesin) and papA (pilin) allele type, agglutination phenotypes, virulence factor genotypes, and randomly amplified polymorphic DNA and pulsed-field gel electrophoresis fingerprints were analyzed and compared with those of human ExPEC controls. The 16 canine strains contained predominantly papG allele III. Agglutination phenotypes segregated strictly according to papG allele status and were homogeneous among strains with the same papG allele profile irrespective of their human versus canine origin. Canine and human PapG variant III peptide sequences were highly homologous, without host species-specific differences. The most prevalent canine papA allele was F48, a novel variant recently identified among human urosepsis isolates. In addition to pap, human ExPEC-associated virulence genes detected among the canine strains included sfa/focDE, sfaS, fyuA, hlyA, cnf1, cdtB, kpsMT-II and -III, rfc, traT, ompT, and a marker for a pathogenicity-associated island from archetypal human ExPEC strain CFT073. Molecular fingerprinting confirmed the fecal origin of all but one canine urine isolate and showed one pair of O6 canine urine and fecal isolates to be extremely similar to an O6 human urosepsis isolate with which they shared all other genotypic and phenotypic characteristics analyzed. These data demonstrate that canine ExPEC strains are similar to, and in some instances essentially indistinguishable from, human ExPEC strains, which implicates dogs and their feces as potential reservoirs of E. coli with infectious potential for humans.
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305. |
Binsztein N,
Pichel M,
( 2000 ) CS22, a novel human enterotoxigenic Escherichia coli adhesin, is related to CS15. PMID : 10816474 : DOI : 10.1128/iai.68.6.3280-3285.2000 PMC : PMC97580 Abstract >>
Enterotoxigenic Escherichia coli (ETEC) expresses a broad spectrum of O:H antigens. Serogroup O20 is one of the most prevalent among the ETEC strains lacking any of the defined colonization factors (CFs), in Argentina. An O20:H- strain, ARG-3, adhered to Caco-2 cells and exhibited a thermoregulated 15.7-kDa protein band upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). An antiserum against this protein inhibited ARG-3 adhesion to Caco-2 cells and bound to very thin fibrilla-like structures on the bacterial surface. A 15.7-kDa protein-defective mutant failed to adhere to Caco-2 cells and lacked immunogold-labeled surface structures. The N-terminal amino acid sequence of the structural subunit showed 95% homology to that of CS15 of ETEC (former antigen 8786) and 65% homology with fimbria SEF14 of Salmonella enterica serovar Enteritidis. Nevertheless, the molecular size of ARG-3 adhesin was different from that of CS15, as revealed by SDS-PAGE and mass spectrometry. Both proteins are immunologically related, yet not identical, since an antiserum against the 15.7-kDa protein reacted solely with ARG-3 after absorption with bacteria bearing CS15. Moreover, only under low stringency conditions could DNA from strain ARG-3 be amplified by PCR using primers derived from the nfaA sequence of CS15. Thus, from the DNA sequence obtained from the ARG-3 PCR product, it could be deduced that the subunit protein differed in 30 residues from that of CS15. ARG-3 adhesin was found in 60% of the O20:H- CF-negative ETEC strains from Argentina; however, it appeared restricted to this serotype. We propose the designation CS22 for the herein identified nonfimbrial adhesin of human ETEC.
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306. |
Kido N,
( 2000 ) A single amino acid substitution in a mannosyltransferase, WbdA, converts the Escherichia coli O9 polysaccharide into O9a: generation of a new O-serotype group. PMID : 10762260 : DOI : 10.1128/jb.182.9.2567-2573.2000 PMC : PMC111322 Abstract >>
wbdA is a mannosyltransferase gene that is involved in synthesis of the Escherichia coli O9a polysaccharide, a mannose homopolymer with a repeating unit of 2-alphaMan-1,2-alphaMan-1,3-alphaMan-1, 3-alphaMan-1. The equivalent structural O polysaccharide in the E. coli O9 and Klebsiella O3 strains is 2-alphaMan-1,2-alphaMan-1, 2-alphaMan-1,3-alphaMan-1,3-alphaMan-1, with an excess of one mannose in the 1,2 linkage. We have cloned wbdA genes from these O9 and O3 strains and shown by genetic and functional studies that wbdA is the only gene determining the O-polysaccharide structure of O9 or O9a. Based on functional analysis of chimeric genes and site-directed mutagenesis, we showed that a single amino acid substitution, C55R, in WbdA of E. coli O9 converts the O9 polysaccharide into O9a. DNA sequencing revealed the substitution to be conserved in other E. coli O9a strains. The reverse substitution, R55C, in WbdA of E. coli O9a resulted in lipopolysaccharide synthesis showing no ladder profile instead of the conversion of O9a to O9. This suggests that more than one amino acid substitution in WbdA is required for conversion from O9a to O9.
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307. |
Pfaff-McDonough SJ,
Horne SM,
( N/A ) Cloning and sequencing of the iss gene from a virulent avian Escherichia coli. PMID : 10737659 : Abstract >>
Control of colibacillosis is important to the poultry industry. We have found that the presence of a gene for increased serum survival, iss, is strongly correlated with Escherichia coli isolated from birds with colibacillosis. Therefore, the iss gene and its protein product, Iss, are potential targets for detection and control of avian colibacillosis. The iss gene was amplified from a virulent avian E. coli isolate and sequenced. The sequences of the gene and the predicted protein product were compared with those of iss from a human E. coli isolate and lambda bor. The iss gene from the avian E. coli isolate has 96.8% identity with the iss gene from the human E. coli isolate and 89.4% identity with lambda bor. The Iss protein from the avian isolate has 87% identity with Iss from the human isolate and 90% identity with Bor. The low identity between the two Iss proteins is because of a frame-shift in their respective coding sequences. In sum, iss from this avian E. coli isolate is very similar to iss from a human E. coli isolate, but because of a frameshift mutation in the coding sequence of iss from the human E. coli isolate, Iss proteins from avian and human E. coli isolates have only 87% identity. The strong association of iss with E. coli isolated from birds with colibacillosis, suggests that this sequence be studied for its value as a marker or target to be used in colibacillosis control.
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308. |
Shibata N,
Shibayama K,
Yagi T,
Kurokawa H,
( 2000 ) A new SHV-derived extended-spectrum beta-lactamase (SHV-24) that hydrolyzes ceftazidime through a single-amino-acid substitution (D179G) in the -loop. PMID : 10817740 : DOI : 10.1128/aac.44.6.1725-1727.2000 PMC : PMC89944 Abstract >>
A new SHV-derived extended-spectrum beta-lactamase (SHV-24) conferring high-level resistance to ceftazidime but not cefotaxime and cefazolin was identified in Japan. This enzyme was encoded by a transferable 150-kb plasmid from an Escherichia coli clinical isolate. The pI and K(m) for CAZ of this enzyme were 7.5 and 30 microM, respectively. SHV-24 was found to have a D179G substitution in the Omega-loop of the enzyme.
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309. |
Rosche TM,
Kim PD,
( 2000 ) Identification of a potential membrane-targeting region of the replication initiator protein (TrfA) of broad-host-range plasmid RK2. PMID : 10783300 : DOI : 10.1006/plas.2000.1467 Abstract >>
Plasmid RK2 codes for two species of the replication initiator protein TrfA (33 and 44 kDa). Both polypeptides are strongly associated with membrane fractions of Escherichia coli host cells (W. Firshein and P. Kim, Mol. Microbiol. 23, 1-10, 1997). We investigated the role of a 12-amino-acid hydrophobic region (HR) in the membrane association of TrfA. Epitope-tagged polypeptide fragments of TrfA that contained HR were expressed and found to be associated with membrane fractions. Site-directed mutagenesis of trfA revealed that changes of specific amino acids in HR can affect both TrfA association with the membrane and its ability to support replication of an RK2 oriV plasmid in vivo. These results are consistent with the hypothesis that membrane association of TrfA is functionally relevant and that the HR region of TrfA is involved in membrane association and DNA replication in vivo.
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310. |
Noguchi N,
Takada K,
Katayama J,
Emura A,
Sasatsu M,
( 2000 ) Regulation of transcription of the mph(A) gene for macrolide 2'-phosphotransferase I in Escherichia coli: characterization of the regulatory gene mphR(A). PMID : 10960087 : DOI : 10.1128/jb.182.18.5052-5058.2000 PMC : PMC94651 Abstract >>
The synthesis of macrolide 2'-phosphotransferase I [Mph(A)], which inactivates erythromycin, is inducible by erythromycin. The expression of high-level resistance to erythromycin requires the mph(A) and mrx genes, which encode Mph(A) and an unidentified protein, respectively. We have studied the mphR(A) gene, which regulates the inducible expression of mph(A). An analysis of the synthesis of Mph(A) in minicells and results of a complementation test indicated that mphR(A) is located downstream from mrx and that its product, MphR(A), represses the production of Mph(A). DNA sequencing indicated that the mph(A), mrx, and mphR(A) genes exist as a cluster that begins with mph(A) and that the deduced amino acid sequence of MphR(A) can adopt an alpha-helix-turn-alpha-helix structure. To study the regulation of gene expression by MphR(A), we performed Northern blotting and primer extension. A transcript of 2. 9 kb that corresponded to the transcript of mph(A) through mphR(A) was detected, and its level was elevated upon exposure of cells to erythromycin. Gel mobility shift assays and DNase I footprinting indicated that MphR(A) binds specifically to the promoter region of mph(A), and the amount of DNA shifted as a results of the binding of MphR(A) decreased as the concentration of erythromycin was increased. These results indicate that transcription of the mph(A)-mrx-mphR(A) operon is negatively regulated by the binding of a repressor protein, MphR(A), to the promoter of the mph(A) gene and is activated upon inhibition of binding of MphR(A) to the promoter in the presence of erythromycin.
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311. |
Hoffman JA,
Badger JL,
Zhang Y,
Huang SH,
Kim KS,
( 2000 ) Escherichia coli K1 aslA contributes to invasion of brain microvascular endothelial cells in vitro and in vivo. PMID : 10948126 : DOI : 10.1128/iai.68.9.5062-5067.2000 PMC : PMC101739 Abstract >>
Neonatal Escherichia coli meningitis remains a devastating disease, with unacceptably high morbidity and mortality despite advances in supportive care measures and bactericidal antibiotics. To further our ability to improve the outcome of affected neonates, a better understanding of the pathogenesis of the disease is necessary. To identify potential bacterial genes which contribute to E. coli invasion of the blood-brain barrier, a cerebrospinal fluid isolate of E. coli K1 was mutagenized with TnphoA. TnphoA mutant 27A-6 was found to have a significantly decreased ability to invade brain microvascular endothelial cells compared to the wild type. In vivo, 32% of the animals infected with mutant 27A-6 developed meningitis, compared to 82% of those infected with the parent strain, despite similar levels of bacteremia. The DNA flanking the TnphoA insertion in 27A-6 was cloned and sequenced and determined to be homologous to E. coli K-12 aslA (arylsulfatase-like gene). The deduced amino acid sequence of the E. coli K1 aslA gene product shows homology to a well-characterized arylsulfatase family of enzymes found in eukaryotes, as well as prokaryotes. Two additional aslA mutants were constructed by targeted gene disruption and internal gene deletion. Both of these mutants demonstrated decreased invasion phenotypes, similar to that of TnphoA mutant 27A-6. Complementation of the decreased-invasion phenotypes of these mutants was achieved when aslA was supplied in trans. This is the first demonstration that this locus contributes to invasion of the blood-brain barrier by E. coli K1.
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312. |
Lindler LE,
Fleckenstein JM,
( 2000 ) Identification of a gene within a pathogenicity island of enterotoxigenic Escherichia coli H10407 required for maximal secretion of the heat-labile enterotoxin. PMID : 10768971 : DOI : 10.1128/iai.68.5.2766-2774.2000 PMC : PMC97486 Abstract >>
Studies of the pathogenesis of enterotoxigenic Escherichia coli (ETEC) have largely centered on extrachromosomal determinants of virulence, in particular the plasmid-encoded heat-labile (LT) and heat-stable enterotoxins and the colonization factor antigens. ETEC causes illnesses that range from mild diarrhea to severe cholera-like disease. These differences in disease severity are not readily accounted for by our current understanding of ETEC pathogenesis. Here we demonstrate that Tia, a putative adhesin of ETEC H10407, is encoded on a large chromosomal element of approximately 46 kb that shares multiple features with previously described E. coli pathogenicity islands. Further analysis of the region downstream from tia revealed the presence of several candidate open reading frames (ORFs) in the same transcriptional orientation as tia. The putative proteins encoded by these ORFs bear multiple motifs associated with bacterial secretion apparatuses. An in-frame deletion in one candidate gene identified here as leoA (labile enterotoxin output) resulted in marked diminution of secretion of the LT enterotoxin and lack of fluid accumulation in a rabbit ileal loop model of infection. Although previous studies have suggested that E. coli lacks the capacity to secrete LT, our studies show that maximal release of LT from the periplasm of H10407 is dependent on one or more elements encoded on a pathogenicity island.
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313. |
Girardeau JP,
Bertin Y,
( 2000 ) Epidemiological study of pap genes among diarrheagenic or septicemic Escherichia coli strains producing CS31A and F17 adhesins and characterization of Pap(31A) fimbriae. PMID : 10747134 : PMC : PMC86476 Abstract >>
The association of the pap operon with the CS31A and F17 adhesins was studied with 255 Escherichia coli strains isolated from calves, lambs, or humans with diarrhea. The three classes of PapG adhesin with different receptor binding preferences were also screened. The pap operon was associated with 50 and 36% of human strains that produced CS31A and ovine strains that produced F17, respectively. Among the bovine isolates, the pap operon was detected in 61% of the CS31A-positive isolates and 72% of the strains that produce both CS31A and F17. The class II adhesin gene was present in bovine (20%) and ovine (71%) isolates. Both class II and III adhesins were genetically associated with 36% of the human strains. The highest prevalence of the pap operon was observed among E. coli strains that produce additional adhesins involved in the binding of bacteria to intestinal cells. Among the bovine isolates, the reference strain for CS31A and F17c was found to be positive for the pap operon. Phenotypic and genotypic characterizations were undertaken. Pap(31A) appeared as fine and flexible fimbriae surrounding the bacteria but did not mediate adhesion to calf intestinal villi. Pap(31A) production was optimal with bacteria cultured on minimal growth media and repressed by addition of exogenous leucine. The deduced amino acid sequence of the PapA(31A) structural subunit showed 57 to 97% identity with the different P-related structural subunits produced by E. coli strains isolated from pigs with septicemia or humans with urinary tract infections. None of the three papG allelic variants was detected, but a homologous papG gene was present in the chromosome of strain 31A.
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314. |
Armstrong GD,
Clark CG,
Boodhoo A,
Pannu NS,
Ling H,
( 2000 ) A mutant Shiga-like toxin IIe bound to its receptor Gb(3): structure of a group II Shiga-like toxin with altered binding specificity. PMID : 10745005 : Abstract >>
Shiga-like toxins (SLTs) are produced by the pathogenic strains of Escherichia coli that cause hemorrhagic colitis and hemolytic uremic syndrome. These diseases in humans are generally associated with group II family members (SLT-II and SLT-IIc), whereas SLT-IIe (pig edema toxin) is central to edema disease of swine. The pentameric B-subunit component of the majority of family members binds to the cell-surface glycolipid globotriaosyl ceramide (Gb(3)), but globotetraosyl ceramide (Gb(4)) is the preferred receptor for SLT-IIe. A double-mutant of the SLT-IIe B subunit that reverses two sequence differences from SLT-II (GT3; Gln65-->Glu, Lys67-->Gln, SLT-I numbering) has been shown to bind more strongly to Gb(3) than to Gb(4). To understand the molecular basis of receptor binding and specificity, we have determined the structure of the GT3 mutant B pentamer, both in complex with a Gb(3) analogue (2.0 A resolution; R = 0.155, R(free) = 0.194) and in its native form (2.35 A resolution; R = 0.187, R(free) = 0.232). These are the first structures of a member of the medically important group II Shiga-like toxins to be reported. The structures confirm the previous observation of multiple binding sites on each SLT monomer, although binding site 3 is not occupied in the GT3 structure. Analysis of the binding properties of mutants suggests that site 3 is a secondary Gb(4)-binding site. The two mutated residues are located appropriately to interact with the extra betaGalNAc residue on Gb(4). Differences in the binding sites provide a molecular basis for understanding the tissue specificities and pathogenic mechanisms of members of the SLT family.
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315. |
Mainil J,
Charlier G,
Raymond I,
Pohl P,
Boullier S,
Nougayrède JP,
Marchès O,
( 2000 ) Role of tir and intimin in the virulence of rabbit enteropathogenic Escherichia coli serotype O103:H2. PMID : 10722617 : DOI : 10.1128/iai.68.4.2171-2182.2000 PMC : PMC97401 Abstract >>
Attaching and effacing (A/E) rabbit enteropathogenic Escherichia coli (REPEC) strains belonging to serogroup O103 are an important cause of diarrhea in weaned rabbits. Like human EPEC strains, they possess the locus of enterocyte effacement clustering the genes involved in the formation of the A/E lesions. In addition, pathogenic REPEC O103 strains produce an Esp-dependent but Eae (intimin)-independent alteration of the host cell cytoskeleton characterized by the formation of focal adhesion complexes and the reorganization of the actin cytoskeleton into bundles of stress fibers. To investigate the role of intimin and its translocated coreceptor (Tir) in the pathogenicity of REPEC, we have used a newly constructed isogenic tir null mutant together with a previously described eae null mutant. When human HeLa epithelial cells were infected, the tir mutant was still able to induce the formation of stress fibers as previously reported for the eae null mutant. When the rabbit epithelial cell line RK13 was used, REPEC O103 produced a classical fluorescent actin staining (FAS) effect, whereas both the eae and tir mutants were FAS negative. In a rabbit ligated ileal loop model, neither mutant was able to induce A/E lesions. In contrast to the parental strain, which intimately adhered to the enterocytes and destroyed the brush border microvilli, bacteria of both mutants were clustered in the mucus without reaching and damaging the microvilli. The role of intimin and Tir was then analyzed in vivo by oral inoculation of weaned rabbits. Although both mutants were still present in the intestinal flora of the rabbits 3 weeks after oral inoculation, neither mutant strain induced any clinical signs or significant weight loss in the inoculated rabbits whereas the parental strain caused the death of 90% of the inoculated rabbits. Nevertheless, an inflammatory infiltrate was present in the lamina propria of the rabbits infected with both mutants, with an inflammatory response greater for the eae null mutant. In conclusion, we have confirmed the role of intimin in virulence, and we have shown, for the first time, that Tir is also a key factor in vivo for pathogenicity.
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316. |
Xun L,
( 2000 ) Characterization of 4-hydroxyphenylacetate 3-hydroxylase (HpaB) of Escherichia coli as a reduced flavin adenine dinucleotide-utilizing monooxygenase. PMID : 10653707 : DOI : 10.1128/aem.66.2.481-486.2000 PMC : PMC91852 Abstract >>
4-Hydroxyphenylacetate 3-hydroxylase (HpaB and HpaC) of Escherichia coli W has been reported as a two-component flavin adenine dinucleotide (FAD)-dependent monooxygenase that attacks a broad spectrum of phenolic compounds. However, the function of each component in catalysis is unclear. The large component (HpaB) was demonstrated here to be a reduced FAD (FADH(2))-utilizing monooxygenase. When an E. coli flavin reductase (Fre) having no apparent homology with HpaC was used to generate FADH(2) in vitro, HpaB was able to use FADH(2) and O(2) for the oxidation of 4-hydroxyphenylacetate. HpaB also used chemically produced FADH(2) for 4-hydroxyphenylacetate oxidation, further demonstrating that HpaB is an FADH(2)-utilizing monooxygenase. FADH(2) generated by Fre was rapidly oxidized by O(2) to form H(2)O(2) in the absence of HpaB. When HpaB was included in the reaction mixture without 4-hydroxyphenylacetate, HpaB bound FADH(2) and transitorily protected it from rapid autoxidation by O(2). When 4-hydroxyphenylacetate was also present, HpaB effectively competed with O(2) for FADH(2) utilization, leading to 4-hydroxyphenylacetate oxidation. With sufficient amounts of HpaB in the reaction mixture, FADH(2) produced by Fre was mainly used by HpaB for the oxidation of 4-hydroxyphenylacetate. At low HpaB concentrations, most FADH(2) was autoxidized by O(2), causing uncoupling. However, the coupling of the two enzymes' activities was increased by lowering FAD concentrations in the reaction mixture. A database search revealed that HpaB had sequence similarities to several proteins and gene products involved in biosynthesis and biodegradation in both bacteria and archaea. This is the first report of an FADH(2)-utilizing monooxygenase that uses FADH(2) as a substrate rather than as a cofactor.
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317. |
Lindner H,
Glatter O,
Oberer M,
( 1999 ) Thermodynamic properties and DNA binding of the ParD protein from the broad host-range plasmid RK2/RP4 killing system. PMID : 10661868 : DOI : 10.1515/BC.1999.181 Abstract >>
ParD is a small, acidic protein from the partitioning system of the plasmid RK2/RP4. The ParD protein exhibits specific DNA binding activity and, as the antidote component of a toxin-antidote plasmid addiction system, ParD forms a tight complex in solution with its toxin antagonist, the ParE protein. Unopposed ParE acts as a toxin that causes growth retardation and killing of plasmid cured cells. ParD negatively autoregulates its expression by binding to an operator sequence in the parDE promoter region. This DNA binding activity is crucial for the regulation of the relative abundance of toxin and antidote which ultimately determines life or death for the bacterial host and its daughter cells. In light scattering studies and gel filtration chromatography we observed the existence of a stable dimer of ParD in solution. The stoichiometry of ParD-DNA complex formation appeared to be 4:1, the molecular mass of the complex was 72.1 kDa. The alpha-helical content of ParD as determined by CD-spectrometry was 35%. The protein exhibited high thermostability with a T(M) of 64 degrees C and deltaH of 25 kcal/mol as shown by differential scanning calorimetry. Upon complex formation the T(M) increased by 10 degrees C. The thermal unfolding of the ParD protein was highly reversible as observed in repeated DSC scans of the same sample. The recovery of the native fold was proven by CD-spectroscopy.
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318. |
Lopata M,
Biery MC,
( 2000 ) A minimal system for Tn7 transposition: the transposon-encoded proteins TnsA and TnsB can execute DNA breakage and joining reactions that generate circularized Tn7 species. PMID : 10704304 : DOI : 10.1006/jmbi.2000.3558 Abstract >>
In the presence of ATP and Mg(2+), the bacterial transposon Tn7 translocates via a cut and paste mechanism executed by the transposon-encoded proteins TnsA+TnsB+TnsC+TnsD. We report here that in the presence of Mn(2+), TnsA+TnsB alone can execute the DNA breakage and joining reactions of Tn7 recombination. ATP is not essential in this minimal system, revealing that this cofactor is not directly involved in the chemical steps of recombination. In both the TnsAB and TnsABC+D systems, recombination initiates with double-strand breaks at each transposon end that cut Tn7 away from flanking donor DNA. In the minimal system, breakage occurs predominantly at a single transposon end and the subsequent end-joining reactions are intramolecular, with the exposed 3' termini of a broken transposon end joining near the other end of the Tn7 element in the same donor molecule to form circular transposon species. In contrast, in TnsABC+D recombination, breaks occur at both ends of Tn7 and the two ends join to a target site on a different DNA molecule to form an intermolecular simple insertion. This demonstration of the capacity of TnsAB to execute breakage and joining reactions supports the view that these proteins form the Tn7 transposase.
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319. |
McNamara BP,
Lai LC,
Malstrom C,
Scaletsky IC,
Klapproth JM,
( 2000 ) A large toxin from pathogenic Escherichia coli strains that inhibits lymphocyte activation. PMID : 10722613 : DOI : 10.1128/iai.68.4.2148-2155.2000 PMC : PMC97397 Abstract >>
The mechanisms by which bacteria resist cell-mediated immune responses to cause chronic infections are largely unknown. We report the identification of a large gene present in enteropathogenic strains of Escherichia coli (EPEC) that encodes a toxin that specifically inhibits lymphocyte proliferation and interleukin-2 (IL-2), IL-4, and gamma interferon production in response to a variety of stimuli. Lymphostatin, the product of this gene, is predicted to be 366 kDa and shares significant homology with the catalytic domains of the large clostridial cytotoxins. A mutant EPEC strain that has a disruption in this gene lacks the ability to inhibit lymphokine production and lymphocyte proliferation. Enterohemorrhagic E. coli strains of serotype O157:H7 possess a similar gene located on a large plasmid. Loss of the plasmid is associated with loss of the ability to inhibit IL-2 expression while transfer of the plasmid to a nonpathogenic strain of E. coli is associated with gain of this activity. Among 89 strains of E. coli and related bacteria tested, lifA sequences were detected exclusively in strains capable of attaching and effacing activity. Lymphostatin represents a new class of large bacterial toxins that blocks lymphocyte activation.
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320. |
Miyahara M,
( 1999 ) Escherichia coli O157 strains which caused Japanese outbreaks have residues of bacteriophage sequences. PMID : 10746172 : DOI : 10.1248/bpb.22.1372 Abstract >>
Twelve strains of Escherichia coli O157 which caused outbreaks in Japan were used as DNA sources. The sequences of the gene encoding the Shiga toxin 2 in all 12 strains were almost identical and the sequences downstream of this gene were similar to that of bacteriophage 933W.
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321. |
Aguilar C,
Ayala G,
Estrada MA,
Silva J,
( 2000 ) TLA-1: a new plasmid-mediated extended-spectrum beta-lactamase from Escherichia coli. PMID : 10722503 : DOI : 10.1128/aac.44.4.997-1003.2000 PMC : PMC89804 Abstract >>
Escherichia coli R170, isolated from the urine of an infected patient, was resistant to expanded-spectrum cephalosporins, aztreonam, ciprofloxacin, and ofloxacin but was susceptible to amikacin, cefotetan, and imipenem. This particular strain contained three different plasmids that encoded two beta-lactamases with pIs of 7.0 and 9.0. Resistance to cefotaxime, ceftazidime, aztreonam, trimethoprim, and sulfamethoxazole was transferred by conjugation from E. coli R170 to E. coli J53-2. The transferred plasmid, RZA92, which encoded a single beta-lactamase, was 150 kb in length. The cefotaxime resistance gene that encodes the TLA-1 beta-lactamase (pI 9.0) was cloned from the transconjugant by transformation to E. coli DH5alpha. Sequencing of the bla(TLA-1) gene revealed an open reading frame of 906 bp, which corresponded to 301 amino acid residues, including motifs common to class A beta-lactamases: (70)SXXK, (130)SDN, and (234)KTG. The amino acid sequence of TLA-1 shared 50% identity with the CME-1 chromosomal class A beta-lactamase from Chryseobacterium (Flavobacterium) meningosepticum; 48.8% identity with the VEB-1 class A beta-lactamase from E. coli; 40 to 42% identity with CblA of Bacteroides uniformis, PER-1 of Pseudomonas aeruginosa, and PER-2 of Salmonella typhimurium; and 39% identity with CepA of Bacteroides fragilis. The partially purified TLA-1 beta-lactamase had a molecular mass of 31.4 kDa and a pI of 9.0 and preferentially hydrolyzed cephaloridine, cefotaxime, cephalothin, benzylpenicillin, and ceftazidime. The enzyme was markedly inhibited by sulbactam, tazobactam, and clavulanic acid. TLA-1 is a new extended-spectrum beta-lactamase of Ambler class A.
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322. |
Nakano M,
Nair GB,
Terai A,
Yamamoto S,
Kurazono H,
( 2000 ) Characterization of a putative virulence island in the chromosome of uropathogenic Escherichia coli possessing a gene encoding a uropathogenic-specific protein. PMID : 10702359 : DOI : 10.1006/mpat.1999.0331 Abstract >>
This study was initiated to search for a homologue of the Vibrio cholerae zot gene in uropathogenic Escherichia coli (UPEC) using a specific DNA probe. The faint signal obtained at low stringency with some UPEC strains associated with prostatitis cases prompted us to examine UPEC strains by PCR using primers designed from the conserved regions of the proteins of the Zot group of putative NTPases containing the classical NTP binding motif. This led to the discovery of a DNA fragment in UPEC strains which hybridized with a probe designed from the PCR. Further analysis of this DNA fragment revealed an ORF which was designated as uropathogenic specific protein (Usp). The gene encoding Usp was 1038 bp long and codes for 346 amino acids with an appropriate SD sequence. Upstream and downstream analysis of usp revealed motifs of prokaryotic consensus promoters and three small ORFs with SDs and ribosome binding sites transcribed in the same direction of usp. The proximity of these set of genes in a specific area of the bacterial chromosome resembling a block of genes preferentially associated with UPEC coupled with the presence of a motif matching that of a Tn3 transposon family lead us to believe that this could be an hitherto unknown pathogenicity island.
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323. |
Morabito S,
Caprioli A,
Scheef J,
Schmidt H,
( 2000 ) A new Shiga toxin 2 variant (Stx2f) from Escherichia coli isolated from pigeons. PMID : 10698793 : DOI : 10.1128/aem.66.3.1205-1208.2000 PMC : PMC91964 Abstract >>
We have isolated Shiga toxin (Stx)-producing Escherichia coli (STEC) strains from the feces of feral pigeons which contained a new Stx2 variant gene designated stx(2f). This gene is most similar to sltIIva of patient E. coli O128:B12 isolate H.I.8. Stx2f reacted only weakly with commercial immunoassays. The prevalence of STEC organisms carrying the stx(2f) gene in pigeon droppings was 12.5%. The occurrence of a new Stx2 variant in STEC from pigeons enlarges the pool of Stx2 variants and raises the question whether horizontal gene transfer to E. coli pathogenic to humans may occur.
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324. |
Kreuzinger N,
Kavka GG,
Grillenberger S,
Farnleitner AH,
( 2000 ) Simultaneous detection and differentiation of Escherichia coli populations from environmental freshwaters by means of sequence variations in a fragment of the beta-D-glucuronidase gene. PMID : 10742209 : DOI : 10.1128/aem.66.4.1340-1346.2000 PMC : PMC91990 Abstract >>
A PCR-based denaturing-gradient gel electrophoresis (DGGE) approach was applied to a partial sequence of the beta-D-glucuronidase gene (uidA) for specific detection and differentiation of Escherichia coli populations according to their uidA sequence variations. Detection of sequence variations by PCR-DGGE and by PCR with direct sequencing correlated perfectly. Screening of 50 E. coli freshwater isolates and reference strains revealed 11 sequence types, showing nine polymorphic sites and an average number of pairwise differences between alleles of the uidA gene fragments (screened fragment length, 126 bp) of 2.3%. Among the analyzed strains a range of dominating to more rarely and/or uniquely observed E. coli sequence types was revealed. PCR-DGGE applied to fecally polluted river water samples simultaneously detected E. coli and generated a fingerprint of the mixed populations by separating the polymorphic uidA amplicons. No significant differences between non-cultivation-based and cultivation-based profiles were observed, suggesting that at least some members of all occurring sequence types could be cultivated. As E. coli is frequently used as a fecal indicator, this work is considered an important step towards a new, practical tool for the differentiation and tracing of fecal pollution in all kinds of waters.
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325. |
Kim PD,
( 2000 ) Isolation of an inner membrane-derived subfraction that supports in vitro replication of a mini-RK2 plasmid in Escherichia coli. PMID : 10692384 : DOI : 10.1128/jb.182.6.1757-1760.2000 PMC : PMC94476 Abstract >>
Previous results have demonstrated that the inner, but not the outer, membrane fraction of Escherichia coli is the site of membrane-associated DNA replication of plasmid RK2, a broad-host-range plasmid capable of replication in a wide variety of gram-negative hosts (K. Michaels, J. Mei, and W. Firshein, Plasmid 32:19-31, 1994). To resolve the inner membrane replication site further, the procedure of Ishidate et al. (K. Ishidate, E. S. Creeger, J. Zrike, S. Deb, G. Glauner, T. J. MacAlister, and L. I. Rothfield, J. Biol. Chem. 261:428-443, 1986) was used to separate the inner membrane into a number of subfractions, of which only one, a small subfraction containing only 10% of the entire membrane, was found to synthesize DNA inhibited by antibody prepared against the plasmid-encoded initiation protein TrfA. This is the same subfraction that was also found to bind oriV and TrfA to the greatest extent in filter binding assays (J. Mei, S. Benashski, and W. Firshein, J. Bacteriol. 177:6766-6772, 1995).
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326. |
Thomson CJ,
Adrian PV,
( 2000 ) New gene cassettes for trimethoprim resistance, dfr13, and Streptomycin-spectinomycin resistance, aadA4, inserted on a class 1 integron. PMID : 10639362 : DOI : 10.1128/aac.44.2.355-361.2000 PMC : PMC89683 Abstract >>
In a previous survey of 357 trimethoprim-resistant isolates of aerobic gram-negative bacteria from commensal fecal flora, hybridization experiments showed that 25% (90 of 357) of the isolates failed to hybridize to specific oligonucleotide probes for dihydrofolate reductase types 1, 2b, 3, 5, 6, 7, 8, 9, 10, and 12. Subsequent cloning and sequencing of a plasmid-borne trimethoprim resistance gene from one of these isolates revealed a new dihydrofolate reductase gene, dfr13, which occurred as a cassette integrated in a site-specific manner in a class 1 integron. The gene product shared 84% amino acid identity with dfr12 and exhibited a trimethoprim inhibition profile similar to that of dfr12. Gene probing experiments with an oligonucleotide probe specific for this gene showed that 12.3% (44 of 357) of the isolates which did not hybridize to probes for other dihydrofolate reductases hybridized to this probe. Immediately downstream of dfr13, a new cassette, an aminoglycoside resistance gene of the class AADA ?ANT(3")(9)-I, which encodes streptomycin-spectinomycin resistance, was identified. This gene shares 57% identity with the consensus aadA1 (ant(3")-Ia) and has been called aadA4 (ant(3")-Id). The 3' end of the aadA4 cassette was truncated by IS26, which was contiguous with a truncated form of Tn3. On the same plasmid, pUK2381, a second copy of IS26 was associated with sul2, which suggests that both integrase and transposase activities have played major roles in the arrangement and dissemination of antibiotic resistance genes dfr13, aadA4, bla(TEM-1), and sul2.
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327. |
Schmidt H,
Morabito S,
Karch H,
Oswald E,
( 2000 ) Typing of intimin genes in human and animal enterohemorrhagic and enteropathogenic Escherichia coli: characterization of a new intimin variant. PMID : 10603369 : DOI : 10.1128/iai.68.1.64-71.2000 PMC : PMC97102 Abstract >>
Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) produce the characteristic "attaching and effacing" (A/E) lesion of the brush border. Intimin, an outer membrane protein encoded by eae, is responsible for the tight association of both pathogens with the host cell. Several eae have been cloned from different EPEC and EHEC strains isolated from humans and animals. These sequences are conserved in the N-terminal region but highly variable in the last C-terminal 280 amino acids (aa), where the cell binding activity is localized. Based on these considerations, we developed a panel of specific primers to investigate the eae heterogeneity of the variable 3' region by using PCR amplification. We then investigated the distribution of the known intimin types in a large collection of EPEC and EHEC strains isolated from humans and different animal species. The existence of a yet-unknown family of intimin was suspected because several EHEC strains, isolated from human and cattle, did not react with any of the specific primer pairs, although these strains were eae positive when primers amplifying the conserved 5' end were used. We then cloned and sequenced the eae present in one of these strains (EHEC of serotype O103:H2) and subsequently designed a PCR primer that recognizes in a specific manner the variable 3' region of this new intimin type. This intimin, referred to as "epsilon," was present in human and bovine EHEC strains of serogroups O8, O11, O45, O103, O121, and O165. Intimin epsilon is the largest intimin cloned to date (948 aa) and shares the greatest overall sequence identity with intimin beta, although analysis of the last C-terminal 280 aa suggests a greater similarity with intimins alpha and gamma.
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328. |
Kuhar I,
( 1999 ) Transcription regulation of the colicin K cka gene reveals induction of colicin synthesis by differential responses to environmental signals. PMID : 10572143 : PMC : PMC103702 Abstract >>
Colicin-producing strains occur frequently in natural populations of Escherichia coli, and colicinogenicity seems to provide a competitive advantage in the natural habitat. A cka-lacZ fusion was used to study the regulation of expression of the colicin K structural gene. Expression is growth phase dependent, with high activity in the late stationary phase. Nutrient depletion induces the expression of cka due to an increase in ppGpp. Temperature is a strong signal for cka expression, since only basal-level activity was detected at 22 degrees C. Mitomycin C induction demonstrates that cka expression is regulated to a lesser extent by the SOS response independently of ppGpp. Increased osmolarity induces a partial increase, while the global regulator integration host factor inhibits expression in the late stationary phase. Induction of cka was demonstrated to be independent of the cyclic AMP-Crp complex, carbon source, RpoS, Lrp, H-NS, pH, and short-chain fatty acids. In contrast to colicin E1, cka expression is independent of catabolite repression and is partially affected by anaerobiosis only upon SOS induction. These results indicate that while different colicins are expressed in response to some common signals such as nutrient depletion, the expression of individual colicins could be further influenced by specific environmental cues.
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329. |
Sandvang D,
( 1999 ) Novel streptomycin and spectinomycin resistance gene as a gene cassette within a class 1 integron isolated from Escherichia coli. PMID : 10582907 : PMC : PMC89612 Abstract >>
The aadA genes, encoding resistance to streptomycin and spectinomycin, have been found as gene cassettes in different gram-negative and gram-positive bacterial species. The present study has revealed the sequence of a new gene, aadA5, integrated as a gene cassette together with the trimethoprim resistance gene dfr7 in a class 1 integron. The integron was located on a plasmid and was identified in a pathogenic porcine Escherichia coli isolate.
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330. |
Li B,
LeClerc JE,
( 1999 ) Promiscuous origin of a chimeric sequence in the Escherichia coli O157:H7 genome. PMID : 10601221 : PMC : PMC94221 Abstract >>
A novel sequence of 2.9 kb in the intergenic region between the mutS and rpoS genes of Escherichia coli O157:H7 and closely related strains replaces a sequence of 6.1 kb in E. coli K-12 strains. At the same locus in Shigella dysenteriae type 1, a sequence identical to that in O157:H7 is bounded by the IS1 insertion sequence element. Extensive polymorphism in the mutS-rpoS chromosomal region is indicative of horizontal transfer events.
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331. |
Bradford PA,
( 1999 ) Automated thermal cycling is superior to traditional methods for nucleotide sequencing of bla(SHV) genes. PMID : 10582889 : PMC : PMC89594 Abstract >>
Genes encoding SHV-1 and SHV-2 were sequenced by different methods. Nucleotide sequencing of the coding strand by standard dideoxy-chain termination methods resulted in errors in the interpretation of the nucleotide sequence and the derived amino acid sequence in two main regions which corresponded to nucleotide and amino acid changes that had been reported previously. The automated thermal cycling method was clearly superior and consistently resulted in the correct sequences for these genes.
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332. |
Sunde M,
( 1999 ) Characterization of integrons in Escherichia coli of the normal intestinal flora of swine. PMID : 10647086 : DOI : 10.1089/mdr.1999.5.279 Abstract >>
Multiresistant Escherichia coli isolates of the normal intestinal flora of healthy fattening pigs were examined for the presence of integron class 1 by XL (extra long) PCR. The class 1 integron was detected in 17 isolates originating from 14 healthy animals on seven different farms. One isolate contained two class 1 integrons. The inserted gene cassettes were characterized by DNA sequencing and PCR. The ant(3")-Ia gene responsible for resistance to streptomycin/spectinomycin was inserted in all integrons detected. Fifteen isolates contained this gene cassette as the only inserted cassette. Three isolates contained integrons with two gene cassettes. Two isolates contained integrons with the trimethoprim resistance gene dfr1 and one isolate contained the oxa1 beta-lactamase gene upstream to the ant(3")-Ia gene. Detection of these three different resistance gene cassettes in bacteria from swine shows that cassettes occurring in integrons in human clinical isolates also appear in bacteria of the normal intestinal flora of healthy swine. Two integron-harboring strains were obtained from each of three different animals. These strains were probably not clonal derivatives of each other, suggesting the existence of different multiresistance clones within the intestinal normal flora of one specific animal. The oxa1 nucleotide sequence found in E. coli from swine differ by seven nucleotides from the oxa1 nucleotide sequence of the gene from the R-plasmid RGN238. The fact that these two sequences are not identical might indicate that the two genes have evolved separately in different surroundings from the common ancestor. Transmissible plasmids of approximately 200 kb containing integron class I were detected in eight of the isolates when conjugation experiments were performed with E. coli DH5 as recipient strain. The transfer frequency ranged from 4x10(-4) to 6x10(-2) transconjugants per recipient cell. This study shows that the enteric commensals of domestic animals may be considered as a reservoir of integron-containing transmissible plasmids and gene cassettes that might be transferable to the pathogens of swine and to important zoonotic bacteria associated with the enteric flora of swine such as Salmonella typhimurium DT104.
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333. |
Grant TH,
Nicholls L,
( 2000 ) Identification of a novel genetic locus that is required for in vitro adhesion of a clinical isolate of enterohaemorrhagic Escherichia coli to epithelial cells. PMID : 10652089 : DOI : 10.1046/j.1365-2958.2000.01690.x Abstract >>
Enterohaemorrhagic Escherichia coli (EHEC) are food-borne intestinal pathogens with a low infectious dose. Adhesion of some EHEC strains to epithelial cells is attributed, in part, to intimin, but other factors may be required for the intestinal colonizing ability of these bacteria. In order to identify additional adherence factors of EHEC, we generated transposon mutants of a clinical EHEC isolate of serotype O111:H-, which displayed high levels of adherence to cultured Chinese hamster ovary (CHO) cells. One mutant was markedly deficient in CHO cell adherence, human red blood cell agglutination and autoaggregation. Sequence analysis of the gene disrupted in this mutant revealed a 9669 bp novel chromosomal open reading frame (ORF), which was designated efa1, for EHEC factor for adherence. efa1 displayed 28% amino acid identity with the predicted product of a recently described ORF from the haemolysin-encoding plasmid of EHEC O157:H7. The amino termini of the putative products of these two genes exhibit up to 38% amino acid similarity to Clostridium difficile toxins A and B. efa1 occurred within a novel genetic locus, at least 15 kb in length, which featured a low G+C content, several insertion sequence homologues and a homologue of the Shigella flexneri enterotoxin ShET2. DNA probes prepared from different regions of efa1 hybridized with all of 116 strains of attaching-effacing E. coli (AEEC) of a variety of serotypes, including enteropathogenic E. coli (EPEC) and EHEC, but with none of 91 non-AEEC strains. Nevertheless, efa1 was not required for the attachment-effacement phenotype, and the efa1 locus was not physically linked to the locus for enterocyte effacement (LEE) pathogenicity island, which is responsible for this phenotype in EPEC. These findings suggest that efa1 encodes a novel virulence-associated determinant of AEEC, which contributes to the adhesive capacity of these bacteria.
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334. |
McIver CJ,
White PA,
( 2000 ) Characterisation of two new gene cassettes, aadA5 and dfrA17. PMID : 10620677 : DOI : 10.1111/j.1574-6968.2000.tb08906.x Abstract >>
Escherichia coli INS33 was isolated from the urinary tract of an infected patient. It was resistant to ampicillin, chloramphenicol, spectinomycin, streptomycin, sulfafurazole, tetracycline and trimethoprim. PCR screening revealed the presence of a class 1 integron that harboured two new gene cassettes, designated dfrA17 and aadA5. The new dfrA17 cassette was 91% identical to the known dfrA7 cassette. The aadA5 cassette was 95% identical over the first 830 bp to aadA4, but lacked the IS26 element found at the 3' end of this truncated cassette. Cloning and expression of the cassette region demonstrated that dfrA17 conferred high level resistance to trimethoprim but aadA5 conferred resistance to spectinomycin but not to streptomycin.
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335. |
Wang Y,
( 2000 ) Effect of rpoS mutations on stress-resistance and invasion of brain microvascular endothelial cells in Escherichia coli K1. PMID : 10620673 : DOI : 10.1111/j.1574-6968.2000.tb08902.x Abstract >>
Escherichia coli K1 strains are predominant in causing neonatal meningitis. We have shown that invasion of brain microvascular endothelial cells (BMEC) is a prerequisite for E. coli K1 crossing of the blood-brain barrier. BMEC invasion by E. coli K1 strain RS218, however, has been shown to be significantly greater with stationary-phase cultures than with exponential-phase cultures. Since RpoS participates in regulating stationary-phase gene expression, the present study examined a possible involvement of RpoS in E. coli K1 invasion of BMEC. We found that the cerebrospinal fluid isolates of E. coli K1 strains RS218 and IHE3034 have a nonsense mutation in their rpoS gene. Complementation with the E. coli K12 rpoS gene significantly increased the BMEC invasion of E. coli K1 strain IHE3034, but failed to significantly increase the invasion of another E. coli K1 strain RS218. Of interest, the recovery of E. coli K1 strains following environmental insults was 10-100-fold greater on Columbia blood agar than on LB agar, indicating that growing medium is important for viability of rpoS mutants after environmental insults. Taken together, our data suggest that the growth-phase-dependent E. coli K1 invasion of BMEC is affected by RpoS and other growth-phase-dependent regulatory mechanisms.
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336. |
Russo TA,
Carlino UB,
Fasching C,
O'Bryan TT,
Scheutz F,
Stell AL,
Johnson JR,
( 2000 ) Analysis of the F antigen-specific papA alleles of extraintestinal pathogenic Escherichia coli using a novel multiplex PCR-based assay. PMID : 10678978 : DOI : 10.1128/iai.68.3.1587-1599.2000 PMC : PMC97319 Abstract >>
Polymorphisms in PapA, the major structural subunit and antigenic determinant of P fimbriae of extraintestinal pathogenic Escherichia coli, are of considerable epidemiological, phylogenetic, and immunotherapeutic importance. However, to date, no method other than DNA sequencing has been generally available for their detection. In the present study, we developed and rigorously validated a novel PCR-based assay for the 11 recognized variants of papA and then used the new assay to assess the prevalence, phylogenetic distribution, and bacteriological associations of the papA alleles among 75 E. coli isolates from patients with urosepsis. In comparison with conventional F serotyping, the assay was extremely sensitive and specific, evidence that papA sequences are highly conserved within each of the traditionally recognized F serotypes despite the diversity observed among F types. In certain strains, the assay detected serologically occult copies of papA, of which some were shown to represent false-negative serological results and others were shown to represent the presence of nonfunctional pap fragments. Among the urosepsis isolates, the assay revealed considerable segregation of papA alleles according to O:K:H serotype, consistent with vertical transmission within clones, but with exceptions which strongly suggested horizontal transfer of papA alleles between lineages. Sequencing of papA from two strains that were papA positive by probe and PCR but F negative in the new PCR assay led to the discovery of two novel papA variants, one of which was actually more prevalent among the urosepsis isolates than were several of the known papA alleles. These findings provide novel insights into the papA alleles of extraintestinal pathogenic E. coli and indicate that the F PCR assay represents a versatile new molecular tool for epidemiological and phylogenetic investigations which should make rapid, specific detection of papA alleles available to any laboratory with PCR capability.
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337. |
Schnappinger D,
Hillen W,
Orth P,
( 2000 ) Structural basis of gene regulation by the tetracycline inducible Tet repressor-operator system. PMID : 10700280 : DOI : 10.1038/73324 Abstract >>
The tetracycline repressor (TetR) regulates the most abundant resistance mechanism against the antibiotic tetracycline in grain-negative bacteria. The TetR protein and its mutants are commonly used as control elements to regulate gene expression in higher eukaryotes. We present the crystal structure of the TetR homodimer in complex with its palindromic DNA operator at 2.5 A resolution. Comparison to the structure of TetR in complex with the inducer tetracycline-Mg2+ allows the mechanism of induction to be deduced. Inducer binding in the repressor core initiates conformational changes starting with C-terminal unwinding and shifting of the short helix a6 in each monomer. This forces a pendulum-like motion of helix a4, which increases the separation of the attached DNA binding domains by 3 A, abolishing the affinity of TetR for its operator DNA.
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338. |
Díaz E,
Galán B,
( 2000 ) Functional analysis of the small component of the 4-hydroxyphenylacetate 3-monooxygenase of Escherichia coli W: a prototype of a new Flavin:NAD(P)H reductase subfamily. PMID : 10633095 : DOI : 10.1128/jb.182.3.627-636.2000 PMC : PMC94324 Abstract >>
Escherichia coli W uses the aromatic compound 4-hydroxyphenylacetate (4-HPA) as a sole source of carbon and energy for growth. The monooxygenase which converts 4-HPA into 3,4-dihydroxyphenylacetate, the first intermediate of the pathway, consists of two components, HpaB (58.7 kDa) and HpaC (18.6 kDa), encoded by the hpaB and hpaC genes, respectively, that form a single transcription unit. Overproduction of the small HpaC component in E. coli K-12 cells has facilitated the purification of the protein, which was revealed to be a homodimer that catalyzes the reduction of free flavins by NADH in preference to NADPH. Subsequently, the reduced flavins diffuse to the large HpaB component or to other electron acceptors such as cytochrome c and ferric ion. Amino acid sequence comparisons revealed that the HpaC reductase could be considered the prototype of a new subfamily of flavin:NAD(P)H reductases. The construction of a fusion protein between the large HpaB oxygenase component and the choline-binding domain of the major autolysin of Streptococcus pneumoniae allowed us to develop a rapid method to efficiently purify this highly unstable enzyme as a chimeric CH-HpaB protein, which exhibited a 4-HPA hydroxylating activity only when it was supplemented with the HpaC reductase. These results suggest the 4-HPA 3-monooxygenase of E. coli W as a representative member of a novel two-component flavin-diffusible monooxygenase (TC-FDM) family. Relevant features on the evolution and structure-function relationships of these TC-FDM proteins are discussed.
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339. |
Grafková J,
St?pánek V,
Sobotková L,
( 1999 ) Indigenous plasmids in a production line of strains for penicillin G acylase derived from Escherichia coli W. PMID : 10664880 : DOI : 10.1007/bf02818544 Abstract >>
Three indigenous plasmids designated pRK1, pRK2 and pRK3 were identified among producers of penicillin G acylase (PGA) derived from the strain Escherichia coli W ATCC 9637. Their size and copy number (CN) in E. coli W were determined (kb; CN): pRK1 (80; 3.4), pRK2 (5.1; 71), and pRK3 (4.8; 13.7). Strain E. coli RE2 harboring these plasmids was used for selection of strains with reduced number of plasmids: the strain RE3 without plasmid pRK1 and the plasmid-less strain cERE3 were isolated. Indigenous plasmids did not code for the resistance determinants against 23 antibiotics and 10 heavy metals.
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340. |
Tsuda J,
Kato T,
Kita K,
( 1999 ) Evidence of horizontal transfer of the EcoO109I restriction-modification gene to Escherichia coli chromosomal DNA. PMID : 10542186 : PMC : PMC94149 Abstract >>
A DNA fragment carrying the genes coding for EcoO109I endonuclease and EcoO109I methylase, which recognize the nucleotide sequence 5'-(A/G)GGNCC(C/T)-3', was cloned from the chromosomal DNA of Escherichia coli H709c. The EcoO109I restriction-modification (R-M) system was found to be inserted between the int and psu genes from satellite bacteriophage P4, which were lysogenized in the chromosome at the P4 phage attachment site of the corresponding leuX gene observed in E. coli K-12 chromosomal DNA. The sid gene of the prophage was inactivated by insertion of one copy of IS21. These findings may shed light on the horizontal transfer and stable maintenance of the R-M system.
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341. |
Hall RM,
Liebert CA,
( 1999 ) Transposon Tn21, flagship of the floating genome. PMID : 10477306 : PMC : PMC103744 Abstract >>
The transposon Tn21 and a group of closely related transposons (the Tn21 family) are involved in the global dissemination of antibiotic resistance determinants in gram-negative facultative bacteria. The molecular basis for their involvement is carriage by the Tn21 family of a mobile DNA element (the integron) encoding a site-specific system for the acquisition of multiple antibiotic resistance genes. The paradigm example, Tn21, also carries genes for its own transposition and a mercury resistance (mer) operon. We have compiled the entire 19,671-bp sequence of Tn21 and assessed the possible origins and functions of the genes it contains. Our assessment adds molecular detail to previous models of the evolution of Tn21 and is consistent with the insertion of the integron In2 into an ancestral Tn501-like mer transposon. Codon usage analysis indicates distinct host origins for the ancestral mer operon, the integron, and the gene cassette and two insertion sequences which lie within the integron. The sole gene of unknown function in the integron, orf5, resembles a puromycin-modifying enzyme from an antibiotic producing bacterium. A possible seventh gene in the mer operon (merE), perhaps with a role in Hg(II) transport, lies in the junction between the integron and the mer operon. Analysis of the region interrupted by insertion of the integron suggests that the putative transposition regulator, tnpM, is the C-terminal vestige of a tyrosine kinase sensor present in the ancestral mer transposon. The extensive dissemination of the Tn21 family may have resulted from the fortuitous association of a genetic element for accumulating multiple antibiotic resistances (the integron) with one conferring resistance to a toxic metal at a time when clinical, agricultural, and industrial practices were rapidly increasing the exposure to both types of selective agents. The compendium offered here will provide a reference point for ongoing observations of related elements in multiply resistant strains emerging worldwide.
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342. |
Staendner LH,
Manning PA,
Duthy TG,
( 1999 ) CS5 pilus biosynthesis genes from enterotoxigenic Escherichia coli O115:H40. PMID : 10482530 : PMC : PMC94109 Abstract >>
We have sequenced the entire region of DNA required for the biosynthesis of CS5 pili from enterotoxigenic Escherichia coli O115:H40 downstream of the major subunit gene, designated csfA (for coli surface factor five A). Five more open reading frames (ORFs) (csfB, csfC, csfE, csfF, and csfD) which are transcribed in the same direction as the major subunit and are flanked by a number of insertion sequence regions have been identified. T7 polymerase-mediated overexpression of the cloned csf ORFs confirmed protein sizes based on the DNA sequences that encode them. The expression of only the csf region in E. coli K-12 resulted in the hemagglutination of human erythrocytes and the cell surface expression of CS5 pili, suggesting that the cluster contains all necessary information for CS5 pilus biogenesis and function.
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343. |
Yasuda Y,
Tochikubo K,
Taniguchi T,
( 1999 ) The gene encoding the prepilin peptidase involved in biosynthesis of pilus colonization factor antigen III (CFA/III) of human enterotoxigenic Escherichia coli. PMID : 10553678 : DOI : 10.1111/j.1348-0421.1999.tb01220.x Abstract >>
The assembly of pilus colonization factor antigen III (CFA/III) of human enterotoxigenic Escherichia coli requires the processing of CFA/III major pilin (CofA) by a peptidase, likely another type IV pilus formation system. Western blot analysis of CofA reveals that CofA is produced initially as a 26.5-kDa preform pilin (prepilin) and then processed to 20.5-kDa mature pilin by a prepilin peptidase. This processing is essential for exportation of the CofA from the cytoplasm to the periplasm. In this experiment, the structural gene, cofP, encoding CFA/III prepilin peptidase which cleavages at the Gly-30-Met-31 junction of CofA was identified, and the nucleotide sequence of the gene was determined. CofP consists of 819 bp encoding a 273-amino acid protein with a relative molecular mass of 30,533 Da. CofP is predicted to be localized in the inner membrane based on its hydropathy index. The amino acid sequence of CofP shows a high degree of homology with other prepilin peptidases which play a role in the assembly of type IV pili in several gram-negative bacteria.
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344. |
Caprioli A,
Huter V,
Zanial M,
Knutton S,
Batchelor M,
( 1999 ) Development of a universal intimin antiserum and PCR primers. PMID : 10565891 : PMC : PMC85821 Abstract >>
Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) constitute a significant risk to human health worldwide. A hallmark of both pathogens is their ability to produce characteristic attaching-and-effacing (A/E) lesions in intestinal epithelial cells. Genes encoding A/E lesion formation map to a chromosomal pathogenicity island termed the locus of enterocyte effacement (LEE). Intimin, an LEE-encoded bacterial adhesion molecule, mediates the intimate bacterium-host cell interaction characteristic of A/E lesions. On the basis of characterization of the C-terminal 280-amino-acid cell binding domain of intimin (Int280(661-939)), four distinct Int280 types (types alpha, beta, gamma, and delta) have been identified. Importantly, Int280alpha and Int280beta antisera specifically recognized their respective intimin types. Using a conserved region of the intimin molecule (Int(388-667)) and primers synthesized to generate the recombinant Int(388-667), we have now generated universal intimin antiserum and PCR primers that are reactive with the different intimin types expressed by both human and animal A/E lesion-forming strains. Use of immunogold electron microscopy to visualize intimin on the surfaces of EPEC and EHEC strains revealed, in general, a uniform distribution on the bacterial cell surface. However, a filamentous staining pattern was observed with a few strains expressing intimin gamma. Cloning of the intimin eae gene from one such strain (strain ICC57) into strain CVD206, an EPEC strain which harbors a null deletion in eae, produced a uniform intimin staining pattern indicating that, if the filamentous staining pattern defines a filamentous form of intimin gamma, it is dependent upon the genetic background of the strain and is not a feature of the intimin molecule.
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345. |
Stuchlík S,
Burian J,
( 1999 ) Replication control of a small cryptic plasmid of Escherichia coli. PMID : 10556028 : DOI : 10.1006/jmbi.1999.3266 Abstract >>
The role of the RepA initiator protein in replication and copy-number control of pKL1, a small cryptic plasmid of Escherichia coli, was elucidated. The identified ori region encompasses a copy-number control element (cop) and an active single-strand initiation signal (ssi), n'-pasH, which were essential for efficient plasmid replication. The cop region also harbors a region of plasmid incompatibility, inc, encompassing a stem-loop structure, the repA promoter, Prep, as well as two distinct RepA binding sites, BD-1 and BD-2. RepA was shown to bind to these sites quite differently, binding primarily as a monomer or dimer to BD-1 to initiate RepA transcription and plasmid replication, and as higher oligomers to BD-2 to autoregulate repA transcription, the balance being reflected in plasmid copy number. An active integration host factor (IHF) binding sequence was located in the cop region and plasmid replication was shown to be dependent on host IHF encoding genes himA and himD. Low concentrations of IHF predisposed the cop region to RepA binding, although when highly expressed in trans RepA effectively displaced bound IHF and it overcame IHF dependency. Incompatibility was shown to be due to the titration of RepA at the cop locus but could be easily overridden by excess RepA. Both RepA binding sites were required to maintain incompatibility and effective pKL1 replication. Neither antisense RNA nor iterons were found to be involved in pKL1 regulation, thus pKL1 is a novel example of autoregulation of DNA replication. When produced in excess from a helper plasmid, RepA induced pKL1 replication to unusually high levels (>2500 copies/cell). In addition, pKL1 replication could be artificially modulated and a wide range of copy numbers maintained.
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346. |
Jose J,
Maurer J,
( 1999 ) Characterization of the essential transport function of the AIDA-I autotransporter and evidence supporting structural predictions. PMID : 10559167 : PMC : PMC94176 Abstract >>
The current model for autodisplay suggests a mechanism that allows a passenger protein to be translocated across the outer membrane by coordinate action of a C-terminal beta-barrel and its preceding linking region. The passenger protein, linker, and beta-barrel are together termed the autotransporter, while the linker and beta-barrel are here referred to as the translocation unit (TU). We characterized the minimal TU necessary for autodisplay with the adhesin-involved-in-diffuse-adherence (AIDA-I) autotransporter. The assumed beta-barrel structure at the C terminus of the AIDA-I autotransporter was studied by constructing a set of seven AIDA-I-cholera toxin B subunit fusion proteins containing various portions of AIDA-I. Surface exposure of the cholera toxin B moiety was assessed by dot blot experiments and trypsin accessibility of the chimeric proteins expressed in Escherichia coli JK321 or UT5600. Export of cholera toxin B strictly depended on a complete predicted beta-barrel region. The absolute necessity for export of a linking region and its influence on expression as an integral part of the TU was also demonstrated. The different electrophoretic mobilities of native and denatured chimeras indicated that the proposed beta-barrel resides within the C-terminal 312 amino acids of AIDA-I. Together these data provide evidence for the predicted beta-barrel structure and support our formerly proposed model of membrane topology of the AIDA-I autotransporter.
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347. |
Wang SY,
Lai XH,
( 1999 ) Expression of cytotoxicity by potential pathogens in the standard Escherichia coli collection of reference (ECOR) strains. PMID : 10589739 : DOI : 10.1099/00221287-145-11-3295 Abstract >>
The standard Escherichia coli collection of reference (ECOR) strains was examined for ability to exert cytotoxicity towards mammalian cells. A group of strains with functional haemolysin expression caused strong cytotoxicity and detachment in J774 macrophage cells as measured by lactate dehydrogenase release and as observed under a microscope. The expression of haemolysin was monitored by using antisera recognizing the E. coli alpha-haemolysin, the HlyA protein, and by quantitative haemolysis assays. The presence of the hlyA gene, which may be part of a pathogenicity island, was also confirmed. These analyses revealed that different ECOR strains express quantitatively different levels of haemolysin. One putative enteroaggregative E. coli (EAEC) strain was also found in the ECOR collection. The EAEC strain was characterized by the clump formation assay, PCR amplification of the EAEC DNA probe sequence and confirmative sequence analysis of the amplified fragment. The EAEC heat-stable enterotoxin 1 gene, astA, was found in 14% (10/72) of the ECOR strains and a consensus sequence for astA was proposed by comparing these sequences with those from pathogens. The astA gene appeared to be plasmid-located. Based on evidence from the work of other laboratories and from the present findings, it is concluded that the ECOR collection contains strains that may represent pathogenic E. coli. It is noted that caution is necessary when handling or disposing of those potentially pathogenic ECOR strains.
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348. |
Zhao D,
Li C,
( 1999 ) Crystal structure of colicin E3 immunity protein: an inhibitor of a ribosome-inactivating RNase. PMID : 10574790 : Abstract >>
Colicins are antibiotic-like proteins of Escherichia coli that kill related strains. Colicin E3 acts as an RNase that specifically cleaves 16S rRNA, thereby inactivating the ribosomes in the infected cell. The producing organism is protected against colicin E3 by a specific inhibitor, the immunity protein Im3, which forms a tight 1:1 complex with colicin E3 and renders it inactive. Crystallographic studies on colicin E3 and Im3 have been undertaken to unravel the structural basis for the ribonucleolytic activity and its inhibition. The crystal structure of Im3 has been determined to a resolution of 1.8 A. The structure consists of a four-standard antiparallel beta sheet flanked by three alpha helices on one side of the sheet. Thr7, Phe9, Phe16 and Phe74 form a hydrophobic cluster on the surface of the protein in the vicinity of Cys47. This cluster is part of a putative binding pocket which also includes nine polar residues. The putative binding pocket of Im3 is the probable site of interaction with colicin E3. The six acidic residues in the pocket may interact with some of the numerous basic residues of colicin E3. The involvement of hydrophobic moieties in the binding is consistent with the observation that the tight complex can only be dissociated by denaturation. The structure of Im3 resembles those of certain nucleic acid binding proteins, in particular domain II of topoisomerase I and RNA-binding proteins that contain the ribonucleoprotein (RNP) sequence motif. This observation suggests that Im3 has a nucleic acid binding function in addition to binding colicin E3.
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349. |
Paton AW,
( 1999 ) Molecular characterization of the locus encoding biosynthesis of the lipopolysaccharide O antigen of Escherichia coli serotype O113. PMID : 10531250 : PMC : PMC96976 Abstract >>
Shiga toxigenic Escherichia coli (STEC) strains are a diverse group of organisms capable of causing severe gastrointestinal disease in humans. Within the STEC family, eae-positive STEC strains, particularly those belonging to serogroups O157 and O111, appear to have greater virulence for humans. However, in spite of being eae negative, STEC strains belonging to serogroup O113 have frequently been associated with cases of severe STEC disease, including hemolytic-uremic syndrome (HUS). Western blot analysis with convalescent-phase serum from a patient with HUS caused by an O113:H21 STEC strain indicated that human immune responses were directed principally against lipopolysaccharide O antigen. Accordingly, the serum was used to isolate a clone expressing O113 O antigen from a cosmid library of O113:H21 DNA constructed in E. coli K-12. Sequence analysis indicated that the O113 O-antigen biosynthesis (rfb) locus contains a cluster of nine genes which may be cotranscribed. Comparison with sequence databases identified candidate genes for four glycosyl transferases, an O-acetyl transferase, an O-unit flippase, and an O-antigen polymerase, as well as copies of galE and gnd. Two additional, separately transcribed genes downstream of the O113 rfb region were predicted to encode enzymes involved in synthesis of activated sugar precursors, one of which (designated wbnF) was essential for O113 O-antigen synthesis, and so is clearly a part of the O113 rfb locus. Interestingly, expression of O113 O antigen by E. coli K-12 significantly increased in vitro adherence to both HEp-2 and Henle 407 cells.
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350. |
Feldman MF,
Marolda CL,
( 1999 ) Genetic organization of the O7-specific lipopolysaccharide biosynthesis cluster of Escherichia coli VW187 (O7:K1). PMID : 10517601 : DOI : 10.1099/00221287-145-9-2485 Abstract >>
In previous studies the authors cloned and characterized the DNA sequence of the regions at both ends of the O7-specific lipopolysaccharide (LPS) biosynthesis cluster of Escherichia coli VW187 (O7:K1), and identified the biosynthetic genes for dTDP-rhamnose and GDP-mannose, as well as one of the candidate glycosyltransferases. In this work the complete DNA sequence of a 6.9 kb intervening region is presented. Seven new ORFs were identified. All the functions required for the synthesis and transfer of the O7 LPS were assigned on the basis of complementation experiments of transposon insertion mutants, and amino acid sequence homology to proteins involved in LPS synthesis of other bacteria. Of the seven ORFs, two encoded membrane proteins that were homologous to the O-antigen translocase (Wzx) and polymerase (Wxy), two were involved in the biosynthesis of dTDP-N-acetylviosamine, and the remaining three showed homologies to sugar transferases. The O antigen chain length regulator gene wzz was also identified in the vicinity of the O7 polysaccharide cluster. O7-specific DNA primers were designed and tested for serotyping of O7 E. coli strains.
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351. |
Kaper JB,
Jarvis KG,
Ruiz-Tagle A,
Gómez DC,
Viboud GI,
( 1999 ) Identification of lngA, the structural gene of longus type IV pilus of enterotoxigenic Escherichia coli. PMID : 10439420 : DOI : 10.1099/13500872-145-7-1809 Abstract >>
Human enterotoxigenic Escherichia coli (ETEC) produces a type IV pilus termed longus which is encoded on large plasmids in association with colonization factor antigens (CFAs) and enterotoxins. A plasmid-derived 7 kbp BamHI DNA fragment hybridizing with an oligonucleotide probe designed from the amino-terminal amino acid sequence of the denatured 22 kDa structural longus pilin subunit was subcloned and sequenced. DNA sequencing analysis revealed an open reading frame, designated lngA, whose predicted amino acid sequence matched perfectly the N-terminal sequence of LngA obtained by Edman degradation. lngA is the first gene described of the longus gene cluster. Cloned lngA encoded and expressed a prepilin protein of 236 residues with a calculated mass of 25.17 kDa. The prepilin is apparently processed into a mature pilin of 206 residues with a calculated mass of 21.5 kDa. The predicted peptide sequence of lngA showed 78.8 and 37% identity to CFA/III pilin (CofA) of ETEC and the toxin-coregulated pilus (TcpA) of Vibrio cholerae. Peptide sequence homology between lngA and cofA was more prominent towards the amino terminus than within the carboxy region. Like other type IV pilins, LngA contains two cysteine residues towards the carboxy-terminal region. Transmission electron microscopy and immunoblot analysis of ETEC strains expressing either longus or CFA/III detected antigenic differences between native and denatured epitopes of these pili. In addition, differential regulation of pilus expression was identified when ETEC strains were grown in different media. Our data indicate that longus and CFA/III are two distinct but yet highly related type IV pili of ETEC.
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352. |
Rakin A,
Fischer D,
Schubert S,
( 1999 ) Characterization of the integration site of Yersinia high-pathogenicity island in Escherichia coli. PMID : 10518744 : DOI : 10.1111/j.1574-6968.1999.tb08756.x Abstract >>
The high-pathogenicity island (HPI) of virulent Yersiniae consists of (i) a functional core encoding for biosynthesis and uptake of the siderophore yersiniabactin and (ii) a 5- to 13-kb AT-rich region of unknown function. This Yersinia HPI has been shown to be widely distributed among different pathotypes of Escherichia coli. In this study, the insertion site of the HPI was defined in three different E. coli strains: The enteroaggregative E. coli (EAggEC) strain 17-2, the uropathogenic (UPEC) E. coli strain 536, and the probiotic E. coli DSM6601. We demonstrated that in all three E. coli isolates the HPI is associated with the asnT tRNA (5'-extremity) and truncated in the AT-rich region (3'-extremity) since the 17-bp direct repeat (DR) of the asn tRNA that flanks the HPI in Yersinia is missing in E. coli. Moreover, in comparison to the HPI-negative E. coli K-12 strain, a uniform deletion must have taken place in the E. coli chromosome adjacent to the 3'-border of the HPI.
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353. |
Kim KS,
Huang SH,
Wass CA,
Stins MF,
( 1999 ) The gene locus yijP contributes to Escherichia coli K1 invasion of brain microvascular endothelial cells. PMID : 10456927 : PMC : PMC96805 Abstract >>
Most cases of Escherichia coli meningitis develop as a result of hematogenous spread, but it is not clear how circulating E. coli crosses the blood-brain barrier. A TnphoA mutant of E. coli K1 RS218 was shown to be significantly less invasive than its parent strain in bovine and human brain microvascular endothelial cells (BMEC), which constitute the blood-brain barrier. More importantly, traversal of the blood-brain barrier was significantly less with this mutant than with the parent strain in newborn rats with experimental hematogenous meningitis. A DNA segment containing the TnphoA insertion site was cloned from RS218, and the cloned DNA complemented the TnphoA mutant in invasion of BMEC. Nucleotide sequence revealed a near identity to that of a hypothetical yijP gene (also called f577) in the E. coli K-12 genome. Sequence analysis indicated that the E. coli K1 yijP gene likely encodes a 66. 6-kDa membrane protein. Deletion and complementation experiments indicated that the yijP gene was involved in E. coli K1 invasion of BMEC, i.e., the invasive ability of E. coli K1 was significantly reduced after yijP was deleted and was restored by complementation with a plasmid containing the yijP open reading frame. This is the first demonstration that the yijP gene locus plays a role in the pathogenesis of E. coli K1 meningitis.
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354. |
Lengyel Z,
Tsunekawa H,
( 1999 ) Cloning, sequencing, and expression of cscA invertase from Escherichia coli B-62. PMID : 10446718 : Abstract >>
We have isolated a 2.5-kb DNA fragment from plasmid pST5R7 encoding a sucrose utilization system from Escherichia coli B-62 which confers a sucrose-fermenting phenotype to transformed E. coli K-12 strains. DNA-sequence determination revealed one full-length open reading frame 98% identical to cscA, the sucrose-hydrolase (invertase) gene of the csc regulon from E. coli EC3132. Functional characterization indicates that high-level expression and limited periplasmic release of invertase is responsible for the sucrose-fermenting capacity of transformed E. coli K-12 strains carrying cscA.
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355. |
Worsham LM,
Ernst-Fonberg ML,
( 1999 ) HlyC, the internal protein acyltransferase that activates hemolysin toxin: roles of various conserved residues in enzymatic activity as probed by site-directed mutagenesis. PMID : 10413532 : DOI : 10.1021/bi9905617 Abstract >>
Hemolysin, a toxic protein produced by pathogenic Escherichia coli, is one of a family of homologous toxins and toxin-processing proteins produced by Gram-negative bacteria. HlyC, an internal protein acyltransferase, converts it from nontoxic prohemolysin to toxic hemolysin. Acyl-acyl carrier protein is the essential acyl donor. The acyltransferase reaction progresses through formation of a binary complex between acyl-ACP and HlyC to a reactive acyl-HlyC intermediate [Trent, M. S., Worsham, L. M., and Ernst-Fonberg, M. L. (1998) Biochemistry 37, 4644-4655]. The homologous acyltransferases of the family have a number of conserved amino acid residues that may be catalytically important. Experiments to illuminate the reaction mechanism were done. The formation of an acyl-enzyme intermediate suggested that the reaction likely proceeded through two partial reactions. The reversibility of the first partial reaction was shown by using separately subcloned, purified, and expressed substrates and enzyme. The effects of single site-directed mutations of conserved residues of HlyC on different portions of reaction progress (binary complex formation, acyl-enzyme formation, and enzyme activity, including kinetic parameters) were determined. Mutations of His23, the only residue essential for activity, formed normal binary complexes but were unable to form acyl-HlyC. The same was seen with S20A, a mutant with greatly impaired activity. Mutation of two conserved tyrosines separately to glycines results in greatly impaired binary complex and acyl-HlyC formation, but mutation of those residues to phenylalanines restored behavior to wild-type.
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356. |
Mobashery S,
Maveyraud L,
Bulychev A,
Golemi D,
Cabantous S,
( 1999 ) X-ray structure of the Asn276Asp variant of the Escherichia coli TEM-1 beta-lactamase: direct observation of electrostatic modulation in resistance to inactivation by clavulanic acid. PMID : 10423234 : DOI : 10.1021/bi990758z Abstract >>
The clinical use of beta-lactam antibiotics combined with beta-lactamase inactivators, such as clavulanate, has resulted in selection of beta-lactamases that are insensitive to inactivation by these molecules. Therefore, therapeutic combinations of an enzyme inactivator and a penicillin are harmless for bacteria harboring such an enzyme. The TEM beta-lactamase variants are the most frequently encountered enzymes of this type, and presently, 20 variants are designated as inhibitor-resistant TEM ("IRT") enzymes. Three mutations appear to account for the phenotype of the majority of IRT enzymes, one of them being the Asn276Asp substitution. In this study, we have characterized the kinetic properties of the inhibition process of the wild-type TEM-1 beta-lactamase and of its Asn276Asp variant with the three clinically used inactivators, clavulanic acid (clavulanate), sulbactam, and tazobactam, and we report the X-ray structure for the mutant variant at 2.3 A resolution. The changes in kinetic parameters for the interactions of the inhibitors with the wild-type and the mutant enzymes were more pronounced for clavulanate, and relatively inconsequential for sulbactam and tazobactam. The structure of the Asn276Asp mutant enzyme revealed a significant movement of Asp276 and the formation of a salt bridge of its side chain with the guanidinium group of Arg244, the counterion of the inhibitor carboxylate. A water molecule critical for the inactivation chemistry by clavulanate, which is observed in the wild-type enzyme structure, is not present in the crystal structure of the mutant variant. Such structural changes favor the turnover process over the inactivation chemistry for clavulanate, with profound phenotypic consequences. The report herein represents the best studied example of inhibitor-resistant beta-lactamases.
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357. |
Czeczulin J,
Eslava C,
Henderson IR,
( 1999 ) Characterization of pic, a secreted protease of Shigella flexneri and enteroaggregative Escherichia coli. PMID : 10531204 : PMC : PMC96930 Abstract >>
We have identified and characterized a secreted protein, designated Pic, which is encoded on the chromosomes of enteroaggregative Escherichia coli (EAEC) 042 and Shigella flexneri 2457T. The product of the pic gene is synthesized as a 146.5-kDa precursor molecule which is processed at the N and C termini during secretion, allowing the release of a mature protein (109.8 kDa) into the culture supernatant. The deduced amino acid sequence of Pic shows high homology to autotransporter proteins, particularly a subgroup termed the SPATEs (serine protease autotransporters of the Enterobacteriaceae). Present in all members of this subgroup is a motif similar to the active sites of certain serine proteases. Pic catalyzes gelatin degradation, which can be abolished by disruption of the predicted proteolytic active site. Functional analysis of the Pic protein implicates this factor in mucinase activity, serum resistance, and hemagglutination. Our data suggest that Pic may be a multifunctional protein involved in enteric pathogenesis.
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358. |
Althorpe NJ,
Roscoe RA,
Bates S,
( 1999 ) Expression of leading region genes on IncI1 plasmid ColIb-P9: genetic evidence for single-stranded DNA transcription. PMID : 10537187 : DOI : 10.1099/00221287-145-10-2655 Abstract >>
The leading region of a plasmid is the first sector to enter the recipient cell in bacterial conjugation. This sector of IncI1 plasmid ColIb-P9 includes genes that are transcribed in a transient pulse early in the conjugatively infected cell to promote establishment of the immigrant plasmid. Evidence is presented that the burst of gene expression is regulated by a process which is independent of a repressor but dependent on the orientation of the genes on the unique plasmid strand transferred in conjugation. The nucleotide sequence of 11.7 kb of the leading region was determined and found to contain 10 ORFs; all are orientated such that the template strand for transcription corresponds to the transferred strand. The leading region contains three dispersed repeats of a sequence homologous to a novel promoter in ssDNA described by H. Masai & K. Arai (1997, Cell 89, 897-907). It is proposed that the repeats are promoters that form in the transferring strand of ColIb to support transient transcription of genes transferred early in conjugation.
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359. |
Jouve M,
Lalioui L,
( 1999 ) Molecular cloning and characterization of the afa-7 and afa-8 gene clusters encoding afimbrial adhesins in Escherichia coli strains associated with diarrhea or septicemia in calves. PMID : 10496877 : PMC : PMC96852 Abstract >>
The afa gene clusters, which encode proteins involved in adhesion to epithelial cells, from Escherichia coli strains associated with urinary and intestinal infections in humans have been characterized. Pathogenic isolates of bovine and porcine origin that possess afa-related sequences have recently been described. We report in this work the cloning and characterization of the afa-7 and afa-8 gene clusters from bovine isolates. Hybridization and sequencing experiments revealed that despite similarity in genetic organization, the afa-7 and afa-8 genes, and the well-characterized afa-3 operon expressed by human-pathogenic isolates, correspond to three different members of the afa family of gene clusters. However, like the afa-3 gene cluster, both the afa-7 and afa-8 gene clusters were found to encode an afimbrial adhesin (AfaE) and an invasin (AfaD). The AfaD peptides encoded by the three gene clusters were only 45% identical, but functional complementation experiments indicated that they belong to the same family of invasins. Hemagglutination and adhesion assays demonstrated that the AfaE-VII and AfaE-VIII adhesins bind to different receptors and that these receptors are not the human decay-accelerating factor recognized to be the receptor of all previously described AfaE adhesins. The AfaE-VIII adhesin is very similar to the M agglutinin of human-uropathogenic strains. We used PCR assays to screen 25 bovine strains for afaD and afaE genes of either the afa-7 or afa-8 gene cluster. The afa-8 gene cluster was highly prevalent in bovine isolates previously reported to carry afa-related sequences (23 of 24 strains), particularly in strains producing cytotoxic necrotizing factors (16 of 16 strains). The location of the afa-8 gene cluster on the plasmids or chromosome of these isolates suggests that it could be carried by a mobile element, facilitating its dissemination among bovine-pathogenic E. coli strains.
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360. |
Hesslinger C,
( 1998 ) The tdcE gene in Escherichia coli strain W3110 is separated from the rest of the tdc operon by insertion of IS5 elements. PMID : 10520749 : Abstract >>
Unlike other Escherichia coli K-12 strains, W3110 contains multiple copies of the insertion sequence IS5. Some of these IS5 elements have been involved in tandem duplication of a portion of the chromosome which includes, amonst others, the tdcABC-DEFG operon genes. The nucleotide sequence and insertion site of one of these elements, IS5P, was determined. It was shown that IS5P has inserted within the coding sequence of the tdcA gene and is flanked, not by the remaining portion of the tdcA gene, but by the extreme 3' end of the tdcD gene. In other E. coli K-12 strains the tdcD gene and three other genes, tdcE, tdcF and tdcG, all form part of the tdc operon. Our results demonstrate that during the duplication event the tdcABCgenes have been amplified and separated from the remaining genes tdcE, tdcF and tdcG of the operon, which are each present in single copy.
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361. |
Zhang D,
Zhang W,
Schmidt H,
Schubert S,
Karch H,
( 1999 ) A genomic island, termed high-pathogenicity island, is present in certain non-O157 Shiga toxin-producing Escherichia coli clonal lineages. PMID : 10531259 : PMC : PMC96985 Abstract >>
Shiga toxin-producing Escherichia coli (STEC) strains cause a wide spectrum of diseases in humans. In this study, we tested 206 STEC strains isolated from patients for potential virulence genes including stx, eae, and enterohemorrhagic E. coli hly. In addition, all strains were examined for the presence of another genetic element, the high-pathogenicity island (HPI). The HPI was first described in pathogenic Yersinia species and encodes the pesticin receptor FyuA and the siderophore yersiniabactin. The HPI was found in the genome of distinct clonal lineages of STEC, including all 31 eae-positive O26:H11/H(-) strains and 7 of 12 eae-negative O128:H2/H(-) strains. In total, the HPI was found in 56 (27.2%) of 206 STEC strains. However, it was absent from the genome of all 37 O157:H7/H(-), 14 O111:H(-), 13 O103:H2, and 13 O145:H(-) STEC isolates, all of which were positive for eae. Polypeptides encoded by the fyuA gene located on the HPI could be detected by using immunoblot analysis in most of the HPI-positive STEC strains, suggesting the presence of a functional yersiniabactin system. The HPI in STEC was located next to the tRNA gene asnT. In contrast to the HPI of other pathogenic enterobacteria, the HPI of O26 STEC strains shows a deletion at its left junction, leading to a truncated integrase gene int. We conclude from this study that the Yersinia HPI is disseminated among certain clonal subgroups of STEC strains. The hypothesis that the HPI in STEC contributes to the fitness of the strains in certain ecological niches rather than to their pathogenic potential is discussed.
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362. |
Carlino UB,
Russo TA,
( 1999 ) Identification of genes in an extraintestinal isolate of Escherichia coli with increased expression after exposure to human urine. PMID : 10496910 : PMC : PMC96885 Abstract >>
The identification of genes with increased expression in vivo may lead to the identification of novel or unrecognized virulence traits and/or recognition of environmental signals involved in modulating gene expression. Our laboratory is studying an extraintestinal isolate of Escherichia coli as a model pathogen. We had previously used human urine ex vivo to identify the unrecognized urovirulence genes guaA and argC and to establish that arginine and guanine (or derivatives) were limiting in this body fluid (T. A. Russo et al., Mol. Microbiol. 22:217-229, 1996). In this study, we have continued with this approach and identified three additional genes that have increased expression in human urine relative to Luria-Bertani (LB) medium. Expression of ure1 (urine-responsive element) is increased a mean of 47.6-fold in urine but completely suppressed by exogenous glucose. This finding suggests that ure1 is regulated by catabolite repression and that limiting glucose in urine is a regulatory signal. ure1 is present in the E. coli K-12 genome, but its function is unknown. Although disruption of ure1 results in diminished growth in human urine, limiting concentrations of amino acids, nucleosides, or iron (Fe), or changes in osmolarity or pH do not affect the expression of ure1. Therefore, Ure1 appears to have a role independent of the synthesis or uptake of these nutrients and does not appear to be involved in osmoprotection. iroN(E. coli) is a novel E. coli gene with 77% DNA homology to a catecholate siderophore receptor gene recently identified in Salmonella. Its expression is increased a mean of 27.2-fold in urine and is repressed by exogenous Fe and a urinary pH of 5.0. This finding supports the contention that Fe is a limiting element in urine and that alteration of pH can affect gene expression. It is linked to the P-pilus (prs) and F1C fimbrial (foc) gene clusters on a pathogenicity island and appears to have been acquired by IS1230-mediated horizontal transmission. The homologous iroN(E. coli) sequence is significantly more prevalent in urinary tract and blood isolates of E. coli compared to fecal isolates. Last, the expression of ArtJ, an arginine periplasmic binding protein, is increased a mean of 16.6-fold in urine. This finding implicates arginine concentrations as limited in urine and, in combination with previous data demonstrating that argC is important for urovirulence, suggests that the ability of E. coli to synthesize or acquire arginine is important for urovirulence. ure1, iroN(E. coli), and artJ all have increased expression in human blood and ascites relative to LB medium as well. The identification of these genes increases our understanding of regulatory signals present in human urine, blood, and ascites. Ure1, IroN(E. coli), and ArtJ also warrant further evaluation as virulence traits both within and outside the urinary tract.
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363. |
Sasakawa C,
Schoolnik GK,
Wu CY,
Tatsuno I,
Katayama E,
( 1999 ) A novel chromosomal locus of enteropathogenic Escherichia coli (EPEC), which encodes a bfpT-regulated chaperone-like protein, TrcA, involved in microcolony formation by EPEC. PMID : 10447884 : DOI : 10.1046/j.1365-2958.1999.01522.x Abstract >>
The bfpTVW operon, also known as the per operon, of enteropathogenic Escherichia coli (EPEC) is required for the transcriptional activation of the bfp operon, which encodes the major subunit and assembly machinery of bundle-forming pili (BFP). An immobilized T7-tagged BfpT fusion protein that binds specifically to upstream promoter sequences of bfpA and eae was used to 'fish out' from a promoter library other EPEC chromosomal fragments that are bound by the BfpT protein. After screening for promoters exhibiting bfpTVW-dependent expression, one was identified that was positively regulated by bfpTVW and that is not present in the chromosomes of two non-virulent E. coli laboratory strains, DH5alpha and HB101. Further analysis of this positively regulated promoter in EPEC showed that it resided within a 4.9 kb sequence that is not present in E. coli K12. This locus, located downstream of the potB gene, was found to contain four open reading frames (ORFs): bfpTVW-activated promoter was localized upstream of ORF1. An ORF1 knockout mutant produced less of the BFP structural subunit (BfpA) and formed smaller than normal adherent microcolonies on cultured epithelial cells; however, this mutation did not affect bfp transcription. An ORF1-His6 fusion protein specifically bound the preprocessed and mature forms of the BfpA protein and thus appears to stabilize the former within the cytoplasmic compartment. ORF1 therefore is a newly isolated EPEC chromosomal gene that encodes a chaperone-like protein involved in the production of BFP. Hence, ORF1 was designated trcA (bfpT-regulated chaperone-like protein gene). The TrcA protein also specifically bound 39 kDa and 90 kDa proteins that are expressed by EPEC but not by E. coli K12. The 90 kDa protein was revealed to be intimin, a protein product of the eae gene, which is required for the EPEC attaching/effacing phenotype, suggesting a direct interaction of TrcA with intimin in the cytoplasmic compartment.
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364. |
Collighan RJ,
Woodward MJ,
( 1999 ) Non-curliation of Escherichia coli O78:K80 isolates associated with IS1 insertion in csgB and reduced persistence in poultry infection. PMID : 10386375 : DOI : 10.1111/j.1574-6968.1999.tb13627.x Abstract >>
The elaboration of curli fimbriae by Escherichia coli is associated with the development of a lacy colony morphology when grown on colonisation factor antigen agar at 25 degrees C. Avian colisepticaemia E. coli isolates screened for curliation by this culture technique showed lacy and smooth colonial morphologies and the genetic basis of the non-curliated smooth colonial phenotype was analysed. Two smooth E. coli O78:K80 isolates possessed about 40 copies of the IS1 element within their respective genomes of which one copy insertionally inactivated the csgB gene, the nucleator gene for curli fibril formation. One of these two isolates also possessed a defective rpoS gene which is a known regulator of curli expression. In the day-old chick model, both smooth isolates were as invasive as a known virulent O78:K80 isolate as determined by extent of liver and spleen colonisation post oral inoculation but were less persistent in terms of caecal colonisation.
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365. |
Worsham LM,
Ernst-Fonberg ML,
( 1999 ) HlyC, the internal protein acyltransferase that activates hemolysin toxin: the role of conserved tyrosine and arginine residues in enzymatic activity as probed by chemical modification and site-directed mutagenesis. PMID : 10393560 : DOI : 10.1021/bi990138y Abstract >>
Internal fatty acylation of proteins is a recognized means of modifying biological behavior. Escherichia coli hemolysin A (HlyA), a toxic protein, is transcribed as a nontoxic protein and made toxic by internal acylation of two lysine residue epsilon-amino groups; HlyC catalyzes the acyl transfer from acyl-acyl carrier protein (ACP), the obligate acyl donor. Conserved residues among the respective homologous C proteins that activate 13 different RTX (repeats in toxin) toxins of which HlyA is the prototype likely include some residues that are important in catalysis. Possible roles of two conserved tyrosines and two conserved arginines were investigated by noting the effects of chemical modifiers and site-directed mutagenesis. TNM modification of HlyC at pH 8.0 led to extensive inhibition that was prevented by the presence of the substrate myristoyl-ACP but not by the product, ACPSH. NAI had no effect. Y70G and Y150G greatly diminished enzyme activity, whereas mutations Y70F and Y150F exhibited wild-type activity. Modification of arginine residues with PG markedly lowered acyltransferase activity with moderate protection by both myristoyl-ACP and ACPSH. Under optimum conditions, four separate mutations of the two conserved arginine residues (R24A, R24K, R87A, and R87K) had little effect on acyltransferase activity.
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366. |
Gaggero C,
Laviña M,
( 1999 ) The structural gene for microcin H47 encodes a peptide precursor with antibiotic activity. PMID : 10471561 : PMC : PMC89443 Abstract >>
Microcin H47 is a bactericidal antibiotic produced by a naturally occurring Escherichia coli strain isolated in Uruguay. The microcin genetic system is located in the chromosome and extends over a 10-kb DNA segment containing the genes required for microcin synthesis, secretion, and immunity. The smallest microcin synthesis gene, mchB, was sequenced and shown to encode a highly hydrophobic peptide. An mchB-phoA gene fusion, which directed the synthesis of a hybrid bifunctional protein with both PhoA and microcin H47-like activities, was isolated. The results presented herein lead us to propose that microcin H47 is indeed a ribosomally synthesized peptide antibiotic and that its peptide precursor already has antibiotic activity of the same specificity as that of mature microcin.
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367. |
Rosato A,
Barker PD,
Faraone-Mennella J,
Banci L,
Bertini I,
( 1999 ) The solution structure of oxidized Escherichia coli cytochrome b562. PMID : 10393541 : DOI : 10.1021/bi982785f Abstract >>
The solution structure of the oxidized, paramagnetic form of cytochrome b562 from Escherichia coli (106 amino acids) is here reported as obtained from 1653 meaningful NOEs (from a total of 2051 unique NOEs), 33 (3)JHNHalpha values, and 339 pseudocontact shifts. The structure displays the typical four-helix bundle motif, and a disordered loop between helices alpha2 and alpha3, as found in the solid state. The solution structure has a conformation intermediate between the two independent solid-state molecules, although different orientations are observed for a few residues. The magnetic susceptibility tensor is similar to that of cytochrome c, which has the same ligands, although the anisotropy is somewhat smaller. This difference in the electronic structure is consistent with the thermal accessibility in cytochrome b562 of states with S > 1/2. The structure is also compared with the solution structure of the apoprotein, and some information on the role of the cofactor on the protein folding and mobility is obtained. Helix alpha4 seems to be the most sensitive to the chemical environment in terms of structure and mobility. The pKa values affecting the hyperfine-shifted signals are also discussed. Quite intriguing is the comparison of the structure of cytochrome b562 with the available structures of cytochromes c' which display a similar folding motif and similar pKa values but very little sequence similarity.
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368. |
Waksman G,
Hultgren SJ,
Dodson KW,
Fütterer K,
Pinkner JS,
( 1999 ) Structural basis of chaperone function and pilus biogenesis. PMID : 10446050 : DOI : 10.1126/science.285.5430.1058 Abstract >>
Many Gram-negative pathogens assemble architecturally and functionally diverse adhesive pili on their surfaces by the chaperone-usher pathway. Immunoglobulin-like periplasmic chaperones escort pilus subunits to the usher, a large protein complex that facilitates the translocation and assembly of subunits across the outer membrane. The crystal structure of the PapD-PapK chaperone-subunit complex, determined at 2.4 angstrom resolution, reveals that the chaperone functions by donating its G(1) beta strand to complete the immunoglobulin-like fold of the subunit via a mechanism termed donor strand complementation. The structure of the PapD-PapK complex also suggests that during pilus biogenesis, every subunit completes the immunoglobulin-like fold of its neighboring subunit via a mechanism termed donor strand exchange.
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369. |
Mainil J,
Jacquemin E,
Devrin AC,
Pirson V,
( 1999 ) Heterogeneity of the eae genes in attaching/effacing Escherichia coli from cattle: comparison with human strains. PMID : 10422693 : Abstract >>
Enteropathogenic (EPEC) and enterohaemorrhagic (EHEC) Escherichia coli isolated from cattle were studied by DNA colony hybridization to subtype their intimin-encoding (eae) gene with probes derived from the variable parts of the eae alpha gene of the human EPEC strain E2348/69, the eae gamma gene of the human O157:H7 EHEC strain ATCC43888, and the eae beta gene of the bovine O26:H- EHEC strain 193, whose eae gene was first cloned and sequenced during this work. The EPEC and EHEC had been isolated from diarrhoeic calves (143 EPEC and 48 EHEC) and from healthy animals at the slaughterhouse (10 EPEC and 34 EHEC). The 191 bovine EPEC and EHEC isolated from diseased calves were positive with the Eae beta probe (55 and 27% respectively) and with the Eae gamma probe (9 and 73% respectively), whereas 52 EPEC (36%) were negative with the Eae alpha, Eae beta, and Eae gamma probes. The results were different for the 44 bovine EPEC and EHEC isolated from healthy cattle at slaughterhouses: most tested positive with the Eae gamma probe (80 and 82% respectively) and the remaining (20 and 18% respectively) with the Eae beta probe. Nine O26 human EHEC tested positive with the Eae beta probe and seven O111 with the Eae gamma probe. The bovine and human EPEC and EHEC belonging to these two serogroups gave identical results: the 18 bovine and human O26 isolates tested positive with the Eae beta probe, whereas the 13 O111 isolates were positive with the Eae gamma probe. In contrast, the isolates belonging to other serogroups (O5, O15, O18, O20, and O118) gave more variable results. The eae beta and eae gamma, but not the eae alpha, variants were thus distributed amongst bovine EPEC and EHEC. The eae beta variant seemed to be more frequently associated with the presence of clinical signs in calves, but one third of EPEC from diarrhoeic calves carried an eae gene variant other than the alpha, beta, or gamma variants. In addition, the use of these gene probes did not enable differentiation between bovine and human EHEC belonging to the same O serogroup.
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370. |
Yuan HS,
Chak KF,
Liao CC,
Ku WY,
( 1999 ) The crystal structure of the DNase domain of colicin E7 in complex with its inhibitor Im7 protein. PMID : 10368275 : Abstract >>
Colicin E7 (ColE7) is one of the bacterial toxins classified as a DNase-type E-group colicin. The cytotoxic activity of a colicin in a colicin-producing cell can be counteracted by binding of the colicin to a highly specific immunity protein. This biological event is a good model system for the investigation of protein recognition. The crystal structure of a one-to-one complex between the DNase domain of colicin E7 and its cognate immunity protein Im7 has been determined at 2.3 A resolution. Im7 in the complex is a varied four-helix bundle that is identical to the structure previously determined for uncomplexed Im7. The structure of the DNase domain of ColE7 displays a novel alpha/beta fold and contains a Zn2+ ion bound to three histidine residues and one water molecule in a distorted tetrahedron geometry. Im7 has a V-shaped structure, extending two arms to clamp the DNase domain of ColE7. One arm (alpha1(*)-loop12-alpha2(*); where * represents helices in Im7) is located in the region that displays the greatest sequence variation among members of the immunity proteins in the same subfamily. This arm mainly uses acidic sidechains to interact with the basic sidechains in the DNase domain of ColE7. The other arm (loop 23-alpha3(*)-loop 34) is more conserved and it interacts not only with the sidechain but also with the mainchain atoms of the DNase domain of ColE7. The protein interfaces between the DNase domain of ColE7 and Im7 are charge-complementary and charge interactions contribute significantly to the tight and specific binding between the two proteins. The more variable arm in Im7 dominates the binding specificity of the immunity protein to its cognate colicin. Biological and structural data suggest that the DNase active site for ColE7 is probably near the metal-binding site.
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371. |
Schmidt H,
Frosch M,
Karch H,
( 1999 ) The large plasmids of Shiga-toxin-producing Escherichia coli (STEC) are highly variable genetic elements. PMID : 10376815 : DOI : 10.1099/13500872-145-5-1005 Abstract >>
Shiga-toxin-producing Escherichia coli (STEC) of different serotypes are known to harbour large plasmids. The aim of this study was to investigate, using the example of the plasmid-encoded serine protease EspP, whether these plasmids are a uniform genetic element present in STEC. Examination of 201 diarrhoeagenic E. coli strains using a newly developed espP-specific PCR showed that espP is specific for STEC and present in 57% of STEC belonging to 16 different serotypes. The espP genes of the 16 STEC serotypes varied to a certain extent, as shown by nucleotide sequence and restriction enzyme analyses, but the DNA regions adjacent to the espP gene were completely different. When two further STEC-plasmid markers, the catalase-peroxidase gene katP and the enterohaemorrhagic E. coli-haemolysin gene EHEC-hlyA were included, many combinations of the three markers were found, depending in part on the serotype. In addition, strains possessing none of the three markers still harboured large plasmids. In the most prevalent STEC serogroup, O157, it was observed that the plasmid of sorbitol-fermenting STEC O157:H- lacks the espP and katP genes although both genes are present in the plasmid of the non-sorbitol-fermenting STEC O157:H7. The EHEC-hlyA gene, however, is present in both. In conclusion, this study shows that the large plasmids of STEC are not uniform genetic elements but heterogeneous in both their gene composition and arrangement.
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372. |
Baldwin GS,
Brady RL,
Halford SE,
Sessions RB,
( 1999 ) Structural analysis of a mutational hot-spot in the EcoRV restriction endonuclease: a catalytic role for a main chain carbonyl group. PMID : 10446231 : DOI : 10.1093/nar/27.17.3438 PMC : PMC148585 Abstract >>
Following random mutagenesis of the Eco RV endonuclease, a high proportion of the null mutants carry substitutions at Gln69. Such mutants display reduced rates for the DNA cleavage step in the reaction pathway, yet the crystal structures of wild-type Eco RV fail to explain why Gln69 is crucial for activity. In this study, crystal structures were determined for two mutants of Eco RV, with Leu or Glu at residue 69, bound to specific DNA. The structures of the mutants are similar to the native protein and no function can be ascribed to the side chain of the amino acid at this locus. Instead, the structures of the mutant proteins suggest that the catalytic defect is due to the positioning of the main chain carbonyl group. In the enzyme-substrate complex for Eco RV, the main chain carbonyl of Gln69 makes no interactions with catalytic functions but, in the enzyme-product complex, it coordinates a metal ion bound to the newly liberated 5'-phosphate. This re-positioning may be hindered in the mutant proteins. Molecular dynamics calculations indicate that the metal on the phosphoryl oxygen interacts with the carbonyl group upon forming the pentavalent intermediate during phosphodiester hydrolysis. A main chain carbonyl may thus play a role in catalysis by Eco RV.
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373. |
Trabulsi LR,
Keller R,
Bortolini MR,
( 1999 ) Lack of expression of bundle-forming pili in some clinical isolates of enteropathogenic Escherichia coli (EPEC) is due to a conserved large deletion in the bfp operon. PMID : 10481102 : DOI : 10.1111/j.1574-6968.1999.tb08723.x Abstract >>
Enteropathogenic Escherichia coli (EPEC) produces a plasmid-encoded type IV pilus, called the bundle-forming pilus (BFP), involved in the formation of the localized adhesion onto epithelial cells. In this study, we demonstrate that clinical isolates of serotypes O128ab:H2 and O119:H2 contain a ca. 13-kb deletion in the bfp operon, resulting in a lack of expression of these pili. An IS sequence with homology to the IS66 of Agrobacterium tumefaciens replaced the deleted bfp genes. These results suggest that the bfp operon was deleted through a transpositional event and that other adherence factors may mediate attachment of these bacteria to the host cells.
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374. |
Davies DR,
Mahnke Braam L,
Reznikoff WS,
( 1999 ) The three-dimensional structure of a Tn5 transposase-related protein determined to 2.9-A resolution. PMID : 10207011 : DOI : 10.1074/jbc.274.17.11904 Abstract >>
Transposon Tn5 employs a unique means of self-regulation by expressing a truncated version of the transposase enzyme that acts as an inhibitor. The inhibitor protein differs from the full-length transposase only by the absence of the first 55 N-terminal amino acid residues. It contains the catalytic active site of transposase and a C-terminal domain involved in protein-protein interactions. The three-dimensional structure of Tn5 inhibitor determined to 2.9-A resolution is reported here. A portion of the protein fold of the catalytic core domain is similar to the folds of human immunodeficiency virus-1 integrase, avian sarcoma virus integrase, and bacteriophage Mu transposase. The Tn5 inhibitor contains an insertion that extends the beta-sheet of the catalytic core from 5 to 9 strands. All three of the conserved residues that make up the "DDE" motif of the active site are visible in the structure. An arginine residue that is strictly conserved among the IS4 family of bacterial transposases is present at the center of the active site, suggesting a catalytic motif of "DDRE." A novel C-terminal domain forms a dimer interface across a crystallographic 2-fold axis. Although this dimer represents the structure of the inhibited complex, it provides insight into the structure of the synaptic complex.
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375. |
Broll H,
Beutin L,
( 1999 ) Absence of DNA sequence homology with genes of the Escherichia coli hemB locus in Shiga-toxin producing E. coli (STEC) O157 strains. PMID : 10234826 : DOI : 10.1111/j.1574-6968.1999.tb13554.x Abstract >>
By molecular cloning of chromosomal DNA of a human faecal Escherichia coli O6:non-motile strain, we identified a 1350-bp DNA segment which is commonly present in laboratory and wild-type E. coli strains but had no homology to DNA of Shiga-toxin producing E. coli O157, O145 and enteropathogens E. coli O55 strains. The nucleotide sequence of the 1350-bp segment cloned on plasmid pEO67 was determined (GenBank accession number AF087670) and a 97.2% sequence homology was found to a region of the E. coli hemB locus with an unknown gene function. The introduction of pEO67 into an STEC O157:H- strain had a stimulating effect on the growth of the recipient strain which was most expressed when bacteria were grown in iron depleted M9 medium with hemin added as the exogenous iron source. This growth effect was not observed with E. coli K-12 carrying pEO67. We suggest that the cloned gene is involved in iron uptake of E. coli and that the alteration in this part of the hemB locus is clonally inherited in genetically closely related STEC O157 and O55 strains.
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376. |
Skurray RA,
( 1999 ) Nucleotide sequence of the F plasmid leading region. PMID : 10366527 : DOI : 10.1006/plas.1999.1390 Abstract >>
The entire nucleotide sequence of the first DNA segment of the conjugative F plasmid to enter the recipient cell, the leading region, is described. Analysis of the sequence provides further evidence that products encoded within the 13.2-kb leading region are likely to be expressed and perform functions associated with the transferred strand in the recipient cell.
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377. |
Nataro JP,
Whittam TS,
Henderson IR,
Navarro-Garcia F,
( 1999 ) Phylogenetic analysis of enteroaggregative and diffusely adherent Escherichia coli. PMID : 10338471 : PMC : PMC96572 Abstract >>
The phylogenetics of the various pathotypes of diarrheagenic Escherichia coli are not completely understood. In this study, we identified several plasmid and chromosomal genes in the pathogenic enteroaggregative E. coli (EAEC) prototype strain 042 and determined the prevalence of these loci among EAEC and diffusely adherent E. coli strains. The distribution of these genes is analyzed within an evolutionary framework provided by the characterization of allelic variation in housekeeping genes via multilocus enzyme electrophoresis. Our data reveal that EAEC strains are heterogeneous with respect to chromosomal and plasmid-borne genes but that the majority harbor a member of a conserved family of virulence plasmids. Comparison of plasmid and chromosomal relatedness of strains suggests clonality of chromosomal markers and a limited transfer model of plasmid distribution.
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378. |
Ciaccio M,
Farías RN,
Solbiati JO,
Salomón RA,
( 1999 ) Sequence analysis of the four plasmid genes required to produce the circular peptide antibiotic microcin J25. PMID : 10198038 : PMC : PMC93700 Abstract >>
A 4.8-kb plasmid region carrying the four genes mcjABCD necessary for production of and immunity to the cyclic peptide antibiotic microcin J25 (MccJ25) has been sequenced. mcjA encodes the primary structure of MccJ25 as a precursor endowed with an N-terminal extension of 37 amino acids. The products of mcjB and mcjC are thought to be involved in microcin maturation, which implies cleavage of McjA and head-tail linkage of the 21-residue pro-MccJ25. The predicted McjD polypeptide, which is highly similar to several ABC exporters, was found to be required for MccJ25 secretion, thus explaining its ability to confer immunity to MccJ25.
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379. |
Henderson D,
( 1999 ) The MobA-linked primase is the only replication protein of R1162 required for conjugal mobilization. PMID : 10217797 : PMC : PMC93748 Abstract >>
Cells newly transformed with plasmid R1162 DNA were used as donors in conjugal matings to determine if the plasmid replication genes are necessary for transfer. An intact system for vegetative replication is not required for transfer at normal frequency, but the plasmid primase, in the form linked to the nickase, must be present in donor cells.
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380. |
Vlassi M,
Kokkinidis M,
( 1998 ) Structural parameters for proteins derived from the atomic resolution (1.09 A) structure of a designed variant of the ColE1 ROP protein. PMID : 10089502 : DOI : 10.1107/s0907444998002492 Abstract >>
The crystal structure of a designed variant of the ColE1 repressor of primer (ROP) protein has been refined with SHELXL93 to a resolution of 1.09 A. The final model with 510 non-H protein atoms, 576 H atoms in calculated positions and 114 water molecules converged to a standard R factor of 10% using unrestrained blocked full-matrix refinement. For all non-H atoms six-parameter anisotropic thermal parameters have been refined. The majority of atomic vibrations have a preferred orientation which is approximately perpendicular to the bundle axis; analysis with the TLS method [Schomaker & Trueblood (1968). Acta Cryst. B24, 63-77] showed a relatively good agreement between the individual atomic displacements and a rigid-body motion of the protein. Disordered residues with multiple conformations form clusters on the surface of the protein; six C-terminal residues have been omitted from the refined model due to disorder. Part of the solvent structure forms pentagonal or hexagonal clusters which bridge neighbouring protein molecules. Some water molecules are also conserved in wild-type ROP. The unrestrained blocked full-matrix least-squares refinement yielded reliable estimates of the standard deviations of the refined parameters. Comparison of these parameters with the stereochemical restraints used in various protein refinement programs showed statistically significant differences. These restraints should be adapted to the refinement of macromolecules by taking into account parameters determined from atomic resolution protein structures.
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381. |
Rich C,
Forestier C,
Joly B,
Sapena F,
( 1999 ) Characterization of enteroaggregative Escherichia coli isolates. PMID : 10220881 : DOI : 10.1111/j.1574-6968.1999.tb13484.x Abstract >>
Forty enteraggregative Escherichia coli (EAggEC) previously characterized by their ability to adhere to HEp-2 cells or/and their hybridization with the 1-kb EAggEC DNA probe were investigated for the presence of adherence factors and heat-stable enterotoxin (EAST1)-encoding genes. Only 45% of the isolates harbored the EAST1-encoding genes as detected by polymerase chain reaction. None of them hybridized with an AAF/II-encoding gene specific DNA probe and 35% (14/40) were positive in a PCR assay using primers specific for aggC, an accessory gene of the AAF/I-encoding operon. Cloning and sequence analysis of the aggA variant from one isolate, EAggEC 457, revealed 68.9% identity between its deduced amino acid sequence and those of the aggA product from the AAF/I-producing reference strain, E. coli 17.2. No major protein subunit was detected at the surface of EAggEC 457 compared to the bacterial surface extract of E. coli 17.2.
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382. |
Lee MD,
Bolton L,
Sanchez S,
Schmidt D,
Petrosko P,
( 1999 ) Development of primers to O-antigen biosynthesis genes for specific detection of Escherichia coli O157 by PCR. PMID : 10388689 : PMC : PMC91442 Abstract >>
The chemical composition of each O-antigen subunit in gram-negative bacteria is a reflection of the unique DNA sequences within each rfb operon. By characterizing DNA sequences contained with each rfb operon, a diagnostic serotype-specific probe to Escherichia coli O serotypes that are commonly associated with bacterial infections can be generated. Recently, from an E. coli O157:H7 cosmid library, O-antigen-positive cosmids were identified with O157-specific antisera. By using the cosmid DNAs as probes, several DNA fragments which were unique to E. coli O157 serotypes were identified by Southern analysis. Several of these DNA fragments were subcloned from O157-antigen-positive cosmids and served as DNA probes in Southern analysis. One DNA fragment within plasmid pDS306 which was specific for E. coli O157 serotypes was identified by Southern analysis. The DNA sequence for this plasmid revealed homology to two rfb genes, the first of which encodes a GDP-mannose dehydratase. These rfb genes were similar to O-antigen biosynthesis genes in Vibrio cholerae and Yersinia enterocolitica serotype O:8. An oligonucleotide primer pair was designed to amplify a 420-bp DNA fragment from E. coli O157 serotypes. The PCR test was specific for E. coli O157 serotypes. PCR detected as few as 10 cells with the O157-specific rfb oligonucleotide primers. Coupled with current enrichment protocols, O157 serotyping by PCR will provide a rapid, specific, and sensitive method for identifying E. coli O157.
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383. |
Reingold J,
Lee MD,
Maurer J,
( 1999 ) Identification of a new Escherichia coli She haemolysin homolog in avian E. coli. PMID : 10227474 : DOI : 10.1016/s0378-1135(98)00310-1 Abstract >>
Haemolysin is one type of virulence factor that assists in the pathogenesis of Escherichia coli. Currently, hemolytic activity in E. coli has been attributed to haemolysin genes found in either uropathogenic or enterohemorrhagic E. coli. Both haemolysins are classified as RTX toxins because they both have repeats in toxin domains and share similar operon organization, sequence homology, and mechanisms of action. Haemolytic avian E. coli isolates, however, lack either E. coli haemolysin gene. To investigate the avian E. coli haemolysin, a genomic library was made from an avian pathogenic E. coli. A haemolytic clone that was isolated was shown to contain homology with sheA, an E. coli K- 12 gene which causes haemolysis when present in high copy number. The cloned haemolysin gene, hlyE, lacked the conserved amino acid sequence and accessory genes common to all RTX toxins. DNA hybridizations and polymerase chain reaction amplifications showed that the nucleotide sequences homologous to hlyE were not present in a collection of three O157: H7 E. coli, five haemolytic canine uropathogenic E. coli, one haemolytic O26 E. coli, and three haemolytic avian pathogenic E. coli. Thus we have identified a new E. coli haemolysin distinct from the RTX haemolysins and have shown that some avian pathogenic E. coli possess a haemolysin with no apparent homology to hlyE or RTX haemolysins.
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384. |
Amils R,
Ruiz-Santa-Quiteria JA,
de Greve H,
( 1999 ) Characterization of nonenterotoxigenic Escherichia coli strains producing F17 fimbriae isolated from diarrheic lambs and goat kids. PMID : 10203489 : PMC : PMC84778 Abstract >>
Forty-five ovine and caprine nonenterotoxigenic Escherichia coli strains producing F17-related fimbriae were characterized with respect to the fimbrial structural subunit and adhesin subtypes produced. In addition, several characteristics related to the virulence of strains producing F17 fimbriae were studied. Most of the strains (73%) possessed the f17cA structural subunit gene, whereas the f17aA and f17dA genes were detected only on three (6%) and two (4%) strains, respectively. The f17bA gene was not detected. All but one of these strains possessed the f17G genes of the adhesin subfamily II. The only strain having the f17G gene of subfamily I possessed the structural subunit gene f17dA. Sequencing of the f17A and f17G genes of four selected strains confirmed the association of f17cA and f17dA structural subunit genes with the f17G genes of the adhesin subfamily II. These results indicated that adhesins of the subfamily II are prominent among ovine and caprine isolates and that they are indistinctly associated with the F17 structural subunit subtypes on these field strains. CS31A- and CNF2-related genes were not detected. Most of the strains adhered in vitro to ovine intestinal brush borders (36 of 45) and agglutinated the erythrocytes of different species in the presence of D-mannose (39 of 45). F17-positive strains produced colicin V (57%) and were resistant to the bactericidal effect of serum (91%) in significantly higher percentages than F17-negative strains (34% produced colicin V, and 66% were serum resistant). Thus, most of the studied ovine and caprine strains showed phenotypic characteristics of septicemic strains.
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385. |
Stuckey JA,
( 1999 ) Crystal structure of a phospholipase D family member. PMID : 10074947 : DOI : 10.1038/6716 Abstract >>
The first crystal structure of a phospholipase D (PLD) family member has been determined at 2.0 A resolution. The PLD superfamily is defined by a common sequence motif, HxK(x)4D(x)6GSxN, and includes enzymes involved in signal transduction, lipid biosynthesis, endonucleases and open reading frames in pathogenic viruses and bacteria. The crystal structure suggests that residues from two sequence motifs form a single active site. A histidine residue from one motif acts as a nucleophile in the catalytic mechanism, forming a phosphoenzyme intermediate, whereas a histidine residue from the other motif appears to function as a general acid in the cleavage of the phosphodiester bond. The structure suggests that the conserved lysine residues are involved in phosphate binding. Large-scale genomic sequencing revealed that there are many PLD family members. Our results suggest that all of these proteins may possess a common structure and catalytic mechanism.
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386. |
Trent MS,
Worsham LM,
( 1999 ) HlyC, the internal protein acyltransferase that activates hemolysin toxin: role of conserved histidine, serine, and cysteine residues in enzymatic activity as probed by chemical modification and site-directed mutagenesis. PMID : 10079090 : DOI : 10.1021/bi982491u Abstract >>
HlyC is an internal protein acyltransferase that activates hemolysin, a toxic protein produced by pathogenic Escherichia coli. Acyl-acyl carrier protein (ACP) is the essential acyl donor. Separately subcloned, expressed, and purified prohemolysin A (proHlyA), HlyC, and [1-14C]myristoyl-ACP have been used to study the conversion of proHlyA to HlyA [Trent, M. S., Worsham, L. M., and Ernst-Fonberg, M. L. (1998) Biochemistry 37, 4644-4655]. HlyC and hemolysin belong to a family of at least 13 toxins produced by Gram-negative bacteria. The homologous acyltransferases of the family show a number of conserved residues that are possible candidates for participation in acyl transfer. Specific chemical reagents and site-directed mutagenesis showed that neither the single conserved cysteine nor the three conserved serine residues were required for enzyme activity. Treatment with the reversible histidine-modifying diethyl pyrocarbonate (DEPC) inhibited acyltransferase activity, and acyltransferase activity was restored following hydroxylamine treatment. The substrate myristoyl-ACP protected HlyC from DEPC inhibition. These findings and spectral absorbance changes suggested that histidine, particularly a histidine proximal to the substrate binding site, was essential for enzyme activity. Site-directed mutageneses of the single conserved histidine residue, His23, to alanine, cysteine, or serine resulted in each instance in complete inactivation of the enzyme.
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387. |
Shannon KP,
French GL,
( 1999 ) Carbapenem resistance in Escherichia coli associated with plasmid-determined CMY-4 beta-lactamase production and loss of an outer membrane protein. PMID : 10223937 : PMC : PMC89134 Abstract >>
Three cefoxitin-resistant Escherichia coli isolates from stool specimens of a patient with leukemia were either resistant, intermediate, or sensitive to imipenem. Conjugation experiments showed that cefoxitin resistance, but not imipenem resistance, was transferable. All isolates were shown by isoelectric focusing to produce two beta-lactamases with isoelectric points of 5.4 (TEM-1, confirmed by sequencing of a PCR product) and >8.5 (consistent with a class C beta-lactamase). The gene coding for the unknown beta-lactamase was cloned and sequenced and revealed an enzyme which had 99.9% sequence identity with the plasmid-determined class C beta-lactamase CMY-2. The cloned beta-lactamase gene differed from blaCMY-2 at one nucleotide position that resulted in an amino acid change, tryptophan to arginine at position 221. We propose that this enzyme be designated CMY-4. Both the imipenem-resistant and -intermediate isolates lacked a 38-kDa outer membrane protein (OMP) that was present in the imipenem-sensitive isolate. The lack of an OMP alone did not explain the difference in carbapenem susceptibilities observed. However, measurement of beta-lactamase activities (including measurements under conditions where TEM-1 beta-lactamase was inhibited) indicated that the imipenem-intermediate isolate expressed six- to eightfold less beta-lactamase than did the other isolates. This study illustrates that carbapenem resistance in E. coli can arise from high-level expression of plasmid-mediated class C beta-lactamase combined with an OMP deficiency. Furthermore, in the presence of an OMP deficiency, the level of expression of a plasmid-mediated class C beta-lactamase is an important factor in determining whether E. coli isolates are fully resistant to carbapenems.
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388. |
Fairbrother JM,
Dubreuil JD,
Harel J,
( 1999 ) Cloning and characterization of the esp region from a dog attaching and effacing Escherichia coli strain 4221 and detection of EspB protein-binding to HEp-2 cells. PMID : 10339811 : DOI : 10.1111/j.1574-6968.1999.tb13571.x Abstract >>
The espA, espB and espD genes from enteropathogenic Escherichia coli were previously shown to be essential for triggering the signal transduction in infected host cells. We have cloned and determined the nucleotide sequences of the espA, espB and espD homologues from an E. coli strain (4221) isolated from a dog which manifested the attaching and effacing lesions in the small intestine. This strain is designated as a dog enteropathogenic E. coli. When comparing predicted amino acid sequences to those of the corresponding proteins from enteropathogenic E. coli O127, enterohemorrhagic E. coli serotype O26, enterohemorrhagic E. coli O157 and rabbit enteropathogenic E. coli, the EspADEPEC protein showed the same level of similarity (75% identity) with EspA of enteropathogenic E. coli O127 and rabbit enteropathogenic E. coli. The EspBDEPEC protein showed the highest similarity with the EspB of enteropathogenic E. coli O127 (99% identity). The EspDDEPEC protein showed 88% identity with the EspDEPEC. We constructed and purified a maltose-binding fusion protein containing the product of the entire espBDEPEC gene of the dog enteropathogenic E. coli strain 4221. Purified maltose-binding protein-EspBDEPEC fusion protein was shown to bind efficiently to HEp-2 cells in a localized fashion as shown by immunofluorescence microscopy. In addition, when the dog enteropathogenic E. coli strain 4221 was grown in tissue culture medium (DMEM) supplemented with serum, a secreted 36-kDa protein was identified by immunoblot analysis using a polyclonal antiserum against the maltose-binding protein-EspBDEPEC fusion protein.
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389. |
Smajs D,
( 1999 ) Characterization of colicin S4 and its receptor, OmpW, a minor protein of the Escherichia coli outer membrane. PMID : 10348872 : PMC : PMC93827 Abstract >>
Analysis of the nucleotide sequence of an Escherichia coli colicin S4 determinant revealed 76% identity to the pore-forming domain of the colicin A protein, 77% identity to the colicin A immunity protein, and 82% identity to the colicin A lysis protein. The N-terminal region, which is responsible for the Tol-dependent uptake of colicin S4, has 94% identity to the N-terminal region of colicin K. By contrast, the predicted receptor binding domain shows no sequence similarities to other colicins. Mutants that lacked the OmpW protein were resistant to colicin S4.
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390. |
Kurazono H,
Ikeda T,
Shirahata T,
( 1998 ) Detection and genetical characterization of Shiga toxin-producing Escherichia coli from wild deer. PMID : 10037215 : DOI : 10.1111/j.1348-0421.1998.tb02356.x Abstract >>
Shiga toxin (Stx)-producing Escherichia coli (STEC) strains isolated from wild deer in Japan were examined. A total of 43 fecal samples were collected 4 times from 4 different sites around Obihiro City, Hokkaido, Japan, in June and July 1997. Seven STEC strains were isolated by PCR screening, all of them were confirmed by ELISA and Vero cell cytotoxicity assay to be producing only active Stx type 2 (Stx2). Moreover, they seemed to carry the hemolysin and eaeA genes of STEC O157:H7, and some isolates harbored large plasmids which were similar to the 90-kilobase virulence plasmid of STEC O157:H7. Based on their plasmid profiles, antibiotic resistance patterns, PCR-based DNA fingerprinting data obtained by using random amplified polymorphic DNA (RAPD) and the stx2 gene sequences, all isolates were divergent from each other except for 3 isolates from the first and second samplings. A DNA sequence analysis of representative isolates revealed that deer originating STEC strains were closely related to each other, but not to the Stx2-producing STEC strains isolated from a mass outbreak in Obihiro at the same time. A phylogenic analysis of the deduced Stx2 amino acid sequences demonstrated that three distinct clusters existed in the deer originating STEC strains and that the Stx of deer originating STEC was closely associated with that originating from humans, but not those of STEC originating from other animals. These results suggest that STEC contamination of deer carcasses should be considered as a potential source of human infection and adequate sanitary inspection of meat for human consumption is also essential for wild animals.
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391. |
Pallecchi L,
Bartoloni A,
Fiorelli C,
Mantella A,
Di Maggio T,
Gamboa H,
Gotuzzo E,
Kronvall G,
Paradisi F,
Rossolini GM,
( 2007 ) Rapid dissemination and diversity of CTX-M extended-spectrum beta-lactamase genes in commensal Escherichia coli isolates from healthy children from low-resource settings in Latin America. PMID : 17548490 : DOI : 10.1128/AAC.00026-07 PMC : PMC1932529 Abstract >>
A survey carried out in 2005 among members of a healthy population of children living in Bolivia and Peru revealed that fecal carriage of Escherichia coli strains resistant to expanded-spectrum cephalosporins was remarkably increased compared to that observed in the same settings in 2002 (1.7% in 2005 versus 0.1% in 2002). In this work, we demonstrated that this phenomenon was mainly related to the dissemination of CTX-M-type extended-spectrum beta-lactamase (ESBL) determinants among commensal E. coli strains. Of 50 ESBL-producing isolates collected in the 2005 survey, 44 harbored a CTX-M-type and 6 an SHV-type (SHV-2 or SHV-12) ESBL. Compared to 2002 results, an increased diversity of CTX-M-type ESBLs was also observed: members of the CTX-M-1 group (CTX-M-15) emerged in Bolivia (where only CTX-M-2 was observed in 2002), while members of the CTX-M-9 group (CTX-M-14 and CTX-M-24) emerged in Peru (where only CTX-M-15 and CTX-M-2 were observed in 2002). A new CTX-M-2 variant named CTX-M-56 was also detected. Molecular characterization of the CTX-M-producing isolates and gene transfer experiments suggested that different mechanisms could be involved in the spreading of different CTX-M group determinants and revealed that additional resistance determinants for non-beta-lactam antibiotics were preferentially carried by plasmids encoding certain CTX-M variants (CTX-M-15 and variants of the CTX-M-2 group). Three CTX-M-15-encoding conjugative plasmids from Peruvian isolates carried the new fluoroquinolone resistance gene aac(6')-Ib-cr. To our best knowledge, this is the first report of the detection of aac(6')-Ib-cr in Latin America.
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392. |
Arlet G,
Philippon A,
( 1999 ) Sequences of the genes for the TEM-20, TEM-21, TEM-22, and TEM-29 extended-spectrum beta-lactamases. PMID : 10103213 : PMC : PMC89239 Abstract >>
The sequences of the blaTEM genes encoding TEM-20, TEM-21, TEM-22, and TEM-29 extended-spectrum beta-lactamases were determined. Analysis of the deduced amino acid sequences indicated that TEM-20 and TEM-29 were derived from TEM-1 and that TEM-21 and TEM-22 were derived from TEM-2. The substitutions involved were Ser-238 and Thr-182 for TEM-20; His-164 for TEM-29; Lys-104, Arg-153, and Ser-238 for TEM-21; and Lys-104, Gly-237, and Ser-238 for TEM-22. The promoter region of the blaTEM-22 gene was identical to that of blaTEM-3. High-level production of TEM-20 could result from a 135-bp deletion which combined the -35 region of the Pa promoter with the -10 region of the P3 promoter and a G-->T transition in the latter motif.
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393. |
Nelson EC,
Elisha BG,
( 1999 ) Molecular basis of AmpC hyperproduction in clinical isolates of Escherichia coli. PMID : 10103209 : PMC : PMC89235 Abstract >>
DNA sequencing data showed that five clinical isolates of Escherichia coli with reduced susceptibility to ceftazidime, ceftriaxone, and cefotaxime contain an ampC gene that is preceded by a strong promoter. Transcription from the strong promoter was 8- to 18-fold higher than that from the promoter from a susceptible isolate. RNA studies showed that mRNA stability does not play a role in the control of AmpC synthesis.
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394. |
Shimizu T,
Takeda Y,
Tsukamoto T,
( 1999 ) Analysis of the genes responsible for the O-antigen synthesis in enterohaemorrhagic Escherichia coli O157. PMID : 10222209 : DOI : 10.1006/mpat.1998.0253 Abstract >>
The genes responsible for O157 O-antigen synthesis were cloned from Shiga toxin 1 (Stx1)-producing enterohaemorrhagic Escherichia coli O157:H-. Based on the published sequence of the rfbE(EcoO157:H7) gene, the rfbE gene was amplified by PCR and used as the probe. One clone (1-4-1) out of ten agglutinated using the slide agglutination test with O157 antiserum. The DNA sequence of a 31.5 kb fragment in p1-4-1 was determined and found to contain 26 open reading frames (ORFs). These ORFs were organized as two clusters of genes, comprising orf2 to orf16 and orf17 to orf24 regions, which were aligned in opposite directions. The GC contents of orf1, orf12 and orf14 to orf26 were similar to the overall GC content of the E. coli chromosome (51%). However, orf2 to orf11 and orf13 showed a much lower GC content of 30.0 to 39.4%. The minimum region essential for O157 O-antigen expression in E. coli K-12 lay between orf2 and orf13, which corresponded to the region of low GC content in E. coli O157. A colony hybridization test was carried out against reference strains of E. coli representing serogroups O1 to O173 using O157 O-antigen synthesis genes (orf2-orf13) of E. coli O157 as probes. With the exception of orf12, all probes from E. coli O157 O-antigen synthesis gene cluster did not hybridize with DNA from E. coli O1 to O173, except O157 reference strains, under high stringency conditions. These data suggest that the region from orf2 to orf13 has originated from a non-E. coli species with a low GC content.
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395. |
Hung DL,
Hultgren SJ,
( 1998 ) Pilus biogenesis via the chaperone/usher pathway: an integration of structure and function. PMID : 10049807 : DOI : 10.1006/jsbi.1998.4049 Abstract >>
The molecular basis of how pathogenic bacteria cause disease has been studied by blending a well-developed genetic system with X-ray crystallography, protein chemistry, high resolution electron microscopy, and cell biology. Microbial attachment to host tissues is one of the key events in the early stages of most bacterial infections. Attachment is typically mediated by adhesins that are assembled into hair-like fibers called pili on bacterial surfaces. This article focuses on the structure-function correlates of P pili, which are produced by most pyelonephritic strains of Escherichia coli. P pili are assembled via a chaperone/usher pathway. Similar pathways are responsible for the assembly of over 30 adhesive organelles in various Gram-negative pathogens. P pilus biogenesis has been used as a model system to elucidate common themes in bacterial pathogenesis, namely, the protein folding, secretion, and assembly of virulence factors. The structural basis for pilus biogenesis is discussed as well as the function and consequences of microbial attachment.
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396. |
Yamane K,
Wachino J,
Suzuki S,
Kimura K,
Shibata N,
Kato H,
Shibayama K,
Konda T,
Arakawa Y,
( 2007 ) New plasmid-mediated fluoroquinolone efflux pump, QepA, found in an Escherichia coli clinical isolate. PMID : 17548499 : DOI : 10.1128/AAC.00339-07 PMC : PMC2043241 Abstract >>
Plasmid-mediated Qnr and AAC(6')-Ib-cr have been recognized as new molecular mechanisms affecting fluoroquinolone (FQ) resistance. C316, an Escherichia coli strain demonstrating resistance to various FQs, was isolated in Japan. Resistance to FQs was augmented in an E. coli CSH2 transconjugant, but PCR failed to detect qnr genes, suggesting the presence of novel plasmid-mediated FQ resistance mechanisms. Susceptibility tests, DNA manipulation, and analyses of the gene and its product were performed to characterize the genetic determinant. A novel FQ-resistant gene, qepA, was identified in a plasmid, pHPA, of E. coli C316, and both qepA and rmtB genes were mediated by a probable transposable element flanked by two copies of IS26. Levels of resistance to norfloxacin, ciprofloxacin, and enrofloxacin were significantly elevated in E. coli transformants harboring qepA under AcrB-TolC-deficient conditions. QepA showed considerable similarities to transporters belonging to the 14-transmembrane-segment family of environmental actinomycetes. The effect of carbonyl cyanide m-chlorophenylhydrazone (CCCP) on accumulation of norfloxacin was assayed in a qepA-harboring E. coli transformant. The intracellular accumulation of norfloxacin was decreased in a qepA-expressing E. coli transformant, but this phenomenon was canceled by CCCP. The augmented FQ resistance level acquired by the probable intergeneric transfer of a gene encoding a major facilitator superfamily-type efflux pump from some environmental microbes to E. coli was first identified. Surveillance of the qepA-harboring clinical isolates should be encouraged to minimize further dissemination of the kind of plasmid-dependent FQ resistance determinants among pathogenic microbes.
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397. |
Drummelsmith J,
Whitfield C,
( 1999 ) Gene products required for surface expression of the capsular form of the group 1 K antigen in Escherichia coli (O9a:K30). PMID : 10200954 : DOI : 10.1046/j.1365-2958.1999.01277.x Abstract >>
The group 1 K30 antigen from Escherichia coli (O9a:K30) is present on the cell surface as both a capsular structure composed of high-molecular-weight K30 polysaccharide and as short K30 oligosaccharides linked to lipid A-core in a lipopolysaccharide molecule (K30LPS). To determine the molecular processes that are responsible for the two forms of K antigen, the 16 kb chromosomal cps region has been characterized. This region encodes 12 gene products required for the synthesis, polymerization and translocation of the K30 antigen. The gene products include four glycosyltransferases responsible for synthesis of the K30 repeat unit; a PST (1) exporter (Wzx), required to transfer lipid-linked K30 units across the plasma membrane to the periplasmic space; and a K30-antigen polymerase (Wzy). These gene products are typical of those seen in O-antigen biosynthesis gene clusters and they interact with the lipopolysaccharide translocation pathway to express K30LPS on the cell surface. The same gene products also provide the biosynthetic intermediates for the capsule assembly pathway, although they are not in themselves sufficient for synthesis of the K30 capsule. Three additional genes, wza, wzb and wzc, encode homologues to proteins that are encoded by gene clusters involved in expression of a variety of bacterial exopolysaccharides. Mutant analysis indicates that Wza and Wzc are required for wild-type surface expression of the capsular structure but are not essential for polymerization and play no role in the translocation of K30LPS. These surface expression components provide the key feature that distinguishes the assembly systems for O antigens and capsules.
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398. |
Iyer VN,
Walker GC,
Woodgate R,
Cellini C,
( 1999 ) Genetic analysis of the mobilization and leading regions of the IncN plasmids pKM101 and pCU1. PMID : 10198024 : PMC : PMC93686 Abstract >>
The conjugative IncN plasmids pKM101 and pCU1 have previously been shown to contain identical oriT sequences as well as conserved restriction endonuclease cleavage patterns within their tra regions. Complementation analysis and sequence data presented here indicate that these two plasmids encode essentially identical conjugal DNA-processing proteins. This region contains three genes, traI, traJ, and traK, transcribed in the same orientation from a promoter that probably lies within or near the conjugal transfer origin (oriT). Three corresponding proteins were visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and complementation analysis confirmed that this region contains three tra complementation groups. All three proteins resemble proteins of the IncW plasmid R388 and other plasmids thought to have roles in processing of plasmid DNA during conjugation. The hydropathy profile of TraJ suggests a transmembrane topology similar to that of several homologous proteins. Both traK and traI were required for efficient interplasmid site-specific recombination at oriT, while traJ was not required. The leading region of pKM101 contains three genes (stbA, stbB, and stbC), null mutations in which cause elevated levels of plasmid instability. Plasmid instability was observed only in hosts that are proficient in interplasmid recombination, suggesting that this recombination can potentially lead to plasmid loss and that Stb proteins somehow overcome this, possibly via site-specific multimer resolution.
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399. |
Li J,
Selander RK,
Whittam TS,
( 1999 ) Molecular evolution and mosaic structure of alpha, beta, and gamma intimins of pathogenic Escherichia coli. PMID : 10331248 : DOI : 10.1093/oxfordjournals.molbev.a026032 Abstract >>
Two types of pathogenic Escherichia coli, enteropathogenic E. coli (EPEC) and enterohemorrhagic E. coli (EHEC), cause diarrheal disease by disrupting the intestinal environment through the intimate attachment of the bacteria to the intestinal epithelium. This process is mediated by intimin, an outer membrane protein that is homologous to the invasins of pathogenic Yersinia. The intimin (eae) gene is part of a pathogenicity island, a 35-kb segment of DNA that has been acquired independently in different groups of pathogens. Nucleotide sequences of eae of three EPEC and four EHEC strains representing distinct clonal lineages revealed an exceptionally high level of divergence (15%) in the amino acid sequences of alpha, beta, and gamma intimin molecules, most of which is concentrated in the C-terminal region. The gamma intimin sequences from E. coli strains with serotypes O157:H7, O55:H7, and O157:H- are virtually identical, supporting the hypothesis that these bacteria belong to a single clonal lineage. Sequences of beta intimin of EPEC strains of serotypes O111:H2 and O128:H2 show substantial differences from alpha and gamma intimins, indicating that these strains have evolved independently. Strong nonrandom clustering of polymorphic sites indicates that the intimin genes are mosaics, suggesting that protein divergence has been accelerated by recombination and diversifying selection.
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400. |
Rahn A,
Drummelsmith J,
( 1999 ) Conserved organization in the cps gene clusters for expression of Escherichia coli group 1 K antigens: relationship to the colanic acid biosynthesis locus and the cps genes from Klebsiella pneumoniae. PMID : 10094716 : PMC : PMC93651 Abstract >>
Group 1 capsules of Escherichia coli are similar to the capsules produced by strains of Klebsiella spp. in terms of structure, genetics, and patterns of expression. The striking similarities between the capsules of these organisms prompted a more detailed investigation of the cps loci encoding group 1 capsule synthesis. Six strains of K. pneumoniae and 12 strains of E. coli were examined. PCR analysis showed that the clusters in these strains are conserved in their chromosomal locations. A highly conserved block of four genes, orfX-wza-wzb-wzc, was identified in all of the strains. The wza and wzc genes are required for translocation and surface assembly of E. coli K30 antigen. The conservation of these genes points to a common pathway for capsule translocation. A characteristic JUMPstart sequence was identified upstream of each cluster which may function in conjunction with RfaH to inhibit transcriptional termination at a stem-loop structure found immediately downstream of the "translocation-surface assembly" region of the cluster. Interestingly, the sequence upstream of the cps clusters in five E. coli strains and one Klebsiella strain indicated the presence of IS elements. We propose that the IS elements were responsible for the transfer of the cps locus between organisms and that they may continue to mediate recombination between strains.
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401. |
Pearce R,
Roberts IS,
( 1999 ) Genetic organization of the Escherichia coli K10 capsule gene cluster: identification and characterization of two conserved regions in group III capsule gene clusters encoding polysaccharide transport functions. PMID : 10094710 : PMC : PMC93645 Abstract >>
Analysis of the Escherichia coli K10 capsule gene cluster identified two regions, regions 1 and 3, conserved between different group III capsule gene clusters. Region 1 encodes homologues of KpsD, KpsM, KpsT, and KpsE proteins, and region 3 encodes homologues of the KpsC and KpsS proteins. An rfaH mutation abolished K10 capsule production, suggesting that expression of the K10 capsule was regulated by RfaH in a manner analogous to group II capsule gene clusters. An IS3 element and a phiR73-like prophage, both of which may have played a role in the acquisition of group III capsule gene clusters, were detected flanking the K10 capsule genes.
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402. |
Cavin JF,
Diviès C,
( 1999 ) Cloning of branched chain amino acid biosynthesis genes and assays of alpha-acetolactate synthase activities in Leuconostoc mesenteroides subsp. cremoris. PMID : 10229948 : Abstract >>
A genomic library from Leuconostoc mesenteroides subsp. cremoris (Lmc) in Escherichia coli was screened for alpha-acetolactate synthase (ALS) activity using a phenotypic test detecting the production of acetolactate or related C4 derivatives (diacetyl, acetoin or 2,3-butanediol) in the culture. Four recombinant E. coli clones, with plasmids containing overlapping DNA fragments and displaying anabolic ALS activity, were selected. This activity is encoded by an ilvB gene belonging to a putative operon which contains genes highly similar to the genes of the branched chain amino acid (BCAA) operon of Lactococcus lactis subsp. lactis. This putative BCAA operon is not functional as the ilvA gene is interrupted by a single mutation and the strain is auxotrophic for the three BCAAs. Only a very low anabolic ALS activity was present in cell-free extracts of Lmc and no transcript from the ilvB gene could be detected. Instability of ilvB expression in E. coli was the consequence of a frequent IS5 insertion sequence in this gene. Despite the detection of a high catabolic ALS activity in Lmc, no catabolic ALS activity gene could be found in the BCAA gene locus, indicating the presence of a catabolic als gene in the Lmc chromosome that could be absent or not expressed in the screened library.
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403. |
Prunier AL,
Schuch R,
Fernández RE,
Maurelli AT,
( 2007 ) Genetic structure of the nadA and nadB antivirulence loci in Shigella spp. PMID : 17586625 : DOI : 10.1128/JB.00525-07 PMC : PMC1951923 Abstract >>
Comparison of nadA and nadB in 14 Shigella strains and enteroinvasive Escherichia coli versus E. coli showed that at least one locus is altered in all strains. These observations explain the characteristic nicotinic acid auxotrophy of Shigella organisms and are consistent with the previously identified antivirulence nature of these genes for these pathogens.
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404. |
Machado E,
Coque TM,
Cantón R,
Novais A,
Sousa JC,
Baquero F,
Peixe L,
N/A N/A,
( 2007 ) High diversity of extended-spectrum beta-lactamases among clinical isolates of Enterobacteriaceae from Portugal. PMID : 17913717 : DOI : 10.1093/jac/dkm381 Abstract >>
To investigate the occurrence and the diversity of Ambler class A ESBLs among Enterobacteriaceae from different Portuguese clinical settings over a 2 year period (2002-04). One hundred and nine extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae isolates from five geographically distant health institutions in Portugal were studied. ESBLs were characterized by isoelectric focussing, PCR and further sequencing. Antibiotic susceptibility testing, transfer of resistance genes and clonal diversity were determined by standard procedures. Plasmid relatedness was established by comparison of random amplified polymorphic DNA (RAPD) patterns. ESBLs were identified as TEM (46%), SHV (30%), CTX-M (22%) and GES (2%) types; TEM-24, TEM-52, SHV-12 and CTX-M-15 enzymes being the most frequently found. Inter-hospital dissemination of epidemic strains harbouring the most prevalent ESBLs was detected, including the TEM-24-producing Enterobacter aerogenes European epidemic clone. Conjugative transfer of ESBLs was achieved for 67% of isolates and epidemic plasmids containing specific bla genes were detected (bla(CTX-M-15) and bla(TEM-24)). We describe two new ESBLs, SHV-90 (A187T, G238S and E240K) and SHV-91 (P20S and E240K), and a new TEM-type enzyme conferring a phenotype resembling that of a complex mutant TEM beta-lactamase, designated as TEM-154 (M69L and R164S). The broad-spectrum beta-lactamases SHV-26, SHV-36 and TEM-110 were first observed in our country. We describe a complex ESBL epidemiology in Portugal, including widespread dissemination of known strains and plasmids coding for TEM-24 and CTX-M-15 enzymes as observed in other European countries.
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405. |
Torrents E,
Grinberg I,
Gorovitz-Harris B,
Lundström H,
Borovok I,
Aharonowitz Y,
Sjöberg BM,
Cohen G,
( 2007 ) NrdR controls differential expression of the Escherichia coli ribonucleotide reductase genes. PMID : 17496099 : DOI : 10.1128/JB.00440-07 PMC : PMC1951866 Abstract >>
Escherichia coli possesses class Ia, class Ib, and class III ribonucleotide reductases (RNR). Under standard laboratory conditions, the aerobic class Ia nrdAB RNR genes are well expressed, whereas the aerobic class Ib nrdEF RNR genes are poorly expressed. The class III RNR is normally expressed under microaerophilic and anaerobic conditions. In this paper, we show that the E. coli YbaD protein differentially regulates the expression of the three sets of genes. YbaD is a homolog of the Streptomyces NrdR protein. It is not essential for growth and has been renamed NrdR. Previously, Streptomyces NrdR was shown to transcriptionally regulate RNR genes by binding to specific 16-bp sequence motifs, NrdR boxes, located in the regulatory regions of its RNR operons. All three E. coli RNR operons contain two such NrdR box motifs positioned in their regulatory regions. The NrdR boxes are located near to or overlap with the promoter elements. DNA binding experiments showed that NrdR binds to each of the upstream regulatory regions. We constructed deletions in nrdR (ybaD) and showed that they caused high-level induction of transcription of the class Ib RNR genes but had a much smaller effect on induction of transcription of the class Ia and class III RNR genes. We propose a model for differential regulation of the RNR genes based on binding of NrdR to the regulatory regions. The model assumes that differences in the positions of the NrdR binding sites, and in the sequences of the motifs themselves, determine the extent to which NrdR represses the transcription of each RNR operon.
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406. |
Palù G,
Tossi A,
Battistutta R,
D'Acunto MR,
( 1999 ) Crystal structure of the B subunit of Escherichia coli heat-labile enterotoxin carrying peptides with anti-herpes simplex virus type 1 activity. PMID : 10085117 : Abstract >>
Two chimeric proteins, consisting of the B subunit of Escherichia coli heat-labile enterotoxin with different peptides fused to the COOH-terminal ends, have been crystallized and their three-dimensional structure determined. The two extensions correspond to (a) a nonapeptide representing the COOH-terminal sequence of the small subunit of herpes simplex virus type 1 ribonucleotide reductase and (b) a 27-amino acid long peptide, corresponding to the COOH-terminal end of the catalytic subunit (POL) of DNA polymerase from the same virus. Both proteins crystallize in the P41212 space group with one pentameric molecule per asymmetric unit, corresponding to a solvent content of about 75%. The overall conformation of the B subunit pentamer in the two chimeric proteins, which consists of five identical polypeptide chains, is very similar to that in the native AB complex and conforms strictly to 5-fold symmetry. On the contrary, the peptide extensions are essentially disordered: in the case of the nonapeptide, only 5 and 6 amino acids were, respectively, positioned in two monomers, while in the other three only 2 residues are ordered. The extension is fully confined to the surface of the pentamer opposite to the face that interacts with the membrane and consequently it does not interfere with the ability of the B subunit to interact with membrane receptors. Moreover, the conformational flexibility of the two peptide extensions could be correlated to their propensity for proteolytic processing and consequent release of a biologically active molecule into cultured cells.
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407. |
Wachino J,
Shibayama K,
Kurokawa H,
Kimura K,
Yamane K,
Suzuki S,
Shibata N,
Ike Y,
Arakawa Y,
( 2007 ) Novel plasmid-mediated 16S rRNA m1A1408 methyltransferase, NpmA, found in a clinically isolated Escherichia coli strain resistant to structurally diverse aminoglycosides. PMID : 17875999 : DOI : 10.1128/AAC.00926-07 PMC : PMC2168023 Abstract >>
We have isolated a multiple-aminoglycoside-resistant Escherichia coli strain, strain ARS3, and have been the first to identify a novel plasmid-mediated 16S rRNA methyltransferase, NpmA. This new enzyme shared a relatively low level of identity (30%) to the chromosomally encoded 16S rRNA methyltransferase (KamA) of Streptomyces tenjimariensis, an actinomycete aminoglycoside producer. The introduction of a recombinant plasmid carrying npmA could confer on E. coli consistent resistance to both 4,6-disubstituted 2-deoxystreptamines, such as amikacin and gentamicin, and 4,5-disubstituted 2-deoxystreptamines, including neomycin and ribostamycin. The histidine-tagged NpmA elucidated methyltransferase activity against 30S ribosomal subunits but not against 50S subunits and the naked 16S rRNA molecule in vitro. We further confirmed that NpmA is an adenine N-1 methyltransferase specific for the A1408 position at the A site of 16S rRNA. Drug footprinting data indicated that binding of aminoglycosides to the target site was apparently interrupted by methylation at the A1408 position. These observations demonstrate that NpmA is a novel plasmid-mediated 16S rRNA methyltransferase that provides a panaminoglycoside-resistant nature through interference with the binding of aminoglycosides toward the A site of 16S rRNA through N-1 methylation at position A1408.
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408. |
Chong Y,
Lee K,
( 1998 ) Plasmid-encoded AmpC beta-lactamases: how far have we gone 10 years after the discovery? PMID : 10097678 : DOI : 10.3349/ymj.1998.39.6.520 Abstract >>
The dogma that ampC genes are located exclusively on the chromosome was dominant until about 10 years ago. Since 1989 over 15 different plasmid-encoded AmpC beta-lactamases have been reported from several countries. Most of these enzymes evolved in two clusters. The major cluster includes several enzymes with a high similarity to CMY-2, which is the closest related chromosomal AmpC enzyme of Citrobacter freundii. A second cluster centers around CMY-1. It is less homogeneous and not closely related chromosomal AmpC enzymes. Molecular diversification by amino acid substitutions does not usually translate into a change in the resistance phenotype. At this time, CMY-2 appears to be the most prevalent and widely distributed. Further global increase of prevalence and diversity of plasmidic AmpC beta-lactamases have to be anticipated in the next millenium.
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409. |
Rebuffat S,
Salomón RA,
Farías RN,
Prigent Y,
Chiuchiolo MJ,
Barthélémy M,
( 1999 ) The cyclic structure of microcin J25, a 21-residue peptide antibiotic from Escherichia coli. PMID : 10092860 : DOI : 10.1046/j.1432-1327.1999.00085.x Abstract >>
Microcin J25 (MccJ25) is the single representative of the immunity group J of the microcin group of peptide antibiotics produced by Enterobacteriaceae. It induces bacterial filamentation in susceptible cells in a non-SOS-dependent pathway [R. A. Salomon and R. Farias (1992) J. Bacteriol. 174, 7428-7435]. MccJ25 was purified to homogeneity from the growth medium of a microcin-overproducing Escherichia coli strain by reverse-phase HPLC. Based on amino acid composition and absolute configuration determination, liquid secondary ion and electrospray mass spectrometry, extensive two-dimensional NMR, enzymatic and chemical degradations studies, the structure of MccJ25 was elucidated as a 21-residue peptide, cyclo(-Val1-Gly-Ile-Gly-Thr- Pro-Ile-Ser-Phe-Tyr-Gly-Gly-Gly-Ala-Gly-His-Val-Pro-Glu-Tyr-Phe21-). Although MccJ25 showed high resistance to most of endoproteases, linearization by thermolysin occurred from cleavage at the Phe21-Val1 bond and led to a single peptide, MccJ25-L. While MccJ25 exhibited remarkable antibiotic activity towards Salmonella newport and several E. coli strains (minimal inhibitory concentrations ranging between 0.01 and 0.2 microgram.mL-1), the thermolysin-linearized microcin showed a dramatic decrease of the activity, indicating that the cyclic structure is essential for the MccJ25 biological properties. As MccJ25 is ribosomally synthesized as a larger peptide precursor endowed with an N-terminal extremity, the present study shows that removal of this extension and head-tail cyclization of the resulting propeptide are the only post-translational modifications involved in the maturation of MccJ25, that appears as the first cyclic microcin.
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410. |
Bae IK,
Lee YN,
Lee WG,
Lee SH,
Jeong SH,
( 2007 ) Novel complex class 1 integron bearing an ISCR1 element in an Escherichia coli isolate carrying the blaCTX-M-14 gene. PMID : 17517851 : DOI : 10.1128/AAC.00279-07 PMC : PMC1932499 Abstract >>
This work identifies an ISCR1-related bla(CTX-M-14) gene, which has never been reported before, from a clinical isolate of Escherichia coli. The bla(CTX-M-14) gene was preceded by an ISCR1 element that was followed by a class 1 integron containing three different insert gene cassettes, i.e., dfrA12, orfF, and aadA2.
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411. |
Diekmann H,
Ghisla S,
Macheroux P,
Seth O,
Smau L,
( 1999 ) Studies with lysine N6-hydroxylase. Effect of a mutation in the assumed FAD binding site on coenzyme affinities and on lysine hydroxylating activity. PMID : 10064136 : DOI : 10.1515/BC.1999.006 Abstract >>
The proposed FAD binding site of L-lysine N6-hydroxylase (EC 1.14.13.99) exhibits an unusual proline in a position where a highly conserved glycine is found in other FAD dependent hydroxylases. We have studied the role of this proline by mutating it to glycine in [P14G]aerA, which was expressed in Escherichia coli M15-2 and purified to homogeneity. The mutation has marked effects on the affinities of the cofactors FAD and NADPH as well as the substrate, lysine. Compared to the wild-type enzyme, the activity vs. pH profile of the mutant protein indicates a shift of the apparent pK'(a)s (7.8 and 8.7 for wild-type and 6.8 and 7.7 for the P14G-mutant enzyme) and of the activity maximum (pH 8 for wild-type and pH 7 for the P14G-mutant enzyme). While the activity of the mutant enzyme is much lower under conditions found to be optimal for the wild-type enzyme, adjustment of substrate and cofactor concentrations and pH leads to comparable activities for the mutant enzyme. These results suggest that the proline fulfils an important structural role in the proposed FAD binding site.
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412. |
Boor KJ,
Wiedmann M,
Rendano L,
( 1999 ) Characterization of rpoS alleles in Escherichia coli O157:H7 and in other E. coli serotypes. PMID : 10063629 : Abstract >>
The rpoS nucleotide and predicted amino acid sequences from three Escherichia coli O157:H7 isolates were compared with those from three other E. coli isolates, including the likely O157:H7 progenitor, E. coli O55:H7. These clinical and environmental isolates all had identical sigma S amino acid sequences, while laboratory strains K12 and DH1 had three and one amino acid alterations, respectively, in comparison with the majority sequence. To extend the analysis of sigma S sequence conservation to include other Gram-negative bacteria, the E. coli sigma S sequences were compared with those from diverse Gram-negative organisms; sigma S sequence identities ranged from 50.2 to 99.7% among the available sequences. The results further confirm the existence of rpoS alleles among different E. coli strains, although all strains were classified as acid-resistant with survival rates > 10% after 2 h exposure to pH 2.5. It was also found that all E. coli O157:H7 isolates tested had a unique nucleotide at position 543, thus differentiating these strains from other E. coli serotypes.
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413. |
Verger D,
Bullitt E,
Hultgren SJ,
Waksman G,
( 2007 ) Crystal structure of the P pilus rod subunit PapA. PMID : 17511517 : DOI : 10.1371/journal.ppat.0030073 PMC : PMC1868955 DOI : 10.1371/journal.ppat.0030073 PMC : PMC1868955 Abstract >>
P pili are important adhesive fibres involved in kidney infection by uropathogenic Escherichia coli strains. P pili are assembled by the conserved chaperone-usher pathway, which involves the PapD chaperone and the PapC usher. During pilus assembly, subunits are incorporated into the growing fiber via the donor-strand exchange (DSE) mechanism, whereby the chaperone's G1 beta-strand that complements the incomplete immunoglobulin-fold of each subunit is displaced by the N-terminal extension (Nte) of an incoming subunit. P pili comprise a helical rod, a tip fibrillum, and an adhesin at the distal end. PapA is the rod subunit and is assembled into a superhelical right-handed structure. Here, we have solved the structure of a ternary complex of PapD bound to PapA through donor-strand complementation, itself bound to another PapA subunit through DSE. This structure provides insight into the structural basis of the DSE reaction involving this important pilus subunit. Using gel filtration chromatography and electron microscopy on a number of PapA Nte mutants, we establish that PapA differs in its mode of assembly compared with other Pap subunits, involving a much larger Nte that encompasses not only the DSE region of the Nte but also the region N-terminal to it.
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414. |
Han Y,
Lei XG,
( 1999 ) Cloning, sequencing, and expression of an Escherichia coli acid phosphatase/phytase gene (appA2) isolated from pig colon. PMID : 10092520 : DOI : 10.1006/bbrc.1999.0361 Abstract >>
Bacterial strains were isolated from the pig colon to screen for phytase and acid phosphatase activities. Among 93 colonies, Colony 88 had the highest activities for both enzymes and was identified as an Escherichia coli strain. Using primers derived from the E. coli pH 2.5 acid phosphatase appA sequence (Dassa et al. (1990), J. Bacteriol. 172, 5497-5500), we cloned a 1482 bp DNA fragment from the isolate. In spite of 95% homology between the sequenced gene and the appA, 7 amino acids were different in their deduced polypeptides. To characterize the properties and functions of the encoded protein, we expressed the coding region of the isolated DNA fragment and appA in Pichia pastoris, respectively, as r-appA2 and r-appA. The recombinant protein r-appA2, like r-appA and the r-phyA phytase expressed in Aspergillus niger, was able to hydrolyze phosphorus from sodium phytate and p-nitrophenyl phosphate. However, there were distinct differences in their pH profiles, Km and Vmax for the substrates, specific activities of the purified enzymes, and abilities to release phytate phosphorus in soybean meal. In conclusion, the DNA fragment isolated from E. coli in pig colon seems to encode for a new acid phosphatase/phytase and is designated as E. coli appA2.
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415. |
Elias WP,
Nataro JP,
Trabulsi LR,
( 1999 ) Organization of biogenesis genes for aggregative adherence fimbria II defines a virulence gene cluster in enteroaggregative Escherichia coli. PMID : 10074069 : PMC : PMC93575 Abstract >>
Several virulence-related genes have been described for prototype enteroaggregative Escherichia coli (EAEC) strain 042, which has been shown to cause diarrhea in human volunteers. Among these factors are the enterotoxins Pet and EAST and the fimbrial antigen aggregative adherence fimbria II (AAF/II), all of which are encoded on the 65-MDa virulence plasmid pAA2. Using nucleotide sequence analysis and insertional mutagenesis, we have found that the genes required for the expression of each of these factors, as well as the transcriptional activator of fimbrial expression AggR, map to a distinct cluster on the pAA2 plasmid map. The cluster is 23 kb in length and includes two regions required for expression of the AAF/II fimbria. These fimbrial biogenesis genes feature a unique organization in which the chaperone, subunit, and transcriptional activator lie in one cluster, whereas the second, unlinked cluster comprises a silent chaperone gene, usher, and invasin reminiscent of Dr family fimbrial clusters. This plasmid-borne virulence locus may represent an important set of virulence determinants in EAEC strains.
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416. |
Iwasa M,
Morimoto Y,
Kobori H,
Asakura H,
( 1999 ) Detection of Escherichia coli O157:H7 from Musca domestica (Diptera: Muscidae) at a cattle farm in Japan. PMID : 10071501 : DOI : 10.1093/jmedent/36.1.108 Abstract >>
Enterohemorrhagic Escherichia coli (EHEC) O157:H7 was isolated for the first time from Musca domestica L. A total of 310 fly samples was collected from 4 different farms in Obihiro-City, Hokkaido, in the summer and autumn of 1997;5 samples carried E. coli serotype O157:H7. Using ELISA and Vero cell cytotoxicity assay, 3 isolates from 1 cattle farm produced both active Shiga-toxin type 1 (Stx1) and 2 (Stx2). These isolates also carried hemolysin and eaeA genes and harbored the 90-kb virulence plasmid of EHEC O157:H7. Based on plasmid profiles, antibiotic patterns, polymerase chain reaction (PCR)-based DNA finger printing analysis using random amplified polymorphic DNA, pulsed field gel electrophoresis analysis, and DNA sequences of stx1 and stx2, all 3 isolates from fly samples were identical. These results indicate that the house fly is capable of carrying the toxigenic EHEC O157:H7 involved in human disease.
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417. |
Nordmann P,
Chaibi EB,
Labia R,
Naas T,
( 1999 ) Molecular and biochemical characterization of VEB-1, a novel class A extended-spectrum beta-lactamase encoded by an Escherichia coli integron gene. PMID : 10049269 : PMC : PMC89162 Abstract >>
A clinical isolate, Escherichia coli MG-1, isolated from a 4-month-old Vietnamese orphan child, produced a beta-lactamase conferring resistance to extended-spectrum cephalosporins and aztreonam. In a disk diffusion test, a typical synergistic effect between ceftazidime or aztreonam and clavulanic acid was observed along with an unusual synergy between cefoxitin and cefuroxime. The gene for VEB-1 (Vietnamese extended-spectrum beta-lactamase) was cloned and expressed in E. coli JM109. The recombinant plasmid pRLT1 produced a beta-lactamase with a pI of 5.35 and conferred high-level resistance to extended-spectrum (or oxyimino) cephalosporins and to aztreonam. Vmax values for extended-spectrum cephalosporins were uncommonly high, while the affinity of the enzyme for ceftazidime and aztreonam was relatively low. blaVEB-1 showed significant homology at the DNA level with only blaPER-1 and blaPER-2. Analysis of the deduced protein sequence showed that VEB-1 is a class A penicillinase having very low levels of homology with any other known beta-lactamases. The highest percentage of amino acid identity was 38% with PER-1 or PER-2, two uncommon class A extended-spectrum enzymes. Exploration of the genetic environment of blaVEB-1 revealed the presence of gene cassette features, i.e., (i) a 59-base element associated with blaVEB-1; (ii) a second 59-base element just upstream of blaVEB-1, likely belonging to the aacA1-orfG gene cassette; (iii) two core sites (GTTRRRY) on both sides of blaVEB-1; and (iv) a second antibiotic resistance gene 3' of blaVEB-1, aadB. blaVEB-1 may therefore be the first class A extended-spectrum beta-lactamase that is part of a gene cassette, which itself is likely to be located on a class 1 integron, as sulfamide resistance may indicate. Furthermore, blaVEB-1 is encoded on a large (> 100-kb) transferable plasmid found in a Klebsiella pneumoniae MG-2 isolated at the same time from the same patient, indicating a horizontal gene transfer.
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418. |
Vo AT,
van Duijkeren E,
Fluit AC,
Gaastra W,
( 2007 ) Characteristics of extended-spectrum cephalosporin-resistant Escherichia coli and Klebsiella pneumoniae isolates from horses. PMID : 17521833 : DOI : 10.1016/j.vetmic.2007.04.027 Abstract >>
The aim of the present study was to contribute to the knowledge on extended-spectrum beta-lactamases (ESBL's), AmpC beta-lactamases and integrons in Enterobacteriaceae isolated from horses, which is still limited. The susceptibility of 1581 clinical isolates from animals to ceftiofur was tested. Most of these isolates (n=1347) originated from horses. Seven ceftiofur-resistant equine isolates (four Escherichia coli and three Klebsiella pneumoniae) were identified and all seven were multidrug-resistant. These isolates were further studied for the presence of ESBL's, AmpC beta-lactamases and class 1 integrons. The potential for the horizontal transfer of resistance genes among these clinical isolates was also studied. ESBL-type resistance genes were found in five isolates, AmpC-type genes in one isolates and integrons in six isolates. Nucleotide sequence analysis revealed that the isolates carried the bla(CTX-M-1), bla(CMY-2), bla(TEM-1) and/or bla(SHV-1) genes. This is the first report describing the in vitro conjugal transfer of the bla(CTX-M-1) genes from a clinical E. coli isolate to Salmonella isolates. Gene cassettes encoding resistance to aminoglycosides (aadA1, aadA2 and aadA5), and trimethoprim (dfrA1, drfA12 and dfrA17) were found on the integrons present in the isolates. The cassette arrays of the dfrA17-aadA5 and dfrA1-aadA1 genes in the two integrons of a single E. coli isolate have not yet been described before. To our knowledge this is the first report on ESBL's and AmpC beta-lactamases in equine E. coli and Klebsiella isolates.
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419. |
Duquesne S,
Petit V,
Peduzzi J,
Rebuffat S,
( 2007 ) Structural and functional diversity of microcins, gene-encoded antibacterial peptides from enterobacteria. PMID : 17827970 : DOI : 10.1159/000104748 Abstract >>
Microcins are a peculiar class of gene-encoded low-molecular-mass antibacterial peptides secreted by enterobacteria. They contribute to the regulation of microbial competitions within the intestinal microbiota. The genetic systems involved in microcin biosynthesis share a conserved organization. Similar to bacteriocins of Gram-positive bacteria, microcins exert potent antibacterial activity directed against phylogenetically-related bacterial strains, with minimal inhibitory concentrations in the nanomolar range. In contrast to bacteriocins, they display a great structural diversity among the few representatives well characterized until now, that makes difficult the description of microcin subclasses. This review focuses on three microcins, MccE492m that carries a C-terminal posttranslational modification containing a catechol-type siderophore, MccJ25, a cyclic peptide with a unique 'lasso-type' structure and MccC7 or C51, with a common N-formylated heptapeptide-nucleotide structure. We show these microcins exhibit 'Trojan horse' mechanisms of antibacterial activity: either (i) the microcin structure is a mime of an essential element, permitting its recognition by outer membrane receptors used for vital functions in bacteria and further translocation into the periplasmic space, or (ii) it is secreted as a harmless molecule and further processed in susceptible bacteria to form the toxic entity. When inside target bacteria, microcins bind essential enzymes or interact with the inner membrane to form a bacterial killing structure.
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420. |
Horii T,
Ohta M,
Shibayama K,
Sato K,
( 1999 ) Emergence of fosfomycin-resistant isolates of Shiga-like toxin-producing Escherichia coli O26. PMID : 10103182 : PMC : PMC89208 Abstract >>
We evaluated the susceptibilities of 129 Shiga-like toxin-producing Escherichia coli (STEC) isolates to various antibiotics. The numbers of isolates for which MICs were high (> or = 128 micrograms/ml) were as follows: 5 for fosfomycin, 14 for ampicillin, 1 for cefaclor, 6 for kanamycin, 22 for tetracycline, and 2 for doxycycline. For two isolates of STEC O26 MICs of fosfomycin were high (1,024 and 512 micrograms/ml, respectively). Conjugation experiments and glutathione S-transferase assays suggested that the fosfomycin resistance in these isolates was determined not by a plasmid but chromosomally. The amount of active intracellular fosfomycin in these STEC isolates was 100- to 200-fold less than that in E. coli C600 harboring pREFTT47B408 in the presence of either L-alpha-glycerophosphate or glucose-6-phosphate. Cloning, sequencing, and Northern blot analysis demonstrated that the transcriptional level of the murA gene encoding UDP-N-acetylglucosamine enolpyruvoyl transferase in these isolates was greater than that in E. coli C600. Our results suggest that the fosfomycin resistance in these STEC isolates is due to concurrent effects of alteration of the glpT and/or uhp transport systems and of the enhanced transcription of the murA gene.
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421. |
Cookson AL,
Bennett J,
Thomson-Carter F,
Attwood GT,
( 2007 ) Intimin subtyping of Escherichia coli: concomitant carriage of multiple intimin subtypes from forage-fed cattle and sheep. PMID : 17521363 : DOI : 10.1111/j.1574-6968.2007.00755.x Abstract >>
The outer membrane protein, intimin (eae), which mediates bacterial attachment to epithelial cells, is associated with enteropathogenic Escherichia coli and some Shiga toxin-producing E. coli. The eae subtype of E. coli strains isolated from healthy cattle and sheep was identified using a rapid PCR-restriction fragment length polymorphism (RFLP) method to produce profiles that were compared with those generated in silico. The 139 eae-positive E. coli strains were separated into 11 different PCR-RFLP profiles. The most common eae PCR-RFLP type was beta (23.7%), followed by zeta (20.1%), theta (16.5%), iota (12.2%), kappa (8.6%), epsilon (7.2%), gamma (2.9%), nu and beta2 (2.2%) and iota2 (1.4%). Four isolates did not yield a PCR-RFLP amplification product but complete sequencing of the eae gene matched subtype rho. Two different eae variants were isolated from the same swab from 18 different animals and subtype iota was the most 'promiscuous', being isolated with four other eae subtypes from seven separate animals. None of the eae-positive STEC were subtype gamma, which is associated with STEC serogroup O157. This method allowed the rapid identification of eae subtypes and indicates that forage-fed animals possessed a wide diversity of bacterial eae subtypes with a low frequency of eae subtype gamma.
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422. |
Gilmour MW,
Tabor H,
Wang G,
Clark CG,
Tracz DM,
Olson AB,
Mascarenhas M,
Karmali MA,
Mailman T,
Ng LK,
( 2007 ) Isolation and genetic characterization of a coinfection of non-O157 Shiga toxin-producing Escherichia coli. PMID : 17804662 : DOI : 10.1128/JCM.01125-07 PMC : PMC2168521 Abstract >>
A coinfection of O177:NM and O55:H7 Shiga toxin-producing Escherichia coli (STEC) was identified for a child with acute bloody diarrhea and hemolytic uremic syndrome by using culture and serotype-specific molecular reagents. The profile of O157-related genetic islands revealed that the O55:H7 isolate was highly similar to O157 STEC whereas the O177:NM isolate lacked several fimbrial O islands and non-locus-of-enterocyte-effacement effector determinants. However, both STEC serotypes are known to cause serious disease, and the significant repertoire of virulence determinants in both strains made it impossible to determine their individual contributions to the clinical symptoms.
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423. |
Kikuvi GM,
Schwarz S,
Ombui JN,
Mitema ES,
Kehrenberg C,
( 2007 ) Streptomycin and chloramphenicol resistance genes in Escherichia coli isolates from cattle, pigs, and chicken in Kenya. PMID : 17536935 : DOI : 10.1089/mdr.2006.9998 Abstract >>
The aims of this study were to determine the genetic basis of streptomycin and chloramphenicol resistance in 30 Escherichia coli isolates from food animals in Kenya and the role of plasmids in the spread of the resistance. Seven of the 29 streptomycin-resistant isolates harbored both the strA and strB genes. Twenty-one of isolates had the strA, strB, and aadA1 genes. The strA gene was disrupted by a functional trimethoprim gene, dfrA14 in 10 of the 21 isolates harboring the three streptomycin resistance genes. Physical linkage of intact strA and sul2 genes was found in two different plasmids from four isolates. Linkage of cassette-borne aadA1 and dfrA1 genes in class 1 integrons was found in two of the isolates. Chloramphenicol resistance was due to the gene catA1 in all the chloramphenicol resistant isolates. The strB, strA, and catA1 genes were transferable by conjugation and this points to the significance of conjugative resistance plasmids in the spread and persistence of streptomycin and chloramphenicol resistance in food animals in Kenya.
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424. |
Bergfeld AK,
Claus H,
Vogel U,
Mühlenhoff M,
( 2007 ) Biochemical characterization of the polysialic acid-specific O-acetyltransferase NeuO of Escherichia coli K1. PMID : 17519228 : DOI : 10.1074/jbc.M703044200 Abstract >>
Escherichia coli K1 is a leading pathogen in neonatal sepsis and meningitis. The K1 capsule, composed of alpha2,8-linked polysialic acid, represents the major virulence factor. In some K1 strains, phase-variable O-acetylation of the capsular polysaccharide is observed, a modification that is catalyzed by the prophage-encoded O-acetyltransferase NeuO. Phase variation is mediated by changes in the number of heptanucleotide repeats within the 5'-coding region of neuO, and full-length translation is restricted to repeat numbers that are a multiple of three. To understand the biochemical basis of K1 capsule O-acetylation, NeuO encoded by alleles containing 0, 12, 24, and 36 repeats was expressed and purified to homogeneity via a C-terminal hexahistidine tag. All NeuO variants assembled into hexamers and were enzymatically active with a high substrate specificity toward polysialic acid with >14 residues. Remarkably, the catalytic efficiency (k(cat)/K(m)(donor)) increased linearly with increasing numbers of repeats, revealing a new mechanism for modulating NeuO activity. Using homology modeling, we predicted a three-dimensional structure primarily composed of a left-handed parallel beta-helix with one protruding loop. Two amino acids critical for catalytic activity were identified and corresponding alanine substitutions, H119A and W143A, resulted in a complete loss of activity without affecting the oligomerization state. Our results indicate that in NeuO typical features of an acetyltransferase of the left-handed beta-helix family are combined with a unique regulatory mechanism based on variable N-terminal protein extensions formed by tandem copies of an RLKTQDS heptad.
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425. |
Ho PL,
Poon WW,
Loke SL,
Leung MS,
Chow KH,
Wong RC,
Yip KS,
Lai EL,
Tsang KW,
N/A N/A,
( 2007 ) Community emergence of CTX-M type extended-spectrum beta-lactamases among urinary Escherichia coli from women. PMID : 17496058 : DOI : 10.1093/jac/dkm144 Abstract >>
To conduct a territory-wide study of extended-spectrum beta-lactamases (ESBLs) among community isolates of urinary Escherichia coli from women in Hong Kong. Up to 50 consecutive single-patient E. coli isolates, collected from 13 laboratories in 2004, were studied. The ESBLs were characterized by PCR sequencing using specific primers. The epidemiological relationship of the isolates was studied by PFGE and phylogenetic group PCRs. Forty-two ESBL producers were found among 600 consecutive isolates tested. The ESBL prevalence was 7.3% (15/205) for women aged 18-35 years, 5% (11/219) for women aged 36-50 years, 6.3% (4/63) for women aged 51-64 years and 10.6% (12/113) for women aged >or=65 years (P=0.3). The ESBL-producing isolates were often multidrug-resistant and CTX-M-14 was found in 37 isolates, CTX-M-15 in 3 isolates and CTX-M-3 in 2 isolates. PFGE revealed no significant clusters among the ESBL producers. Overall, CTX-M-14 producers were significantly more likely to belong to group D than non-ESBL producers [18/37 (48.6%) versus 13/57 (22.8%), P=0.009]. However, 7 of 13 (53.8%) CTX-M-14 producers from women aged 18-35 years represented phylogenetic group B2, compared with 7 of 24 (29.2%) for women of all other ages (P=0.1). The study documented the community emergence of CTX-M as the predominant ESBL type among urinary isolates from women. The spread of CTX-M enzymes among isolates from young women is concerning and deserves close monitoring.
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426. |
Tsesin N,
Kogan A,
Gdalevsky GY,
Himanen JP,
Cohen-Luria R,
Parola AH,
Goldgur Y,
Almog O,
( 2007 ) The structure of apo tryptophanase from Escherichia coli reveals a wide-open conformation. PMID : 17704565 : DOI : 10.1107/S0907444907036396 Abstract >>
The crystal structure of apo tryptophanase from Escherichia coli (space group F222, unit-cell parameters a = 118.4, b = 120.1, c = 171.2 A) was determined at 1.9 A resolution using the molecular-replacement method and refined to an R factor of 20.3% (R(free) = 23.2%). The structure revealed a significant shift in the relative orientation of the domains compared with both the holo form of Proteus vulgaris tryptophanase and with another crystal structure of apo E. coli tryptophanase, reflecting the internal flexibility of the molecule. Domain shifts were previously observed in tryptophanase and in the closely related enzyme tyrosine phenol-lyase, with the holo form found in an open conformation and the apo form in either an open or a closed conformation. Here, a wide-open conformation of the apo form of tryptophanase is reported. A conformational change is also observed in loop 297-303. The structure contains a hydrated Mg(2+) at the cation-binding site and a Cl(-) ion at the subunit interface. The enzyme activity depends on the nature of the bound cation, with smaller ions serving as inhibitors. It is hypothesized that this effect arises from variations of the coordination geometry of the bound cation.
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427. |
Bergholz TM,
Tarr CL,
Christensen LM,
Betting DJ,
Whittam TS,
( 2007 ) Recent gene conversions between duplicated glutamate decarboxylase genes (gadA and gadB) in pathogenic Escherichia coli. PMID : 17675652 : DOI : 10.1093/molbev/msm163 Abstract >>
Escherichia coli have evolved adaptive systems to resist strongly acidic habitats in part through the production of 2 biochemically identical isoforms of glutamate decarboxylase (GAD), encoded by the gadA and gadB genes. These genes occur in E. coli and other members of the genospecies (e.g., Shigella spp.) and originated as part of a genomic fitness island acquired early in Escherichia evolution. The present duplicated gad loci are widely spaced on the E. coli chromosome, and the 2 genes are 97% similar in sequence. Comparison of the nucleotide sequences of the gadA and gadB in 16 strains of pathogenic E. coli revealed 3.8% and 5.0% polymorphism in the 2 genes, respectively. Alignment of the homologous genes identified a total of 120 variable sites, including 21 fixed nucleotide differences between the loci within the first 82 codons of the genes. Twenty-three phylogenetically informative sites were polymorphic for the same nucleotides in both genes suggesting recent gene conversions or intergenic recombination. Phylogenetic analysis based on the synonymous substitutions per synonymous site indicated 2 cases in which specific gadA and gadB alleles were more closely related to one another than to other alleles at the corresponding locus. The results indicate that at least 3 gene conversion events have occurred after the gad gene duplication in the evolution of E. coli. Despite multiple gene conversion events, the upstream regulatory regions and the 5' end of each gene remains distinct, suggesting that maintaining functionally different gad genes is important in this acid-resistance mechanism in pathogenic E. coli.
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428. |
Young JM,
Park DC,
( 2007 ) Relationships of plant pathogenic enterobacteria based on partial atpD, carA, and recA as individual and concatenated nucleotide and peptide sequences. PMID : 17451899 : DOI : 10.1016/j.syapm.2007.03.002 Abstract >>
Relationships of the genera in the Enterobacteriaceae containing plant pathogenic species: Brenneria, Dickeya, Enterobacter, Erwinia, Pantoea, Pectobacterium, and Samsonia, were investigated by comparison of their nucleotide and peptide sequences of atpD, carA, recA, and the concatenated sequences. Erwinia spp. and Pantoea spp., with Pectobacterium cypripedii, formed a group distinct from other pathogenic taxa. Pectobacterium, Brenneria, Dickeya, and Samsonia formed a contiguous clade. Samsonia was usually concurrent with Pectobacterium. Most Brenneria were also close to Pectobacterium, suggesting that these three taxa might be better represented as a single genus. Brenneria quercina was not closely associated with other members of this genus and may represent a separate genus. The sequences representing Dickeya were distinct, further supporting the generic status of the taxon. Plant pathogenic Enterobacter spp. display such sequence variability that few definite conclusions as to their specific placement could be made. These data highlight the difficulty of drawing reliable and robust taxonomic conclusions based on comparative analysis of sequence data without some independent criterion to calibrate a scale for diversity.
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429. |
Cookson AL,
Bennett J,
Thomson-Carter F,
Attwood GT,
( 2007 ) Molecular subtyping and genetic analysis of the enterohemolysin gene (ehxA) from Shiga toxin-producing escherichia coli and atypical enteropathogenic E. coli. PMID : 17720842 : DOI : 10.1128/AEM.00316-07 PMC : PMC2075064 Abstract >>
Analyses of the distribution of virulence factors among different Escherichia coli pathotypes, including Shiga toxin-producing E. coli (STEC), may provide some insight into the mechanisms by which different E. coli strains cause disease and the evolution of distinct E. coli types. The aim of this study was to examine the DNA sequence of the gene for enterohemolysin, a plasmid-encoded toxin that readily causes the hemolysis of washed sheep erythrocytes, and to assess the distribution of enterohemolysin subtypes among E. coli isolates from various human and animal sources. The 2,997-bp ehxA gene was amplified from 227 (63.8%) of 356 stx- and/or eae-positive E. coli strains isolated from cattle and sheep and from 24 (96.0%) of 25 STEC strains isolated from humans with diarrheal disease. By using PCR and restriction fragment length polymorphism (RFLP) analysis of ehxA, six distinct PCR-RFLP types (A to F) were observed, with strains of subtypes A and C constituting 91.6% of all the ehxA-positive strains. Subtype A was associated mainly with ovine strains with stx only (P < 0.001), and subtype C was associated with bovine eae-positive strains (P < 0.001). Eleven ehxA alleles were fully sequenced, and the phylogenetic analysis indicated the presence of three closely related (>95.0%) ehxA sequence groups, one including eae-positive strains (subtypes B, C, E, and F) and the other two including mainly eae-negative STEC strains (subtypes A and D). In addition to being widespread among STEC strains, stx-negative, eae-positive strains (atypical enteropathogenic E. coli strains) isolated from cattle and sheep have similar ehxA subtypes and hemolytic activities.
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430. |
Pilonieta MC,
Bodero MD,
Munson GP,
( 2007 ) CfaD-dependent expression of a novel extracytoplasmic protein from enterotoxigenic Escherichia coli. PMID : 17496090 : DOI : 10.1128/JB.00131-07 PMC : PMC1951884 Abstract >>
H10407 is a strain of enterotoxigenic Escherichia coli (ETEC) that utilizes CFA/I pili to adhere to surfaces of the small intestine, where it elaborates toxins that cause profuse watery diarrhea in humans. Expression of the CFA/I pilus is positively regulated at the level of transcription by CfaD, a member of the AraC/XylS family. DNase I footprinting revealed that the activator has two binding sites upstream of the pilus promoter cfaAp. One site extends from positions -23 to -56, and the other extends from positions -73 to -103 (numbering relative to the transcription start site of cfaAp). Additional CfaD binding sites were predicted within the genome of H10407 by computational analysis. Two of these sites lie upstream of a previously uncharacterized gene, cexE. In vitro DNase I footprinting confirmed that both sites are genuine binding sites, and cexEp::lacZ reporters demonstrated that CfaD is required for the expression of cexE in vivo. The amino terminus of CexE contains a secretory signal peptide that is removed during translocation across the cytoplasmic membrane through the general secretory pathway. These studies suggest that CexE may be a novel ETEC virulence factor because its expression is controlled by the virulence regulator CfaD, and its distribution is restricted to ETEC.
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431. |
Kiratisin P,
Apisarnthanarak A,
Saifon P,
Laesripa C,
Kitphati R,
Mundy LM,
( 2007 ) The emergence of a novel ceftazidime-resistant CTX-M extended-spectrum beta-lactamase, CTX-M-55, in both community-onset and hospital-acquired infections in Thailand. PMID : 17449211 : DOI : 10.1016/j.diagmicrobio.2007.02.005 Abstract >>
We report a novel CTX-M type of extended-spectrum beta-lactamase (ESBL), designated CTX-M-55, among 7 patients who had infection with ESBL-producing Escherichia coli or Klebsiella pneumoniae at a university hospital in Thailand. The CTX-M-55 ESBL showed reduced susceptibility to ceftazidime. This investigation provides the relevant clinical and molecular epidemiology for the gene encoding for CTX-M-55 in the isolates from these patients.
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432. |
Jobichen C,
Li M,
Yerushalmi G,
Tan YW,
Mok YK,
Rosenshine I,
Leung KY,
Sivaraman J,
( 2007 ) Structure of GrlR and the implication of its EDED motif in mediating the regulation of type III secretion system in EHEC. PMID : 17511515 : DOI : 10.1371/journal.ppat.0030069 PMC : PMC1868957 Abstract >>
Enterohemorrhagic Escherichia coli (EHEC) is a common cause of severe hemorrhagic colitis. EHEC's virulence is dependent upon a type III secretion system (TTSS) encoded by 41 genes. These genes are organized in several operons clustered in the locus of enterocyte effacement. Most of the locus of enterocyte effacement genes, including grlA and grlR, are positively regulated by Ler, and Ler expression is positively and negatively modulated by GrlA and GrlR, respectively. However, the molecular basis for the GrlA and GrlR activity is still elusive. We have determined the crystal structure of GrlR at 1.9 A resolution. It consists of a typical beta-barrel fold with eight beta-strands containing an internal hydrophobic cavity and a plug-like loop on one side of the barrel. Strong hydrophobic interactions between the two beta-barrels maintain the dimeric architecture of GrlR. Furthermore, a unique surface-exposed EDED (Glu-Asp-Glu-Asp) motif is identified to be critical for GrlA-GrlR interaction and for the repressive activity of GrlR. This study contributes a novel molecular insight into the mechanism of GrlR function.
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433. |
Eydallin G,
Morán-Zorzano MT,
Muñoz FJ,
Baroja-Fernández E,
Montero M,
Alonso-Casajús N,
Viale AM,
Pozueta-Romero J,
( 2007 ) An Escherichia coli mutant producing a truncated inactive form of GlgC synthesizes glycogen: further evidences for the occurrence of various important sources of ADPglucose in enterobacteria. PMID : 17719034 : DOI : 10.1016/j.febslet.2007.08.016 Abstract >>
AC70R1-504 Escherichia coli mutants possess a glgC* gene with a nucleotide change resulting in a premature stop codon that renders a truncated, inactive form of GlgC. Cells over-expressing the wild type glgC, but not those over-expressing the AC70R1-504 glgC*, accumulated high ADPglucose and glycogen levels. AC70R1-504 mutants accumulated glycogen, whereas DeltaglgCAP deletion mutants lacking the whole glycogen biosynthetic machinery displayed a glycogen-less phenotype. AC70R1-504 cells with enhanced glycogen synthase activity accumulated high glycogen levels. By contrast, AC70R1-504 cells with high ADPG hydrolase activity accumulated low glycogen. These data further confirm that enterobacteria possess various sources of ADPglucose linked to glycogen biosynthesis.
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434. |
Patikoglou GA,
Westblade LF,
Campbell EA,
Lamour V,
Lane WJ,
Darst SA,
( 2007 ) Crystal structure of the Escherichia coli regulator of sigma70, Rsd, in complex with sigma70 domain 4. PMID : 17681541 : DOI : 10.1016/j.jmb.2007.06.081 PMC : PMC2083641 Abstract >>
The Escherichia coli Rsd protein binds tightly and specifically to the RNA polymerase (RNAP) sigma(70) factor. Rsd plays a role in alternative sigma factor-dependent transcription by biasing the competition between sigma(70) and alternative sigma factors for the available core RNAP. Here, we determined the 2.6 A-resolution X-ray crystal structure of Rsd bound to sigma(70) domain 4 (sigma(70)(4)), the primary determinant for Rsd binding within sigma(70). The structure reveals that Rsd binding interferes with the two primary functions of sigma(70)(4), core RNAP binding and promoter -35 element binding. Interestingly, the most highly conserved Rsd residues form a network of interactions through the middle of the Rsd structure that connect the sigma(70)(4)-binding surface with three cavities exposed on distant surfaces of Rsd, suggesting functional coupling between sigma(70)(4) binding and other binding surfaces of Rsd, either for other proteins or for as yet unknown small molecule effectors. These results provide a structural basis for understanding the role of Rsd, as well as its ortholog, AlgQ, a positive regulator of Pseudomonas aeruginosa virulence, in transcription regulation.
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435. |
Zhang Y,
Laing C,
Steele M,
Ziebell K,
Johnson R,
Benson AK,
Taboada E,
Gannon VP,
( 2007 ) Genome evolution in major Escherichia coli O157:H7 lineages. PMID : 17506902 : DOI : 10.1186/1471-2164-8-121 PMC : PMC1890555 Abstract >>
Genetic analysis of Escherichia coli O157:H7 strains has shown divergence into two distinct lineages, lineages I and II, that appear to have distinct ecological characteristics, with lineage I strains more commonly associated with human disease. In this study, microarray-based comparative genomic hybridization (CGH) was used to identify genomic differences among 31 E. coli O157:H7 strains that belong to various phage types (PTs) and different lineage-specific polymorphism assay (LSPA) types. A total of 4,084 out of 6,057 ORFs were detected in all E. coli O157:H7 strains and 1,751 were variably present or absent. Based on this data, E. coli O157:H7 strains were divided into three distinct clusters, which consisted of 15 lineage I (LSPA type 111111), four lineage I/II (designated in this study) (LSPA type 211111) and 12 lineage II strains (LSPA 222222, 222211, 222212, and 222221), respectively. Eleven different genomic regions that were dominant in lineage I strains (present in > or =80% of lineage I and absent from > or = 92% of lineage II strains) spanned segments containing as few as two and up to 25 ORFs each. These regions were identified within E. coli Sakai S-loops # 14, 16, 69, 72, 78, 83, 85, 153 and 286, Sakai phage 10 (S-loops # 91, 92 and 93) and a genomic backbone region. All four lineage I/II strains were of PT 2 and possessed eight of these 11 lineage I-dominant loci. Several differences in virulence-associated loci were noted between lineage I and lineage II strains, including divergence within S-loop 69, which encodes Shiga toxin 2, and absence of the non-LEE encoded effector genes nleF and nleH1-2 and the perC homologue gene pchD in lineage II strains. CGH data suggest the existence of two dominant lineages as well as LSPA type and PT-related subgroups within E. coli O157:H7. The genomic composition of these subgroups supports the phylogeny that has been inferred from other methods and further suggests that genomic divergence from an ancestral form and lateral gene transfer have contributed to their evolution. The genomic features identified in this study may contribute to apparent differences in the epidemiology and ecology of strains of different E. coli O157:H7 lineages.
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436. |
Hinenoya A,
Nagita A,
Asakura M,
Tsukamoto T,
Ramamurthy T,
Nair GB,
Takeda Y,
Yamasaki S,
( 2007 ) Cytolethal distending toxin (Cdt)-producing escherichia coli isolated from a child with bloody diarrhea in Japan. PMID : 17446683 : DOI : 10.1111/j.1348-0421.2007.tb03917.x Abstract >>
In a retrospective analysis by PCR, the cdtI gene encoding the cytolethal distending toxin (Cdt) was detected in Escherichia coli O2:H12 strain isolated from the bloody diarrheal stool specimen of a child. To our knowledge, this is the first report showing the possible association of Cdt-producing E. coli in Japan, particularly in a child with bloody diarrhea.
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437. |
Persson S,
Olsen KE,
Ethelberg S,
Scheutz F,
( 2007 ) Subtyping method for Escherichia coli shiga toxin (verocytotoxin) 2 variants and correlations to clinical manifestations. PMID : 17446326 : DOI : 10.1128/JCM.02591-06 PMC : PMC1933035 Abstract >>
Shiga toxin 2 (Stx2) from Shiga toxin-producing Escherichia coli (STEC) was subtyped by a method involving partial sequencing of the stxAB2 operon. Of 255 strains from the Danish STEC cohort, all 20 cases of hemolytic-uremic syndrome were associated with subtype Stx2 (11 cases), subtype Stx2c (1 case), or the two combined (8 cases).
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438. |
Jakubauskas A,
Giedriene J,
Bujnicki JM,
Janulaitis A,
( 2007 ) Identification of a single HNH active site in type IIS restriction endonuclease Eco31I. PMID : 17499273 : DOI : 10.1016/j.jmb.2007.04.049 PMC : PMC2754561 Abstract >>
Type IIS restriction endonuclease Eco31I is a "short-distance cutter", which cleaves DNA strands close to its recognition sequence, 5'-GGTCTC(1/5). Previously, it has been proposed that related endonucleases recognizing a common sequence core GTCTC possess two active sites for cleavage of both strands in the DNA substrate. Here, we present bioinformatic identification and experimental evidence for a single nuclease active site. We identified a short region of homology between Eco31I and HNH nucleases, constructed a three-dimensional model of the putative catalytic domain and validated our predictions by random and site-specific mutagenesis. The restriction mechanism of Eco31I is suggested by analogy to the mechanisms of phage T4 endonuclease VII and homing endonuclease I-PpoI. We propose that residues D311 and N334 coordinate the cofactor. H312 acts as a general base-activating water molecule for the nucleophilic attack. K337 together with R340 and D345 are located in close proximity to the active center and are essential for correct folding of catalytic motif, while D345 together with R264 and D273 could be directly involved in DNA binding. We also predict that the Eco31I catalytic domain contains a putative Zn-binding site, which is essential for its structural integrity. Our results suggest that the HNH-like active site is involved in the cleavage of both strands in the DNA substrate. On the other hand, analysis of site-specific mutants in the region, previously suggested to harbor the second active site, revealed its irrelevance to the nuclease activity. Thus, our data argue against the earlier prediction and indicate the presence of a single conserved active site in type IIS restriction endonucleases that recognize common sequence core GTCTC.
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439. |
Stenmark P,
Moche M,
Gurmu D,
Nordlund P,
( 2007 ) The crystal structure of the bifunctional deaminase/reductase RibD of the riboflavin biosynthetic pathway in Escherichia coli: implications for the reductive mechanism. PMID : 17765262 : DOI : 10.1016/j.jmb.2006.12.009 Abstract >>
We have determined the crystal structure of the bi-functional deaminase/reductase enzyme from Escherichia coli (EcRibD) that catalyzes two consecutive reactions during riboflavin biosynthesis. The polypeptide chain of EcRibD is folded into two domains where the 3D structure of the N-terminal domain (1-145) is similar to cytosine deaminase and the C-terminal domain (146-367) is similar to dihydrofolate reductase. We showed that EcRibD is dimeric and compared our structure to tetrameric RibG, an ortholog from Bacillus subtilis (BsRibG). We have also determined the structure of EcRibD in two binary complexes with the oxidized cofactor (NADP(+)) and with the substrate analogue ribose-5-phosphate (RP5) and superposed these two in order to mimic the ternary complex. Based on this superposition we propose that the invariant Asp200 initiates the reductive reaction by abstracting a proton from the bound substrate and that the pro-R proton from C4 of the cofactor is transferred to C1 of the substrate. A highly flexible loop is found in the reductase active site (159-173) that appears to control cofactor and substrate binding to the reductase active site and was therefore compared to the corresponding Met20 loop of E. coli dihydrofolate reductase (EcDHFR). Lys152, identified by comparing substrate analogue (RP5) coordination in the reductase active site of EcRibD with the homologous reductase from Methanocaldococcus jannaschii (MjaRED), is invariant among bacterial RibD enzymes and could contribute to the various pathways taken during riboflavin biosynthesis in bacteria and yeast.
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440. |
Périchon B,
Courvalin P,
Galimand M,
( 2007 ) Transferable resistance to aminoglycosides by methylation of G1405 in 16S rRNA and to hydrophilic fluoroquinolones by QepA-mediated efflux in Escherichia coli. PMID : 17470656 : DOI : 10.1128/AAC.00143-07 PMC : PMC1913276 Abstract >>
Plasmid pIP1206 was detected in Escherichia coli strain 1540 during the screening of clinical isolates of Enterobacteriaceae for high-level resistance to aminoglycosides. The sequence of this IncFI conjugative plasmid of ca. 100 kb was partially determined. pIP1206 carried the rmtB gene for a ribosome methyltransferase that was shown to modify the N7 position of nucleotide G1405, located in the A site of 16S rRNA. It also contained the qepA (quinolone efflux pump) gene that encodes a 14-transmembrane-segment putative efflux pump belonging to the major facilitator superfamily of proton-dependent transporters. Disruption of membrane proton potential by the efflux pump inhibitor carbonyl cyanide m-chlorophenylhydrazone in a transconjugant harboring the qepA gene resulted in elevation of norfloxacin accumulation. The transporter conferred resistance to the hydrophilic quinolones norfloxacin and ciprofloxacin.
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441. |
Robin F,
Delmas J,
Brebion A,
Dubois D,
Constantin JM,
Bonnet R,
( 2007 ) TEM-158 (CMT-9), a new member of the CMT-type extended-spectrum beta-lactamases. PMID : 17709463 : DOI : 10.1128/AAC.00614-07 PMC : PMC2151416 Abstract >>
TEM-158 was found to include the substitutions previously observed for TEM-12 and TEM-35. This enzyme presented hydrolytic activity against ceftazidime and a high level of resistance against clavulanate, which can alter its detection. Its discovery highlights the need for accurate detection methods.
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442. |
Bernasconi C,
Volponi G,
Picozzi C,
Foschino R,
( 2007 ) Use of the tna operon as a new molecular target for Escherichia coli detection. PMID : 17693560 : DOI : 10.1128/AEM.00606-07 PMC : PMC2074986 Abstract >>
A quantitative real-time PCR targeting the tnaA gene was studied to detect Escherichia coli and distinguish E. coli from Shigella spp. These microorganisms revealed high similarity in the molecular organization of the tna operon.
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443. |
Gilmour MW,
Olson AB,
Andrysiak AK,
Ng LK,
Chui L,
( 2007 ) Sequence-based typing of genetic targets encoded outside of the O-antigen gene cluster is indicative of Shiga toxin-producing Escherichia coli serogroup lineages. PMID : 17446284 : DOI : 10.1099/jmm.0.47053-0 PMC : PMC2884935 Abstract >>
Serogroup classifications based upon the O-somatic antigen of Shiga toxin-producing Escherichia coli (STEC) provide significant epidemiological information on clinical isolates. Each O-antigen determinant is encoded by a unique cluster of genes present between the gnd and galF chromosomal genes. Alternatively, serogroup-specific polymorphisms might be encoded in loci that are encoded outside of the O-antigen gene cluster. Segments of the core bacterial loci mdh, gnd, gcl, ppk, metA, ftsZ, relA and metG for 30 O26 STEC strains have previously been sequenced, and comparative analyses to O157 distinguished these two serogroups. To screen these loci for serogroup-specific traits within a broader range of clinically significant serogroups, DNA sequences were obtained for 19 strains of 10 additional STEC serogroups. Unique alleles were observed at the gnd locus for each examined STEC serogroup, and this correlation persisted when comparative analyses were extended to 144 gnd sequences from 26 O-serogroups (comprising 42 O : H-serotypes). These included O157, O121, O103, O26, O5 : non-motile (NM), O145 : NM, O113 : H21, O111 : NM and O117 : H7 STEC; and furthermore, non-toxin encoding O157, O26, O55, O6 and O117 strains encoded distinct gnd alleles compared to STEC strains of the same serogroup. DNA sequencing of a 643 bp region of gnd was, therefore, sufficient to minimally determine the O-antigen of STEC through molecular means, and the location of gnd next to the O-antigen gene cluster offered additional support for the co-inheritance of these determinants. The gnd DNA sequence-based serogrouping method could improve the typing capabilities for STEC in clinical laboratories, and was used successfully to characterize O121 : H19, O26 : H11 and O177 : NM clinical isolates prior to serological confirmation during outbreak investigations.
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444. |
Moulin-Schouleur M,
Répérant M,
Laurent S,
Brée A,
Mignon-Grasteau S,
Germon P,
Rasschaert D,
Schouler C,
( 2007 ) Extraintestinal pathogenic Escherichia coli strains of avian and human origin: link between phylogenetic relationships and common virulence patterns. PMID : 17652485 : DOI : 10.1128/JCM.00037-07 PMC : PMC2045314 Abstract >>
Extraintestinal pathogenic Escherichia coli (ExPEC) strains of human and avian origin show similarities that suggest that the avian strains potentially have zoonotic properties. However, the phylogenetic relationships between avian and human ExPEC strains are poorly documented, so this possibility is difficult to assess. We used PCR-based phylotyping and multilocus sequence typing (MLST) to determine the phylogenetic relationships between 39 avian pathogenic E. coli (APEC) strains of serogroups O1, O2, O18, and O78 and 51 human ExPEC strains. We also compared the virulence genotype and pathogenicity for chickens of APEC strains and human ExPEC strains. Twenty-eight of the 30 APEC strains of serogroups O1, O2, and O18 were classified by MLST into the same subcluster (B2-1) of phylogenetic group B2, whereas the 9 APEC strains of serogroup O78 were in phylogenetic groups D (3 strains) and B1 (6 strains). Human ExPEC strains were closely related to APEC strains in each of these three subclusters. The 28 avian and 25 human strains belonging to phylogenetic subcluster B2-1 all expressed the K1 antigen and presented no significant differences concerning the presence of other virulence factors. Moreover, human strains of this phylogenetic subcluster were highly virulent for chicks, so no host specificity was identified. Thus, APEC strains of serotypes O1:K1, O2:K1, and O18:K1 belong to the same highly pathogenic clonal group as human E. coli strains of the same serotypes isolated from cases of neonatal meningitis, urinary tract infections, and septicemia. These APEC strains constitute a potential zoonotic risk.
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445. |
Bono JL,
Keen JE,
Clawson ML,
Durso LM,
Heaton MP,
Laegreid WW,
( 2007 ) Association of Escherichia coli O157:H7 tir polymorphisms with human infection. PMID : 17718910 : DOI : 10.1186/1471-2334-7-98 PMC : PMC2063500 Abstract >>
Emerging molecular, animal model and epidemiologic evidence suggests that Shiga-toxigenic Escherichia coli O157:H7 (STEC O157) isolates vary in their capacity to cause human infection and disease. The translocated intimin receptor (tir) and intimin (eae) are virulence factors and bacterial receptor-ligand proteins responsible for tight STEC O157 adherence to intestinal epithelial cells. They represent logical genomic targets to investigate the role of sequence variation in STEC O157 pathogenesis and molecular epidemiology. The purposes of this study were (1) to identify tir and eae polymorphisms in diverse STEC O157 isolates derived from clinically ill humans and healthy cattle (the dominant zoonotic reservoir) and (2) to test any observed tir and eae polymorphisms for association with human (vs bovine) isolate source. Five polymorphisms were identified in a 1,627-bp segment of tir. Alleles of two tir polymorphisms, tir 255 T>A and repeat region 1-repeat unit 3 (RR1-RU3, presence or absence) had dissimilar distributions among human and bovine isolates. More than 99% of 108 human isolates possessed the tir 255 T>A T allele and lacked RR1-RU3. In contrast, the tir 255 T>A T allele and RR1-RU3 absence were found in 55% and 57%, respectively, of 77 bovine isolates. Both polymorphisms associated strongly with isolate source (p < 0.0001), but not by pulsed field gel electrophoresis type or by stx1 and stx2 status (as determined by PCR). Two eae polymorphisms were identified in a 2,755-bp segment of 44 human and bovine isolates; 42 isolates had identical eae sequences. The eae polymorphisms did not associate with isolate source. Polymorphisms in tir but not eae predict the propensity of STEC O157 isolates to cause human clinical disease. The over-representation of the tir 255 T>A T allele in human-derived isolates vs the tir 255 T>A A allele suggests that these isolates have a higher propensity to cause disease. The high frequency of bovine isolates with the A allele suggests a possible bovine ecological niche for this STEC O157 subset.
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446. |
Han W,
Liu B,
Cao B,
Beutin L,
Krüger U,
Liu H,
Li Y,
Liu Y,
Feng L,
Wang L,
( 2007 ) DNA microarray-based identification of serogroups and virulence gene patterns of Escherichia coli isolates associated with porcine postweaning diarrhea and edema disease. PMID : 17449692 : DOI : 10.1128/AEM.01820-06 PMC : PMC1932722 Abstract >>
Escherichia coli strains causing postweaning diarrhea (PWD) and edema disease (ED) in pigs are limited to a number of serogroups, with O8, O45, O138, O139, O141, O147, O149, and O157 being the most commonly reported worldwide. In this study, a DNA microarray based on the O-antigen-specific genes of all 8 E. coli serogroups, as well as 11 genes encoding adhesion factors and exotoxins associated with PWD and ED, was developed for the identification of related serogroups and virulence gene patterns. The microarray method was tested against 186 E. coli and Shigella O-serogroup reference strains, 13 E. coli reference strains for virulence markers, 43 E. coli clinical isolates, and 12 strains of other bacterial species and shown to be highly specific with reproducible results. The detection sensitivity was 0.1 ng of genomic DNA or 10(3) CFU per 0.3 g of porcine feces in mock samples. Seventeen porcine feces samples from local hoggeries were examined using the microarray, and the result for one sample was verified by the conventional serotyping methods. This microarray can be readily used to screen for the presence of PWD- and ED-associated E. coli in porcine feces samples.
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447. |
Cheng J,
Liu B,
Bastin DA,
Han W,
Wang L,
Feng L,
( 2007 ) Genetic characterization of the Escherichia coli O66 antigen and functional identification of its wzy gene. PMID : 17342059 : Abstract >>
Escherichia coli is a clonal species, and occurs as both commensal and pathogenic strains, which are normally classified on the basis of their O, H, and K antigens. The O-antigen (O-specific polysaccharide), which consists of a series of oligosaccharide (O-unit) repeats, contributes major antigenic variability to the cell surface. The O-antigen gene cluster of E. coli O66 was sequenced in this study. The genes putatively responsible for the biosynthesis of dTDP-6-deoxy-L-talose and GDP-mannose, as well as those responsible for the transfer of sugars and for O-unit processing were identified based on their homology. The function of the wzy gene was confirmed by the results of a mutation test. Genes specific for E. coli O66 were identified via PCR screening against representatives of 186 E. coli and Shigella O type strains. The comparison of intergenic sequences located between galF and the O-antigen gene cluster in a range of E. coli and Shigella showed that this region may perform an important function in the homologous recombination of the O-antigen gene clusters.
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448. |
Pham HN,
Ohkusu K,
Mishima N,
Noda M,
Monir Shah M,
Sun X,
Hayashi M,
Ezaki T,
( 2007 ) Phylogeny and species identification of the family Enterobacteriaceae based on dnaJ sequences. PMID : 17368802 : DOI : 10.1016/j.diagmicrobio.2006.12.019 Abstract >>
Phylogenetic relations within the family Enterobacteriaceae were analyzed using partial dnaJ sequences of 165 strains belonging to 93 species from 27 enterobacterial genera. The dnaJ phylogeny was in relative agreement with that constructed by 16S rDNA sequences, but more monophyletic groups were obtained from the dnaJ tree than from the 16S rDNA tree. The degree of divergence of the dnaJ gene was approximately 6 times greater than that of 16S rDNA. Also, the dnaJ gene showed the most discriminatory power in comparison with tuf and atpD genes, facilitating clear differentiation of any 2 enterobacterial species by dnaJ sequence analysis. The application of dnaJ sequences to the identification was confirmed by assigning 72 clinical isolates to the correct enterobacterial species. Our data indicate that analysis of the dnaJ gene sequences can be used as a powerful marker for phylogenetic study and identification at the species level of the family Enterobacteriaceae.
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449. |
Chen Y,
Bonnet R,
Shoichet BK,
( 2007 ) The acylation mechanism of CTX-M beta-lactamase at 0.88 a resolution. PMID : 17408273 : DOI : 10.1021/ja0712064 PMC : PMC2577156 Abstract >>
N/A
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450. |
Mruk I,
Kaczorowski T,
( 2007 ) A rapid and efficient method for cloning genes of type II restriction-modification systems by use of a killer plasmid. PMID : 17468281 : DOI : 10.1128/AEM.00119-07 PMC : PMC1932789 Abstract >>
We present a method for cloning restriction-modification (R-M) systems that is based on the use of a lethal plasmid (pKILLER). The plasmid carries a functional gene for a restriction endonuclease having the same DNA specificity as the R-M system of interest. The first step is the standard preparation of a representative, plasmid-borne genomic library. Then this library is transformed with the killer plasmid. The only surviving bacteria are those which carry the gene specifying a protective DNA methyltransferase. Conceptually, this in vivo selection approach resembles earlier methods in which a plasmid library was selected in vitro by digestion with a suitable restriction endonuclease, but it is much more efficient than those methods. The new method was successfully used to clone two R-M systems, BstZ1II from Bacillus stearothermophilus 14P and Csp231I from Citrobacter sp. strain RFL231, both isospecific to the prototype HindIII R-M system.
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451. |
Brockmeyer J,
Bielaszewska M,
Fruth A,
Bonn ML,
Mellmann A,
Humpf HU,
Karch H,
( 2007 ) Subtypes of the plasmid-encoded serine protease EspP in Shiga toxin-producing Escherichia coli: distribution, secretion, and proteolytic activity. PMID : 17704265 : DOI : 10.1128/AEM.00920-07 PMC : PMC2075056 Abstract >>
We investigated the prevalence, distribution, and structure of espP in Shiga toxin-producing Escherichia coli (STEC) and assessed the secretion and proteolytic activity of the encoded autotransporter protein EspP (extracellular serine protease, plasmid encoded). espP was identified in 56 of 107 different STEC serotypes. Sequencing of a 3,747-bp region of the 3,900-bp espP gene distinguished four alleles (espPalpha, espPbeta, espPgamma, and espPdelta), with 99.9%, 99.2%, 95.3%, and 95.1% homology, respectively, to espP of E. coli O157:H7 strain EDL933. The espPbeta, espPgamma, and espPdelta genes contained unique insertions and/or clustered point mutations that enabled allele-specific PCRs; these demonstrated the presence of espPalpha, espPbeta, espPgamma, and espPdelta in STEC isolates belonging to 17, 16, 15, and 8 serotypes, respectively. Among four subtypes of EspP encoded by these alleles, EspPalpha (produced by enterohemorrhagic E. coli [EHEC] O157:H7 and the major non-O157 EHEC serotypes) and EspPgamma cleaved pepsin A, human coagulation factor V, and an oligopeptide alanine-alanine-proline-leucine-para-nitroaniline, whereas EspPbeta and EspPdelta either were not secreted or were proteolytically inactive. The lack of proteolysis correlated with point mutations near the active serine protease site. We conclude that espP is widely distributed among STEC strains and displays genetic heterogeneity, which can be used for subtyping and which affects EspP activity. The presence of proteolytically active EspP in EHEC serogroups O157, O26, O111, and O145, which are bona fide human pathogens, suggests that EspP might play a role as an EHEC virulence factor.
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452. |
Liu Y,
DebRoy C,
Fratamico P,
( 2007 ) Sequencing and analysis of the Escherichia coli serogroup O117, O126, and O146 O-antigen gene clusters and development of PCR assays targeting serogroup O117-, O126-, and O146-specific DNA sequences. PMID : 17452091 : DOI : 10.1016/j.mcp.2007.03.002 Abstract >>
The O-antigen gene clusters of Escherichia coli serogroups O117, O126, and O146 were sequenced, and 11, 10, and 11 open reading frames (ORFs) were identified, respectively. Genes required for O-antigen sugar biosynthesis, sugar transfer, and sugar processing were identified. Multiplex polymerase chain reaction (PCR) assays were developed targeting the wzx and wzy genes present in the O-antigen gene cluster of these serogroups. The assays were highly serogroup specific when tested against strains belonging to serogroups that were isolated from food, humans, animals, and environmental sources, as well as against representative strains belonging to ca. 165 different E. coli O serogroups and a number of non-E. coli bacteria. Thus, the results demonstrate that the wzx and wzy gene sequences were specific to E. coli O117, O126, and O146 and can be used as diagnostic markers for rapid identification and detection of these serogroups.
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453. |
Coldewey SM,
Hartmann M,
Schmidt DS,
Engelking U,
Ukena SN,
Gunzer F,
( 2007 ) Impact of the rpoS genotype for acid resistance patterns of pathogenic and probiotic Escherichia coli. PMID : 17386106 : DOI : 10.1186/1471-2180-7-21 PMC : PMC1852560 Abstract >>
Enterohemorrhagic E. coli (EHEC), a subgroup of Shiga toxin (Stx) producing E. coli (STEC), may cause severe enteritis and hemolytic uremic syndrome (HUS) and is transmitted orally via contaminated foods or from person to person. The infectious dose is known to be very low, which requires most of the bacteria to survive the gastric acid barrier. Acid resistance therefore is an important mechanism of EHEC virulence. It should also be a relevant characteristic of E. coli strains used for therapeutic purposes such as the probiotic E. coli Nissle 1917 (EcN). In E. coli and related enteric bacteria it has been extensively demonstrated, that the alternative sigma factor sigmaS, encoded by the rpoS gene, acts as a master regulator mediating resistance to various environmental stress factors. Using rpoS deletion mutants of a highly virulent EHEC O26:H11 patient isolate and the sequenced prototype EHEC EDL933 (ATCC 700927) of serotype O157:H7 we investigated the impact of a functional rpoS gene for orchestrating a satisfactory response to acid stress in these strains. We then functionally characterized rpoS of probiotic EcN and five rpoS genes selected from STEC isolates pre-investigated for acid resistance. First, we found out that ATCC isolate 700927 of EHEC EDL933 has a point mutation in rpoS, not present in the published sequence, leading to a premature stop codon. Moreover, to our surprise, one STEC strain as well as EcN was acid sensitive in our test environment, although their cloned rpoS genes could effectively complement acid sensitivity of an rpoS deletion mutant. The attenuation of sequenced EHEC EDL933 might be of importance for anyone planning to do either in vitro or in vivo studies with this prototype strain. Furthermore our data supports recently published observations, that individual E. coli isolates are able to significantly modulate their acid resistance phenotype independent of their rpoS genotype.
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454. |
Salerno A,
Delétoile A,
Lefevre M,
Ciznar I,
Krovacek K,
Grimont P,
Brisse S,
( 2007 ) Recombining population structure of Plesiomonas shigelloides (Enterobacteriaceae) revealed by multilocus sequence typing. PMID : 17693512 : DOI : 10.1128/JB.00796-07 PMC : PMC2168737 Abstract >>
Plesiomonas shigelloides is an emerging pathogen that is widespread in the aquatic environment and is responsible for intestinal diseases and extraintestinal infections in humans and other animals. Virtually nothing is known about its genetic diversity, population structure, and evolution, which severely limits epidemiological control. We addressed these questions by developing a multilocus sequence typing (MLST) system based on five genes (fusA, leuS, pyrG, recG, and rpoB) and analyzing 77 epidemiologically unrelated strains from several countries and several ecological sources. The phylogenetic position of P. shigelloides within family Enterobacteriaceae was precisely defined by phylogenetic analysis of the same gene portions in other family members. Within P. shigelloides, high levels of nucleotide diversity (average percentage of nucleotide differences between strains, 1.49%) and genotypic diversity (64 distinct sequence types; Simpson's index, 99.7%) were found, with no salient internal phylogenetic structure. We estimated that homologous recombination in housekeeping genes affects P. shigelloides alleles and nucleotides 7 and 77 times more frequently than mutation, respectively. These ratios are similar to those observed in the naturally transformable species Streptococcus pneumoniae with a high rate of recombination. In contrast, recombination within Salmonella enterica, Escherichia coli, and Yersinia enterocolitica was much less frequent. P. shigelloides thus stands out among members of the Enterobacteriaceae. Its high rate of recombination results in a lack of association between genomic background and O and H antigenic factors, as observed for the 51 serotypes found in our sample. Given its robustness and discriminatory power, we recommend MLST as a reference method for population biology studies and epidemiological tracking of P. shigelloides strains.
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455. |
Vaezeslami S,
Sterling R,
Reznikoff WS,
( 2007 ) Site-directed mutagenesis studies of tn5 transposase residues involved in synaptic complex formation. PMID : 17693501 : DOI : 10.1128/JB.00524-07 PMC : PMC2168436 Abstract >>
Transposition (the movement of discrete segments of DNA, resulting in rearrangement of genomic DNA) initiates when transposase forms a dimeric DNA-protein synaptic complex with transposon DNA end sequences. The synaptic complex is a prerequisite for catalytic reactions that occur during the transposition process. The transposase-DNA interactions involved in the synaptic complex have been of great interest. Here we undertook a study to verify the protein-DNA interactions that lead to synapsis in the Tn5 system. Specifically, we studied (i) Arg342, Glu344, and Asn348 and (ii) Ser438, Lys439, and Ser445, which, based on the previously published cocrystal structure of Tn5 transposase bound to a precleaved transposon end sequence, make cis and trans contacts with transposon end sequence DNA, respectively. By using genetic and biochemical assays, we showed that in all cases except one, each of these residues plays an important role in synaptic complex formation, as predicted by the cocrystal structure.
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456. |
Scherzinger E,
Lurz R,
Otto S,
Dobrinski B,
( 1992 ) In vitro cleavage of double- and single-stranded DNA by plasmid RSF1010-encoded mobilization proteins. PMID : 1738602 : DOI : 10.1093/nar/20.1.41 PMC : PMC310323 Abstract >>
We have used purified RSF1010 mobilization proteins to reproduce in vitro a strand-specific nicking at the plasmid origin of transfer, oriT. In the presence of Mg2+, the proteins MobA (78-kDa form of RSF1010 DNA primase), MobB, and MobC and supercoiled or linear duplex oriT DNA form large amounts of a cleavage complex, which is characterized by its sensitivity to protein-denaturant treatment. Upon addition of SDS to such a complex, a single strand break is generated in the DNA, and MobA is found linked to the 5' nick terminus, presumably covalently. The double-strand nicking activity of MobA requires, in addition to Mg2+, the presence of MobC and is stimulated by the presence of MobB. The nick site has been shown by DNA sequencing to lie at the position cleaved in vivo during transfer, between nucleotides 3138/3139 in the r strand of RSF1010. We have found that MobA will also cleave DNA at sites other than oriT if the DNA is present in single-stranded form. Breakage in this case occurs in the absence of denaturing conditions, and after prolonged incubation, reclosure can be demonstrated.
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457. |
Papagiannitsis CC,
Loli A,
Tzouvelekis LS,
Tzelepi E,
Arlet G,
Miriagou V,
( 2007 ) SCO-1, a novel plasmid-mediated class A beta-lactamase with carbenicillinase characteristics from Escherichia coli. PMID : 17353248 : DOI : 10.1128/AAC.01439-06 PMC : PMC1891400 Abstract >>
A novel class A beta-lactamase (SCO-1) encoded by an 80-kb self-transferable plasmid from Escherichia coli is described. The interaction of SCO-1 with beta-lactams was similar to that of the CARB-type enzymes. Also, SCO-1 exhibited a 51% amino acid sequence identity with the RTG subgroup of chromosomal carbenicillinases (RTG-1, CARB-5, and CARB-8).
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458. |
Tominaga A,
Kutsukake K,
( 2007 ) Expressed and cryptic flagellin genes in the H44 and H55 type strains of Escherichia coli. PMID : 17396015 : Abstract >>
Bacterial H antigens are specified by flagellin molecules, which constitute the flagellar filament. Escherichia coli 781-55 and E2987-73 are the type strains for H44 and H55 antigens, respectively. Unlike E. coli K-12, they possess two flagellin genes, fliC and fllA, on their chromosomes. However, they are monophasic, expressing exclusively the fllA genes, which specify the type antigens. In this study, the flagellin genes were cloned from these strains and their structure and expression were analyzed. It was found that the fliC genes encode apparently intact flagellin subunits but possess inefficient sigma28-dependent promoters, which may result in these genes being silent. The chromosomal locations of the fllA genes are approximately, but not exactly, identical with that of the phase-2 flagellin gene, fljB, of diphasic Salmonella strains. However, unlike the Salmonella fljB gene, the invertible H segment and the fljA gene responsible for the control of flagellar phase variation are both absent from the fllA loci. The fllA genes are highly homologous to the E. coli fliC gene but distantly related to the Salmonella fljB gene. These results suggest a hypothesis that the fllA genes may have emerged by an intra-species lateral transfer of the fliC gene. This hypothesis is further supported by the observation that the fllA genes are flanked by several IS elements and located within cryptic prophage elements.
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459. |
Chen L,
Chen ZL,
Liu JH,
Zeng ZL,
Ma JY,
Jiang HX,
( 2007 ) Emergence of RmtB methylase-producing Escherichia coli and Enterobacter cloacae isolates from pigs in China. PMID : 17353219 : DOI : 10.1093/jac/dkm065 Abstract >>
To investigate the occurrence of 16S rRNA methylases conferring high-level resistance to aminoglycosides in Enterobacteriaceae isolated from two pig farms in China. Enterobacteriaceae isolated from 151 pig rectal swab samples and 9 environmental samples were screened for the presence of the rmtA, rmtB, armA and rmtC genes by PCR and sequencing. Conjugation experiments were carried out to study the transferability of the 16S rRNA methylase genes. All isolates and their transconjugants were tested for susceptibility to antimicrobial agents. The clonal relatedness of RmtB-producing Escherichia coli was assessed by PFGE with XbaI. Of 152 Enterobacteriaceae isolates recovered from pigs, 49 (32%) were positive for the rmtB gene, including 48 E. coli and a single isolate of Enterobacter cloacae. Of the nine Enterobacteriaceae isolates from environmental samples, no 16S rRNA methylase gene was identified. The 49 rmtB-positive isolates showed resistance to ampicillin, tetracycline and trimethoprim and also carried a bla(TEM) gene. Transfer of the rmtB and bla(TEM) genes by conjugation experiments of all 49 isolates was successful, suggesting that the rmtB-containing plasmids in the E. coli and E. cloacae isolates were self-transmissible. Conjugative transfer frequencies varied from 2.2 x 10(-10) to 1.3 x 10(-6) transconjugants per recipient. The transfer of non-aminoglycoside antimicrobial resistance traits was also observed in most cases. Forty-four rmtB-positive E. coli showed 30 different PFGE types. The rmtB gene was detected on conjugative plasmids of porcine E. coli and E. cloacae isolates. Both horizontal gene transfer and clonal spread were responsible for the dissemination of the rmtB gene. The emergence of 16S rRNA methylases in Enterobacteriaceae isolates is described for the first time in China. This is also the first report of rmtB-positive Enterobacteriaceae among healthy food-producing animals.
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460. |
Severinov K,
Semenova E,
Kazakov A,
Kazakov T,
Gelfand MS,
( 2007 ) Low-molecular-weight post-translationally modified microcins. PMID : 17711420 : DOI : 10.1111/j.1365-2958.2007.05874.x Abstract >>
Microcins are a class of ribosomally synthesized antibacterial peptides produced by Enterobacteriaceae and active against closely related bacterial species. While some microcins are active as unmodified peptides, others are heavily modified by dedicated maturation enzymes. Low-molecular-weight microcins from the post-translationally modified group target essential molecular machines inside the cells. In this review, available structural and functional data about three such microcins--microcin J25, microcin B17 and microcin C7-C51--are discussed. While all three low-molecular-weight post-translationally modified microcins are produced by Escherichia coli, inferences based on sequence and structural similarities with peptides encoded or produced by phylogenetically diverse bacteria are made whenever possible to put these compounds into a larger perspective.
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461. |
Divya N,
Grifith E,
Narayana N,
( 2007 ) Structure of the Q67H mutant of R67 dihydrofolate reductase-NADP+ complex reveals a novel cofactor binding mode. PMID : 17473013 : DOI : 10.1110/ps.062740907 PMC : PMC2206676 Abstract >>
Plasmid-encoded bacterial R67 dihydrofolate reductase (DHFR) is a NADPH-dependent enzyme unrelated to chromosomal DHFR in amino acid sequence and structure. R67 DHFR is insensitive to the bacterial drug trimethoprim in contrast to chromosomal DHFR. The crystal structure of Q67H mutant of R67 DHFR bound to NADP(+) has been determined at 1.15 angstroms resolution. The cofactor assumes an extended conformation with the nicotinamide ring bound near the center of the active site pore, the ribose and pyrophosphate group (PP(i)) extending toward the outer pore. The ribonicotinamide exhibits anti conformation as in chromosomal DHFR complexes. The relative orientation between the PP(i) and the nicotinamide ribose differs from that observed in chromosomal DHFR-NADP(+) complexes. The coenzyme displays symmetrical binding mode with several water-mediated hydrogen bonds with the protein besides ionic, stacking, and van der Waals interactions. The structure provides a molecular basis for the observed stoichiometry and cooperativity in ligand binding. The ternary model based on the present structure and the previous R67 DHFR-folate complex provides insight into the catalytic mechanism and indicates that the relative orientation of the reactants in plasmid DHFR is different from that seen in chromosomal DHFRs.
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462. |
Steenbergen SM,
Wrona TJ,
Vimr ER,
( 1992 ) Functional analysis of the sialyltransferase complexes in Escherichia coli K1 and K92. PMID : 1735705 : DOI : 10.1128/jb.174.4.1099-1108.1992 PMC : PMC206402 Abstract >>
The polysialyltransferase (polyST) structural gene, neuS, for poly alpha 2,8sialic acid (PSA) capsule synthesis in Escherichia coli K1 was previously mapped near the kps region 1 and 2 junction (S. M. Steenbergen and E. R. Vimr, Mol. Microbiol. 4:603-611, 1990). Present Southern and colony blot hybridization results confirmed that neuS was a region 2 locus and indicated apparent homology with neuS from E. coli K92, bacteria that synthesize a sialyl alpha 2,8-2,9-linked polymer. A K1- mutant with an insertion mutation in neuS was complemented in trans by K92 neuS, providing direct evidence that neuS encoded the PSA polymerase. A 2.9-kb E. coli K1 kps subclone was sequenced to better characterize polyST. In addition to neuS, the results identified a new open reading frame, designated neuE, the linker sequence between regions 1 and 2, and the last gene of region 1, kpsS. The kpsS translational reading frame was confirmed by sequencing across the junction of a kpsS'-lacZ+ fusion. PolyST was identified by maxicell analysis of nested deletions and coupled in vitro transcription-translation assays. PolyST's derived primary structure predicted a 47,500-Da basic polypeptide without extensive similarity to other known proteins. PolyST activity was increased 31-fold and was membrane localized when neuS was cloned into an inducible expression vector, suggesting, together with the polyST primary structure, that polyST is a peripheral inner membrane glycosyltransferase. However, polyST could not initiate de novo PSA synthesis, indicating a functional requirement for other kps gene products. The existence of a sialyltransferase distinct from polyST was suggested by identification of a potential polyprenyl-binding motif in a C-terminal membrane-spanning domain of the predicted neuE gene product. Direct evidence for a quantitatively minor sialyltransferase activity, which could function to initiate PSA synthesis, was obtained by phenotypic analysis of mutants with multiple defects in sialic acid synthesis, degradation, and polymerization. The results provide an initial molecular description of K1 and K92 sialyltransferase complexes and suggest a possible common function for accessory kps gene products.
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463. |
Korotkova N,
Chattopadhyay S,
Tabata TA,
Beskhlebnaya V,
Vigdorovich V,
Kaiser BK,
Strong RK,
Dykhuizen DE,
Sokurenko EV,
Moseley SL,
( 2007 ) Selection for functional diversity drives accumulation of point mutations in Dr adhesins of Escherichia coli. PMID : 17376081 : DOI : 10.1111/j.1365-2958.2007.05648.x Abstract >>
Immune escape is considered to be the driving force behind structural variability of major antigens on the surface of bacterial pathogens, such as fimbriae. In the Dr family of Escherichia coli adhesins, structural and adhesive functions are carried out by the same subunit. Dr adhesins have been shown to bind decay-accelerating factor (DAF), collagen IV, and carcinoembryonic antigen-related cell adhesion molecules (CEACAMs). We show that genes encoding Dr adhesins from 100 E. coli strains form eight structural groups with a high level of amino acid sequence diversity between them. However, genes comprising each group differ from each other by only a small number of point mutations. Out of 66 polymorphisms identified within the groups, only three were synonymous mutations, indicating strong positive selection for amino acid replacements. Functional analysis of intragroup variants comprising the Dr haemagglutinin (DraE) group revealed that the point mutations result in distinctly different binding phenotypes, with a tendency of increased affinity to DAF, decreased sensitivity of DAF binding to inhibition by chloramphenicol, and loss of binding capability to collagen, CEACAM3 and CEACAM6. Thus, variability by point mutation of major antigenic proteins on the bacterial surface can be a signature of selection for functional modification.
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464. |
Yeh SM,
Koon N,
Squire C,
Metcalf P,
( 2007 ) Structures of the dimerization domains of the Escherichia coli disulfide-bond isomerase enzymes DsbC and DsbG. PMID : 17372350 : DOI : 10.1107/S0907444907003320 Abstract >>
DsbC and DsbG are periplasmic disulfide-bond isomerases, enzymes that facilitate the folding of secreted proteins with multiple disulfide bonds by catalyzing disulfide-bond rearrangement. Both enzymes also have in vitro chaperone activity. The crystal structures of these molecules are similar and both are V-shaped homodimeric modular structures. Each dimeric molecule contains two separate C-terminal thioredoxin-fold domains, joined by hinged helical "stalks" to a single N-terminal dimerization domain formed from the N-terminal 67 residues of each monomer. In this work, the crystal structures of the separate DsbC and DsbG dimerization domains have been determined at resolutions of 2.0 and 1.9 A, respectively. The two structures are both similar to the corresponding domains in the full-length molecules, showing that the dimerization domains fold independently of the catalytic portions of the full-length molecules. Localized structural differences between DsbC and DsbG were observed near the dimer interface and may be relevant to the different functions of the two enzymes.
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465. |
Aidar-Ugrinovich L,
Blanco J,
Blanco M,
Blanco JE,
Leomil L,
Dahbi G,
Mora A,
Onuma DL,
Silveira WD,
Pestana de Castro AF,
( 2007 ) Serotypes, virulence genes, and intimin types of Shiga toxin-producing Escherichia coli (STEC) and enteropathogenic E. coli (EPEC) isolated from calves in S?o Paulo, Brazil. PMID : 17292501 : DOI : 10.1016/j.ijfoodmicro.2006.10.046 Abstract >>
Shiga toxin-producing Escherichia coli (STEC), is the most important recently emerged group of foodborne pathogens. Ruminants, especially cattle, have been implicated as a principal reservoir of STEC, undercooked ground beef and raw milk being the major vehicles of foodborne outbreaks. Enteropathogenic E. coli (EPEC) strains are defined as eae-harboring diarrheagenic E. coli that possess the ability to form A/E lesions on intestinal cells and that do not possess Shiga toxin genes. In order to determine the occurrence, serotypes and virulence markers of STEC and EPEC strains, 546 fecal samples from 264 diarrheic calves and 282 healthy calves in beef farms in S?o Paulo, Brazil, were screened by PCR. STEC and EPEC were isolated in 10% and 2.7% of the 546 animals, respectively. Although IMS test was used, the STEC serotype O157:H7 was not detected. The most frequent serotypes among STEC strains were O7:H10, O22:H16, O111:H(-), O119:H(-) and O174:H21, whereas O26:H11, O123:H11 and O177:H11 were the most prevalent among EPEC strains. In this study, serotypes not previously reported were found among STEC strains: O7:H7, O7:H10, O48:H7, O111:H19, O123:H2, O132:H51, O173:H(-), and O175:H49. The eae gene was detected in 25% of the STEC and 100% of EPEC strains. The intimin type theta/gamma2 was the most frequent among STEC, whereas the intimin beta1 was the most frequent intimin type among EPEC strains. To our knowledge, this is the first report of the occurrence of the new intimin muB in one strain of animal origin. This new intimin was detected in one atypical EPEC strain of serotype O123:H? isolated from diarrheic cattle. The enterohemolysin (ehxA) was detected in 51% of the STEC and 80% of the EPEC strains, whereas STEC autoagglutinating adhesin (saa) virulence gene was detected only in those STEC strains negative for eae gene. All 15 bovine EPEC strains isolated in this study were negative for both eaf and bfp genes. Our data shows that in Brazil cattle are not only a reservoir of STEC and atypical EPEC, but also a potential source of infection in humans, since the important STEC serotypes previously described and associated with severe diseases in humans, such as O111:H(-), O113:H21, O118:H16, and O174:H21 were isolated.
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466. |
Bauer AP,
Dieckmann SM,
Ludwig W,
Schleifer KH,
( 2007 ) Rapid identification of Escherichia coli safety and laboratory strain lineages based on Multiplex-PCR. PMID : 17343689 : DOI : 10.1111/j.1574-6968.2006.00594.x Abstract >>
Escherichia coli K-12, B, C and W strains are the most frequently used bacterial safety and laboratory strains. Lineage-specific DNA fragments were detected by microplate subtractive hybridization and utilized to create a fast differentiation method using a single PCR reaction to differentiate clearly the four lineages and separate them from pathogenic variants. The method has been evaluated on a comprehensive selection of widely used laboratory strains and a variety of pathogenic E. coli representatives. In addition, in silico analysis on all available E. coli genomes and the genomes of the close relatives Shigella and Salmonella confirmed the reliability of the proposed method. A fast identification and differentiation of E. coli safety strains by Multiplex-PCR is a useful tool for researchers and companies to check and monitor their reference stocks.
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467. |
Papagiannitsis CC,
Tzouvelekis LS,
Tzelepi E,
Miriagou V,
( 2007 ) Plasmid-encoded ACC-4, an extended-spectrum cephalosporinase variant from Escherichia coli. PMID : 17664321 : DOI : 10.1128/AAC.00389-07 PMC : PMC2043296 Abstract >>
ACC-4, an omega loop mutant (Val(211)-->Gly) of the Hafnia alvei-derived cephalosporinase ACC-1, was encoded by an Escherichia coli plasmid. The genetic environment of bla(ACC-4) shared similarities with plasmidic regions carrying bla(ACC-1). Kinetics of beta-lactam hydrolysis and levels of resistance to beta-lactams showed that ACC-4 was more effective than ACC-1 against expanded-spectrum cephalosporins.
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468. |
Joo LM,
Macfarlane-Smith LR,
Okeke IN,
( 2007 ) Error-prone DNA repair system in enteroaggregative Escherichia coli identified by subtractive hybridization. PMID : 17351038 : DOI : 10.1128/JB.01764-06 PMC : PMC1913340 Abstract >>
Enteroaggregative Escherichia coli (EAEC) are etiologic agents of diarrhea. The EAEC category is heterogeneous, but most in-depth experimentation has focused on prototypical strain, 042. We hypothesized that 60A, another EAEC strain, might posses virulence or fitness genes that 042 does not have. Through subtractive hybridization we identified 60A-specific sequences, including loci present in other E. coli and phage DNA. One locus thus identified was impB, a LexA repressed error-prone DNA repair gene that has been identified in plasmids from other enteric organisms and which we detected in 21 of 34 EAEC strains. An isogenic 60A impB mutant showed decreased survival and mutagenesis after exposure to UV, as well as bile salt exposure, compared to the wild-type strain, and these phenotypes could be complemented in trans. The EAEC strain 60A imp operon differs structurally from previously described homologs. A cryptic gene, impC, present in other imp operons, is absent from 60A. In addition, transcription of impAB in strain 60A occurs from a promoter that is dissimilar to the previously described impC promoter but is still triggered by UV-mediated damage. In strain 60A the impAB and the aggregative adherence fimbriae I (AAF/I)-encoding genes are on the same large plasmid, and the 60A version of the operon is predominantly seen in AAF/I-positive EAEC. Supplementary imp SOS-inducible error-prone repair systems are common among EAEC even though they are absent in prototypical strain 042.
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469. |
Beutin L,
Wang Q,
Naumann D,
Han W,
Krause G,
Leomil L,
Wang L,
Feng L,
( 2007 ) Relationship between O-antigen subtypes, bacterial surface structures and O-antigen gene clusters in Escherichia coli O123 strains carrying genes for Shiga toxins and intimin. PMID : 17244797 : DOI : 10.1099/jmm.0.46775-0 Abstract >>
Escherichia coli O123 strains express a broad spectrum of phenotypes, H serotypes and virulence markers and are able to colonize and to cause disease in different hosts including humans. In this study, two subtypes of E. coli O123 antigen (group I and group II) have been identified based on their cross-reactions with other E. coli O antigens. Investigation of the relationship between O123 group I and group II strains by O serotyping and Fourier transform infrared spectroscopy of whole bacteria revealed surface structural differences between these two groups of E. coli O123 strains. Nucleotide sequence analysis of the O-antigen gene clusters of two E. coli O123 strains representing O123 group I and group II revealed no change at the amino acid level. These findings indicate that the differences in the surface structures of group I and group II strains are not related to genetic heterogeneity in their O-antigen gene clusters. A PCR assay based on O123 antigen-specific wzx and wzy genes was developed and found to be suitable for reliable detection of all subtypes of E. coli O123 strains, which bears an advantage over traditional serological detection.
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470. |
Grape M,
Sundström L,
Kronvall G,
( 2007 ) Two new dfr genes in trimethoprim-resistant integron-negative Escherichia coli isolates. PMID : 17307981 : DOI : 10.1128/AAC.01273-06 PMC : PMC1855533 Abstract >>
Two Escherichia coli isolates resistant to trimethoprim but negative for integrons carried two new resistance genes, dfrA24 and dfrA26, remotely similar to one another and to the cassette-independent genes dfrA8 and dfrA9. The dfrA24 gene was not associated with known mobile elements, while dfrA26 was associated with the CR1 common region.
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471. |
Haney SA,
Platko JV,
Oxender DL,
Calvo JM,
( 1992 ) Lrp, a leucine-responsive protein, regulates branched-chain amino acid transport genes in Escherichia coli. PMID : 1729203 : DOI : 10.1128/jb.174.1.108-115.1992 PMC : PMC205683 Abstract >>
We investigated the relationship between two regulatory genes, livR and lrp, that map near min 20 on the Escherichia coli chromosome. livR was identified earlier as a regulatory gene affecting high-affinity transport of branched-chain amino acids through the LIV-I and LS transport systems, encoded by the livJ and livKHMGF operons. lrp was characterized more recently as a regulatory gene of a regulon that includes operons involved in isoleucine-valine biosynthesis, oligopeptide transport, and serine and threonine catabolism. The expression of each of these livR- and lrp-regulated operons is altered in cells when leucine is added to their growth medium. The following results demonstrate that livR and lrp are the same gene. The lrp gene from a livR1-containing strain was cloned and shown to contain two single-base-pair substitutions in comparison with the wild-type strain. Mutations in livR affected the regulation of ilvIH, an operon known to be controlled by lrp, and mutations in lrp affected the regulation of the LIV-I and LS transport systems. Lrp from a wild-type strain bound specifically to several sites upstream of the ilvIH operon, whereas binding by Lrp from a livR1-containing strain was barely detectable. In a strain containing a Tn10 insertion in lrp, high-affinity leucine transport occurred at a high, constitutive level, as did expression from the livJ and livK promoters as measured by lacZ reporter gene expression. Taken together, these results suggest that Lrp acts directly or indirectly to repress livJ and livK expression and that leucine is required for this repression. This pattern of regulation is unusual for operons that are controlled by Lrp.
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472. |
Zapata G,
Crowley JM,
Vann WF,
( 1992 ) Sequence and expression of the Escherichia coli K1 neuC gene product. PMID : 1729218 : DOI : 10.1128/jb.174.1.315-319.1992 PMC : PMC205711 Abstract >>
The nucleotide sequence of the neuC gene of the Escherichia coli K1 capsule gene cluster encodes a protein with a predicted molecular weight of 44,210 containing 391 amino acids. A chimeric protein with beta-galactosidase fused to the carboxy terminus of the neuC gene product (P7) was constructed and purified. Its amino-terminal sequence confirmed the prediction from the nucleotide sequence that the neuC gene overlaps the distal end of the neuA gene by a single base pair. Both the neuA and neuC genes are coexpressed under the control of a single upstream T7 or tac promoter, suggesting that neuA and neuC are part of an operon.
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473. |
Lim D,
( 1991 ) Structure of two retrons of Escherichia coli and their common chromosomal insertion site. PMID : 1722556 : DOI : 10.1111/j.1365-2958.1991.tb00810.x Abstract >>
It has been shown that certain strains of myxobacteria and of Escherichia coli have a genetic element encoding a reverse transcriptase (RT). This element, called a 'retron', produces a covalently linked RNA-DNA compound (msDNA-RNA). Here, I report the complete nucleotide sequence of retron EC-86, the retron in E. coli B, together with its flanking regions. Retron EC-86 contains genes for msDNA-RNA (msd, and msr), a gene for RT (ret) and a gene for an open reading frame whose function is unknown. The upstream junction is composed of the sequence GCGCGCGC, but there are no direct or inverted repeats at the retron-host junctions. It is also shown that another retron of E. coli, EC-67, which was isolated originally from the clinical strain CL1 and was later found to be present also in a clinical E. coli isolate from Brazil, is inserted at the same chromosomal site as retron EC-86. Retron EC-67 contains only msd, msr, and ret. I suggest that these two retrons were independently inserted into the same site of their host strains via a novel mechanism of integration.
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474. |
Oberer M,
Zangger K,
Gruber K,
Keller W,
( 2007 ) The solution structure of ParD, the antidote of the ParDE toxin antitoxin module, provides the structural basis for DNA and toxin binding. PMID : 17656583 : DOI : 10.1110/ps.062680707 PMC : PMC2203376 Abstract >>
ParD is the antidote of the plasmid-encoded toxin-antitoxin (TA) system ParD-ParE. These modules rely on differential stabilities of a highly expressed but labile antidote and a stable toxin expressed from one operon. Consequently, loss of the coding plasmid results in loss of the protective antidote and poisoning of the cell. The antidote protein usually also exhibits an autoregulatory function of the operon. In this paper, we present the solution structure of ParD. The repressor activity of ParD is mediated by the N-terminal half of the protein, which adopts a ribbon-helix-helix (RHH) fold. The C-terminal half of the protein is unstructured in the absence of its cognate binding partner ParE. Based on homology with other RHH proteins, we present a model of the ParD-DNA interaction, with the antiparallel beta-strand being inserted into the major groove of DNA. The fusion of the N-terminal DNA-binding RHH motif to the toxin-binding unstructured C-terminal domain is discussed in its evolutionary context.
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475. |
Cunneen MM,
Reeves PR,
( 2007 ) The Yersinia kristensenii O11 O-antigen gene cluster was acquired by lateral gene transfer and incorporated at a novel chromosomal locus. PMID : 17400574 : DOI : 10.1093/molbev/msm058 Abstract >>
We have sequenced the O-antigen gene clusters for the Escherichia coli O98 and Yersinia kristensenii O11 O antigens. The basic structures of these O antigens are identical, and the sequence data indicate that Y. kristensenii O11 gained its O-antigen gene cluster by lateral gene transfer (LGT). Escherichia coli O98 has a typical O-antigen gene cluster between galF and gnd as is usual in E. coli. However, the O-antigen gene cluster of Y. kristensenii O11 is not located at the traditional Yersinia O-antigen gene cluster locus, between hemH and gsk, but at a novel chromosomal locus between aroA and cmk where it is flanked by remnant galF and gnd genes that indicate the probable source of the gene cluster. Phylogenetic analysis indicated that the source was not E. coli itself but a species in the Escherichia, Salmonella, and Klebsiella group of genera. Although other O-antigen studies imply LGT on the basis of the hypervariability of the loci and GC content, this report also identifies a potential donor and provides evidence for the mechanism involved. Remnant insertion sequence (IS) sequences flank the galF and gnd remnants and suggest that LGT of the gene cluster was IS mediated.
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476. |
Duquesne S,
Destoumieux-Garzón D,
Zirah S,
Goulard C,
Peduzzi J,
Rebuffat S,
( 2007 ) Two enzymes catalyze the maturation of a lasso peptide in Escherichia coli. PMID : 17656316 : DOI : 10.1016/j.chembiol.2007.06.004 Abstract >>
Microcin J25 (MccJ25) is a gene-encoded lasso peptide secreted by Escherichia coli which exerts a potent antibacterial activity by blocking RNA polymerase. Here we demonstrate that McjB and McjC, encoded by genes in the MccJ25 gene cluster, catalyze the maturation of MccJ25. Requirement for both McjB and McjC was shown by gene inactivation and complementation assays. Furthermore, the conversion of the linear precursor McjA into mature MccJ25 was obtained in vitro in the presence of McjB and McjC, all proteins being produced by recombinant expression in E. coli. Analysis of the amino acid sequences revealed that McjB could possess proteolytic activity, whereas McjC would be the ATP/Mg(2+)-dependent enzyme responsible for the formation of the Gly1-Glu8 amide bond. Finally, we show that putative lasso peptides are widespread among Proteobacteria and Actinobacteria.
|
477. |
Harms K,
de Vries J,
Wackernagel W,
( 2007 ) A double kill gene cassette for the positive selection of transforming non-selective DNA segments in Acinetobacter baylyi BD413. PMID : 17250911 : DOI : 10.1016/j.mimet.2006.12.006 Abstract >>
Natural transformation has been widely used for the monitoring of DNA in biological and environmental samples. These assays depended on selectable traits on the tested DNA. We have now developed a transformation assay system in which recombinational removal of a cassette with two conditional kill genes (hok and sacB) from the recipient genome provides positive selection for non-selective DNA. The cassette was integrated into the Acinetobacter baylyi BD413 chromosome within trpE and was flanked by two segments of non-selective test DNA, which in this study were from a T-DNA construct previously used to generate a transgenic potato. Genes for tetracycline and spectinomycin/streptomycin resistance located at the sides of the cassette allowed to maintain selection pressure against spontaneous loss of the cassette. Plasmid DNA containing the T-DNA gave transformation frequencies ranging linearly from 10(-4) per recipient (at 1 mug DNA ml(-1)) down to 10(-7) (1 ng DNA ml(-1)) by selecting for survivors after activation of both kill functions. Transformation depended on the two flanking homologous segments for recombinational exchange. DNA of the transgenic potato also gave positive scores in spite of the about 10(5)-fold dilution of T-DNA by potato DNA. False positives having a spontaneous deletion of hok and sacB occurred at a frequency of 1.8x10(-9) per cell but could be distinguished by PCR from real transformants. Thus, the system is suitable for detection of transformation frequencies down to about 10(-9). Hok and sacB as well as the regulatory system used (LacI-lac operator and T5 promoter) are known to function in many organisms suggesting wide applicability of the cassette for positive selection.
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478. |
Lehmacher A,
Bockemühl J,
( 2007 ) L-Sorbose utilization by virulent Escherichia coli and Shigella: different metabolic adaptation of pathotypes. PMID : 17382590 : DOI : 10.1016/j.ijmm.2007.01.007 Abstract >>
The frequency of L-Sorbose utilization differs significantly between pathotypes of Escherichia coli and Shigella from 93% to 0%. Among 266 strains tested, this frequency increased in the order Shigella, enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC), Shiga toxin-producing E. coli (STEC), enteroaggregative E. coli, enteropathogenic E. coli (EPEC), and neonatal bacterial meningitis (NBM) E. coli. This suggests an association of pathomechanism with the capability to degrade L-Sorbose. The use of a selective agar, containing L-Sorbose and antibiotics, facilitated the isolation of L-Sorbose-non-utilizing ETEC from stool specimens of patients. The sor operon, comprising seven genes in the order sorCDFBAME, confers L-Sorbose utilization. Surprisingly, L-Sorbose-non-degrading Shigella harbored all genes of the sor operon indicating L-Sorbose-utilizing E. coli as ancestor. Additionally, strains of several EIEC and STEC serotypes harbored an inactivated sor operon. These L-Sorbose-non-utilizing Shigella, EIEC, and STEC showed significantly reduced amounts of transcripts as examined for sorC and sorD. Common surface antigens, types of intimin gene, and hemolysin gene as well as use of L-Sorbose suggested the relatedness of attaching and effacing O26:H11 and O55:H7 EPEC and STEC, respectively. pepE and yibC genes flank the sor operon of E. coli and Shigella strains. Surprisingly, one O7:K1:H- NBM E. coli harbored an aroE-homologous gene between its sor operon and pepE as in Klebsiella pneumoniae suggesting a horizontal gene transfer. In conclusion, L-Sorbose utilization of virulent E. coli and Shigella is characterized by different adaptation that represents a valuable tool for evolutionary and diagnostic analysis of related patho- and serotypes.
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479. |
Bogaerts P,
Galimand M,
Bauraing C,
Deplano A,
Vanhoof R,
De Mendonca R,
Rodriguez-Villalobos H,
Struelens M,
Glupczynski Y,
( 2007 ) Emergence of ArmA and RmtB aminoglycoside resistance 16S rRNA methylases in Belgium. PMID : 17224412 : DOI : 10.1093/jac/dkl527 Abstract >>
16S rRNA methylase-mediated high-level resistance to aminoglycosides has been reported recently in clinical isolates of Gram-negative bacilli only from a limited number of countries. This study was conducted to investigate the occurrence of this type of resistance in clinical isolates of Enterobacteriaceae from two Belgian hospitals and the characteristics of the strains. We screened for high-level gentamicin, tobramycin and amikacin resistance in clinical isolates of Enterobacteriaceae consecutively collected between 2000 and 2005 at two laboratories by PCR for the armA, rmtA and rmtB 16S rRNA methylase genes. The beta-lactamase presence in the strains was also determined by phenotypic and genotypic methods. Overall armA genes were detected in 18 Klebsiella pneumoniae, Escherichia coli, Enterobacter aerogenes, Enterobacter cloacae and Citrobacter amalonaticus whereas rmtB was detected in a single E. coli isolate. The rmtA gene was not found. All 16S rRNA methylase-bearing strains produced extended-spectrum beta-lactamases (ESBLs), predominantly type CTX-M-3, as well as various types of beta-lactamases. In the majority of the strains, the armA gene was carried by conjugative plasmids of the IncL/M incompatibility group whereas rmtB was borne by an IncFI plasmid. This is the first report of the emergence of 16S rRNA methylases in Enterobacteriaceae in Belgium. The rapid spread of multidrug-resistant isolates producing both ESBLs and 16S rRNA methylases raises clinical concern and may become a major therapeutic threat in the future.
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480. |
Van Molle I,
Joensuu JJ,
Buts L,
Panjikar S,
Kotiaho M,
Bouckaert J,
Wyns L,
Niklander-Teeri V,
De Greve H,
( 2007 ) Chloroplasts assemble the major subunit FaeG of Escherichia coli F4 (K88) fimbriae to strand-swapped dimers. PMID : 17368480 : DOI : 10.1016/j.jmb.2007.02.051 DOI : 10.1016/j.jmb.2007.02.051 Abstract >>
F4 fimbriae encoded by the fae operon are the major colonization factors associated with porcine neonatal and postweaning diarrhoea caused by enterotoxigenic Escherichia coli (ETEC). Via the chaperone/usher pathway, the F4 fimbriae are assembled as long polymers of the major subunit FaeG, which also possesses the adhesive properties of the fimbriae. Intrinsically, the incomplete fold of fimbrial subunits renders them unstable and susceptible to aggregation and/or proteolytic degradation in the absence of a specific periplasmic chaperone. In order to test the possibility of producing FaeG in plants, FaeG expression was studied in transgenic tobacco plants. FaeG was directed to different subcellular compartments by specific targeting signals. Targeting of FaeG to the chloroplast results in much higher yields than FaeG targeting to the endoplasmic reticulum or the apoplast. Two chloroplast-targeted FaeG variants were purified from tobacco plants and crystallized. The crystal structures show that chloroplasts circumvent the absence of the fimbrial assembly machinery by assembling FaeG into strand-swapped dimers. Furthermore, the structures reveal how FaeG combines the structural requirements of a major fimbrial subunit with its adhesive role by grafting an additional domain on its Ig-like core.
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481. |
Zou Y,
Li C,
Brunzelle JS,
Nair SK,
( 2007 ) Molecular basis for substrate selectivity and specificity by an LPS biosynthetic enzyme. PMID : 17371001 : DOI : 10.1021/bi061056u Abstract >>
Diversity in the polysaccharide component of lipopolysaccharide (LPS) contributes to the persistence and pathogenesis of Gram-negative bacteria. The Nudix hydrolase GDP-mannose mannosyl hydrolase (Gmm) contributes to this diversity by regulating the concentration of mannose in LPS biosynthetic pathways. Here, we present seven high-resolution crystal structures of Gmm from the enteropathogenic E. coli strain O128: the structure of the apo enzyme, the cocrystal structure of Gmm bound to the product Mg2+-GDP, two cocrystal structures of precatalytic and turnover complexes of Gmm-Ca2+-GDP-alpha-d-mannose, and three cocrystal structures of an inactive mutant (His-124 --> Leu) Gmm bound to substrates GDP-alpha-d-mannose, GDP-alpha-d-glucose, and GDP-beta-l-fucose. These crystal structures help explain the molecular basis for substrate specificity and promiscuity and provide a structural framework for reconciling previously determined kinetic parameters. Unexpectedly, these structures reveal concerted changes in the enzyme structure that result in the formation of a catalytically competent active site only in the presence of the substrate/product. These structural views of the enzyme may provide a rationale for the design of inhibitors that target the biosynthesis of LPS by pathogenic bacteria.
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482. |
Galani I,
Souli M,
Chryssouli Z,
Giamarellou H,
( 2007 ) Detection of CTX-M-15 and CTX-M-33, a novel variant of CTX-M-15, in clinical Escherichia coli isolates in Greece. PMID : 17234386 : DOI : 10.1016/j.ijantimicag.2006.11.010 Abstract >>
N/A
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483. |
Liu JH,
Wei SY,
Ma JY,
Zeng ZL,
Lü DH,
Yang GX,
Chen ZL,
( 2007 ) Detection and characterisation of CTX-M and CMY-2 beta-lactamases among Escherichia coli isolates from farm animals in Guangdong Province of China. PMID : 17314033 : DOI : 10.1016/j.ijantimicag.2006.12.015 Abstract >>
The objective of this study was to characterise the beta-lactamase genes of cephalosporin-resistant Escherichia coli isolated from farm animals in Guangdong Province of China. Of 592 E. coli isolates recovered from farm animals from 2003-2005, 50 (8.4%) showed cephalosporin resistance. Polymerase chain reaction and sequencing analysis showed that 14 isolates (2.4%) from chickens, ducks, pigs and partridges were positive for the bla(CTX-M) gene (10 for bla(CTX-M-14) and 4 for bla(CTX-M-27)). CMY-2 was detected for the first time in mainland China in six E. coli isolates (1.0%) from chickens and goose. Except for one isolate, bla(CTX-M)- and bla(CMY-2)-containing isolates also harboured the bla(TEM-1b) gene. Conjugation experiments demonstrated that the bla(CTX-M) and bla(TEM) genes could be transferred to E. coli DH5alpha. Pulsed-field gel electrophoresis (PFGE) showed that the 14 CTX-M-producing isolates belonged to 12 different types. Two isolates (one from a chicken, the other from a pig) containing CTX-M-14 showed indistinguishable PFGE patterns, indicating clonal dissemination of this strain among animals from different farms. This study describes for the first time the emergence of CTX-M- and CMY-2-producing E. coli among farm animals in China, with the CTX-M-9 group being the predominant extended-spectrum beta-lactamase detected.
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484. |
Leclerc S,
Boerlin P,
Gyles C,
Dubreuil JD,
Mourez M,
Fairbrother JM,
Harel J,
( N/A ) paa, originally identified in attaching and effacing Escherichia coli, is also associated with enterotoxigenic E. coli. PMID : 17125971 : DOI : 10.1016/j.resmic.2006.09.004 Abstract >>
Previous studies on virotypes and antimicrobial resistance in a collection of porcine enterotoxigenic Escherichia coli (ETEC) O149 strains from Quebec revealed an increase in the number of multiresistant strains (in particular to tetracycline) and the appearance of new virulence factors with time. Among these factors is paa (for porcine attaching- and effacing-associated), originally identified in a porcine enteropathogenic strain, but also present in enterohemorrhagic E. coli O157:H7. In the present study, the association of paa with other ETEC virulence genes, its conservation and expression were investigated in the O149 ETEC collection. All 37 paa-positive strains possessed estB, elt, astA and faeG, and more than half also carried the estA gene, defining two main virotypes, estA(+) and estA(-). Most strains were tetA- or tetB-positive, or both. paa is carried on high molecular weight plasmids. On tetA plasmids, paa is mostly found with enterotoxin gene estA and autotransporter gene sepA. Paa, a 30 kDa protein, is highly conserved and expressed in these strains. Moreover, paaETEC and porcine EPEC/EHEC contain IS signatures, suggesting that paa could be derived from a common ancestor. All these observations suggest a broader role than previously assessed in virulence for paa.
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485. |
Bakker D,
Vader CE,
Roosendaal B,
Mooi FR,
Oudega B,
de Graaf FK,
( 1991 ) Structure and function of periplasmic chaperone-like proteins involved in the biosynthesis of K88 and K99 fimbriae in enterotoxigenic Escherichia coli. PMID : 1713284 : DOI : 10.1111/j.1365-2958.1991.tb00761.x Abstract >>
The nucleotide sequence of faeE and fanE, two genes involved in the biosynthesis of K88 and K99 fimbriae, respectively, was determined and the amino acid sequence of the FaeE and FanE proteins was deduced. Immunoblotting of subcellular fractions with an antiserum raised against purified FaeE confirmed that FaeE is located in the periplasm. Indications were obtained that FaeE functions as a chaperone-like protein. Its interaction with the fimbrial subunit (FaeG) in the periplasm stabilized this polypeptide and prevents its degradation by the cell-envelope protease DegP. Furthermore, FaeE prevents the formation of FaeG multimers which cannot be incorporated into fimbriae. The reactions of the FaeE/FaeG dimers with a set of monoclonal antibodies directed against the various epitopes present on K88 fimbriae revealed that the fimbrial subunits associated with FaeE were present in a conformation resembling their native configuration. Indications about the domains in FaeG involved in the interaction with FaeE are discussed.
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486. |
Keenleyside WJ,
Jayaratne P,
MacLachlan PR,
Whitfield C,
( 1992 ) The rcsA gene of Escherichia coli O9:K30:H12 is involved in the expression of the serotype-specific group I K (capsular) antigen. PMID : 1729226 : DOI : 10.1128/jb.174.1.8-16.1992 PMC : PMC205669 Abstract >>
Escherichia coli produces two distinct types of capsular polysaccharide (designated groups I and II), which are distinguished by chemical, physical, and genetic characteristics. The K30 capsular antigen is a member of the group I, or heat-stable, capsules. We have cloned rcsA from E. coli O9:K30 and determined the nucleotide sequence. The rcsAK30 sequence is virtually identical to the rcsAK-12 sequence (V. Stout, A. Torres-Cabassa, M. R. Maurizi, D. Gutnick, and S. Gottesman, J. Bacteriol. 173:1738-1747, 1991). RcsAK-12 is a transcriptional activator involved in expression of the extracellular polysaccharide colanic acid in E. coli K-12. rcsAK30 complemented an rcsAK-12 mutation and activated colanic acid synthesis in E. coli K-12 strains. However, in E. coli K30, increasing the levels of RcsA by introducing multicopy rcsAK30 or a Lon mutation resulted in elevated synthesis of the K30 capsular polysaccharide; no colanic acid was detected. E. coli K-12 strains in which the chromosomal his region was replaced by that from E. coli K30 were able to synthesize K30 capsular polysaccharide. These K-12/K30 hybrid strains did not produce colanic acid, suggesting that the genes for synthesis of colanic acid and the K30 capsular polysaccharide may be allelic. rcsA sequences were also detected in the group II strains E. coli K1 and K5. Introduction of rcsAK30 into group II strains resulted in activation of colanic acid biosynthesis rather than the group II capsule. Given the role of RcsA in other members of the family Enterobacteriaceae, our results provide further evidence that this protein may be a relatively widespread regulatory component for the synthesis of enterobacterial extracellular polysaccharides.
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487. |
Galani I,
Souli M,
Koratzanis E,
Koratzanis G,
Chryssouli Z,
Giamarellou H,
( 2007 ) Emerging bacterial pathogens: Escherichia coli, Enterobacter aerogenes and Proteus mirabilis clinical isolates harbouring the same transferable plasmid coding for metallo-beta-lactamase VIM-1 in Greece. PMID : 17255145 : DOI : 10.1093/jac/dkl508 Abstract >>
N/A
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488. |
Castanheira M,
Pereira AS,
Nicoletti AG,
Pignatari AC,
Barth AL,
Gales AC,
( 2007 ) First report of plasmid-mediated qnrA1 in a ciprofloxacin-resistant Escherichia coli strain in Latin America. PMID : 17220425 : DOI : 10.1128/AAC.00780-06 PMC : PMC1855464 Abstract >>
Among 144 ciprofloxacin-resistant Escherichia coli isolated in Brazil, one (0.69%) QnrA1-producing isolate was detected. The qnrA1 gene was associated with ISCR1. The QnrA1 determinant was carried on a 41-kb conjugative plasmid, which also carried a FOX-type cephalosporinase encoding gene and a class 1 integron with the aadB and catB3 cassettes. This is the first report of a qnrA-carrying isolate in a Latin American country.
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489. |
Robin F,
Delmas J,
Schweitzer C,
Tournilhac O,
Lesens O,
Chanal C,
Bonnet R,
( 2007 ) Evolution of TEM-type enzymes: biochemical and genetic characterization of two new complex mutant TEM enzymes, TEM-151 and TEM-152, from a single patient. PMID : 17220412 : DOI : 10.1128/AAC.01058-06 PMC : PMC1855492 Abstract >>
Two clinical isolates of Escherichia coli, CF1179 and CF1295, were isolated from a patient hospitalized in the hematology unit of the University Hospital of Clermont-Ferrand, Clermont-Ferrand, France. They were resistant to penicillin-clavulanate combinations and to ceftazidime. The double-disk synergy test was positive only for isolate CF1179. Molecular comparison of the isolates showed that they were clonally related. E. coli recombinant strains exhibiting the resistance phenotype of the clinical strains were obtained by cloning. The clones corresponding to strains CF1179 and CF1295 produced TEM-type beta-lactamases with pI values of 5.7 and 5.3, respectively. Sequencing analysis revealed two novel blaTEM genes encoding closely related complex mutant TEM enzymes, designated TEM-151 (pI 5.3) and TEM-152 (pI 5.7). These two genes also harbored a new promoter region which presented a 9-bp deletion. The two novel beta-lactamases differed from the parental enzyme, TEM-1, by the substitution Arg164His, previously observed in extended-spectrum beta-lactamases (ESBLs), and by the substitutions Met69Val and Asn276Asp, previously observed in the inhibitor-resistant penicillinase TEM-36/IRT-7. They differed by two amino acid substitutions: TEM-152 harbored a Glu240Lys ESBL-type substitution and TEM-151 had an Ala284Gly substitution. Functional analysis of TEM-151 and TEM-152 showed that both enzymes had hydrolytic activity against ceftazidime (kcat, 5 and 16 s-1, respectively). TEM-152 was more resistant than TEM-151 to the inhibitor clavulanic acid (50% inhibitory concentrations, 1 versus 0.17 microM). These results confirm the evolution of TEM-type enzymes toward complex enzymes harboring the two kinds of substitutions which confer an extended spectrum of action against beta-lactam antibiotics and resistance to inhibitors.
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490. |
Zienkiewicz M,
Kern-Zdanowicz I,
Go?ebiewski M,
Zyliñska J,
Mieczkowski P,
Gniadkowski M,
Bardowski J,
Ceg?owski P,
( 2007 ) Mosaic structure of p1658/97, a 125-kilobase plasmid harboring an active amplicon with the extended-spectrum beta-lactamase gene blaSHV-5. PMID : 17220406 : DOI : 10.1128/AAC.00772-06 PMC : PMC1855452 Abstract >>
Escherichia coli isolates recovered from patients during a clonal outbreak in a Warsaw, Poland, hospital in 1997 produced different levels of an extended-spectrum beta-lactamase (ESBL) of the SHV type. The beta-lactamase hyperproduction correlated with the multiplication of ESBL gene copies within a plasmid. Here, we present the complete nucleotide sequence of plasmid p1658/97 carried by the isolates recovered during the outbreak. The plasmid is 125,491 bp and shows a mosaic structure in which all modules constituting the plasmid core are homologous to those found in plasmids F and R100 and are separated by segments of homology to other known regions (plasmid R64, Providencia rettgeri genomic island R391, Vibrio cholerae STX transposon, Klebsiella pneumoniae or E. coli chromosomes). Plasmid p1658/97 bears two replication systems, IncFII and IncFIB; we demonstrated that both are active in E. coli. The presence of an active partition system (sopABC locus) and two postsegregational killing systems (pemIK and hok/sok) indicates that the plasmid should be stably maintained in E. coli populations. The conjugative transfer is ensured by the operons of the tra and trb genes. We also demonstrate that the plasmidic segment undergoing amplification contains the blaSHV-5 gene and is homologous to a 7.9-kb fragment of the K. pneumoniae chromosome. The amplicon displays the structure of a composite transposon of type I.
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491. |
Rijavec M,
Budic M,
Mrak P,
Müller-Premru M,
Podlesek Z,
Zgur-Bertok D,
( 2007 ) Prevalence of ColE1-like plasmids and colicin K production among uropathogenic Escherichia coli strains and quantification of inhibitory activity of colicin K. PMID : 17122402 : DOI : 10.1128/AEM.01780-06 PMC : PMC1800769 Abstract >>
Colicin K exhibited pronounced inhibitory activity against uropathogenic Escherichia coli (UPEC) strains. Low prevalence of colicin K production and a relatively high prevalence of ColE1-like plasmids were determined among 215 UPEC strains from Slovenia. Sequencing of the colicin K-encoding pColK-K235 revealed a mosaic structure and the presence of the insertion sequence IS2.
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492. |
Hogg RW,
Voelker C,
Von Carlowitz I,
( 1991 ) Nucleotide sequence and analysis of the mgl operon of Escherichia coli K12. PMID : 1719366 : DOI : 10.1007/bf00267469 Abstract >>
The nucleotide sequence of the Escherichia coli K12 beta-methylgalactoside transport operon, mgl, was determined. Primer extension analysis indicated that the synthesis of mRNA initiates at guanine residue 145 of the determined sequence. The operon contains three open reading frames (ORF). The operator proximal ORF, mglB, encodes the galactose binding protein, a periplasmic protein of 332 amino acids including the 23 residue amino-terminal signal peptide. Following a 62 nucleotide spacer, the second ORF, mglA, is capable of encoding a protein of 506 amino acids. The amino-terminal and carboxyl-terminal halves of this protein are homologous to each other and each half contains a putative nucleotide binding site. The third ORF, mglC, is capable of encoding a hydrophobic protein of 336 amino acids which is thought to generate the transmembrane pore. The overall organization of the mglBAC operon and its potential to encode three proteins is similar to that of the ara FGH high affinity transport operon, located approximately 1 min away on the E. coli K12 chromosome.
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493. |
Beutin L,
Strauch E,
( 2007 ) Identification of sequence diversity in the Escherichia coli fliC genes encoding flagellar types H8 and H40 and its use in typing of Shiga toxin-producing E. coli O8, O22, O111, O174, and O179 strains. PMID : 17135431 : DOI : 10.1128/JCM.01627-06 PMC : PMC1829044 Abstract >>
Flagellar type H8 is associated with many strains of pathogenic Shiga toxin-producing Escherichia coli (STEC), such as O8, O22, O111, O174, and O179 strains. Serological typing of the H8 antigen is limited to motile strains only and suffers from cross-reactivity between flagellar H8 and H40 antigens. In order to develop a method useful for typing of motile and nonmotile STEC O111 and other strains, we have analyzed the flagellar antigen (fliC) genes in representative E. coli H8 and H40 types. Two genotypes of the fliC gene encoding H8 (the fliC-H8 gene) were identified. Genotype fliC-H8a was found to be conserved in STEC O111, O174, and O179 strains; and type fliC-H8b was associated with STEC O8 and O22 strains. Sequence variations were also found in the genetically closely related fliC-H40 gene, although the latter was not found to be associated with STEC strains. A PCR was developed for the specific identification of the fliC-H8 and the fliC-H40 genes in motile and nonmotile E. coli strains. Digestion of PCR products with HhaI resulted in restriction fragment length polymorphisms (RFLPs) which were associated with genotypes fliC-H8a and -H8b as well as with genotypes fliC-H40a and -H40b. The fliC-specific PCR/RFLP typing method was suitable for the rapid typing of motile and nonmotile STEC O8, O22, O111, O174, and O179 strains from different sources whose fliC-H8 genotypes were found to be highly conserved. The fliC genotyping method is advantageous over serotyping and is useful for epidemiological investigations and studies of the evolution of STEC clones.
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494. |
Feng L,
Perepelov AV,
Zhao G,
Shevelev SD,
Wang Q,
Senchenkova SN,
Shashkov AS,
Geng Y,
Reeves PR,
Knirel YA,
Wang L,
( 2007 ) Structural and genetic evidence that the Escherichia coli O148 O antigen is the precursor of the Shigella dysenteriae type 1 O antigen and identification of a glucosyltransferase gene. PMID : 17185542 : DOI : 10.1099/mic.0.2006/001107-0 Abstract >>
Shigella dysenteriae type 1 is the most virulent serotype of Shigella. Enterotoxigenic Escherichia coli O148 is pathogenic and can cause diarrhoea. The following structure was established for the tetrasaccharide repeating unit of the E. coli O148 O antigen: -->3)-alpha-L-Rhap-(1-->3)-alpha-L-Rhap-(1-->2)-alpha-D-Glcp-(1-->3)-alpha-D-GlcpNAc-(1-->. This differs from the structure reported earlier for S. dysenteriae type 1 by having a glucose (Glc) residue in place of a galactose (Gal) residue. The two bacteria also have the same genes for O antigen synthesis, with the same organization and high level of DNA identity, except that in S. dysenteriae type 1 wbbG is interrupted by a deletion, and a galactosyltransferase gene wbbP located on a plasmid is responsible for the transfer of galactose to make a novel antigenic epitope of the O antigen. The S. dysenteriae type 1 O antigen was reconstructed by replacing the E. coli O148 wbbG gene with the wbbP gene, and it had the LPS structure and antigenic properties of S. dysenteriae type 1, indicating that the S. dysenteriae type 1 O antigen evolved from that of E. coli O148. It was also confirmed that wbbG of E. coli O148 is a glucosyltransferase gene, and two serotype-specific genes of E. coli O148 and S. dysenteriae type 1 were identified.
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495. |
Denich K,
Blyn LB,
Craiu A,
Braaten BA,
Hardy J,
Low DA,
O'Hanley PD,
( 1991 ) DNA sequences of three papA genes from uropathogenic Escherichia coli strains: evidence of structural and serological conservation. PMID : 1682251 : PMC : PMC258967 Abstract >>
Pyelonephritis-associated pili (Pap) are important in the pathogenesis of ascending, unobstructive Escherichia coli-caused renal infections because these surface bacterial organelles mediate digalactoside-specific binding to host uroepithelial cells. Pap are composed of many different polypeptides, of which only the tip proteins mediate specific binding. The PapA moiety polymerizes to form the bulk of the pilus structure and has been employed in vaccines despite its lack of Gal alpha(1-4)Gal receptor specificity. Animal recipients of PapA pilus-based vaccines are protected against experimental pyelonephritis caused by homologous and heterologous Gal-Gal-binding uropathogenic E. coli strains. Specific PapA immunoglobulin G antibodies in urine are correlated with protection in these infection models. The nucleotide sequences of the gene encoding PapA were determined for three E. coli clones expressing F7(1), F7(2), and F9 pili and were compared with corresponding sequences for other F serotypes. Specific rabbit antisera were employed in enzyme-linked immunosorbent assays to study the cross-reactivity between Gal-Gal pili purified from recombinant strains expressing F7(1), F7(2), F9, or F13 pili and among 60 Gal-Gal-binding wild-type strains. We present data which corroborate the concept that papA genes are highly homologous and encode proteins which exhibit greater than 70% homology among pili of different serotypes. The differences primarily occur in the cysteine-cysteine loop and variable regions and constitute the basis for serological diversity of these pili. Although there are differences in primary structures among these pili, antisera raised against pili of one serotype cross-reacted frequently with many other Gal-Gal pili of different serotypes. Furthermore, antisera raised against pili of the F13 serotype cross-reacted strongly or moderately with 52 (86%) of 60 wild-type Gal-Gal-binding E. coli strains. These data suggest that there are common immunogenic domains among these proteins. These additional data further support the hypothesis that broadly cross-protective PapA pilus vaccines for the immunoprophylaxis of pyelonephritis might be developed.
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496. |
Dong C,
Beis K,
Nesper J,
Brunkan-Lamontagne AL,
Clarke BR,
Whitfield C,
Naismith JH,
( 2006 ) Wza the translocon for E. coli capsular polysaccharides defines a new class of membrane protein. PMID : 17086202 : DOI : 10.1038/nature05267 PMC : PMC3315050 Abstract >>
Many types of bacteria produce extracellular polysaccharides (EPSs). Some are secreted polymers and show only limited association with the cell surface, whereas others are firmly attached to the cell surface and form a discrete structural layer, the capsule, which envelopes the cell and allows the bacteria to evade or counteract the host immune system. EPSs have critical roles in bacterial colonization of surfaces, such as epithelia and medical implants; in addition some EPSs have important industrial and biomedical applications in their own right. Here we describe the 2.26 A resolution structure of the 340 kDa octamer of Wza, an integral outer membrane lipoprotein, which is essential for group 1 capsule export in Escherichia coli. The transmembrane region is a novel alpha-helical barrel. The bulk of the Wza structure is located in the periplasm and comprises three novel domains forming a large central cavity. Wza is open to the extracellular environment but closed to the periplasm. We propose a route and mechanism for translocation of the capsular polysaccharide. This work may provide insight into the export of other large polar molecules such as DNA and proteins.
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497. |
Yang J,
Nie H,
Chen L,
Zhang X,
Yang F,
Xu X,
Zhu Y,
Yu J,
Jin Q,
( 2007 ) Revisiting the molecular evolutionary history of Shigella spp. PMID : 17160643 : DOI : 10.1007/s00239-006-0052-8 Abstract >>
The theory that Shigella is derived from multiple independent origins of Escherichia coli (Pupo et al. 2000) has been challenged by recent findings that the virulence plasmids (VPs) and the chromosomes share a similar evolutionary history (Escobar-Paramo et al. 2003), which suggests that an ancestral VP entered an E. coli strain only once, which gave rise to Shigella spp. In an attempt to resolve these conflicting theories, we constructed three phylogenetic trees in this study: a robust chromosomal tree using 23 housekeeping genes from 46 strains of Shigella and enteroinvasive E. coli (EIEC), a chromosomal tree using 4 housekeeping genes from 19 EcoR strains and 46 Shigella/EIEC strains, and a VP tree using 5 genes outside of the VP cell-entry region from 38 Shigella/EIEC strains. Both chromosomal trees group Shigella into three main clusters and five outliers, and strongly suggest that Shigella has multiple origins within E. coli. Most strikingly, the VP tree shows that the VPs from two main Shigella clusters, C1 and C2, are more closely related, which contradicts the chromosomal trees that place C2 and C3 next to each other but C1 at a distance. Additionally, we have identified a complete tra operon of the F-plasmid in the genome sequence of an EIEC strain and found that two other EIEC strains are also likely to possess a complete tra operon. All lines of evidence support an alternative multiorigin theory that transferable diverse ancestral VPs entered diverse origins of E. coli multiple times during a prolonged period of time, resulting in Shigella species with diverse genomes but similar pathogenic properties.
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498. |
Morinaga N,
Yahiro K,
Matsuura G,
Watanabe M,
Nomura F,
Moss J,
Noda M,
( 2007 ) Two distinct cytotoxic activities of subtilase cytotoxin produced by shiga-toxigenic Escherichia coli. PMID : 17101670 : DOI : 10.1128/IAI.01336-06 PMC : PMC1828409 Abstract >>
Subtilase cytotoxin (SubAB) is a recently identified AB5 subunit toxin produced by Shiga-toxigenic Escherichia coli. The A subunit is thought to be a subtilase-like, serine protease, whereas the B subunit binds to the toxin receptor on the cell surface. We cloned the genes from a clinical isolate; the toxin was produced as His-tagged proteins. SubAB induced vacuolation at concentrations greater than 1 microg/ml after 8 h, in addition to the reported cytotoxicity induced at a ng/ml level after 48 h. Vacuolation was induced with the B, but not the A, subunit and was dependent on V-type ATPase. The cytotoxicity of SubAB at low concentrations was associated with the inhibition of protein synthesis; the 50% inhibitory dose was approximately 1 ng/ml. The A subunit, containing serine 272, which is thought to be a part of the catalytic triad of a subtilase-like serine protease, plus the B subunit was necessary for this activity, both in vivo and in vitro. SubAB did not cleave azocasein, bovine serum albumin, ovalbumin, or synthetic peptides. These data suggest that SubAB is a unique AB toxin: first, the B subunit alone can induce vacuolation; second, the A subunit containing serine 272 plus the B subunit inhibited protein synthesis, both in vivo and in vitro; and third, the A subunit proteolytic activity may have a strict range of substrate specificity.
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499. |
Monzingo AF,
Ozburn A,
Xia S,
Meyer RJ,
Robertus JD,
( 2007 ) The structure of the minimal relaxase domain of MobA at 2.1 A resolution. PMID : 17157875 : DOI : 10.1016/j.jmb.2006.11.031 PMC : PMC1894915 Abstract >>
The plasmid R1162 encodes proteins that enable its conjugative mobilization between bacterial cells. It can transfer between many different species and is one of the most promiscuous of the mobilizable plasmids. The plasmid-encoded protein MobA, which has both nicking and priming activities on single-stranded DNA, is essential for mobilization. The nicking, or relaxase, activity has been localized to the 186 residue N-terminal domain, called minMobA. We present here the 2.1 A X-ray structure of minMobA. The fold is similar to that seen for two other relaxases, TraI and TrwC. The similarity in fold, and action, suggests these enzymes are evolutionary homologs, despite the lack of any significant amino acid similarity. MinMobA has a well- defined target DNA called oriT. The active site metal is observed near Tyr25, which is known to form a phosphotyrosine adduct with the substrate. A model of the oriT substrate complexed with minMobA has been made, based on observed substrate binding to TrwC and TraI. The model is consistent with observations of substrate base specificity, and provides a rationalization for elements of the likely enzyme mechanism.
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500. |
Barak Y,
Ackerley DF,
Dodge CJ,
Banwari L,
Alex C,
Francis AJ,
Matin A,
( 2006 ) Analysis of novel soluble chromate and uranyl reductases and generation of an improved enzyme by directed evolution. PMID : 17088379 : DOI : 10.1128/AEM.01334-06 PMC : PMC1636143 Abstract >>
Most polluted sites contain mixed waste. This is especially true of the U.S. Department of Energy (DOE) waste sites which hold a complex mixture of heavy metals, radionuclides, and organic solvents. In such environments enzymes that can remediate multiple pollutants are advantageous. We report here evolution of an enzyme, ChrR6 (formerly referred to as Y6), which shows a markedly enhanced capacity for remediating two of the most serious and prevalent DOE contaminants, chromate and uranyl. ChrR6 is a soluble enzyme and reduces chromate and uranyl intracellularly. Thus, the reduced product is at least partially sequestered and nucleated, minimizing the chances of reoxidation. Only one amino acid change, (Tyr)128(Asn), was responsible for the observed improvement. We show here that ChrR6 makes Pseudomonas putida and Escherichia coli more efficient agents for bioremediation if the cellular permeability barrier to the metals is decreased.
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501. |
Besser TE,
Shaikh N,
Holt NJ,
Tarr PI,
Konkel ME,
Malik-Kale P,
Walsh CW,
Whittam TS,
Bono JL,
( 2007 ) Greater diversity of Shiga toxin-encoding bacteriophage insertion sites among Escherichia coli O157:H7 isolates from cattle than in those from humans. PMID : 17142358 : DOI : 10.1128/AEM.01035-06 PMC : PMC1800756 Abstract >>
Escherichia coli O157:H7, a zoonotic human pathogen for which domestic cattle are a reservoir host, produces a Shiga toxin(s) (Stx) encoded by bacteriophages. Chromosomal insertion sites of these bacteriophages define three principal genotypes (clusters 1 to 3) among clinical isolates of E. coli O157:H7. Stx-encoding bacteriophage insertion site genotypes of 282 clinical and 80 bovine isolates were evaluated. A total of 268 (95.0%) of the clinical isolates, but only 41 (51.3%) of the bovine isolates, belonged to cluster 1, 2, or 3 (P < 0.001). Thirteen additional genotypes were identified in isolates from both cattle and humans (four genotypes), from only cattle (seven genotypes), or from only humans (two genotypes). Two other markers previously associated with isolates from cattle or with clinical isolates showed similar associations with genotype groups within bovine isolates; the tir allele sp-1 and the Q933W allele were under- and overrepresented, respectively, among cluster 1 to 3 genotypes. Stx-encoding bacteriophage insertion site typing demonstrated that there is broad genetic diversity of E. coli O157:H7 in the bovine reservoir and that numerous genotypes are significantly underrepresented among clinical isolates, consistent with the possibility that there is reduced virulence or transmissibility to humans of some bovine E. coli O157:H7 genotypes.
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502. |
Barlow RS,
Gobius KS,
( 2006 ) Diverse class 2 integrons in bacteria from beef cattle sources. PMID : 17065187 : DOI : 10.1093/jac/dkl423 Abstract >>
The purpose of this study was to determine the diversity of class 2 integrons in bacteria isolated from beef cattle sources. The variable regions of a subset of 11 class 2 integron-containing bacteria were analysed by PCR and DNA sequencing for the presence of novel rearrangements. A total of six different class 2 integron arrays were identified and four of these were fully characterized. Three of the four arrays characterized have been previously described; however the remaining array is unlike previously described class 2 integrons. The novel class 2 integron was found in Providencia stuartii and contains an apparently functional class 2 integrase. Examination of the variable region of the P. stuartii integron identified nine open reading frames, mostly of unknown function, and represents the first report of a class 2 integron without inserted antibiotic resistance gene cassettes. This study has identified a novel class 2 integron found in P. stuartii that contains an apparently functional naturally occurring class 2 integrase. Further investigation of this novel class 2 integron is required to determine the impact of a functional class 2 integrase upon the evolution of class 2 integrons.
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503. |
Yang J,
Baldi DL,
Tauschek M,
Strugnell RA,
Robins-Browne RM,
( 2007 ) Transcriptional regulation of the yghJ-pppA-yghG-gspCDEFGHIJKLM cluster, encoding the type II secretion pathway in enterotoxigenic Escherichia coli. PMID : 17085567 : DOI : 10.1128/JB.01115-06 PMC : PMC1797218 Abstract >>
The gene cluster gspCDEFGHIJKLM codes for various structural components of the type II secretion pathway which is responsible for the secretion of heat-labile enterotoxin by enterotoxigenic Escherichia coli (ETEC). In this work, we used a variety of molecular approaches to elucidate the transcriptional organization of the ETEC type II secretion system and to unravel the mechanisms by which the expression of these genes is controlled. We showed that the gspCDEFGHIJKLM cluster and three other upstream genes, yghJ, pppA, and yghG, are cotranscribed and that a promoter located in the upstream region of yghJ plays a major role in the expression of this 14-gene transcriptional unit. Transcription of the yghJ promoter was repressed 168-fold upon a temperature downshift from 37 degrees C to 22 degrees C. This temperature-induced repression was mediated by the global regulatory proteins H-NS and StpA. Deletion mutagenesis showed that the promoter region encompassing positions -321 to +301 relative to the start site of transcription of yghJ was required for full repression. The yghJ promoter region is predicted to be highly curved and bound H-NS or StpA directly. The binding of H-NS or StpA blocked transcription initiation by inhibiting promoter open complex formation. Unraveling the mechanisms of regulation of type II secretion by ETEC enhances our understanding of the pathogenesis of ETEC and other pathogenic varieties of E. coli.
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504. |
Ito K,
Iida M,
Yamazaki M,
Moriya K,
Moroishi S,
Yatsuyanagi J,
Kurazono T,
Hiruta N,
Ratchtrachenchai OA,
( 2007 ) Intimin types determined by heteroduplex mobility assay of intimin gene (eae)-positive Escherichia coli strains. PMID : 17229860 : DOI : 10.1128/JCM.01103-06 PMC : PMC1829111 Abstract >>
We developed a quick genetic approach to screen variants of the intimin gene (eae) by using a heteroduplex mobility assay (HMA) that targets the 5' conserved region of eae. The eae variants were categorized into 4 major HMA types and 10 minor subtypes.
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505. |
Sheshko V,
Hejnova J,
Rehakova Z,
Sinkora J,
Faldyna M,
Alexa P,
Felsberg J,
Nemcova R,
Bomba A,
Sebo P,
( 2006 ) HlyA knock out yields a safer Escherichia coli A0 34/86 variant with unaffected colonization capacity in piglets. PMID : 17064280 : DOI : 10.1111/j.1574-695X.2006.00140.x Abstract >>
Escherichia coli A0 34/86 (O83:K24:H31) has been successfully used for prophylactic and therapeutic intestinal colonization of premature and newborn infants, with the aim of preventing nosocomial infections. Although E. coli A0 34/86 was described as a nonpathogenic commensal, partial sequencing revealed that its genome harbours gene clusters highly homologous to virulence determinants of different types of E. coli, including closely linked genes of the alpha-haemolysin operon (hlyCABD) and for the cytotoxic necrotizing factor (cnf1). A haemolysin-deficient mutant (Delta hlyA) of E. coli A0 34/86 was generated and its colonization capacity was determined. The results show that a single dose of the A0 34/86 wild-type or Delta hlyA strains resulted in efficient intestinal colonization of newborn conventional piglets, and that this was still considerable after several weeks. No difference was observed between the wild-type and the mutant strains, showing that haemolysin expression does not contribute to intestinal colonization capacity of E. coli A0 34/86. Safety experiments revealed that survival of colostrum-deprived gnotobiotic newborn piglets was substantially higher upon colonization by the nonhaemolytic strain than following inoculation by its wild-type ancestor. We suggest that the E. coli A0 34/86 Delta hlyA mutant may represent a safer prophylactic and/or immunomodulatory tool with unaffected colonization capacity.
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506. |
Zhang W,
Mellmann A,
Sonntag AK,
Wieler L,
Bielaszewska M,
Tschäpe H,
Karch H,
Friedrich AW,
( 2007 ) Structural and functional differences between disease-associated genes of enterohaemorrhagic Escherichia coli O111. PMID : 17157559 : DOI : 10.1016/j.ijmm.2006.10.004 Abstract >>
We analysed 72 clinical isolates of enterohaemorrhagic Escherichia coli (EHEC) O111 from patients with diarrhoea or haemolytic uraemic syndrome (HUS) isolated during the period from 1955 to 2005 and identified six motile strains (flagellar antigens 8, 10 and 11); the remaining 66 (92%) were nonmotile (NM) and could not be typed by conventional H serotyping. To improve subtyping methodologies, we determined genotypes of the flagellin-encoding fliC. Three fliC genotypes were found which were identical to those of motile EHEC O111 with H antigens 8, 10 and 11 and designated fliC(H8), fliC(H10) and fliC(H11). The IS629 insertion element was present, identically located, in six epidemiologically unrelated isolates with fliC(H8). The prevalence of the fliC genotypes in the 72 EHEC O111 strains were fliC(H8) (89%), fliC(H10) (7%) and fliC(H11) (4%). Within these fliC genotypes, a high degree of homogeneity for the presence of disease-associated genes was found. The adhesins-encoding genes eae and efa-1 were present in all strains with fliC(H8) and fliC(H11), but absent from strains with fliC(H10). The latter strains have not been reported previously. Strains with fliC(H10) and fliC(H11), but not those with fliC(H8), retained intact cadA and cadC loci and decarboxylated lysine. Three different stx genotypes including stx(1), stx(2) and stx(1)/stx(2) were determined among the 72 EHEC O111. We observed a significant increase over time in the frequency of strains harbouring both stx(1) and stx(2). The presence of stx(2) both alone and in combination with stx(1) was significantly (chi(2)=23.16, P<0.00001, CI(95) [2.29; 9.76]) associated with HUS. Therefore, the emergence of EHEC O111 should be monitored carefully. We conclude that EHEC O111 strains can be differentiated using specific loci required for motility, adherence, Stx production, and lysine decarboxylation. The divergence within EHEC O111 makes it possible to subtype these emerging pathogens in the laboratory thereby providing a basis for further investigations into their ecological niches and survival capabilities.
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507. |
Gerdes K,
Thisted T,
Martinussen J,
( 1990 ) Mechanism of post-segregational killing by the hok/sok system of plasmid R1: sok antisense RNA regulates formation of a hok mRNA species correlated with killing of plasmid-free cells. PMID : 1707122 : DOI : 10.1111/j.1365-2958.1990.tb02029.x Abstract >>
The hok/sok system of plasmid R1, which mediates plasmid stabilization via killing of plasmid-free segregants, encodes two genes: hok and sok. The hok gene product is a potent cell-killing protein. The expression of hok is regulated post-transcriptionally by the sok gene-encoded repressor, an antisense RNA complementary to the hok mRNA leader region. We show here that the hok mRNA is very stable, while the sok RNA decays rapidly. We also observe a new hok mRNA species which is 70 nucleotides shorter in the 3'-end than the full-length hok transcript. The appearance of the truncated hok mRNA was found to be regulated by the sok antisense RNA. Furthermore, the presence of the truncated hok mRNA was found to be correlated with efficient expression of the Hok protein. On the basis of these findings, we propose an extended model in order to explain the killing of plasmid-free segregants by the hok/sok system.
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508. |
Kosti? T,
Weilharter A,
Rubino S,
Delogu G,
Uzzau S,
Rudi K,
Sessitsch A,
Bodrossy L,
( 2007 ) A microbial diagnostic microarray technique for the sensitive detection and identification of pathogenic bacteria in a background of nonpathogens. PMID : 17123456 : DOI : 10.1016/j.ab.2006.09.026 Abstract >>
A major challenge in microbial diagnostics is the parallel detection and identification of low-bundance pathogens within a complex microbial community. In addition, a high specificity providing robust, reliable identification at least at the species level is required. A microbial diagnostic microarray approach, using single nucleotide extension labeling with gyrB as the marker gene, was developed. We present a novel concept applying competitive oligonucleotide probes to improve the specificity of the assay. Our approach enabled the sensitive and specific detection of a broad range of pathogenic bacteria. The approach was tested with a set of 35 oligonucleotide probes targeting Escherichia coli, Shigella spp., Salmonella spp., Aeromonas hydrophila, Vibrio cholerae, Mycobacterium avium, Mycobacterium tuberculosis, Helicobacter pylori, Proteus mirabilis, Yersinia enterocolitica, and Campylobacter jejuni. The introduction of competitive oligonucleotides in the labeling reaction successfully suppressed cross-reaction by closely related sequences, significantly improving the performance of the assay. Environmental applicability was tested with environmental and veterinary samples harboring complex microbial communities. Detection sensitivity in the range of 0.1% has been demonstrated, far below the 5% detection limit of traditional microbial diagnostic microarrays.
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509. |
Woodford N,
Reddy S,
Fagan EJ,
Hill RL,
Hopkins KL,
Kaufmann ME,
Kistler J,
Palepou MF,
Pike R,
Ward ME,
Cheesbrough J,
Livermore DM,
( 2007 ) Wide geographic spread of diverse acquired AmpC beta-lactamases among Escherichia coli and Klebsiella spp. in the UK and Ireland. PMID : 17110393 : DOI : 10.1093/jac/dkl456 Abstract >>
To determine the distribution of acquired AmpC beta-lactamases in 173 isolates of Escherichia coli and Klebsiella spp. submitted to the UK's national reference laboratory for antibiotic resistance. MICs were determined and interpreted according to BSAC guidelines. Candidate isolates were those resistant to cefotaxime and/or ceftazidime, irrespective of addition of clavulanic acid. Genes encoding six phylogenetic groups of acquired AmpC enzymes were sought by PCR. Selected isolates were compared by pulsed-field gel electrophoresis (PFGE), and one bla(AmpC) amplicon was sequenced. Genes encoding acquired AmpC enzymes were detected in 67 (49%) candidate E. coli and 21 (55%) Klebsiella spp. Sixty isolates produced CIT-type enzymes, 14 had ACC types, 11 had FOX types and 3 had DHA enzymes. The low-level cephalosporin resistance of the remaining isolates (n = 85; 49%) was inferred to result from reduced permeability or, in E. coli, from hyperexpression of chromosomal ampC. Twenty-four E. coli isolates from one hospital produced a CIT-type enzyme, with 20 of these additionally producing a group 1 CTX-M extended-spectrum beta-lactamase. PFGE indicated that these isolates belonged to UK epidemic strain A, which normally produces CTX-M-15, but no acquired AmpC. Sequencing a representative bla(AmpC) amplicon indicated that in one centre this strain had acquired a novel CMY-2 variant, designated CMY-23. Diverse acquired AmpC enzymes occur in E. coli and Klebsiella spp. isolates in the UK and Ireland, with CIT types the most common. Producers are geographically scattered, but with some local outbreaks. Acquisition of a CMY-2-like enzyme by E. coli epidemic strain A suggests that these enzymes may be poised to become an important public health issue.
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510. |
Xu L,
Evans J,
Ling T,
Nye K,
Hawkey P,
( 2007 ) Rapid genotyping of CTX-M extended-spectrum beta-lactamases by denaturing high-performance liquid chromatography. PMID : 17210774 : DOI : 10.1128/AAC.01088-06 PMC : PMC1855489 Abstract >>
Denaturing high-performance liquid chromatography (dHPLC) is a powerful technique which has been used extensively to detect genetic variation. This is the first report of the application of dHPLC for rapid genotyping of bacterial beta-lactamase genes. The technique was specifically developed to genotype members of all blaCTX-M DNA homology groups. Thirteen well-defined blaCTX-M extended-spectrum beta-lactamase (ESBL)-producing strains were used to develop and optimize the dHPLC genotyping assay. Further evaluation was carried out with a blinded panel of 62 clinical isolates. The results of blaCTX-M genotyping achieved by dHPLC were comparable to the typing results obtained by DNA sequencing. Applying the newly developed dHPLC-based genotyping method, we successfully genotyped all 73 blaCTX-M ESBL-producing strains from the 4-month survey study. Furthermore, we found the first reported cases in the United Kingdom of clinically significant disease caused by CTX-M-14- and CTX-M-1-producing Escherichia coli strains. We conclude that the novel dHPLC assay is highly accurate, rapid, and cost-effective for the genotyping of blaCTX-M-producing ESBLs and has great potential for determining the clinical relevance of different and new blaCTX-M genotypes, as well as for epidemiological studies and surveillance programs.
|
511. |
Swinger KK,
Rice PA,
( 2007 ) Structure-based analysis of HU-DNA binding. PMID : 17097674 : DOI : 10.1016/j.jmb.2006.10.024 PMC : PMC1945228 Abstract >>
HU and IHF are prokaryotic proteins that induce very large bends in DNA. They are present in high concentrations in the bacterial nucleoid and aid in chromosomal compaction. They also function as regulatory cofactors in many processes, such as site-specific recombination and the initiation of replication and transcription. HU and IHF have become paradigms for understanding DNA bending and indirect readout of sequence. While IHF shows significant sequence specificity, HU binds preferentially to certain damaged or distorted DNAs. However, none of the structurally diverse HU substrates previously studied in vitro is identical with the distorted substrates in the recently published Anabaena HU(AHU)-DNA cocrystal structures. Here, we report binding affinities for AHU and the DNA in the cocrystal structures. The binding free energies for formation of these AHU-DNA complexes range from approximately 10-14.5 kcal/mol, representing K(d) values in the nanomolar to low picomolar range, and a maximum stabilization of at least approximately 6.3 kcal/mol relative to complexes with undistorted, non-specific DNA. We investigated IHF binding and found that appropriate structural distortions can greatly enhance its affinity. On the basis of the coupling of structural and relevant binding data, we estimate the amount of conformational strain in an IHF-mediated DNA kink that is relieved by a nick (at least 0.76 kcal/mol) and pinpoint the location of the strain. We show that AHU has a sequence preference for an A+T-rich region in the center of its DNA-binding site, correlating with an unusually narrow minor groove. This is similar to sequence preferences shown by the eukaryotic nucleosome.
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512. |
Power ML,
Ferrari BC,
Littlefield-Wyer J,
Gordon DM,
Slade MB,
Veal DA,
( 2006 ) A naturally occurring novel allele of Escherichia coli outer membrane protein A reduces sensitivity to bacteriophage. PMID : 16980421 : DOI : 10.1128/AEM.01040-06 PMC : PMC1694255 Abstract >>
A novel Escherichia coli outer membrane protein A (OmpA) was discovered through a proteomic investigation of cell surface proteins. DNA polymorphisms were localized to regions encoding the protein's surface-exposed loops which are known phage receptor sites. Bacteriophage sensitivity testing indicated an association between bacteriophage resistance and isolates having the novel ompA allele.
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513. |
Williams LE,
Detter C,
Barry K,
Lapidus A,
Summers AO,
( 2006 ) Facile recovery of individual high-molecular-weight, low-copy-number natural plasmids for genomic sequencing. PMID : 16820486 : DOI : 10.1128/AEM.00354-06 PMC : PMC1489313 Abstract >>
Sequencing of the large (>50 kb), low-copy-number (<5 per cell) plasmids that mediate horizontal gene transfer has been hindered by the difficulty and expense of isolating DNA from individual plasmids of this class. We report here that a kit method previously devised for purification of bacterial artificial chromosomes (BACs) can be adapted for effective preparation of individual plasmids up to 220 kb from wild gram-negative and gram-positive bacteria. Individual plasmid DNA recovered from less than 10 ml of Escherichia coli, Staphylococcus, and Corynebacterium cultures was of sufficient quantity and quality for construction of high-coverage libraries, as shown by sequencing five native plasmids ranging in size from 30 kb to 94 kb. We also report recommendations for vector screening to optimize plasmid sequence assembly, preliminary annotation of novel plasmid genomes, and insights on mobile genetic element biology derived from these sequences. Adaptation of this BAC method for large plasmid isolation removes one major technical hurdle to expanding our knowledge of the natural plasmid gene pool.
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514. |
Praszkier J,
Wei T,
Siemering K,
Pittard J,
( 1991 ) Comparative analysis of the replication regions of IncB, IncK, and IncZ plasmids. PMID : 1706708 : DOI : 10.1128/jb.173.7.2393-2397.1991 PMC : PMC207792 Abstract >>
Minireplicons from the I-complex plasmids R387 (IncK) and pIE545 (IncZ) were constructed, and the nucleotide sequences of their replication regions were compared with that of the B plasmid, pMU720. The coding sequence of the putative replication protein, RepA, of each plasmid was located. RepA of K and B plasmids were homologous, whereas RepA of Z resembled RepA1 of FII plasmid. Sequences upstream of RepA were conserved in the three I-complex plasmids. Group B and Z plasmids were incompatible.
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515. |
Verger D,
Miller E,
Remaut H,
Waksman G,
Hultgren S,
( 2006 ) Molecular mechanism of P pilus termination in uropathogenic Escherichia coli. PMID : 17082819 : DOI : 10.1038/sj.embor.7400833 PMC : PMC1794691 DOI : 10.1038/sj.embor.7400833 PMC : PMC1794691 Abstract >>
P pili are important adhesive fibres that are assembled by the conserved chaperone-usher pathway. During pilus assembly, the subunits are incorporated into the growing fibre by the donor-strand exchange mechanism, whereby the beta-strand of the chaperone, which complements the incomplete immunoglobulin fold of each subunit, is displaced by the amino-terminal extension of an incoming subunit in a zip-in-zip-out exchange process that is initiated at the P5 pocket, an exposed hydrophobic pocket in the groove of the subunit. In vivo, termination of P pilus growth requires a specialized subunit, PapH. Here, we show that PapH is incorporated at the base of the growing pilus, where it is unable to undergo donor-strand exchange. This inability is not due to a stronger PapD-PapH interaction, but to a lack of a P5 initiator pocket in the PapH structure, suggesting that PapH terminates pilus growth because it is lacking the initiation point by which donor-strand exchange proceeds.
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516. |
Lintermans PF,
Bertels A,
Schlicker C,
Deboeck F,
Charlier G,
Pohl P,
Norgren M,
Normark S,
van Montagu M,
De Greve H,
( 1991 ) Identification, characterization, and nucleotide sequence of the F17-G gene, which determines receptor binding of Escherichia coli F17 fimbriae. PMID : 1675211 : DOI : 10.1128/jb.173.11.3366-3373.1991 PMC : PMC207947 Abstract >>
Enterotoxigenic Escherichia coli strains express fimbriae which mediate binding to intestinal mucosal cells. The F17 fimbriae mediate binding to N-acetylglucosamine-containing receptors present on calf intestinal mucosal cells. These fimbriae consist of F17-A subunit peptides. Analysis of the F17 gene cluster indicated that at least the F17-A, F17-C, F17-D, and F17-G genes are indispensable to obtain adhesive F17 fimbriae (unpublished data). Genetic evidence is presented that the F17-G protein, a minor fimbrial component, is required for the binding of the F17 fimbriae to the intestinal villi. The F17-G gene was cloned and sequenced. An open reading frame of 1,032 bp encoding a polypeptide of 344 amino acids, starting with a signal sequence of 22 residues, was localized. The F17-G mutant strain produced F17 fimbriae which were morphologically identical to the fimbriae purified from strains which contained the intact F17 gene cluster. However, this F17-G mutant could no longer adhere to calf villi. The F17-G locus was shown to act in trans: transformation of the F17-G mutant strain, still expressing the genes F17-A, F17-C, and F17-D, with a vector expressing the F17-G gene restored the binding activity of this mutant strain.
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517. |
Rosen BP,
( N/A ) The plasmid-encoded arsenical resistance pump: an anion-translocating ATPase. PMID : 1704144 : Abstract >>
An anion-translocating ATPase has been identified as the product of the arsenical resistance operon of resistance plasmid R773. When expressed in Escherichia coli this ATP-driven oxyanion pump catalyses extrusion of the oxyanions arsenite, antimonite and arsenate. Maintenance of a low intracellular concentration of oxyanion produces resistance to the toxic agents. The pump is composed of two polypeptides, the products of the arsA and arsB genes. This two-subunit enzyme produces resistance to arsenite and antimonite. A third gene, arsC, expands the substrate specificity to allow for arsenate pumping and resistance.
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518. |
Woods R,
Schneider D,
Winkworth CL,
Riley MA,
Lenski RE,
( 2006 ) Tests of parallel molecular evolution in a long-term experiment with Escherichia coli. PMID : 16751270 : DOI : 10.1073/pnas.0602917103 PMC : PMC1482574 Abstract >>
The repeatability of evolutionary change is difficult to quantify because only a single outcome can usually be observed for any precise set of circumstances. In this study, however, we have quantified the frequency of parallel and divergent genetic changes in 12 initially identical populations of Escherichia coli that evolved in identical environments for 20,000 cell generations. Unlike previous analyses in which candidate genes were identified based on parallel phenotypic changes, here we sequenced four loci (pykF, nadR, pbpA-rodA, and hokB/sokB) in which mutations of unknown effect had been discovered in one population, and then we compared the substitution pattern in these "blind" candidate genes with the pattern found in 36 randomly chosen genes. Two candidate genes, pykF and nadR, had substitutions in all 11 other populations, and the other 2 in several populations. There were very few cases, however, in which the exact same mutations were substituted, in contrast to the findings from conceptually related work performed with evolving virus populations. No random genes had any substitutions except in four populations that evolved defects in DNA repair. Tests of four different statistical aspects of the pattern of molecular evolution all indicate that adaptation by natural selection drove the parallel changes in these candidate genes.
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519. |
Ouyang Z,
Isaacson R,
( 2006 ) Identification and characterization of a novel ABC iron transport system, fit, in Escherichia coli. PMID : 16982838 : DOI : 10.1128/IAI.00866-06 PMC : PMC1698097 Abstract >>
A putative ABC transporter, fit, with significant homology to several bacterial iron transporters was identified in Escherichia coli. The E. coli fit system consists of six genes designated fitA, -B, -C, -D, -E, and -R. Based on DNA sequence analysis, fit encodes an outer membrane protein (FitA), a periplasmic binding protein (FitE), two permease proteins (FitC and -D), an ATPase (FitB), and a hypothetical protein (FitR). Introduction of the E. coli fit system into E. coli strain K-12 increased intracellular iron content and transformed bacteria were more sensitive to streptonigrin, which suggested that fit transports iron in E. coli. Expression of fit was studied using a lacZ reporter assay. A functional, bidirectional promoter was identified in the intergenic region between genes fitA and fitB. The expression of the E. coli fit system was found to be induced by iron limitation and repressed when Fe(2+) was added to minimal medium. Several fit mutants were created in E. coli using an in vitro transposon mutagenesis strategy. Mutations in fit did not affect bacterial growth in iron-restricted media. Using a growth promotion test, it was found that fit was not able to transport enterobactin, ferrichrome, transferrin, and lactoferrin in E. coli.
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520. |
Zaleski P,
Wolinowska R,
Strzezek K,
Lakomy A,
Plucienniczak A,
( 2006 ) The complete sequence and segregational stability analysis of a new cryptic plasmid pIGWZ12 from a clinical strain of Escherichia coli. PMID : 16828160 : DOI : 10.1016/j.plasmid.2006.05.004 Abstract >>
A new cryptic plasmid from a multi-resistant, multi-plasmid clinical strain of Escherichia coli has been isolated. The sequence of the 4072-base-pair pIGWZ12 (GenBank Accession No. DQ311641) was determined and analyzed. Two open-reading frames that code for proteins involved in plasmid mobilization and initiation of replication were identified. The putative origin of replication possesses all characteristic features of the theta mechanism for replicating plasmids. pIGWZ12 is stably maintained without selective pressure in bacterial cultures (for up to 80 generations), making it a good candidate for engineering a new cloning vector.
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521. |
Hontz JS,
Villar-Lecumberri MT,
Potter BM,
Yoder MD,
Dreyfus LA,
Laity JH,
( 2006 ) Differences in crystal and solution structures of the cytolethal distending toxin B subunit: Relevance to nuclear translocation and functional activation. PMID : 16809347 : DOI : 10.1074/jbc.M603727200 Abstract >>
Cytolethal distending toxin (CDT) induces cell cycle arrest and apoptosis in eukaryotic cells, which are mediated by the DNA-damaging CdtB subunit. Here we report the first x-ray structure of an isolated CdtB subunit (Escherichia coli-II CdtB, EcCdtB). In conjunction with previous structural and biochemical observations, active site structural comparisons between free and holotoxin-assembled CdtBs suggested that CDT intoxication is contingent upon holotoxin disassembly. Solution NMR structural and 15N relaxation studies of free EcCdtB revealed disorder in the interface with the CdtA and CdtC subunits (residues Gly233-Asp242). Residues Leu186-Thr209 of EcCdtB, which encompasses tandem arginine residues essential for nuclear translocation and intoxication, were also disordered in solution. In stark contrast, nearly identical well defined alpha-helix and beta-strand secondary structures were observed in this region of the free and holotoxin CdtB crystallographic models, suggesting that distinct changes in structural ordering characterize subunit disassembly and nuclear localization factor binding functions.
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522. |
Lacher DW,
Steinsland H,
Blank TE,
Donnenberg MS,
Whittam TS,
( 2007 ) Molecular evolution of typical enteropathogenic Escherichia coli: clonal analysis by multilocus sequence typing and virulence gene allelic profiling. PMID : 17098897 : DOI : 10.1128/JB.01472-06 PMC : PMC1797380 Abstract >>
Enteropathogenic Escherichia coli (EPEC) infections are a leading cause of infantile diarrhea in developing nations. Typical EPEC isolates are differentiated from other types of pathogenic E. coli by two distinctive phenotypes, attaching effacement and localized adherence. The genes specifying these phenotypes are found on the locus of enterocyte effacement (LEE) and the EPEC adherence factor (EAF) plasmid. To describe how typical EPEC has evolved, we characterized a diverse collection of strains by multilocus sequence typing (MLST) and performed restriction fragment length polymorphism (RFLP) analysis of three virulence genes (eae, bfpA, and perA) to assess allelic variation. Among 129 strains representing 20 O-serogroups, 21 clonal genotypes were identified using MLST. RFLP analysis resolved nine eae, nine bfpA, and four perA alleles. Each bfpA allele was associated with only one perA allele class, suggesting that recombination has not played a large role in shuffling the bfpA and perA loci between separate EAF plasmids. The distribution of eae alleles among typical EPEC strains is more concordant with the clonal relationships than the distribution of the EAF plasmid types. These results provide further support for the hypothesis that the EPEC pathotype has evolved multiple times within E. coli through separate acquisitions of the LEE island and EAF plasmid.
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523. |
Prosseda G,
Latella MC,
Casalino M,
Nicoletti M,
Michienzi S,
Colonna B,
( 2006 ) Plasticity of the P junc promoter of ISEc11, a new insertion sequence of the IS1111 family. PMID : 16788177 : DOI : 10.1128/JB.00332-06 PMC : PMC1483014 Abstract >>
We describe identification and functional characterization of ISEc11, a new insertion sequence that is widespread in enteroinvasive E. coli (EIEC), in which it is always present on the virulence plasmid (pINV) and very frequently also present on the chromosome. ISEc11 is flanked by subterminal 13-bp inverted repeats (IRs) and is bounded by 3-bp terminal sequences, and it transposes with target specificity without generating duplication of the target site. ISEc11 is characterized by an atypical transposase containing the DEDD motif of the Piv/MooV family of DNA recombinases, and it is closely related to the IS1111 family. Transposition occurs by formation of minicircles through joining of the abutted ends and results in assembly of a junction promoter (P juncC) containing a -10 box in the interstitial sequence and a -35 box upstream of the right IR. A natural variant of ISEc11 (ISEc11p), found on EIEC pINV plasmids, contains a perfect duplication of the outermost 39 bp of the right end. Upon circularization, ISEc11p forms a junction promoter (P juncP) which, despite carrying -10 and -35 boxes identical to those of P juncC, exhibits 30-fold-greater strength in vivo. The discovery of only one starting point in primer extension experiments rules out the possibility that there are alternative promoter sites within the 39-bp duplication. Analysis of in vitro-generated transcripts confirmed that at limiting RNA polymerase concentrations, the activity of P juncP is 20-fold higher than the activity of P juncC. These observations suggest that the 39-bp duplication might host cis-acting elements that facilitate the binding of RNA polymerase to the promoter.
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524. |
Johnson JR,
Owens KL,
Clabots CR,
Weissman SJ,
Cannon SB,
( 2006 ) Phylogenetic relationships among clonal groups of extraintestinal pathogenic Escherichia coli as assessed by multi-locus sequence analysis. PMID : 16820314 : DOI : 10.1016/j.micinf.2006.02.007 Abstract >>
The evolutionary origins of extraintestinal pathogenic Escherichia coli (ExPEC) remain uncertain despite these organisms' relevance to human disease. A valid understanding of ExPEC phylogeny is needed as a framework against which the observed distribution of virulence factors and clinical associations can be analyzed. Accordingly, phylogenetic relationships were defined by multi-locus sequence analysis among 44 representatives of selected ExPEC clonal groups and the E. coli Reference (ECOR) collection. Recombination, which significantly obscured the phylogenetic signal for several strains, was dealt with by excluding strains or specific sequences. Conflicting overall phylogenies, and internal phylogenies for virulence-associated phylogenetic group B2, were inferred depending on the specific dataset (i.e., how extensively purged of recombination), outgroup (Salmonella enterica and/or Escherichia fergusonii), and analysis method (neighbor joining, maximum parsimony, maximum likelihood, or Bayesian likelihood). Nonetheless, the major E. coli phylogenetic groups A, B1, and B2 were consistently well resolved, as was a major sub-component of group D and an ECOR 37-O157:H7 clade. Moreover, nine important ExPEC clonal groups within groups B2 and D, characterized by serotypes O6:K2:H1, O18:K1:H7, O6:H31, and O4:K+:H+ (from group B2), and O1:K1:H-, O7:K1:H-, O157:K+:H (non-7), O15:K52:H1, and O11/17/77:K52:H18 ("clonal group A") (from group D), were consistently well resolved, regardless of clinical background (cystitis, pyelonephritis, neonatal meningitis, sepsis, or fecal), host group, geographical origin, and virulence profile. Among the group B2-derived clonal groups the O6:K2:H1 clade appeared basal. Within group D, "clonal group A" and the O15:K52:H1 clonal group were consistently placed with ECOR 47 and ECOR 44, respectively, as nearest neighbors. These findings clarify phylogenetic relationships among key ExPEC clonal groups but also emphasize that recombination appears to obscure the oldest evolutionary relationships, despite extensive targeted sequencing and use of a wide range of analysis techniques.
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525. |
Rosen BP,
Weigel U,
Monticello RA,
Edwards BP,
( 1991 ) Molecular analysis of an anion pump: purification of the ArsC protein. PMID : 1703401 : DOI : 10.1016/0003-9861(91)90312-7 Abstract >>
The ars operon of resistance plasmid R773 encodes an anion-translocating ATPase which catalyzes extrusion of the oxyanions arsenite, antimonite, and arsenate, thus providing resistance to the toxic compounds. Although both arsenite and arsenate contain arsenic, they have different chemical properties. In the absence of the arsC gene the pump transports arsenite and antimonite, oxyanions with the +III oxidation state of arsenic or antimony. The complex neither transports nor provides resistance to arsenate, the oxyanion of the +V oxidation state of arsenic. The arsC gene encodes a 16-kDa polypeptide, the ArsC protein, which alters the substrate specificity of the pump to allow for recognition and transport of the alternate substrate arsenate. The arsC gene was cloned behind a strong promoter and expressed at high levels. The ArsC protein was purified and crystallized.
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526. |
Robin F,
Delmas J,
Archambaud M,
Schweitzer C,
Chanal C,
Bonnet R,
( 2006 ) CMT-type beta-lactamase TEM-125, an emerging problem for extended-spectrum beta-lactamase detection. PMID : 16801418 : DOI : 10.1128/AAC.01639-05 PMC : PMC1489774 Abstract >>
The clinical strain Escherichia coli TO799 was resistant to penicillin-clavulanate combinations and ceftazidime and was not reproducibly detected as an extended-spectrum beta-lactamase (ESBL) according to the standards of the Clinical Laboratory Standards Institute (CLSI; formerly NCCLS) and the national guidelines of the French Society for Microbiology (Comit? de l'Antibiogramme de la Soci?t? Fran?aise de Microbiologie). A novel beta-lactamase, designated TEM-125, was responsible for this phenotype. TEM-125 harbors a complex association of mutations previously described in the ESBL TEM-12 and in the inhibitor-resistant beta-lactamase TEM-39. TEM-125 is the first complex mutant TEM to present hydrolytic activity against ceftazidime (kcat, 3.7 s(-1)) together with a high level of resistance to clavulanate (50% inhibitory concentration, 13.6 microM). The discovery of such an ESBL, which is difficult to detect by the usual ESBL detection methods, confirms the emergence of a complex mutant TEM subgroup and highlights the need to evaluate detection methods so as to avoid possible therapeutic failures.
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527. |
Pinkner JS,
Remaut H,
Buelens F,
Miller E,
Aberg V,
Pemberton N,
Hedenström M,
Larsson A,
Seed P,
Waksman G,
Hultgren SJ,
Almqvist F,
( 2006 ) Rationally designed small compounds inhibit pilus biogenesis in uropathogenic bacteria. PMID : 17098869 : DOI : 10.1073/pnas.0606795103 PMC : PMC1693844 DOI : 10.1073/pnas.0606795103 PMC : PMC1693844 Abstract >>
A chemical synthesis platform with broad applications and flexibility was rationally designed to inhibit biogenesis of adhesive pili assembled by the chaperone-usher pathway in Gram-negative pathogens. The activity of a family of bicyclic 2-pyridones, termed pilicides, was evaluated in two different pilus biogenesis systems in uropathogenic Escherichia coli. Hemagglutination mediated by either type 1 or P pili, adherence to bladder cells, and biofilm formation mediated by type 1 pili were all reduced by approximately 90% in laboratory and clinical E. coli strains. The structure of the pilicide bound to the P pilus chaperone PapD revealed that the pilicide bound to the surface of the chaperone known to interact with the usher, the outer-membrane assembly platform where pili are assembled. Point mutations in the pilicide-binding site dramatically reduced pilus formation but did not block the ability of PapD to bind subunits and mediate their folding. Surface plasmon resonance experiments confirmed that the pilicide interfered with the binding of chaperone-subunit complexes to the usher. These pilicides thus target key virulence factors in pathogenic bacteria and represent a promising proof of concept for developing drugs that function by targeting virulence factors.
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528. |
Bhagwat AA,
Tan J,
Sharma M,
Kothary M,
Low S,
Tall BD,
Bhagwat M,
( 2006 ) Functional heterogeneity of RpoS in stress tolerance of enterohemorrhagic Escherichia coli strains. PMID : 16820496 : DOI : 10.1128/AEM.02842-05 PMC : PMC1489321 Abstract >>
The stationary-phase sigma factor (RpoS) regulates many cellular responses to environmental stress conditions such as heat, acid, and alkali shocks. On the other hand, mutations at the rpoS locus have frequently been detected among pathogenic as well as commensal strains of Escherichia coli. The objective of this study was to perform a functional analysis of the RpoS-mediated stress responses of enterohemorrhagic E. coli strains from food-borne outbreaks. E. coli strains belonging to serotypes O157:H7, O111:H11, and O26:H11 exhibited polymorphisms for two phenotypes widely used to monitor rpoS mutations, heat tolerance and glycogen synthesis, as well as for two others, alkali tolerance and adherence to Caco-2 cells. However, these strains synthesized the oxidative acid resistance system through an rpoS-dependent pathway. During the transition from mildly acidic growth conditions (pH 5.5) to alkaline stress (pH 10.2), cell survival was dependent on rpoS functionality. Some strains were able to overcome negative regulation by RpoS and induced higher beta-galactosidase activity without compromising their acid resistance. There were no major differences in the DNA sequences in the rpoS coding regions among the tested strains. The heterogeneity of rpoS-dependent phenotypes observed for stress-related phenotypes was also evident in the Caco-2 cell adherence assay. Wild-type O157:H7 strains with native rpoS were less adherent than rpoS-complemented counterpart strains, suggesting that rpoS functionality is needed. These results show that some pathogenic E. coli strains can maintain their acid tolerance capability while compromising other RpoS-dependent stress responses. Such adaptation processes may have significant impact on a pathogen's survival in food processing environments, as well in the host's stomach and intestine.
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529. |
Stakenborg T,
Vandekerchove D,
Mariën J,
Laevens H,
Imberechts H,
Peeters J,
( 2006 ) Protection of rabbits against enteropathogenic Escherichia coli (EPEC) using an intimin null mutant. PMID : 16796739 : DOI : 10.1186/1746-6148-2-22 PMC : PMC1544329 Abstract >>
Diarrhea and mortality resulting from infections with enteropathogenic Escherichia coli (EPEC) are of major economic importance in the rabbit meat industry. There is a growing need for an effective vaccine to cope with these problems and to reduce the use of antibiotics. EPEC are characterized by an attaching and effacing virulence mechanism. This is partly mediated by the intimate binding between an adhesin, called intimin, and a translocated receptor (Tir) of prokaryote origin. We constructed an intimin deletion mutant of the rabbit EPEC (REPEC) wild-type strain 97/241.6 (bio-/serogroup 3-/O15) and examined its protective capacity. After verifying its complete loss of virulence, we used the attenuated strain in vaccination-challenge experiments in which complete protection against a homologous, but virulent, strain was observed. The attenuated strain was able to persist in the intestinal lumen, where it elicited an immune response against EPEC-related virulence proteins, as was shown using an EspB-specific ELISA. Despite the priming of an immune response and the generation of specific antibodies, the intimin mutant was not able to fully protect rabbits against challenges with REPEC strains of other bio-/serogroups. These data indicate that protection against REPEC infections is at least partly bio-/serogroup dependent and a multivalent vaccine may be needed for protection against the full range of REPEC types. Such a combination vaccine may be developed using intimin null mutants, as the latter were clearly shown to be safe and effective against homologous infections.
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530. |
Hejnova J,
Pages D,
Rusniok C,
Glaser P,
Sebo P,
Buchrieser C,
( 2006 ) Specific regions of genome plasticity and genetic diversity of the commensal Escherichia coli A0 34/86. PMID : 17049458 : DOI : 10.1016/j.ijmm.2006.06.007 Abstract >>
Escherichia coli A0 34/86 (O83:K24:H31) is a commensal strain that has been used for prophylactic and therapeutic colonization of the intestine of newborn infants. To identify traits specific for E. coli A0 34/86, we used a minimal tiling set of 148 BAC clones of A0 34/86 genomic DNA, to construct restriction-digested BAC arrays. Hybridization with genomic DNA from four E. coli strains (CFT073; O157:H7; K12 and Nissle 1917) allowed selection of two BAC clones that were sequenced to identify A0 34/86-specific regions. Genes for the yersiniabactin siderophore system, several proteins homologous to Salmonella enterica serovar Typhimurium vitamin B12 synthesis proteins, as well as genes necessary for the degradation of propanediol, the pix fimbriae determinant and genes coding for a putative phosphoglycerate transport system present also on pathogenicity island V of E. coli strain 536 were all identified in E. coli A0 34/86. This comparative analysis underlines the important genome heterogeneity between E. coli strains.
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531. |
Paton AW,
Beddoe T,
Thorpe CM,
Whisstock JC,
Wilce MC,
Rossjohn J,
Talbot UM,
Paton JC,
( 2006 ) AB5 subtilase cytotoxin inactivates the endoplasmic reticulum chaperone BiP. PMID : 17024087 : DOI : 10.1038/nature05124 Abstract >>
AB5 toxins are produced by pathogenic bacteria and consist of enzymatic A subunits that corrupt essential eukaryotic cell functions, and pentameric B subunits that mediate uptake into the target cell. AB5 toxins include the Shiga, cholera and pertussis toxins and a recently discovered fourth family, subtilase cytotoxin, which is produced by certain Shiga toxigenic strains of Escherichia coli. Here we show that the extreme cytotoxicity of this toxin for eukaryotic cells is due to a specific single-site cleavage of the essential endoplasmic reticulum chaperone BiP/GRP78. The A subunit is a subtilase-like serine protease; structural studies revealed an unusually deep active-site cleft, which accounts for its exquisite substrate specificity. A single amino-acid substitution in the BiP target site prevented cleavage, and co-expression of this resistant protein protected transfected cells against the toxin. BiP is a master regulator of endoplasmic reticulum function, and its cleavage by subtilase cytotoxin represents a previously unknown trigger for cell death.
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532. |
Roos V,
Schembri MA,
Ulett GC,
Klemm P,
( 2006 ) Asymptomatic bacteriuria Escherichia coli strain 83972 carries mutations in the foc locus and is unable to express F1C fimbriae. PMID : 16735742 : DOI : 10.1099/mic.0.28711-0 Abstract >>
Escherichia coli is the most common organism associated with asymptomatic bacteriuria (ABU). In contrast to uropathogenic E. coli (UPEC), which causes symptomatic urinary tract infection (UTI), very little is known about the mechanisms by which these strains colonize the urinary tract. Bacterial adhesion conferred by specific surface-associated adhesins is normally considered as a prerequisite for colonization of the urinary tract. The prototype ABU E. coli strain 83972 was originally isolated from a girl who had carried it asymptomatically for 3 years. This study characterized the molecular status of one of the primary adhesion factors known to be associated with UTI, namely F1C fimbriae, encoded by the foc gene cluster. F1C fimbriae recognize receptors present in the human kidney and bladder. Expression of the foc genes was found to be up-regulated in human urine. It was also shown that although strain 83972 contains a seemingly intact foc gene cluster, F1C fimbriae are not expressed. Sequencing and genetic complementation revealed that the focD gene, encoding a component of the F1C transport and assembly system, was non-functional, explaining the inability of strain 83972 to express this adhesin. The data imply that E. coli 83972 has lost its ability to express this important colonization factor as a result of host-driven evolution. The ancestor of the strain seems to have been a pyelonephritis strain of phylogenetic group B2. Strain 83972 therefore represents an example of bacterial adaptation from pathogenicity to commensalism through virulence factor loss.
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533. |
Houman F,
Diaz-Torres MR,
Wright A,
( 1990 ) Transcriptional antitermination in the bgl operon of E. coli is modulated by a specific RNA binding protein. PMID : 1698125 : DOI : 10.1016/0092-8674(90)90392-r Abstract >>
Regulation of bgl operon expression in E. coli occurs by a mechanism involving antitermination of transcription at two termination sites within the operon. The bglG gene product is absolutely required for this process. Here we provide evidence that BglG is an RNA binding protein that recognizes a specific sequence located just upstream of each of the terminators. The sequence was delimited using a series of specific oligonucleotide probes. Mutational analysis of this sequence indicates that the protein requires a specific RNA secondary structure for recognition. We propose that BglG prevents transcription termination by binding to nascent RNA and blocking formation of the terminator structure.
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534. |
Korth MJ,
Schneider RA,
Moseley SL,
( 1991 ) An F41-K88-related genetic determinant of bovine septicemic Escherichia coli mediates expression of CS31A fimbriae and adherence to epithelial cells. PMID : 1675627 : PMC : PMC258015 Abstract >>
A genetic determinant related to that encoding the F41 fimbrial adhesin was cloned from a bovine septicemic isolate of Escherichia coli. This determinant was found to mediate expression of morphologically distinct fimbriae in E. coli HB101. The gene encoding the fimbrial subunit protein was identified, and the nucleotide sequence was determined. Homology with the amino-terminal amino acid sequence of CS31A (J. Girardeau, M. Der Vartanian, J. Ollier, and M. Contrepois, Infect. Immun. 56:2180-2188, 1988) was observed, suggesting that this determinant encodes expression of the CS31A fimbrial antigen. The CS31A subunit gene was found to share extensive homology in its signal sequence to the subunit genes encoding the F41 and K88 adhesins. No apparent homology between the mature F41 and CS31A subunits was identified. However, substantial relatedness to the K88 fimbrial subunit was observed. Analysis of the protein products encoded by the CS31A, F41, and K88 determinants in maxicells established that despite extensive genetic similarities between the determinants, each encodes a distinct profile of proteins. E. coli HB101 harboring the cloned CS31A determinant was found to adhere to epithelial cells in a tissue culture assay, suggesting a role for CS31A in adherence. A CS31A-specific DNA hybridization probe detected homologous sequences among enterotoxigenic as well as septicemic E. coli isolates from calves.
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535. |
Hopkins KL,
Deheer-Graham A,
Threlfall EJ,
Batchelor MJ,
Liebana E,
( 2006 ) Novel plasmid-mediated CTX-M-8 subgroup extended-spectrum beta-lactamase (CTX-M-40) isolated in the UK. PMID : 16697557 : DOI : 10.1016/j.ijantimicag.2006.03.003 Abstract >>
N/A
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536. |
Shi L,
Zheng M,
Xiao Z,
Asakura M,
Su J,
Li L,
Yamasaki S,
( 2006 ) Unnoticed spread of class 1 integrons in gram-positive clinical strains isolated in Guangzhou, China. PMID : 16785718 : DOI : 10.1111/j.1348-0421.2006.tb03815.x Abstract >>
A total of 46 gram-positive bacteria isolated from clinical specimens collected in China were subjected to PCR analysis with the intI1-specific primers, and the intI1-positive strains were further analyzed for their resistance gene cassette. All isolates possessed the class 1 integron in their genomes and the array of gene cassettes was dhfrXII-orfF-aadA2, which is very similar to other organisms except in one isolate carrying an additional copy of the class 1 integron containing the aadA2 gene cassette. Altogether, the results indicate that the class 1 integron is widespread in gram-positive clinical strains isolated in Guangzhou, China.
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537. |
Bae IK,
Lee YN,
Hwang HY,
Jeong SH,
Lee SJ,
Kwak HS,
Song W,
Kim HJ,
Youn H,
( 2006 ) Emergence of CTX-M-12 extended-spectrum beta-lactamase-producing Escherichia coli in Korea. PMID : 17003059 : DOI : 10.1093/jac/dkl397 Abstract >>
To characterize CTX-M-12 extended-spectrum beta-lactamase (ESBL) produced by clinical Escherichia coli isolates and to investigate its genetic environment. Antimicrobial susceptibilities were determined by disc diffusion and agar dilution methods, and the double-disc synergy test was carried out. Detection of genes encoding class A beta-lactamases was performed by PCR amplification, and the genetic environments of the bla(CTX-M-12) genes were investigated by PCR and sequencing of the regions surrounding the genes. Kinetic parameters were determined from purified CTX-M-12. Sequence data for the CTX-M-1 cluster from three clinical E. coli isolates indicated the presence of CTX-M-12. An ISEcp1 insertion sequence was located 49 bp upstream of bla(CTX-M-12) in all three E. coli isolates. CTX-M-12 had a more potent hydrolytic activity against cefotaxime than against ceftazidime and was encoded on a self-transferable approximately 18 kbp plasmid. This work shows that CTX-M-12, which confers high-level resistance to cefotaxime but not to ceftazidime, has emerged in Korea. The bla(CTX-M-12) gene was associated with an upstream ISEcp1 insertion sequence.
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538. |
Cota E,
Jones C,
Simpson P,
Altroff H,
Anderson KL,
du Merle L,
Guignot J,
Servin A,
Le Bouguénec C,
Mardon H,
Matthews S,
( 2006 ) The solution structure of the invasive tip complex from Afa/Dr fibrils. PMID : 16965519 : DOI : 10.1111/j.1365-2958.2006.05375.x PMC : PMC2628978 DOI : 10.1111/j.1365-2958.2006.05375.x PMC : PMC2628978 Abstract >>
Afa/Dr family of adhesins are produced by pathogenic Escherichia coli strains that are especially prevalent in chronic diarrhoeal and recurrent urinary tract infections. Most notably, they are found in up to 50% of cystitis cases in children and 30% of pyelonephritis in pregnant women. Afa/Dr adhesins are capped surface fibrils that mediate recognition of the host and subsequent bacterial internalization. Using the newly solved three-dimensional structure of the minimal invasive complex (AfaDE) combined with biochemical and cellular assays, we reveal the architecture of the fibrillar cap and identify a novel mode of synergistic integrin recognition.
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539. |
Stummeyer K,
Schwarzer D,
Claus H,
Vogel U,
Gerardy-Schahn R,
Mühlenhoff M,
( 2006 ) Evolution of bacteriophages infecting encapsulated bacteria: lessons from Escherichia coli K1-specific phages. PMID : 16689790 : DOI : 10.1111/j.1365-2958.2006.05173.x Abstract >>
Bacterial capsules are not only important virulence factors, but also provide attachment sites for bacteriophages that possess capsule degrading enzymes as tailspike proteins. To gain insight into the evolution of these specialized viruses, we studied a panel of tailed phages specific for Escherichia coli K1, a neuroinvasive pathogen with a polysialic acid capsule. Genome sequencing of two lytic K1-phages and comparative analyses including a K1-prophage revealed that K1-phages did not evolve from a common ancestor. By contrast, each phage is related to a different progenitor type, namely T7-, SP6-, and P22-like phages, and gained new host specificity by horizontal uptake of an endosialidase gene. The new tailspikes emerged by combining endosialidase domains with the capsid binding module of the respective ancestor. For SP6-like phages, we identified a degenerated tailspike protein which now acts as versatile adaptor protein interconnecting tail and newly acquired tailspikes and demonstrate that this adapter utilizes an N-terminal undecapeptide interface to bind otherwise unrelated tailspikes. Combining biochemical and sequence analyses with available structural data, we provide new molecular insight into basic mechanisms that allow changes in host specificity while a conserved head and tail architecture is maintained. Thereby, the present study contributes not only to an improved understanding of phage evolution and host-range extension but may also facilitate the on purpose design of therapeutic phages based on well-characterized template phages.
|
540. |
Cheng J,
Wang Q,
Wang W,
Wang Y,
Wang L,
Feng L,
( 2006 ) Characterization of E. coli O24 and O56 O antigen gene clusters reveals a complex evolutionary history of the O24 gene cluster. PMID : 17072668 : DOI : 10.1007/s00284-006-0032-7 Abstract >>
O antigen is part of the lipopolysaccharide present in the outer membrane of Gram-negative bacteria. It has many different forms, which are almost entirely due to genetic variations of O antigen gene clusters. In this study, the O antigen gene clusters of E. coli O24 and O56 were sequenced, and all genes were assigned functions on the basis of homology. Comparison of O antigen gene clusters indicated that E. coli O24 O antigen gene cluster has possibly arisen from the E. coli O56 gene cluster, through inactivation of two glycosyltransferase genes and acquisition of two new genes from E. coli O157 and O152, respectively. The insertion sequence elements seemed to play important roles for the assembly of the O24 O antigen gene cluster. This is the first time that the evolutionary history of a multi-origin O antigen gene cluster is clearly demonstrated. Genes specific to E. coli O24 and O56 were also identified, which may be used for development of DNA-based serotyping schemes.
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541. |
Merlo LM,
Sadowsky MJ,
Ferguson JA,
Dean AM,
( 2006 ) The argRB of Escherichia coli is rare in isolates obtained from natural sources. PMID : 16797147 : DOI : 10.1016/j.gene.2006.04.002 Abstract >>
A single nucleotide polymorphism between Escherichia coli strains K12 and B is known to alter the mechanism by which the arginine repressor regulates arginine biosynthesis, from a regulated system in E. coli K12 to a deregulated system in E. coli B. Laboratory experiments have demonstrated that the different regulatory strategies are selectively favored under different environmental conditions. In this study we analyzed 537 E. coli strains and show that the argR allele in E. coli B, which causes deregulation, is rare in isolates obtained from natural sources. Moreover, sequence analysis of 85 strains shows no evidence of selection at the arginine repressor locus. This illustrates that analysis of sequence data is insufficient to detect selection of uncommon alleles in rare environments.
|
542. |
Nagy G,
Altenhoefer A,
Knapp O,
Maier E,
Dobrindt U,
Blum-Oehler G,
Benz R,
Emody L,
Hacker J,
( 2006 ) Both alpha-haemolysin determinants contribute to full virulence of uropathogenic Escherichia coli strain 536. PMID : 16787757 : DOI : 10.1016/j.micinf.2006.02.029 Abstract >>
Uropathogenic Escherichia coli strain 536 possesses two intact copies of the alpha-haemolysin determinant localised on distinct pathogenicity islands. The coding regions of the two hlyCABD operons are conserved; however, upstream sequences are entirely dissimilar. Consequently, expression of the encoded toxin molecules in vitro is highly different. On the other hand, the contribution of the individual determinants to the strain's virulence is the same. Isogenic mutants lacking individual hly determinants have a similar increase in LD50 value in a mouse model of urinary tract infection. Mouse lung toxicity as well as in vitro assays reveals a significant decrease in acute cytotoxicity of both mutants in comparison to the parent wild-type strain; however, the two hly mutants do not significantly differ from each other in these respects. Single channel recordings show no difference in electrophysiological characteristics of the pores formed by the individual HlyA molecules on synthetic planar lipid membranes. Nor do the paralogues have any target cell preference in an in vitro cytotoxicity assay. Our data suggest that the two hly paralogues encode identical toxin functions; however, due to different regulation of expression, they participate at distinct stages of the infectious process. Interestingly, the unrelated uropathogenic E. coli strain J96 shares the same two hly alleles, suggesting that acquisition of the two paralogues accorded a selective evolutionary advantage.
|
543. |
Rath D,
Jawali N,
( 2006 ) Loss of expression of cspC, a cold shock family gene, confers a gain of fitness in Escherichia coli K-12 strains. PMID : 16980479 : DOI : 10.1128/JB.00471-06 PMC : PMC1595533 Abstract >>
The CspA family of cold shock genes in Escherichia coli K-12 includes nine paralogs, cspA to cspI. Some of them have been implicated in cold stress adaptation. Screening for mutations among common laboratory E. coli strains showed a high degree of genetic diversity in cspC but not in cspA and cspE. This diversity in cspC was due to a wide spectrum of variations including insertions of IS elements, deletion, and point mutation. Northern analysis of these mutants showed loss of cspC expression in all but one case. Further analysis of the loss-of-function cspC mutants showed that they have a fitness advantage in broth culture after 24 h over their isogenic wild-type derivatives. Conversely, introduction of mutated cspC alleles conferred a competitive fitness advantage to AB1157, a commonly used laboratory strain. This provides the evidence that loss of cspC expression is both necessary and sufficient to confer a gain of fitness as seen in broth culture over 24 h. Together, these results ascribe a novel role in cellular growth at 37 degrees C for CspC, a member of the cold shock domain-containing protein family.
|
544. |
Jordi BJ,
van Vliet AH,
Willshaw GA,
van der Zeijst BA,
Gaastra W,
( 1991 ) Analysis of the first two genes of the CS1 fimbrial operon in human enterotoxigenic Escherichia coli of serotype 0139:H28. PMID : 1679404 : DOI : 10.1016/0378-1097(91)90607-c Abstract >>
An oligonucleotide, derived from the N-terminal amino acid sequence of the CS1 fimbrial subunit protein was used to identify the subunit gene on recombinant plasmid pDEP23 containing the structural genes of the CS1 fimbrial operon. The nucleotide sequence of the subunit gene (csoA), encoding a protein of 171 amino acids, was determined. Flanking it upstream, a gene (csoB) encoding a protein of 238 amino acids was found. The CsoB and CsoA proteins are homologous to the CfaA and CfaB proteins in the CFA/I fimbrial operon. For all the CS1 producing strains investigated the structural genes are located on plasmids. Like CFA/I fimbriae, CS1 fimbriae are only expressed in the presence of a positive regulator, CfaD for CFA/I and Rns for CS1, respectively. The promoter region upstream of the csoB gene was cloned in front of the promoterless alkaline phosphatase (phoA) gene of the promoter-probe vector pCB267. PhoA activity was enhanced approximately two-fold by the introduction of compatible plasmids containing either rns or cfaD.
|
545. |
Xie Y,
Yao Y,
Kolisnychenko V,
Teng CH,
Kim KS,
( 2006 ) HbiF regulates type 1 fimbriation independently of FimB and FimE. PMID : 16790777 : DOI : 10.1128/IAI.02058-05 PMC : PMC1489709 Abstract >>
Type 1 fimbriae have been suggested to play a role in the pathogenesis of extraintestinal Escherichia coli infection. Type 1 fimbriation in E. coli is phase variable and known to be dependent upon FimB and FimE, the two recombinases that invert the molecular switch fimS and control the expression of the downstream fim operon. Here we showed that HbiF, a novel site-specific recombinase, inverted fimS independently of FimB and FimE. HbiF-mediated fimS inversion appeared to be predominantly switching from "off" (termed OFF) to "on" (termed ON) orientation. This is different from the fimS inversion mediated by either FimB (bidirectional ON to OFF and OFF to ON) or FimE (unidirectional ON to OFF). Constitutive expression of the hbiF gene in E. coli resulted in a fimS-locked-ON phenotype, which resulted in the pathogenic E. coli K1 strain being incapable of inducing a high degree of bacteremia in neonatal rats. Discovery of HbiF-mediated OFF-to-ON fimS switching provides a new opportunity to develop a strategy for the prevention and therapy of extraintestinal E. coli infection including bacteremia and meningitis.
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546. |
Hsu MY,
Inouye M,
Inouye S,
( 1990 ) Retron for the 67-base multicopy single-stranded DNA from Escherichia coli: a potential transposable element encoding both reverse transcriptase and Dam methylase functions. PMID : 1701261 : DOI : 10.1073/pnas.87.23.9454 PMC : PMC55184 Abstract >>
The region (retron-Ec67) required for the biosynthesis of a branched-RNA-linked multicopy single-stranded DNA (msDNA-Ec67) from a clinical isolate of Escherichia coli was mapped at a position equivalent to 19 min on the K-12 chromosome. The element containing the retron consisted of a unique 34-kilobase sequence that was flanked by direct repeats of a 26-base-pair sequence found in the K-12 chromosomal DNA. This suggests that the 34-kilobase element was probably integrated into the E. coli genome by a mechanism related to transposition or phage integration. In the 34-kilobase sequence an open reading frame of 285 residues was found, which displays 44% sequence identity with the E. coli Dam methylase. Interestingly, there are three GATC sequences, the site of Dam methylation, in the promoter region of the gene for reverse transcriptase.
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547. |
Narayana N,
( 2006 ) High-resolution structure of a plasmid-encoded dihydrofolate reductase: pentagonal network of water molecules in the D2-symmetric active site. PMID : 16790925 : DOI : 10.1107/S0907444906014764 Abstract >>
R67 plasmid-encoded dihydrofolate reductase (R67 DHFR) is an NADPH-dependent homotetrameric enzyme that catalyzes the reduction of dihydrofolate to tetrahydrofolate. The amino-acid sequence and molecular architecture of R67 DHFR and its inhibitory properties toward folate analogues are different from those of chromosomal DHFR. Here, the crystal structure of R67 DHFR refined using 1.1 A resolution data is presented. Blocked full-matrix least-squares refinement without restraints resulted in a final R factor of 11.4%. The anisotropic atomic displacement parameters analyzed by Rosenfield matrices and translation-libration-screw validation suggested four quasi-rigid domains. A total of ten Calpha-H...O hydrogen bonds were identified between the beta-strands. There is reasonable structural evidence that His62 is not protonated in the tetramer, which is in accord with previous pH-profile studies. The side chain of Gln67 that protrudes into the active site exhibits dual conformation, a feature noticed for the first time owing to the availability of atomic resolution data. The R67 DHFR active site is unique: it has D2 symmetry and is a large active site with a pentagonal network of water molecules and exposure of backbone atoms to solvent; the central pore is favorable for planar ring-stacking interactions. The geometrical shape, overall symmetry, local asymmetry and waters appear to dominate the binding of ligands, catalysis and inhibition.
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548. |
Jones CH,
Tuckman M,
Murphy E,
Bradford PA,
( 2006 ) Identification and sequence of a tet(M) tetracycline resistance determinant homologue in clinical isolates of Escherichia coli. PMID : 17015654 : DOI : 10.1128/JB.00705-06 PMC : PMC1636245 Abstract >>
The presence of the tetracycline resistance determinant tet(M) in human clinical isolates of Escherichia coli is described for the first time in this report. The homologue was >99% identical to the tet(M) genes reported to occur in Lactobacillus plantarum, Neisseria meningitidis, and Streptococcus agalactiae, and 3% of the residues in its deduced amino acid sequence diverge from tet(M) of Staphylococcus aureus. Sequence analysis of the regions immediately flanking the gene revealed that sequences upstream of tet(M) in E. coli have homology to Tn916; however, a complete IS26 insertion element was present immediately upstream of the promoter element. Downstream from the termination codon is an insertion sequence that was homologous to the ISVs1 element reported to occur in a plasmid from Vibrio salmonicida that has been associated with another tetracycline resistance determinant, tet(E). Results of mating experiments demonstrated that the E. coli tet(M) gene was on a mobile element so that resistance to tetracycline and minocycline could be transferred to a susceptible strain by conjugation. Expression of the cloned tet(M) gene, under the control of its own promoter, provided tetracycline and minocycline resistance to the E. coli host.
|
549. |
Ndjonka D,
Bell CE,
( 2006 ) Structure of a hyper-cleavable monomeric fragment of phage lambda repressor containing the cleavage site region. PMID : 16934834 : DOI : 10.1016/j.jmb.2006.07.026 PMC : PMC1896146 Abstract >>
The key event in the switch from lysogenic to lytic growth of phage lambda is the self-cleavage of lambda repressor, which is induced by the formation of a RecA-ssDNA-ATP filament at a site of DNA damage. Lambda repressor cleaves itself at the peptide bond between Ala111 and Gly112, but only when bound as a monomer to the RecA-ssDNA-ATP filament. Here we have designed a hyper-cleavable fragment of lambda repressor containing the hinge and C-terminal domain (residues 101-229), in which the monomer-monomer interface is disrupted by two point mutations and a deletion of seven residues at the C terminus. This fragment crystallizes as a monomer and its structure has been determined to 1.8 A resolution. The hinge region, which bears the cleavage site, is folded over the active site of the C-terminal oligomerization domain (CTD) but with the cleavage site flipped out and exposed to solvent. Thus, the structure represents a non-cleavable conformation of the repressor, but one that is poised for cleavage after modest rearrangements that are presumably stabilized by binding to RecA. The structure provides a unique snapshot of lambda repressor in a conformation that sheds light on how its self-cleavage is tempered in the absence of RecA, as well as a framework for interpreting previous genetic and biochemical data concerning the RecA-mediated cleavage reaction.
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550. |
Whale AD,
Garmendia J,
Gomes TA,
Frankel G,
( 2006 ) A novel category of enteropathogenic Escherichia coli simultaneously utilizes the Nck and TccP pathways to induce actin remodelling. PMID : 16681840 : DOI : 10.1111/j.1462-5822.2006.00682.x Abstract >>
Enterohaemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC) induce drastic reorganization of the microfilament cytoskeleton. EHEC and EPEC translocate Tir (translocated intimin receptor) which, once inserted into the host plasma membrane, binds the bacterial outer membrane adhesin intimin. Tir(EPEC) then becomes tyrosine phosphorylated facilitating the recruitment and site-specific binding of the eukaryotic adaptor Nck, which in turn binds and activates the Wiskott-Aldrich syndrome protein (N-WASP), leading to actin-related protein 2/3 (Arp2/3) complex-mediated actin polymerization. In contrast, Tir(EHEC) has no Nck binding site; instead, EHEC utilizes the translocated effector TccP (Tir-cytoskeleton coupling protein) to bind and activate N-WASP. Here we report a novel class of EPEC that translocates both TccP and Tir(EPEC)-like effector molecules. Consistent with these characteristics, we show that both the Tir-Nck and Tir:TccP actin remodelling pathways function simultaneously during infection, making this a novel and versatile EPEC category.
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551. |
Pomba C,
Mendonça N,
Costa M,
Louro D,
Baptista B,
Ferreira M,
Correia JD,
Caniça M,
( 2006 ) Improved multiplex PCR method for the rapid detection of beta-lactamase genes in Escherichia coli of animal origin. PMID : 16678992 : DOI : 10.1016/j.diagmicrobio.2006.03.005 Abstract >>
We developed 2 variants, A and B, of a multiplex polymerase chain reaction method for detecting the beta-lactam resistance genes bla(TEM), bla(SHV), and bla(OXA) in 122 uropathogenic Escherichia coli animal strains. Method B yielded 98% specificity and 100% sensitivity, and method A yielded 100% and 89%, respectively. Variant B was more accurate (99%) than A (94%).
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552. |
Lu Y,
Iyoda S,
Satou H,
Satou H,
Itoh K,
Saitoh T,
Watanabe H,
( 2006 ) A new immunoglobulin-binding protein, EibG, is responsible for the chain-like adhesion phenotype of locus of enterocyte effacement-negative, shiga toxin-producing Escherichia coli. PMID : 16988252 : DOI : 10.1128/IAI.00724-06 PMC : PMC1594913 Abstract >>
Shiga toxin-producing Escherichia coli (STEC) are important enteropathogens causing severe diseases such as hemorrhagic colitis and hemolytic-uremic syndrome in humans. The majority of STEC strains of serogroups O157, O26, or O111 associated with severe cases of these diseases possess a pathogenicity island termed the locus of enterocyte effacement (LEE). LEE, which is responsible for the formation of attaching-and-effacing lesions on intestinal epithelial cells, is important for the full virulence of STEC. Nonetheless, LEE-negative STEC strains have repeatedly been reported to be associated with severe diseases in humans. In this study, we characterized adhesion to cultured epithelial cells of certain LEE-negative STEC isolated from humans with or without bloody diarrhea. Several LEE-negative STEC belonging to serogroup O91 showed an unusual, chain-like adhesion pattern to HEp-2 cells. Using Tn5-based transposon mutagenesis, we identified the gene essential for the chain-like adhesion phenotype of this O91 STEC strain. Sequence analysis of the Tn5-inserted allele identified a novel chromosomal open reading frame (ORF) encoding a polypeptide with a high degree of similarity to the E. coli immunoglobulin-binding (Eib) proteins EibA, -C, -D, -E, and -F. Therefore, the ORF was designated EibG. Laboratory E. coli strain MC4100 transformed with a multicopy plasmid carrying eibG showed chain-like adhesion to HEp-2 cells, and whole-cell lysates of the strain bound to human-derived immunoglobulin G (IgG) Fc and IgA. These results indicate that EibG acts as an IgG Fc- and IgA-binding protein, as well as an adhesin of LEE-negative STEC.
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553. |
Cook PD,
Thoden JB,
Holden HM,
( 2006 ) The structure of GDP-4-keto-6-deoxy-D-mannose-3-dehydratase: a unique coenzyme B6-dependent enzyme. PMID : 16943443 : DOI : 10.1110/ps.062328306 PMC : PMC2242600 Abstract >>
L-colitose is a 3,6-dideoxysugar found in the O-antigens of some Gram-negative bacteria such as Escherichia coli and in marine bacteria such as Pseudoalteromonas tetraodonis. The focus of this investigation, GDP-4-keto-6-deoxy-D-mannose-3-dehydratase, catalyzes the third step in colitose production, which is the removal of the hydroxyl group at C3' of GDP-4-keto-6-deoxymannose. It is an especially intriguing PLP-dependent enzyme in that it acts as both a transaminase and a dehydratase. Here we present the first X-ray structure of this enzyme isolated from E. coli Strain 5a, type O55:H7. The two subunits of the protein form a tight dimer with a buried surface area of approximately 5000 A2. This is a characteristic feature of the aspartate aminotransferase superfamily. Although the PLP-binding pocket is formed primarily by one subunit, there is a loop, delineated by Phe 240 to Glu 253 in the second subunit, that completes the active site architecture. The hydrated form of PLP was observed in one of the enzyme/cofactor complexes described here. Amino acid residues involved in anchoring the cofactor to the protein include Gly 56, Ser 57, Asp 159, Glu 162, and Ser 183 from one subunit and Asn 248 from the second monomer. In the second enzyme/cofactor complex reported, a glutamate ketimine intermediate was found trapped in the active site. Taken together, these two structures, along with previously reported biochemical data, support the role of His 188 as the active site base required for catalysis.
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554. |
Sonnevend A,
Al Dhaheri K,
Mag T,
Herpay M,
Kolodziejek J,
Nowotny N,
Usmani A,
Sheikh FA,
Pál T,
( 2006 ) CTX-M-15-producing multidrug-resistant enteroaggregative Escherichia coli in the United Arab Emirates. PMID : 16700710 : DOI : 10.1111/j.1469-0691.2006.01413.x Abstract >>
Extended-spectrum beta-lactamase (ESBL) production was demonstrated in five independent, multidrug-resistant isolates of enteroaggregative Escherichia coli (EAEC) from the United Arab Emirates, representing 11.3% of the EAEC isolates recovered during 1 year. All five isolates carried the bla(CTX-M-15) and the bla(TEM-1) genes, the former positioned 48 bp downstream of an ISecp1 element. In two isolates, the bla(CTX-M-15)and bla(TEM-1) genes were located on a 95-kb plasmid. This is the first detailed description and characterisation of ESBL production in enteroaggregative E. coli and also the first report of CTX-M-producing organisms encountered on the Arabian Peninsula.
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555. |
Hopkins KL,
Deheer-Graham A,
Karisik E,
Batchelor MJ,
Liebana E,
Threlfall EJ,
( 2006 ) New plasmid-mediated AmpC beta-lactamase (CMY-21) in Escherichia coli isolated in the UK. PMID : 16716570 : DOI : 10.1016/j.ijantimicag.2006.03.020 Abstract >>
N/A
|
556. |
Skyberg JA,
Johnson TJ,
Johnson JR,
Clabots C,
Logue CM,
Nolan LK,
( 2006 ) Acquisition of avian pathogenic Escherichia coli plasmids by a commensal E. coli isolate enhances its abilities to kill chicken embryos, grow in human urine, and colonize the murine kidney. PMID : 16954398 : DOI : 10.1128/IAI.00363-06 PMC : PMC1695531 Abstract >>
We have found an avian pathogenic Escherichia coli (APEC) plasmid, pAPEC-O2-ColV, which contains many of the genes associated with APEC virulence and also shows similarity in content to a plasmid and pathogenicity island of human uropathogenic E. coli (UPEC). To test the possible role of this plasmid in virulence, it was transferred by conjugation along with a large R plasmid, pAPEC-O2-R, into a commensal avian E. coli strain. The transconjugant was compared to recipient strain NC, UPEC strain HE300, and donor strain APEC O2 using various assays, including lethality for chicken embryos, growth in human urine, and ability to cause urinary tract infection in mice. The transconjugant killed significantly more chicken embryos than did the recipient. In human urine, APEC O2 grew at a rate equivalent to that of UPEC strain HE300, and the transconjugant showed significantly increased growth compared to the recipient. The transconjugant also significantly outcompeted the recipient in colonization of the murine kidney. These findings suggest that APEC plasmids, such as pAPEC-O2-ColV, contribute to the pathogenesis of avian colibacillosis. Moreover, since avian E. coli and their plasmids may be transmitted to humans, evaluation of APEC plasmids as possible reservoirs of urovirulence genes for human UPEC may be warranted.
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557. |
Delmas J,
Breysse F,
Devulder G,
Flandrois JP,
Chomarat M,
( 2006 ) Rapid identification of Enterobacteriaceae by sequencing DNA gyrase subunit B encoding gene. PMID : 16626902 : DOI : 10.1016/j.diagmicrobio.2006.02.003 Abstract >>
Real-time polymerase chain reaction and sequencing were used to characterize a 506-bp-long DNA fragment internal to the gyrB gene (gyrBint). The sequences obtained from 32 Enterobacteriaceae-type strains and those available in the Genbank nucleotide sequence database (n = 24) were used as a database to identify 240 clinical enterobacteria isolates. Sequence analysis of the gyrBint fragment of 240 strains showed that gyrBint constitutes a discriminative target sequence to differentiate between Enterobacteriaceae species. Comparison of these identifications with those obtained by phenotypic methods (Vitek 1 system and/or Rapid ID 32E; bioM?rieux, Marcy l'Etoile, France) revealed discrepancies essentially with genera Citrobacter and Enterobacter. Most of the strains identified as Enterobacter cloacae by phenotypic methods were identified as Enterobacter hormaechei strains by gyrBint sequencing. The direct sequencing of gyrBint would be useful as a complementary tool in the identification of clinical Enterobacteriaceae isolates.
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558. |
Faubladier M,
Cam K,
Bouché JP,
( 1990 ) Escherichia coli cell division inhibitor DicF-RNA of the dicB operon. Evidence for its generation in vivo by transcription termination and by RNase III and RNase E-dependent processing. PMID : 1691299 : DOI : 10.1016/0022-2836(90)90325-G Abstract >>
We have established that the long non-coding intercistronic region of the dicB operon of Escherichia coli expresses a trans-acting division inhibitor specified by a region dicF, at most 65 nucleotides-long. The present study deals with the processing of dicBF operon mRNA in vivo, and identifies the dicF gene product as a 53 nucleotide RNA species. A sequence at the end of DicF resembles, and behaves as, a Rho-independent terminator, but further processing of readthrough transcripts, presumably by RNase III, followed by a limited 3' to 5' degradation, appears to generate additional DicF-RNA 3' ends. For the 5' end of DicF-RNA, our results show that a 190 nucleotide precursor DicF-RNA species is formed by cleavage at an RNase III site, while the 53 nucleotide minimal DicF-RNA is generated by further processing requiring the presence of an active form of RNase E in vivo. These data indicate that an untranslated product derived from an operon RNA can have a regulatory activity by affecting cell division.
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559. |
Velázquez L,
Camarena L,
Reyes JL,
Bastarrachea F,
( 1991 ) Mutations affecting the Shine-Dalgarno sequences of the untranslated region of the Escherichia coli gltBDF operon. PMID : 1673677 : DOI : 10.1128/jb.173.10.3261-3264.1991 PMC : PMC207927 Abstract >>
Individual mutations which affected each of the two Shine-Dalgarno sequences at the 5' untranslated region of the gltB gene of Escherichia coli were characterized. They were isolated in plasmids carrying a gltB'-'lacZ protein fusion preceded by the regulatory region of the gltBDF operon. Subcloning and nucleotide sequencing of approximately 1,206 bp of DNA encompassing the gltBDF regulatory region showed that the mutations affected the first base at each of the two identical Shine-Dalgarno sequences, SD1 and SD2, located 40 and 8 bases, respectively, upstream from the putative gltB open reading frame. Only mutation gltB2r227, an adenine in place of a guanine, affecting the first base of SD2, lowered beta-galactosidase expression significantly, i.e., about fivefold. The results suggest that SD2 is the preferred functional site at which ribosomes initiate gltB mRNA translation.
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560. |
Mammeri H,
Poirel L,
Fortineau N,
Nordmann P,
( 2006 ) Naturally occurring extended-spectrum cephalosporinases in Escherichia coli. PMID : 16801449 : DOI : 10.1128/AAC.01633-05 PMC : PMC1489779 Abstract >>
Genetic and functional characterization of the cephalosporinases produced by 65 clonally unrelated clinical Escherichia coli isolates revealed genetic diversity of the ampC genes and showed that Gln287, Cys287, Pro296, Leu298, and Phe350 substitutions were involved in extension of the hydrolysis spectrum to include ceftazidime and cefepime.
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561. |
Léveillé S,
Caza M,
Johnson JR,
Clabots C,
Sabri M,
Dozois CM,
( 2006 ) Iha from an Escherichia coli urinary tract infection outbreak clonal group A strain is expressed in vivo in the mouse urinary tract and functions as a catecholate siderophore receptor. PMID : 16714573 : DOI : 10.1128/IAI.00107-06 PMC : PMC1479231 Abstract >>
Virulence factors of pathogenic Escherichia coli belonging to a recently emerged and disseminated clonal group associated with urinary tract infection (UTI), provisionally designated clonal group A (CGA), have not been experimentally investigated. We used a mouse model of ascending UTI with CGA member strain UCB34 in order to identify genes of CGA that contribute to UTI. iha was identified to be expressed by strain UCB34 in the mouse kidney using selective capture of transcribed sequences. iha from strain UCB34 demonstrated a siderophore receptor phenotype when cloned in a catecholate siderophore receptor-negative E. coli K-12 strain, as shown by growth promotion experiments and uptake of (55)Fe complexed to enterobactin or its linear 2, 3-dihydroxybenzoylserine (DHBS) siderophore derivatives. Siderophore-mediated growth promotion by Iha was TonB dependent. Growth and iron uptake were more marked with linear DHBS derivatives than with purified enterobactin. The reported phenotype of adherence to epithelial cells conferred by expressing iha from a multicopy cloning vector in a poorly adherent E. coli K-12 host strain was confirmed to be specific to iha, in comparison with other siderophore receptor genes. iha expression was regulated by the ferric uptake regulator Fur and by iron availability, as shown by real-time reverse transcriptase PCR. In a competitive infection experiment using the mouse UTI model, wild-type strain UCB34 significantly outcompeted an isogenic iha null mutant. Iha thus represents a Fur-regulated catecholate siderophore receptor that, uniquely, exhibits an adherence-enhancing phenotype and is the first described urovirulence factor identified in a CGA strain.
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562. |
Jang SB,
Jeong MS,
Carter RJ,
Holbrook EL,
Comolli LR,
Holbrook SR,
( 2006 ) Novel crystal form of the ColE1 Rom protein. PMID : 16699189 : DOI : 10.1107/S0907444906012388 DOI : 10.1107/S0907444906012388 Abstract >>
The RNA I modulator protein (Rom) acts as a co-regulator of ColE1 plasmid copy number by binding to RNA kissing hairpins and stabilizing their interaction. The structure of Rom has been determined in a new crystal form from X-ray diffraction data to 2.5 A resolution. In this structure, a dimer of the 57-amino-acid protein is found in the asymmetric unit. Each subunit consists almost entirely of two antiparallel alpha-helices joined by a short hairpin bend. The dimer contains a non-crystallographic twofold axis and forms a highly regular four-alpha-helical bundle. The structural packing in this novel crystal form is different from previously known Rom structures. The asymmetric unit contains one dimer, giving a crystal volume per protein weight (V(M)) of 1.83 A(3) Da(-1) and a low solvent content of 30%. Strong packing interactions and low solvation are characteristic of the structure. The Rom protein was cocrystallized with the Tar-Tar* kissing hairpin RNA. Although the electron-density maps do not show bound RNA, altered conformations in the side chains of Rom that are known to be involved in RNA binding have been identified. These results provide additional information about Rom protein conformational flexibility and suggest that the presence of a highly charged polymer such as RNA can promote tight packing of an RNA-binding protein, even when the RNA itself is not observed in the crystal.
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563. |
Hama C,
Takizawa T,
Moriwaki H,
Urasaki Y,
Mizobuchi K,
( 1990 ) Organization of the replication control region of plasmid ColIb-P9. PMID : 1690704 : DOI : 10.1128/jb.172.4.1983-1991.1990 PMC : PMC208695 Abstract >>
We identified a 1,845-base-pair sequence that contains essential information for the autonomous replication and regulation of the 93-kilobase-pair IncI alpha group ColIb-P9 plasmid. Biochemical and genetic analyses revealed that this sequence specifies at least two structural genes, designated repZ and inc. The repZ gene encodes a protein with a molecular weight of 39,000, which probably functions as an initiator for the ColIb-P9 replicon. The inc gene that phenotypically governs the incompatibility encodes an RNA with a size of about 70 bases. This small RNA acts in trans to repress the expression of repZ, thereby functioning to maintain a constant copy number of the ColIb-P9 replicon in host cells.
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564. |
García-Aljaro C,
Muniesa M,
Jofre J,
Blanch AR,
( 2006 ) Newly identified bacteriophages carrying the stx2g Shiga toxin gene isolated from Escherichia coli strains in polluted waters. PMID : 16630267 : DOI : 10.1111/j.1574-6968.2006.00213.x Abstract >>
Shiga toxin-converting bacteriophages are the main vehicle involved in horizontal transmission of stx1 and stx2 genes, which has led to the current spread of stx genes among a high number of Escherichia coli serotypes and other enterobacteria. Several stx gene variants have been described, although some of the variants have never been isolated from inducible bacteriophages. In this study, two stx2g-carrying bacteriophages induced from two different E. coli O2:H25 strains isolated from different wastewater samples were characterized. These bacteriophages when transduced into Shigella sonnei retained their ability to produce Stx2 protein. They had similar but not identical DNA restriction patterns, and similar host ranges. Electron microscopy studies showed that they had isometric capsids with short tails, which resembled phage 933W. However, DNA cross-hybridization studies showed that phage 933W was not closely related to stx2g bacteriophages. This first description of stx2g-carrying bacteriophages enlarges the list of stx-carrying bacteriophages involved in horizontal transmission of stx genes in the environment.
|
565. |
Tisa LS,
Rosen BP,
( 1990 ) Molecular characterization of an anion pump. The ArsB protein is the membrane anchor for the ArsA protein. PMID : 1688427 : Abstract >>
R-factor mediated bacterial resistance to arsenical salts occurs by active extrusion of the toxic oxyanions from cells of gram negative bacteria. The ars operon of the conjugative plasmid R773 encodes an anion pump. The pump has two polypeptide components. The catalytic subunit, the ArsA protein, is an oxyanion-stimulated ATPase. The membrane component, the ArsB protein, has been localized in the inner membrane of Escherichia coli. The ArsA and ArsB proteins have been postulated to form a membrane complex which functions as an anion-translocating ATPase. In this study evidence is presented showing that expression of the arsB gene is required to anchor the ArsA protein to the inner membrane. Binding studies with purified ArsA to membranes with and without the arsB gene product confirm this requirement. Membranes of uncA mutants containing both the ArsA and ArsB proteins exhibit arsenite(antimonite)-stimulated ATPase activity. These results support the model in which the ArsA protein is the catalytic energy transducing component of the anion pump, whereas the integral membrane ArsB protein serves as both the anion channel and membrane binding site for the ArsA protein.
|
566. |
van Die I,
Kramer C,
Hacker J,
Bergmans H,
Jongen W,
Hoekstra W,
( N/A ) Nucleotide sequence of the genes coding for minor fimbrial subunits of the F1C fimbriae of Escherichia coli. PMID : 1683712 : Abstract >>
F1C fimbriae allow uropathogenic Escherichia coli to adhere to specific epithelial surfaces. This adhesive property is probably due to the presence of minor fimbrial components in F1C fimbriae. The foc gene cluster encoding F1C fimbriae has been cloned, as described previously. Here we present the nucleotide sequence (2081 bp) coding for the F1C minor fimbrial subunits. The structural genes code for polypeptides of 175 (FocF), 166 (FocG), and 300 (FocH) amino acids. The deduced amino acids of the F1C minor subunits were compared with the reported sequences of the minor subunits of other types of fimbriae. The data show that the Foc minor subunits are highly homologous to the corresponding Sfa proteins, whereas homology to the minor subunits of type 1 and P fimbriae is much lower.
|
567. |
Avgustin JA,
Grabnar M,
( 2007 ) Sequence analysis of the plasmid pColG from the Escherichia coli strain CA46. PMID : 16870252 : DOI : 10.1016/j.plasmid.2006.05.010 Abstract >>
The complete 4715 nucleotide sequence of the plasmid pColG from the Escherichia coli strain CA46, which was originally assumed to code for colicin G activity, has been determined. Based on the nucleotide sequence homology of the 1828bp replication region, with an average G+C content of 48%, pColG was classified as a ColE1-like plasmid. Computer assisted analysis of the remaining 2887bp nucleotide sequence with an average G+C content of 34% revealed three putative OFRs. To find out whether one or all of the three ORFs code for a possible bacteriocin, a DNA fragment encompassing these ORFs has been cloned and the recombinant colonies tested for bacteriocin production. None of the colonies had an inhibitory activity against E. coli strains DH5, HB101 and MC4100. The assumption that the plasmid pColG from the E. coli strain CA46 codes for a bacteriocin thus could not be confirmed.
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568. |
Schifferli DM,
Beachey EH,
Taylor RK,
( 1991 ) 987P fimbrial gene identification and protein characterization by T7 RNA polymerase-induced transcription and TnphoA mutagenesis. PMID : 1673018 : DOI : 10.1111/j.1365-2958.1991.tb01826.x Abstract >>
The 987P fimbrial gene cluster has been previously cloned as a 12 kb fragment from prototype strain 987. Gene products encoded by the whole clone were analysed by utilizing an in vivo system based on the induction of transcription by T7 RNA polymerase. The sensitivity of this technique permitted us to identify new proteins involved in 987P fimbriation. In total, eight proteins were detected, their genes (fasA to fasH) were mapped and their orientation of transcription determined. Several of the gene products demonstrated typical properties of exported proteins. Precursor and processed forms could be correlated after inhibiting protein transport with ethanol. The detection of enzymatically active fusion proteins after TnphoA (Tn5IS50L::phoA) mutagenesis supported and complemented these results. One protein encoded by the 12kb fragment was found not to be related to fimbriation but rather the product of the STla gene, identified as a component of a Tn1681-like transposon.
|
569. |
Lymberopoulos MH,
Houle S,
Daigle F,
Léveillé S,
Brée A,
Moulin-Schouleur M,
Johnson JR,
Dozois CM,
( 2006 ) Characterization of Stg fimbriae from an avian pathogenic Escherichia coli O78:K80 strain and assessment of their contribution to colonization of the chicken respiratory tract. PMID : 16952934 : DOI : 10.1128/JB.00453-06 PMC : PMC1595498 Abstract >>
In a previous study, ecs-3, a sequence from avian pathogenic Escherichia coli (APEC) O78:K80 strain chi7122, was found to be expressed in vivo in infected chicken tissues. The region encompassing ecs-3 carries a fimbrial gene cluster that is a putative ortholog of the stg fimbrial gene cluster of Salmonella enterica serovar Typhi. This APEC fimbrial gene cluster, which we have termed stg, is a member of a distinct group of related fimbriae that are located in the glmS-pstS intergenic region of certain E. coli and S. enterica strains. Under the control of the pBAD promoter, the production of Stg fimbriae was demonstrated by Western blotting and immunogold electron microscopy with E. coli K-12. Transcriptional fusions suggest that stg expression is influenced by the carbohydrate source and decreased by the addition of iron and that Fur plays a role in the regulation of stg expression. stg sequences were associated with APEC O78 isolates, and stg was phylogenetically distributed among E. coli reference strains and clinical isolates from human urinary tract infections. Stg fimbriae contributed to the adherence of a nonfimbriated E. coli K-12 strain to avian lung sections and human epithelial cells in vitro. Coinfection experiments with APEC strain chi7122 and an isogenic Deltastg mutant demonstrated that compared to the wild-type parent, the Deltastg mutant was less able to colonize air sacs, equally able to colonize lungs, and able to more effectively colonize tracheas of infected chickens. Stg fimbriae, together with other adhesins, may therefore contribute to the colonization of avian respiratory tissues by certain APEC strains.
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570. |
Brunder W,
Karch H,
Schmidt H,
( 2006 ) Complete sequence of the large virulence plasmid pSFO157 of the sorbitol-fermenting enterohemorrhagic Escherichia coli O157:H- strain 3072/96. PMID : 16887390 : DOI : 10.1016/j.ijmm.2006.05.005 Abstract >>
The large virulence plasmid pSFO157 of sorbitol-fermenting E. coli O157:H(-) strain 3072/96 has a size of 121,239bp and contains 96 open reading frames >50bp. It is therefore 29,162bp larger (ca. 32%) than plasmid pO157 of E. coli O157:H7 strain EDL933. Major differences between the plasmids are the absence of katP, espP, and toxB in pSFO157 and, instead of these, the presence of the sfp fimbriae gene cluster and a large part of an F-plasmid transfer region, the latter accounting for most of the additional DNA. The differences in the order of the genes and their composition, as well as the presence of a number of replication-associated genes and mobile genetic elements suggests that the large E. coli O157 virulence plasmids have a complex evolutionary origin.
|
571. |
Goussard S,
Sougakoff W,
Mabilat C,
Bauernfeind A,
Courvalin P,
( 1991 ) An IS1-like element is responsible for high-level synthesis of extended-spectrum beta-lactamase TEM-6 in Enterobacteriaceae. PMID : 1665171 : DOI : 10.1099/00221287-137-12-2681 Abstract >>
Resistance of Escherichia coli strain HB251 to the newer beta-lactam antibiotics, in particular ceftazidime and aztreonam, results from production of the extended-spectrum beta-lactamase TEM-6. The corresponding structural gene, bla(T)-6, and its promoter region were amplified by the polymerase chain reaction. Analysis of the sequence of the amplification product showed that bla(T)-6 differed by two nucleotide substitutions from bla(T)-1, the gene encoding TEM-1 penicillinase in plasmid pBR322. The mutations led to the substitution of a lysine for a glutamic acid at position 102 and of a histidine for an arginine at position 162 of the unprocessed TEM-1 protein. The presence of a 116 bp DNA insert upstream from bla(T)-6 resulted in the creation of hybrid promoter P6 in which the -10 region was that of TEM-1 promoter P3 whereas the -35 canonical sequence TTGACA was provided by the right end of the insert. P6 was found to be 10 times more active than P3 and to confer higher levels of antibiotic resistance upon the host. Analysis of the sequence of the insert indicated that the 116 bp fragment is related to insertion sequence IS1 but differs from it by three internal deletions that removed regions encoding the transposase. The distribution of the IS1-like element in clinical isolates of Enterobacteriaceae was studied by the polymerase chain reaction and by DNA-DNA hybridization. The element appeared to be widespread and was detected in strains producing TEM-6 or other TEM variants.
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572. |
Lee K,
Lee M,
Shin JH,
Lee MH,
Kang SH,
Park AJ,
Yong D,
Chong Y,
( 2006 ) Prevalence of plasmid-mediated AmpC beta-lactamases in Escherichia coli and Klebsiella pneumoniae in Korea. PMID : 16584308 : DOI : 10.1089/mdr.2006.12.44 Abstract >>
Cefoxitin-resistant Escherichia coli and Klebsiella pneumoniae are relatively prevalent in Korea, suggesting dissemination of plasmid-mediated AmpC beta-lactamases. In this study, 238 isolates of cefoxitin-resistant E. coli and K. pneumoniae (not including subspecies ozaenae and rhinoscleromatis) were collected in 2003 from 16 Korean hospitals. The prevalence of plasmid-mediated AmpC beta-lactamases was determined by PCR. The AmpC gene alleles detected in E. coli and K. pneumoniae were bla(DHA-1), 10 (8.6%) and 93 (76.2%); bla(CMY-1)-like, 14 (12.1%) and 2 (1.6%); and bla(CMY-2)-like, 38 (32.7%) and 1 (0.8%) isolates, respectively. The genes identified were bla(DHA-1), bla(CMY10)-like, and bla(CMY-2)-like, and a new variant, bla(CMY-18). Plasmidmediated AmpC gene allele-positive isolates were present both in large city and in small province hospitals, as well as in isolates from outpatients. The proportions of plasmid-mediated AmpC gene-positive isolates were similar in both expanded spectrum beta-lactamase (ESBL)-producing and -nonproducing isolates. In conclusion, DHA-1, CMY-2-like, and CMY-10-like plasmid-mediated AmpC beta-lactamase-producing K. pneumoniae and E. coli isolates are widely disseminated in both large city and small province hospitals. Absence of bla(CMY-1) and detection of a novel variant of bla(CMY-2), bla(CMY-18), indicate continued evolution of the prototype genes. Similar proportions of plasmid-mediated AmpC gene-positive isolates in both ESBL-producing and -nonproducing isolates suggest unhindered future spread of these resistances.
|
573. |
Sharples GJ,
Lloyd RG,
( 1991 ) Resolution of Holliday junctions in Escherichia coli: identification of the ruvC gene product as a 19-kilodalton protein. PMID : 1657895 : DOI : 10.1128/jb.173.23.7711-7715.1991 PMC : PMC212543 Abstract >>
The ruvC gene of Escherichia coli specifies a nuclease that resolves Holliday junction intermediates in genetic recombination (B. Connolly, C.A. Parsons, F.E. Benson, H.J. Dunderdale, G.J. Sharples, R.G. Lloyd, and S.C. West, Proc. Natl. Acad, Sci. USA 88:6063-6067, 1991). The gene was located between aspS and the ruvAB operon by DNA sequencing and deletion analysis of ruvC plasmids and was shown to encode a protein of 18,747 Da. Analysis of the DNA flanking ruvC indicated that the gene is transcribed independently of the LexA-regulated ruvAB operon and is not under direct SOS control. ruvC lies downstream of an open reading frame, orf-33, for a protein which migrates during sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a 33-kDa polypeptide. These two genes probably form an operon. However, expression of ruvC was found to be very poor relative to that of orf-33. A double ribosomal frameshift between these genes is proposed as a possible reason for the low level of RuvC. Two further open reading frames of unknown function were identified, one on either side of the orf-33-ruvC operon.
|
574. |
Swanson TN,
Bilge SS,
Nowicki B,
Moseley SL,
( 1991 ) Molecular structure of the Dr adhesin: nucleotide sequence and mapping of receptor-binding domain by use of fusion constructs. PMID : 1670929 : PMC : PMC257736 Abstract >>
The Dr hemagglutinin of uropathogenic Escherichia coli mediates adherence to the upper urinary tract. E. coli strains which express this adhesin bind to the Dr blood group antigen and mediate mannose-resistant hemagglutination (MRHA). Chloramphenicol inhibits MRHA produced by the Dr hemagglutinin and may act as an analog for the tissue receptor at the adhesin-binding site. The nucleotide sequence of the Dr hemagglutinin fimbrial subunit was determined and found to have significant homology with that of F1845, a fimbrial adhesin associated with diarrhea, and with the afimbrial adhesin AFA-I of uropathogenic E. coli. Chimeric adhesin determinants consisting of the Dr structural subunit and F1845 accessory genes or of the F1845 structural subunit and Dr accessory genes were constructed. The Dr and F1845 determinants were shown to have a close structural relationship, with functional differences concentrated in the fimbrial subunit. Oligonucleotide-directed site-specific mutagenesis was used to facilitate construction of a hybrid adhesin subunit gene containing the amino terminus of F1845 fused to the carboxy terminus of the Dr structural gene. The resulting construct confers chloramphenicol-resistant hemagglutination when introduced into an E. coli strain expressing the cloned Dr hemagglutinin. The chloramphenicol sensitivity or resistant phenotype of MRHA produced by this family of adhesins is determined solely by the fimbrial subunit gene. Domains responsible for the chloramphenicol sensitivity of Dr-mediated MRHA reside within the amino-terminal portion of the fimbrial subunit.
|
575. |
Ziegelin G,
Pansegrau W,
Strack B,
Balzer D,
Kröger M,
Kruft V,
Lanka E,
( 1991 ) Nucleotide sequence and organization of genes flanking the transfer origin of promiscuous plasmid RP4. PMID : 1665997 : Abstract >>
The nucleotide sequence of the relaxase operon and the leader operon which are part of the Tra1 region of the promiscuous plasmid RP4 was determined. These two polycistronic operons are transcribed divergently from an intergenic region of about 360 bp containing the transfer origin and six close-packed genes. A seventh gene completely overlaps another one in a different reading frame. Conjugative DNA transfer proceeds unidirectionally from oriT with the leader operon heading the DNA to be transferred. The traI gene of the relaxase operon includes within its 3' terminal region a promoter controlling the 7.2-kb polycistronic primase operon. Comparative sequence analysis of the closely related IncP plasmid R751 revealed a similarity of 74% at the nucleotide sequence level, indicating that RP4 and R751 have evolved from a common ancestor. The gene organization of relaxase- and leader operons is conserved among the two IncP plasmids. The transfer origins and the genes traJ and traK exhibit greater sequence divergence than the other genes of the corresponding operons. This is conceivable, because traJ and traK are specificity determinants, the products of which can only recognize homologous oriT sequences. Surprisingly, the organization of the IncP relaxase operons resembles that of the virD operon of Agrobacterium tumefaciens plasmid pTiA6 that mediates DNA transfer to plant cells by a process analogous to bacterial conjugation. Furthermore, the IncP TraG proteins and the product of the virD4 gene share extended amino acid sequence similarity, suggesting a functional relationship.
|
576. |
Moszer I,
Glaser P,
Danchin A,
( 1991 ) Multiple IS insertion sequences near the replication terminus in Escherichia coli K-12. PMID : 1665988 : DOI : 10.1016/0300-9084(91)90166-x Abstract >>
In order to assess the feasibility of semi-automatic procedures for large genome sequencing, a fragment of 9.4 kb of Escherichia coli chromosomal DNA isolated at random was sequenced. It was found to map at 30 min on the chromosome map and to harbour two insertion sequences (IS2 and IS30) as well as several putative coding sequences which had no feature in common with known proteins.
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577. |
Hopkins KL,
Batchelor MJ,
Liebana E,
Deheer-Graham AP,
Threlfall EJ,
( 2006 ) Characterisation of CTX-M and AmpC genes in human isolates of Escherichia coli identified between 1995 and 2003 in England and Wales. PMID : 16879949 : DOI : 10.1016/j.ijantimicag.2006.03.027 Abstract >>
CTX-M and AmpC genes in human isolates of Escherichia coli, their genetic environment and their host plasmids were examined. Isolates (n=103) were selected based on resistance (minimum inhibitory concentration (MIC)> or =1 microg/mL) to ceftriaxone and cefotaxime. Polymerase chain reaction (PCR) and sequencing identified 29 isolates containing bla(CTX-M-15), 1 each of bla(CTX-M-2) (a strain originating from Israel) and bla(CTX-M-40), 20 isolates containing bla(CMY-7), 4 bla(CMY-2) and 1 bla(CMY-21). This is the first study of plasmid-mediated AmpC genes in E. coli in the UK. Eleven cefoxitin-resistant, AmpC PCR-negative isolates had ampC promoter region mutations. All bla(CTX-M-15) and 24 of 25 bla(CMY) genes were associated with an ISEcp1-like element. The bla(CTX-M-2) was located in an orf513-bearing class 1 integron. Plasmid restriction digests suggest transfer of genes between different plasmid backbones.
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578. |
Grinberg I,
Shteinberg T,
Gorovitz B,
Aharonowitz Y,
Cohen G,
Borovok I,
( 2006 ) The Streptomyces NrdR transcriptional regulator is a Zn ribbon/ATP cone protein that binds to the promoter regions of class Ia and class II ribonucleotide reductase operons. PMID : 16950922 : DOI : 10.1128/JB.00903-06 PMC : PMC1636249 Abstract >>
Ribonucleotide reductases (RNRs) catalyze the conversion of ribonucleotides to deoxyribonucleotides and are essential for de novo DNA synthesis and repair. Streptomyces spp. contain genes coding for two RNRs, either of which is sufficient for vegetative growth. The class Ia RNR is encoded by the nrdAB genes, and the class II RNR is encoded by nrdJ, which is coexpressed with nrdR. We previously showed that the Streptomyces coelicolor nrdR gene encodes a protein, NrdR, which represses transcription of both sets of RNR genes. NrdR is a member of a highly conserved family of proteins that is confined exclusively to prokaryotes. In this report, we describe a physical and biochemical characterization of the S. coelicolor NrdR protein and show that it is a zinc-ATP/dATP-containing protein that binds to the promoter regions of both Streptomyces RNR operons. The NrdR N terminus contains a zinc ribbon motif that is necessary for binding to the upstream regulatory region of both RNR operons. The latter contains two 16-bp direct repeat sequences, termed NrdR boxes, which are located proximal to, or overlap with, the promoter regions. These experiments support the view that NrdR controls the transcription of RNR genes by binding to the NrdR box sequences. We also show that the central NrdR ATP cone domain binds ATP and dATP and that mutations that abolish ATP/dATP binding significantly reduce DNA binding, suggesting that the ATP cone domain may allosterically regulate NrdR binding. We conclude that NrdR is a widely conserved regulator of RNR genes, binding to specific sequence elements in the promoter region and thereby modulating transcription.
|
579. |
Yi W,
Yao Q,
Zhang Y,
Motari E,
Lin S,
Wang PG,
( 2006 ) The wbnH gene of Escherichia coli O86:H2 encodes an alpha-1,3-N-acetylgalactosaminyl transferase involved in the O-repeating unit biosynthesis. PMID : 16630548 : DOI : 10.1016/j.bbrc.2006.03.181 Abstract >>
O-repeating unit biosynthesis is the first committed step in lipopolysaccharide (LPS) biosynthesis in a variety of gram-negative bacteria. The wbnH gene was previously proposed to encode a glycosyltransferase involved in O-repeating unit synthesis in Escherichia coli O86:H2 strain. In this work, we provide biochemical evidence to show that wbnH encodes a N-acetylgalactosaminyl transferase (GalNAcT) that catalyzes the transfer of GalNAc from UDP-GalNAc to the GalNAc-pyrophosphate-lipid acceptor. WbnH activity was characterized using a synthetic acceptor substrate GalNAc alpha-PP-O(CH2)11-OPh. The resulting disaccharide product GalNAc-alpha-1,3-GalNAc alpha-PP-O(CH2)11-OPh was analyzed by LC-MS and NMR spectroscopy. Substrate specificity study indicates that pyrophosphate and hydrophobic lipid moiety are structural requirements for WbnH activity. Divalent metal cations are not required for enzyme catalysis, suggesting WbnH belongs to glycosyltransferase GT-B superfamily. Our results complete the characterization of O86 O-unit assembly pathway, and provide the access of chemically defined O-unit substrates for the further investigation of O-antigen biosynthetic mechanism.
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580. |
Brinkley C,
Burland V,
Keller R,
Rose DJ,
Boutin AT,
Klink SA,
Blattner FR,
Kaper JB,
( 2006 ) Nucleotide sequence analysis of the enteropathogenic Escherichia coli adherence factor plasmid pMAR7. PMID : 16926437 : DOI : 10.1128/IAI.01840-05 PMC : PMC1594828 Abstract >>
The complete nucleotide sequence was determined for pMAR7, an enteropathogenic Escherichia coli (EPEC) adherence factor (EAF) plasmid that contains genes encoding a type IV attachment pilus (Bfp) and the global virulence regulator per. Prototypic EAF plasmid pMAR7 is self-transmissible, unlike the smaller EAF plasmid pB171, which has no genes encoding conjugative functions. The tra locus, a highly conserved 33-kb segment found in pMAR7, is similar to the tra (conjugation) region of the F plasmid. ISEc13 copies flanking the pMAR7 tra region could potentially mobilize or delete the tra genes. Hybridization of 134 EPEC strains showed that a complete tra region is present only in strains of the EPEC1 clonal group. This study confirms EPEC's potential for dissemination of virulence attributes by horizontal transfer of the EAF plasmid.
|
581. |
Larsen NA,
Lin H,
Wei R,
Fischbach MA,
Walsh CT,
( 2006 ) Structural characterization of enterobactin hydrolase IroE. PMID : 16922493 : DOI : 10.1021/bi060950i Abstract >>
The proliferation of many pathogenic bacteria is limited by the scarcity of soluble iron in their environment. Many of these bacteria scavenge iron by synthesizing and exporting small molecule siderophores that chelate iron. Iron-bound siderophores are subsequently imported for metabolic processing. Three related serine hydrolases have been characterized biochemically in this pathway: Fes, IroD, and IroE. Here, we report the crystal structure of IroE from uropathogenic Escherichia coli CFT073. The native structure and a complex with diisopropyl fluorophosphonate (DFP, a potent serine hydrolase inhibitor) were determined at 2.3 and 1.4 A resolution, respectively. IroE has the typical alpha/beta-hydrolase fold with an atypical catalytic dyad composed of Ser 189 and His 287. Mutation of either residue was detrimental to catalysis. In addition, rather than the typical oxyanion hole composed of backbone amides, IroE employs the atypical guanidinium moiety of Arg 130. Asp 90 anchors Arg 130 in the active site, and mutation of either residue was likewise detrimental to catalysis. We also compare the structure of IroE to the structure of Fes from Shigella flexneri (PDB entry 2B20). Both enzymes have similar active sites, but Fes has an additional amino-terminal lid domain. These lid domains are proposed to confer specificity to these related hydrolases.
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582. |
Levitz R,
Chapman D,
Amitsur M,
Green R,
Snyder L,
Kaufmann G,
( 1990 ) The optional E. coli prr locus encodes a latent form of phage T4-induced anticodon nuclease. PMID : 1691706 : PMC : PMC551823 Abstract >>
The optional Escherichia coli prr locus restricts phage T4 mutants lacking polynucleotide kinase or RNA ligase. Underlying this restriction is the specific manifestation of the T4-induced anticodon nuclease, an enzyme which triggers the cleavage-ligation of the host tRNALys. We report here the molecular cloning, nucleotide sequence and mutational analysis of prr-associated DNA. The results indicate that prr encodes a latent form of anticodon nuclease consisting of a core enzyme and cognate masking agents. They suggest that the T4-encoded factors of anticodon nuclease counteract the prr-encoded masking agents, thus activating the latent enzyme. The encoding of a tRNA cleavage-ligation pathway by two separate genetic systems which cohabitate E. coli may provide a clue to the evolution of RNA splicing mechanisms mediated by proteins.
|
583. |
Robicsek A,
Strahilevitz J,
Sahm DF,
Jacoby GA,
Hooper DC,
( 2006 ) qnr prevalence in ceftazidime-resistant Enterobacteriaceae isolates from the United States. PMID : 16870791 : DOI : 10.1128/AAC.01647-05 PMC : PMC1538681 Abstract >>
We screened 313 ceftazidime-resistant Enterobacteriaceae isolates obtained in the United States from 1999 to 2004 for all three known qnr genes. A qnr gene was present in 20% of Klebsiella pneumoniae isolates, 31% of Enterobacter sp. isolates, and 4% of Escherichia coli isolates. qnrA and qnrB occurred with equivalent frequencies and, except for qnrB in enterobacters, were stable over time. qnrS was absent.
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584. |
Westerlund B,
van Die I,
Kramer C,
Kuusela P,
Holthöfer H,
Tarkkanen AM,
Virkola R,
Riegman N,
Bergmans H,
Hoekstra W,
( 1991 ) Multifunctional nature of P fimbriae of uropathogenic Escherichia coli: mutations in fsoE and fsoF influence fimbrial binding to renal tubuli and immobilized fibronectin. PMID : 1687325 : DOI : 10.1111/j.1365-2958.1991.tb01856.x Abstract >>
P fimbriae of the F7(1) serotype of Escherichia coli are composed of a major subunit, FsoA, and of three minor proteins named FsoG, FsoE, and FsoF. FsoG is the Gal alpha(1-4)Gal-specific lectin. We assessed mutated recombinant strains each deficient in one fimbrial component for adhesion to frozen sections of rat cortical kidney and to fibronectin immobilized on glass. Rat kidney lacks the Gal alpha(1-4)Gal-containing glycolipids. The fsoG mutant strain was as adhesive to sections of rat kidney and to fibronectin-coated glass as was the recombinant strain expressing the complete fso gene cluster. The fsoA mutant strain was highly adhesive to fibronectin and to kidney sections. In the rat kidney, the adhesion of these strains was predominantly localized to sites of basolateral membranes of tubuli. The fsoE and the fsoF mutant strains were slightly less adhesive to kidney structures and failed to adhere to fibronectin. The fsoE, fsoF double mutant strain adhered neither to fibronectin nor to kidney sections. None of the fso recombinant strains reacted with soluble fibronectin, suggesting that the interaction is dependent on the conformation of the fibronectin molecules. Recombinant strains expressing the F7(2), F8, F11, F13, and F14 serovariants of the P fimbria also showed adherence to immobilized fibronectin. The results show that in addition to binding to globoseries of glycolipids via the G protein, the P fimbriae of uropathogenic E. coli exhibit a tissue-binding property influenced by fsoE and fsoF gene products and with affinity for basolateral membranes and fibronectin.
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585. |
Arciszewska LK,
McKown RL,
Craig NL,
( 1991 ) Purification of TnsB, a transposition protein that binds to the ends of Tn7. PMID : 1657979 : Abstract >>
We have purified TnsB, a transposition protein encoded by the bacterial transposon Tn7. The purification procedure involves three chromatographic steps (DNA-cellulose, norleucine-Sepharose, and phosphocellulose) and yields milligram quantities of highly purified protein. The apparent molecular mass of denatured TnsB protein is approximately 85 kDa. Gel filtration chromatography and sucrose gradient sedimentation studies indicate that in solution, native TnsB is a monomer of nonspherical shape. Using DNase I protection analysis, we established that TnsB is a sequence-specific DNA-binding protein that recognizes multiple sites in both ends of the transposon. The TnsB binding sites, three in the left end of Tn7 and four in the right end, are highly related in nucleotide sequence and are located in DNA segments that we have previously shown contain cis-acting sequences important for Tn7 transposition. Our results also show that one of the TnsB binding sites overlaps a proposed promoter for the transposition genes of Tn7. These studies suggest that the specific binding of TnsB to the ends of Tn7 mediates recombination and may also regulate the expression of Tn7-encoded transposition genes.
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586. |
Metlitskaya A,
Kazakov T,
Kommer A,
Pavlova O,
Praetorius-Ibba M,
Ibba M,
Krasheninnikov I,
Kolb V,
Khmel I,
Severinov K,
( 2006 ) Aspartyl-tRNA synthetase is the target of peptide nucleotide antibiotic Microcin C. PMID : 16574659 : DOI : 10.1074/jbc.M513174200 Abstract >>
Microcin C is a ribosome-synthesized heptapeptide that contains a modified adenosine monophosphate covalently attached to the C-terminal aspartate. Microcin C is a potent inhibitor of bacterial cell growth. Based on the in vivo kinetics of inhibition of macromolecular synthesis, Microcin C targets translation, through a mechanism that remained undefined. Here, we show that Microcin C is a subject of specific degradation inside the sensitive cell. The product of degradation, a modified aspartyl-adenylate containing an N-acylphosphoramidate linkage, strongly inhibits translation by blocking the function of aspartyl-tRNA synthetase.
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587. |
Siddiqui RA,
Hoischen C,
Holst O,
Heinze I,
Schlott B,
Gumpert J,
Diekmann S,
Grosse F,
Platzer M,
( 2006 ) The analysis of cell division and cell wall synthesis genes reveals mutationally inactivated ftsQ and mraY in a protoplast-type L-form of Escherichia coli. PMID : 16640589 : DOI : 10.1111/j.1574-6968.2006.00237.x Abstract >>
Cell division and cell wall synthesis are tightly linked cellular processes for bacterial growth. A protoplast-type L-form Escherichia coli, strain LW1655F+, indicated that bacteria can divide without assembling a cell wall. However, the molecular basis of its phenotype remained unknown. To establish a first phenotype-genotype correlation, we analyzed its dcw locus, and other genes involved in division of E. coli. The analysis revealed defective ftsQ and mraY genes, truncated by a nonsense and a frame-shift mutation, respectively. Missense mutations were determined in the ftsA and ftsW products yielding amino-acid replacements at conserved positions. FtsQ and MraY, obviously nonfunctional in the L-form, are essential for cell division and cell wall synthesis, respectively, in all bacteria with a peptidoglycan-based cell wall. LW1655F+ is able to survive their loss-of-functions. This points to compensatory mechanisms for cell division in the absence of murein sacculus formation. Hence, this L-form represents an interesting model to investigate the plasticity of cell division in E. coli, and to demonstrate how concepts fundamental for bacterial life can be bypassed.
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588. |
Bhardwaj R,
Majumdar S,
Ganguly NK,
Taneja N,
Dutta S,
Ramamurthy T,
Chakraborti A,
( 2006 ) Characterization of adhesin variants in Indian isolates of enteroaggregative Escherichia coli. PMID : 16640585 : DOI : 10.1111/j.1574-6968.2006.00231.x Abstract >>
Enteroaggregative Escherichia coli (EAEC) are causative agents of diarrhea, being characterized by aggregative adherence to cultured epithelial cells. In this study, phenotypic properties of EAEC were analyzed with respect to AA, hemagglutination, clump and biofilm formation, all of which are mediated by aggregative adherence fimbriae (AAF). The strains were also screened for AAF types, AAF adhesin variants and Dr adhesin by PCR. Of the three known AAF types, AAF/I and AAF/II adhesin variants were identified. An association between the AAF/adhesin genotypes and the subtypes/scores of phenotypic properties was sought and it was observed that strains harboring same adhesins displayed different subtypes/scores and vice versa.
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589. |
Blanco M,
Blanco JE,
Dahbi G,
Mora A,
Alonso MP,
Varela G,
Gadea MP,
Schelotto F,
González EA,
Blanco J,
( 2006 ) Typing of intimin (eae) genes from enteropathogenic Escherichia coli (EPEC) isolated from children with diarrhoea in Montevideo, Uruguay: identification of two novel intimin variants (muB and xiR/beta2B). PMID : 16914645 : DOI : 10.1099/jmm.0.46518-0 Abstract >>
A total of 71 enteropathogenic Escherichia coli (EPEC) strains isolated from children with diarrhoea in Montevideo, Uruguay, were characterized in this study. PCR showed that 57 isolates carried eae and bfp genes (typical EPEC strains), and 14 possessed only the eae gene (atypical EPEC strains). These EPEC strains belonged to 21 O : H serotypes, including eight novel serotypes not previously reported among human EPEC in other studies. However, 72% belonged to only four serotypes: O55:H- (six strains), O111:H2 (13 strains), O111:H- (14 strains) and O119:H6 (18 strains). Nine intimin types, namely, alpha1 (two O142 strains), beta1 (29 strains, including 13 O111:H2 and 14 O111:H-), gamma1 (three O55:H- strains), theta (five strains, including three strains with H40 antigen), kappa (two strains), epsilon1 (one strain), lambda (one strain), muB (six strains of serotypes O55:H51 and O55:H-) and xiR/beta2B (22 strains, including 18 O119:H6) were detected among the 71 EPEC strains. The authors have identified two novel intimin genes (muB and xiR/beta2B) in typical EPEC strains of serotypes O55:H51/H- and O119:H6/H-. The complete nucleotide sequences of the novel muB and xiR/beta2 variant genes were determined. PFGE typing after XbaI DNA digestion was performed on 44 representative EPEC strains. Genomic DNA fingerprinting revealed 44 distinct restriction patterns and the strains were clustered in 12 groups. Only 15 strains clustered in six groups of closely related (similarity>85%) PFGE patterns, suggesting the prevailing clonal diversity among EPEC strains isolated from children with diarrhoea in Montevideo.
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590. |
Escher A,
O'Kane DJ,
Szalay AA,
( 1991 ) The beta subunit polypeptide of Vibrio harveyi luciferase determines light emission at 42 degrees C. PMID : 1685011 : DOI : 10.1007/bf00280295 Abstract >>
The nucleotide sequence of the luxA and luxB genes encoding the alpha beta heterodimeric luciferase from thermotolerant Vibrio harveyi CTP5 was determined. The DNA sequence of the CTP5 luxA and luxB genes is identical to the DNA sequence of the luxA and luxB genes from mesophilic V. harveyi MAV (B 392), with minor exceptions. The sequence differences result in 5 amino acid substitutions in the alpha subunit polypeptide and 7 amino acid substitutions in the beta subunit polypeptide. Escherichia coli cells grown on solid medium and expressing CTP5 or MAV luxAB genes emit similar amounts of light at 37 degrees C, while at 42 degrees C cells containing CTP5 luxAB genes show more than tenfold increased bioluminescence compared to cells with MAV luxAB genes. When grown in liquid medium E. coli cells with CTP5 or MAV luxAB genes emit equivalent amounts of light at 37 degrees C; however, in liquid medium at 42 degrees C cells containing CTP5 luxAB genes show only three times higher bioluminescence than cells with MAV luxAB genes. Expression of T7 promoter-linked hybrid luxAB transcriptional units luxACTP5-luxBMAV and luxAMAV-luxBCTP5 in E. coli reveals that (i) the MAV luxB gene product is responsible for the decreased activity of MAV luciferase at 42 degrees C; (ii) the CTP5 luxB gene encodes the information required for most of the increased activity of CTP5 luciferase relative to MAV luciferase at 42 degrees C; and (iii) E. coli cells containing MAV luxB gene show an increase in bioluminescence when grown in liquid medium at 42 degrees C, which coincides with elevated GroEL chaperonin levels.(ABSTRACT TRUNCATED AT 250 WORDS)
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591. |
Petersen A,
Christensen JP,
Kuhnert P,
Bisgaard M,
Olsen JE,
( 2006 ) Vertical transmission of a fluoroquinolone-resistant Escherichia coli within an integrated broiler operation. PMID : 16650662 : DOI : 10.1016/j.vetmic.2006.03.015 Abstract >>
The epidemiology of an enrofloxacin-resistant Escherichia coli clone was investigated during two separate outbreaks of colibacillosis in the Danish broiler production. In total five flocks were reported affected by the outbreaks. Recorded first-week mortalities were in the range of 1.7-12.7%. The clone was first isolated from dead broilers and subsequently demonstrated in samples from associated hatchers and the parent flock with its embryonated eggs, suggesting a vertical transmission from the parents. The second outbreak involved two broiler flocks unrelated to the affected flocks from the first outbreak. However, the clone could not be demonstrated in the associated parent flock. Furthermore, samplings from grand-parent flocks were negative for the outbreak clone. The clonality was evaluated by plasmid profiling and pulsed-field gel electrophoresis. None of the recognized virulence factors were demonstrated in the outbreak clone by microarray and PCR assay. The molecular background for the fluoroquinolone-resistance was investigated and point mutations in gyrA and parC leading to amino-acid substitutions in quinolone-resistance determining regions of GyrA and ParC were demonstrated. Vertical transmission of enrofloxacin-resistant E. coli from healthy parents resulting in high first-week mortality in the offspring illustrates the potential of the emergence and spreading of fluoroquinolone-resistant bacteria in animal husbandry, even though the use of fluoroquinolones is restricted.
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592. |
Chen Q,
Savarino SJ,
Venkatesan MM,
( 2006 ) Subtractive hybridization and optical mapping of the enterotoxigenic Escherichia coli H10407 chromosome: isolation of unique sequences and demonstration of significant similarity to the chromosome of E. coli K-12. PMID : 16549668 : DOI : 10.1099/mic.0.28648-0 Abstract >>
Enterotoxigenic Escherichia coli (ETEC) is a primary cause of diarrhoea in infants in developing countries and in travellers to endemic regions. While several virulence genes have been identified on ETEC plasmids, little is known about the ETEC chromosome, although it is expected to share significant homology in backbone sequences with E. coli K-12. In the absence of genomic sequence information, the subtractive hybridization method and the more recently described optical mapping technique were carried out to determine the degree of genomic variation between virulent ETEC strain H10407 and the non-pathogenic E. coli K-12 strain MG1655. In one round of PCR-based suppression subtractive hybridization, 153 fragments representing sequences unique to strain H10407 were identified. blast searches indicated that few unique sequences showed homology to known pathogenicity island genes identified in related E. coli pathogens. A total of 65 fragments contained sequences that were either linked to hypothetical proteins or showed no homology to any known sequence in the database. The remaining sequences were either phage or prophage related or displayed homology to classifiable genes that function in various aspects of bacterial metabolism. The 153 unique sequences showed variable distribution across different ETEC strains including ETEC strain B7A, which is attenuated in virulence and lacked several H10407-specific sequences. Restriction-enzyme-based optical maps of strain H10407 were compared to in silico restriction maps of strain MG1655 and related E. coli pathogens. The 5.1 Mb ETEC chromosome was approximately 500 kb greater in length than the chromosome of E. coli K-12, collinear with it and indicated several discrete regions where insertions and/or deletions had occurred relative to the chromosome of strain MG1655. No major inversions, transpositions or gross rearrangements were observed on the ETEC chromosome. Based on comparisons with known genomic sequences and related optical-map-based restriction site similarity, the sequence of the H10407 chromosome is expected to demonstrate approximately 96 % identity with that of E. coli K-12.
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593. |
Cursino L,
Smajs D,
Smarda J,
Nardi RM,
Nicoli JR,
Chartone-Souza E,
Nascimento AM,
( 2006 ) Exoproducts of the Escherichia coli strain H22 inhibiting some enteric pathogens both in vitro and in vivo. PMID : 16553738 : DOI : 10.1111/j.1365-2672.2006.02834.x Abstract >>
The antagonistic activity of the Escherichia coli strain H22 against enteric bacteria was studied both in vitro and in vivo. In vitro, bacterial strains belonging to seven of nine genera of the family Enterobacteriaceae (Enterobacter, Escherichia, Klebsiella, Morganella, Salmonella, Shigella and Yersinia) were inhibited by the strain H22. Six days after simultaneous oral inoculation in germ-free mice, E. coli strain H22 reduced the faecal population of Shigella flexneri 4 to undetectable levels (P < 0.05). In ex vivo assay, inhibitory zones against Sh. flexneri 4 were observed around faecal samples from mice inoculated with E. coli strain H22. The in vitro inhibition of Sh. flexneri 4 was shown to be mediated by microcin C7. In addition to microcin C7, strain H22 was shown to produce aerobactin, new variants of colicins E1 and Ib, and bacteriophage particles with morphology similar to the phages of the family Myoviridae. Altogether, the properties of E. coli H22, observed both under in vitro and in vivo conditions, suggest its potential use as a probiotic strain for livestock and humans. The strain H22 was shown to produce several antimicrobial compounds with inhibitory capabilities against pathogenic or potentially pathogenic enterobacteria.
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594. |
Zhu H,
Pan RJ,
Wang TW,
Shen YL,
Wei DZ,
( 2006 ) Functional solubilization of aggregation-prone TRAIL protein facilitated by coexpressing with protein isoaspartate methyltranferase. PMID : 16575568 : DOI : 10.1007/s00253-006-0383-9 Abstract >>
TRAIL was a tumor-specific protein in development as a novel anticancer therapeutic agent. Generally, when expressed in recombinant Escherichia coli, TRAIL protein was prone to form inclusion bodies. In this study, coexpression of human TRAIL protein and protein isoaspartate methyltranferase (PIMT) from E. coli on plasmid pBV-TRAIL-PCM in E. coli C600 was investigated to overcome the difficulties in soluble expression. The results showed that this PIMT coexpression strategy exerted a positive effect on the TRAIL protein expression in recombinant E. coli, which led to a mean increase in the intracellular concentration of soluble and total protein of TRAIL by 1.57-fold and 1.33-fold, respectively. At the same time, results also suggested that PIMT was a prospective partner for soluble expression of TRAIL protein.
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595. |
Lacher DW,
Steinsland H,
Whittam TS,
( 2006 ) Allelic subtyping of the intimin locus (eae) of pathogenic Escherichia coli by fluorescent RFLP. PMID : 16842363 : DOI : 10.1111/j.1574-6968.2006.00328.x Abstract >>
Intimin is a highly polymorphic protein encoded by the eae gene and plays a crucial role in the attaching-effacing phenotype of diarrheagenic Escherichia coli and related pathogens. We have developed a method to quickly and accurately uncover allelic variation at the eae locus through the use of fluorescent RFLP (fRFLP). Application of fRFLP to 151 eae-positive strains (including the newly described Escherichia albertii) revealed 26 different fRFLP types that correspond to 20 of the 28 previously described eae alleles. Two sequence variants of the gamma, iota, kappa, and zeta alleles and three variants of epsilon were also observed. In addition to being reliable and accurate, the method can be easily adapted to accommodate new eae allelic sequences, as they become known.
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596. |
Nougayrède JP,
Homburg S,
Taieb F,
Boury M,
Brzuszkiewicz E,
Gottschalk G,
Buchrieser C,
Hacker J,
Dobrindt U,
Oswald E,
( 2006 ) Escherichia coli induces DNA double-strand breaks in eukaryotic cells. PMID : 16902142 : DOI : 10.1126/science.1127059 Abstract >>
Transient infection of eukaryotic cells with commensal and extraintestinal pathogenic Escherichia coli of phylogenetic group B2 blocks mitosis and induces megalocytosis. This trait is linked to a widely spread genomic island that encodes giant modular nonribosomal peptide and polyketide synthases. Contact with E. coli expressing this gene cluster causes DNA double-strand breaks and activation of the DNA damage checkpoint pathway, leading to cell cycle arrest and eventually to cell death. Discovery of hybrid peptide-polyketide genotoxins in E. coli will change our view on pathogenesis and commensalism and open new biotechnological applications.
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597. |
de Haan LA,
Willshaw GA,
van der Zeijst BA,
Gaastra W,
( 1991 ) The nucleotide sequence of a regulatory gene present on a plasmid in an enterotoxigenic Escherichia coli strain of serotype O167:H5. PMID : 1685133 : DOI : 10.1016/0378-1097(91)90499-z Abstract >>
A DNA fragment that can functionally substitute for cfaD, the positive regulatory gene involved in expression of CFA/I fimbriae, has recently been cloned from an Escherichia coli strain of serotype O167:H5 that produces CS5 fimbriae. Nucleotide sequence determination showed that the fragment contained a gene, csvR (Coli Surface Virulence factor Regulator) homologous to the cfaD gene, which encoded for a protein of 301 amino acid residues. The csvR gene was found to be located between two different insertion sequences. Comparison of the amino acid sequence of the CsvR and CfaD proteins showed that CsvR is 34 amino acid residues longer at the C-terminus and, in the sequence, it also contains an insertion of two amino acid residues. The similarity between CfaD and Rns, the positive regulator of CS1 and CS2 expression, is much higher (97%) than between CsvR and CfaD (87%). This is reflected by the fact that the level of expression of CFA/I fimbriae induced by CsvR is not as high as when expression is induced by CfaD or Rns.
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598. |
Jansson C,
Sköld O,
( 1991 ) Appearance of a new trimethoprim resistance gene, dhfrIX, in Escherichia coli from swine. PMID : 1659308 : DOI : 10.1128/aac.35.9.1891 PMC : PMC245287 Abstract >>
A new gene, dhfrIX, coding for a trimethoprim-resistant dihydrofolate reductase (DHFR), was found in porcine isolates of Escherichia coli. The new enzyme, DHFR IX, containing 178 amino acids, showed an amino acid similarity of about 26% with DHFR I and the chromosomal DHFR of E. coli K-12. The dhfrIX gene was observed to occur on two distinctly different transferable plasmids, although a fragment of about 2.9 kb, including dhfrIX, had an identical restriction enzyme digestion map in each case. The new plasmid-borne dhfrIX gene mediates resistance to a drug level of only about 250 micrograms/ml, as compared with more than 1,000 micrograms/ml for the more frequently encountered dhfrI gene. The new plasmid-borne trimethoprim resistance gene could have been selected and spread as a consequence of the extensive use of trimethoprim in veterinary practice in Sweden. It will be important to try to follow its possible occurrence in human pathogens as well.
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599. |
Nógrády N,
Pászti J,
Pikó H,
Nagy B,
( 2006 ) Class 1 integrons and their conjugal transfer with and without virulence-associated genes in extra-intestinal and intestinal Escherichia coli of poultry. PMID : 16854651 : DOI : 10.1080/03079450600827007 Abstract >>
Multiple-drug resistance in enteric bacteria is frequently associated with integrons. To determine whether integrons may play a role in avian pathogenic Escherichia coli, isolates of extra-intestinal (n = 27) and intestinal (n = 40) E. coli from dead chicks and turkey poults were analysed for the presence of class 1 integrons and of the virulence-associated genes iss, tsh and colV. Eleven extra-intestinal strains possessed a 1.0 kb class 1 integron with a variable region of aadA1 and were resistant to tetracycline. These traits were indicative of the presence of the Tn21 transposon, which was confirmed by polymerase chain reaction. All extra-intestinal strains had the colV, iss and tsh genes, but none of these genes were cotransferred with the 1.0 kb integron when conjugal transferability was tested. The integron content of the intestinal strains showed considerable variability: one or two of four different class 1 integrons, which varied from 1.0 to 2.4 kb in size, were detected in the 11 strains. The aadA7 gene of the 1.0 kb integron, the dfrA1-aadA1 genes of the 1.6 kb integron and the folA-catB3-aadA5 genes of the 2.4 kb integron were identical to those described by other workers. However, the orfIN682-dhfrV-orfD gene cassette arrangement of the 1.5 kb integron of an intestinal strain of serogroup O5 had no similarity to any previously reported integrons. Conjugal transfer of the 1.6 and 2.4 kb integrons was successful, and in a serogroup O33 intestinal E. coli strain the iss gene was apparently cotransferred with a 1.6 kb integron. The 1.0 and 1.5 kb integrons were not transferable. Our data suggest that intestinal E. coli strains of poultry may be a reservoir for emerging multiresistant strains of avian pathogenic E. coli.
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600. |
Vögele K,
Schwartz E,
Welz C,
Schiltz E,
Rak B,
( 1991 ) High-level ribosomal frameshifting directs the synthesis of IS150 gene products. PMID : 1653413 : DOI : 10.1093/nar/19.16.4377 PMC : PMC328623 Abstract >>
IS150 contains two tandem, out-of-phase, overlapping genes, ins150A and ins150B, which are controlled by the same promoter. These genes encode proteins of 19 and 31 kD, respectively. A third protein of 49 kD is a transframe gene product consisting of domains encoded by both genes. Specific -1 ribosomal frameshifting is responsible for the synthesis of the large protein. Expression of ins150B also involves frameshifting. The IS150 frameshifting signals operate with a remarkably high efficiency, causing about one third of the ribosomes to switch frame. All of the signals required for this process are encoded in a 83-bp segment of the element. The heptanucleotide A AAA AAG and a potential stem-loop-forming sequence mark the frameshifting site. Similar sequence elements are found in -1 frameshifting regions of bacterial and retroviral genes. A mutation within the stem-loop sequence reduces the rate of frameshifting by about 80%. Artificial transposons carrying this mutation transpose at a normal frequency, but form cointegrates at a approximately 100-fold reduced rate.
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601. |
Bielaszewska M,
Prager R,
Zhang W,
Friedrich AW,
Mellmann A,
Tschäpe H,
Karch H,
( 2006 ) Chromosomal dynamism in progeny of outbreak-related sorbitol-fermenting enterohemorrhagic Escherichia coli O157:NM. PMID : 16517637 : DOI : 10.1128/AEM.72.3.1900-1909.2006 PMC : PMC1393231 Abstract >>
Sorbitol-fermenting (SF) enterohemorrhagic Escherichia coli (EHEC) O157:NM (nonmotile) is a unique clone that causes outbreaks of hemorrhagic colitis and hemolytic-uremic syndrome. In well-defined clusters of cases, we have observed significant variability in pulsed-field gel electrophoresis (PFGE) patterns which could indicate coinfection by different strains. An analysis of randomly selected progeny colonies of an outbreak strain after subcultivation demonstrated that they displayed either the cognate PFGE outbreak pattern or one of four additional patterns and were <89% similar. These profound alterations were associated with changes in the genomic position of one of two Shiga toxin 2-encoding genes (stx2) in the outbreak strain or with the loss of this gene. The two stx2 alleles in the outbreak strain were identical but were flanked with phage-related sequences with only 77% sequence identity. Neither of these phages produced plaques, but one lysogenized E. coli K-12 and integrated in yecE in the lysogens and the wild-type strain. The presence of two stx2 genes which correlated with increased production of Stx2 in vitro but not with the clinical outcome of infection was also found in 14 (21%) of 67 SF EHEC O157:NM isolates from sporadic cases of human disease. The variability of PFGE patterns for the progeny of a single colony must be considered when interpreting PFGE patterns in SF EHEC O157-associated outbreaks.
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602. |
Geue L,
Selhorst T,
Schnick C,
Mintel B,
Conraths FJ,
( 2006 ) Analysis of the clonal relationship of shiga toxin-producing Escherichia coli serogroup O165:H25 isolated from cattle. PMID : 16517683 : DOI : 10.1128/AEM.72.3.2254-2259.2006 PMC : PMC1393171 Abstract >>
Variations in time and space of a clonal group of Escherichia coli O165:H25 on a cattle farm were monitored. The virulence marker pattern (stx genes, eae gene, hly(EHEC) gene, katP gene, espP gene, efa gene) suggests that E. coli O165:H25 of bovine origin may represent a risk for human infection.
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603. |
Guo H,
Kong Q,
Cheng J,
Wang L,
Feng L,
( 2005 ) Characterization of the Escherichia coli O59 and O155 O-antigen gene clusters: the atypical wzx genes are evolutionary related. PMID : 15990253 : DOI : 10.1016/j.femsle.2005.05.036 Abstract >>
O-antigens are highly polymorphic. The genes specifically involved in O-antigen synthesis are generally grouped together on the chromosome as a gene cluster. In Escherichia coli, the O-antigen gene clusters are characteristically located between the housekeeping genes galF and gnd. In this study, the O-antigen gene clusters of E. coli O59 and E. coli O155 were sequenced. The former was found to contain genes for GDP-mannose synthesis, glycosyltransferase genes and the O-antigen polymerase gene (wzy), while the latter contained only glycosyltransferase genes and wzy. O unit flippase genes (wzx) were found immediately downstream of the gnd gene, in the region between the gnd and hisI genes in these two strains. This atypical location of wzx has not been reported before, and furthermore these two genes complemented in trans despite the fact that different O-antigen structures are present in E. coli O59 and O155. A putative acetyltransferase gene was found downstream of wzx in both strains. Comparison of the region between gnd and hisI revealed that the wzx and acetyltransferase genes are closely related between E. coli O59 and O155, indicating that the two gene clusters arose recently from a common ancestor. This work provides further evidence for the O-antigen gene cluster having formed gradually, and selection pressure will eventually bring O-antigen genes into a single cluster. Genes specific for E. coli O59 and O155, respectively, were also identified.
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604. |
Sabri M,
Léveillé S,
Dozois CM,
( 2006 ) A SitABCD homologue from an avian pathogenic Escherichia coli strain mediates transport of iron and manganese and resistance to hydrogen peroxide. PMID : 16514154 : DOI : 10.1099/mic.0.28682-0 Abstract >>
An operon encoding a member of the family of ATP-binding cassette (ABC) divalent metal ion transporters, homologous to Salmonella enterica SitABCD, has been identified in the avian pathogenic Escherichia coli (APEC) strain chi7122. The sitABCD genes were located on the virulence plasmid pAPEC-1, and were highly similar at the nucleotide level to the chromosomally encoded sitABCD genes present in Shigella spp. A cloned copy of sitABCD conferred increased growth upon a siderophore-deficient E. coli strain grown in nutrient broth supplemented with the chelator 2,2'-dipyridyl. Ion rescue demonstrated that Sit-mediated growth promotion of this strain was due to the transport of iron. SitABCD mediated increased transport of both iron and manganese as demonstrated by uptake of 55Fe, 59Fe or 54Mn in E. coli K-12 strains deficient for the transport of iron (aroB feoB) and manganese (mntH) respectively. Isotope uptake and transport inhibition studies showed that in the iron transport deficient strain, SitABCD demonstrated a greater affinity for iron than for manganese, and SitABCD-mediated transport was higher for ferrous iron, whereas in the manganese transport deficient strain, SitABCD demonstrated greater affinity for manganese than for iron. Introduction of the APEC sitABCD genes into an E. coli K-12 mntH mutant also conferred increased resistance to the bactericidal effects of hydrogen peroxide. APEC strain chi7122 derivatives lacking either a functional SitABCD or a functional MntH transport system were as resistant to hydrogen peroxide as the wild-type strain, whereas a Deltasit DeltamntH double mutant was more sensitive to hydrogen peroxide. Overall, the results demonstrate that in E. coli SitABCD represents a manganese and iron transporter that, in combination with other ion transport systems, may contribute to acquisition of iron and manganese, and resistance to oxidative stress.
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605. |
Strauch E,
Beutin L,
( 2006 ) Imprecise excision of insertion element IS5 from the fliC gene contributes to flagellar diversity in Escherichia coli. PMID : 16499606 : DOI : 10.1111/j.1574-6968.2006.00100.x Abstract >>
Motile strains of Escherichia coli K12 carrying both a chromosomal fliC-H48 gene and a plasmid encoded fliC-H4 gene express both types of flagellins, which are coassembled into functional flagella. By using flagellar-H48-specific antiserum and a plasmid curing procedure, nonmotile mutants were found that carried an IS5 insertion in the chromosomal fliC-H48 gene. Motile revertants were isolated that showed deletions of the IS5 element together with sections of the fliC-H48 gene resulting in an altered flagellar serotype in these strains. As IS5 elements were found associated with 35 of 53 known H-types in wildtype E. coli strains, this insertion element might play a major role in serotype diversity.
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606. |
Yu Y,
Ji S,
Chen Y,
Zhou W,
Wei Z,
Li L,
Ma Y,
( 2007 ) Resistance of strains producing extended-spectrum beta-lactamases and genotype distribution in China. PMID : 16533535 : DOI : 10.1016/j.jinf.2006.01.014 Abstract >>
To investigate the resistance of Escherichia coli and Klebsiella pneumoniae producing extended-spectrum beta-lactamases (ESBLs) and the genotyping of ESBLs in China. MICs of 12 antibiotics against 50 strains (by random selection) of ESBLs-producing E. coli and K. pneumoniae were determined by E-test. The genotypes of ESBLs were analyzed by PCR, DNA sequencing and isoelectric focusing. The susceptibility rate of 50 isolates was 100% in imipenem, 60%-80% in cefoperazone/sulbactam, ceftazidime and piperacillin/tazobactam, and lower in other antimicrobial agents tested. Only 6.0% of the isolates were sensitive to cefotaxime. Four hundred and forty-seven of 509 isolates had been confirmed the genotype of ESBLs. Four hundred and sixteen strains produced only one type of ESBLs, including CTX-M-14 (271 strains), CTX-M-3 (70 strains), CTX-M-24 (35 strains), CTX-M-22 (8 strains), CTX-M-15 (4 strains), CTX-M-9 (4 strains), CTX-M-28 (3 strains), CTX-M-12 (1 strain), CTX-M-13 (1 strain), CTX-M-27 (1 strain), CTX-M-29 (1 strain), SHV-12 (10 strains), SHV-5 (4 strains), SHV-2 (2 strains), and SHV-9 (1 strain). Thirty isolates carried two or three types of ESBLs, and producing CTX-M-14 and CTX-M-3 together were the most common type. The resistance of E. coli and K. pneumonia producing ESBLs in China was a serious issue and CTX-M type ESBLs were the most common genotype. CTX-M-14 was the predominant genotype. Some isolates produced two or three ESBLs.
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607. |
Rea D,
Fülöp V,
Bugg TD,
Roper DI,
( 2005 ) Expression, purification and preliminary crystallographic analysis of 2,4-dihydroxy-hepta-2-ene-1,7-dioate aldolase (HpcH) from Escherichia coli C. PMID : 16511168 : DOI : 10.1107/S1744309105023079 PMC : PMC1978122 Abstract >>
The gene encoding 2,4-dihydroxy-hepta-2-ene-1,7-dioate (HHED) aldolase (HpcH; EC 4.1.2) from Escherichia coli C was cloned into the high-expression plasmid pProEx-HTa and overexpressed in E. coli BL21 (DE3). The 28 kDa enzyme was purified using immobilized metal-affinity and size-exclusion chromatography prior to crystallization. Crystals were obtained by the hanging-drop vapour-diffusion method at 277 K from a number of screening conditions. Type I crystals grown in a solution containing 0.4 M ammonium dihydrogen phosphate belong to space group R32, with unit-cell parameters a = b = 128.92, c = 175.30 A. Type II crystals grown in a solution containing 0.5 M sodium chloride, 0.1 M sodium citrate pH 5.5 belong to space group I222, with unit-cell parameters a = 133.39, b = 155.39, c = 168.80 A. Complete data sets were collected to 1.6 and 2.0 A from type I and type II crystals, respectively, using synchrotron radiation.
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608. |
Chen SL,
Hung CS,
Xu J,
Reigstad CS,
Magrini V,
Sabo A,
Blasiar D,
Bieri T,
Meyer RR,
Ozersky P,
Armstrong JR,
Fulton RS,
Latreille JP,
Spieth J,
Hooton TM,
Mardis ER,
Hultgren SJ,
Gordon JI,
( 2006 ) Identification of genes subject to positive selection in uropathogenic strains of Escherichia coli: a comparative genomics approach. PMID : 16585510 : DOI : 10.1073/pnas.0600938103 PMC : PMC1424661 Abstract >>
Escherichia coli is a model laboratory bacterium, a species that is widely distributed in the environment, as well as a mutualist and pathogen in its human hosts. As such, E. coli represents an attractive organism to study how environment impacts microbial genome structure and function. Uropathogenic E. coli (UPEC) must adapt to life in several microbial communities in the human body, and has a complex life cycle in the bladder when it causes acute or recurrent urinary tract infection (UTI). Several studies designed to identify virulence factors have focused on genes that are uniquely represented in UPEC strains, whereas the role of genes that are common to all E. coli has received much less attention. Here we describe the complete 5,065,741-bp genome sequence of a UPEC strain recovered from a patient with an acute bladder infection and compare it with six other finished E. coli genome sequences. We searched 3,470 ortholog sets for genes that are under positive selection only in UPEC strains. Our maximum likelihood-based analysis yielded 29 genes involved in various aspects of cell surface structure, DNA metabolism, nutrient acquisition, and UTI. These results were validated by resequencing a subset of the 29 genes in a panel of 50 urinary, periurethral, and rectal E. coli isolates from patients with UTI. These studies outline a computational approach that may be broadly applicable for studying strain-specific adaptation and pathogenesis in other bacteria.
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609. |
Dudley EG,
Abe C,
Ghigo JM,
Latour-Lambert P,
Hormazabal JC,
Nataro JP,
( 2006 ) An IncI1 plasmid contributes to the adherence of the atypical enteroaggregative Escherichia coli strain C1096 to cultured cells and abiotic surfaces. PMID : 16552039 : DOI : 10.1128/IAI.74.4.2102-2114.2006 PMC : PMC1418932 Abstract >>
Enteroaggregative Escherichia coli (EAEC) is defined by a characteristic "stacked-brick" aggregative adherence (AA) pattern to cultured cells. In well-studied EAEC prototype strains (called typical EAEC strains), the AA phenotype requires aggregative adherence fimbriae (AAFs). However, previous studies suggest that known AAF alleles are not found in all EAEC strains. To define mechanisms contributing to adherence in an atypical strain, we studied EAEC strain C1096. An E. coli K12 derivative carrying two plasmids, designated pSERB1 and pSERB2, from C1096 adhered to cell lines and exhibited an AA pattern. Nucleotide sequence analysis of pSERB1 indicated that it is related to plasmids of the IncI1 incompatibility group. These plasmids encode genes involved in pilus-mediated conjugal transfer, as well as pilL-V, which encodes a second pilus of the type IV family. Insertional inactivation of the gene predicted to encode the major type IV pilin subunit (pilS) reduced conjugal transfer of the plasmid by 4 orders of magnitude. Adherence of the mutant strain to polystyrene and to HT29 cells was reduced by approximately 21% and 75%, respectively. In a continuous-flow microfermentor, the pilS inactivation reduced mature biofilm formation on a glass slide by approximately 50%. In addition, the simultaneous presence of both pSERB1 and pSERB2 plasmids promoted pilS-independent biofilm formation. We conclude that the IncI1 plasmid of EAEC C1096 encodes a type IV pilus that contributes to plasmid conjugation, epithelial cell adherence, and adherence to abiotic surfaces. We also observe that AA can be mediated by factors distinct from AAF adhesins.
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610. |
Tietze E,
Brevet J,
( 1991 ) The trimethoprim resistance transposon Tn7 contains a cryptic streptothricin resistance gene. PMID : 1656477 : Abstract >>
The transposon Tn7 codes for a trimethoprim resistance and for a streptomycin/spectinomycin resistance function of the bacterial host cells. Cloning of a restriction fragment of Tn7 into the vector plasmid pUC19 reveals the presence in Tn7 of an additional potential resistance determinant. A streptothricin resistance gene, which appears cryptic in the original Tn7 context becomes activated in the recombinant plasmid upon supplying the promoter function of the lacZ system of pUC19. These results together with previously published sequence data further disclose the modular character in the resistance gene regions of Tn7-like transposons.
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611. |
Orle KA,
Craig NL,
( 1991 ) Identification of transposition proteins encoded by the bacterial transposon Tn7. PMID : 1655576 : DOI : 10.1016/0378-1119(91)90478-t Abstract >>
The bacterial transposon, Tn7, encodes an elaborate array of transposition genes, tnsABCDE. We report here the direct identification of the TnsA, TnsB, TnsC and TnsD polypeptides by immunoblotting. Our results demonstrate that the complexity of the protein information devoted to Tn7 transposition is considerable: the aggregate molecular size of the five Tns polypeptides is about 300 kDa. We also report the sequence of the tnsA gene and of the 5' ends of tnsB and tnsD. This analysis reveals that all five tns genes are oriented in the same direction within Tn7.
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612. |
Cheng D,
Sun H,
Xu J,
Gao S,
( 2006 ) PCR detection of virulence factor genes in Escherichia coli isolates from weaned piglets with edema disease and/or diarrhea in China. PMID : 16567064 : DOI : 10.1016/j.vetmic.2006.02.013 Abstract >>
Fimbriae, toxins and pathogenicity islands (PAIs) are main virulence factors of the pathogenic Escherichia coli strains. To investigate into their prevalence in clinical E. coli isolates associated with porcine postweaning diarrhea (PWD) and/or pig edema disease (ED), 240 isolates were obtained from diseased piglets (140 from PWD, 76 from ED and 24 from ED/PWD) and submitted to PCR detection for genes coding for fimbriae, enterotoxins, shiga toxins, intimin and high-molecular-weight protein 2 (HMWP2). Among the 240 isolates detected, detection rates of the genes for F18, F4, intimin, HMWP2, Stx2e, LTa, STa and STb were 26.25%, 3.75%, 28.33%, 16.67%, 35%, 10.83%, 14.58% and 9.17%, respectively, and 67.92% of the isolates could be assigned into 20 different virulence factor patterns. Further more, F18ab+ STEC are the prevalent pathogens of ED, and F18+ and/or intimin+ STEC/ETEC are the dominant pathogens of ED/PWD, while F18ab+, F4+ and/or intimin+ ETEC and HPI+ and/or LEE+ E. coli are more frequently associated with PWD.
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613. |
Boer R,
Russi S,
Guasch A,
Lucas M,
Blanco AG,
Pérez-Luque R,
Coll M,
de la Cruz F,
( 2006 ) Unveiling the molecular mechanism of a conjugative relaxase: The structure of TrwC complexed with a 27-mer DNA comprising the recognition hairpin and the cleavage site. PMID : 16540117 : DOI : 10.1016/j.jmb.2006.02.018 Abstract >>
TrwC is a DNA strand transferase that catalyzes the initial and final stages of conjugative DNA transfer. We have solved the crystal structure of the N-terminal relaxase domain of TrwC in complex with a 27 base-long DNA oligonucleotide that contains both the recognition hairpin and the scissile phosphate. In addition, a series of ternary structures of protein-DNA complexes with different divalent cations at the active site have been solved. Systematic anomalous difference analysis allowed us to determine unambiguously the nature of the metal bound. Zn2+, Ni2+ and Cu2+ were found to bind the histidine-triad metal binding site. Comparison of the structures of the different complexes suggests two pathways for the DNA to exit the active pocket. They are probably used at different steps of the conjugative DNA-processing reaction. The structural information allows us to propose (i) an enzyme mechanism where the scissile phosphate is polarized by the metal ion facilitating the nucleophilic attack of the catalytic tyrosine, and (ii) a probable sequence of events during conjugative DNA processing that explains the biological function of the relaxase.
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614. |
Fleckenstein JM,
Roy K,
Fischer JF,
Burkitt M,
( 2006 ) Identification of a two-partner secretion locus of enterotoxigenic Escherichia coli. PMID : 16552055 : DOI : 10.1128/IAI.74.4.2245-2258.2006 PMC : PMC1418895 Abstract >>
Enterotoxigenic Escherichia coli (ETEC) remains a formidable cause of diarrheal illness worldwide. At present, there is no vaccine that provides broad-based protection against ETEC. A 'phoA-based self-cloning mutagenesis system, TnphoA.ts, employed to identify novel ETEC surface antigens, led to identification of an ETEC two-partner secretion locus (etpBAC) on the pCS1 virulence plasmid of prototype strain H10407. Cloning and expression of etpBAC in recombinant E. coli LMG194(pJY019) resulted in secretion of a high-molecular-weight (HMW) glycosylated exoprotein. This glycoprotein, EtpA, exhibits linear peptide sequence and predicted structural homologies with known HMW adhesins produced by other two-partner secretion loci. Antibodies directed against recombinant EtpA (anti-rEtpA.6H) recognized an HMW protein in culture supernatants of ETEC strains H10407 and LMG194(pJY019) but not in culture supernatant of strain H10407-P, which lacks the 92-kb pCS1 plasmid, or an isogenic etpA mutant. etpA mutants were deficient in adherence to intestinal epithelial cells in vitro, and anti-rEtpA.6H antibodies inhibited association of H10407 with target epithelial cells. Cloning and expression of etpB in recombinant E. coli were sufficient to confer adherence. Screening of multiple ETEC isolates for the etpBAC locus by colony hybridization and by EtpA immunoblotting suggested that EtpA is one of the most common antigens secreted by these pathogens. Together, these results indicate that the newly identified ETEC two-partner secretion locus directs the secretion of a high-molecular-weight glycosylated protein, EtpA, that in concert with the putative EtpB transporter participates in adherence of H10407 to epithelial cells, thereby expanding the repertoire of potential ETEC virulence proteins and vaccine candidates.
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615. |
Waleron K,
Waleron M,
Osipiuk J,
Podhajska AJ,
Lojkowska E,
( 2006 ) Identification of a DNA restriction-modification system in Pectobacterium carotovorum strains isolated from Poland. PMID : 16430511 : DOI : 10.1111/j.1365-2672.2005.02766.x Abstract >>
Polish isolates of pectinolytic bacteria from the species Pectobacterium carotovorum were screened for the presence of a DNA restriction-modification (R-M) system. Eighty-nine strains of P. carotovorum were isolated from infected potato plants. Sixty-six strains belonged to P. carotovorum ssp. atrosepticum and 23 to P. carotovorum ssp. carotovorum. The presence of restriction enzyme Pca17AI, which is an isoschizomer of EcoRII endonuclease, was observed in all isolates of P. c. atrosepticum but not in P. c. carotovorum. The biochemical properties, PCR amplification, and sequences of the Pca17AI restriction endonuclease and methyltransferase genes were compared with the prototype EcoRII R-M system genes. Only when DNA isolated from cells of P. c. atrosepticum was used as a template, amplification of a 680 bp homologous to the gene coding EcoRII endonuclease. Endonuclease Pca17AI, having a relatively low temperature optimum, was identified. PCR amplification revealed that the nucleotide sequence of genes for EcoRII and Pca17AI R-M are different. Dcm methylation was observed in all strains of Pectobacterium and other Erwinia species tested. The sequence of a DNA fragment coding Dcm methylase in P. carotovorum was different from that of Escherichia coli. Pca17AI is the first psychrophilic isoschizomer of EcoRII endonuclease. The presence of specific Dcm methylation in chromosomal DNA isolated from P. carotovorum is described for the first time. A 680 bp PCR product, unique for P. c. atrosepticum strains, could serve as a molecular marker for detection of these bacteria in environmental samples.
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616. |
Morales C,
Lee MD,
Hofacre C,
Maurer JJ,
( 2004 ) Detection of a novel virulence gene and a Salmonella virulence homologue among Escherichia coli isolated from broiler chickens. PMID : 15992275 : DOI : 10.1089/fpd.2004.1.160 Abstract >>
Despite the diversity of Escherichia coli pathotypes, there are many virulence genes common to isolates from food animals and humans, suggesting that opportunity exists for genetic exchange between human and animal isolates to create the next emerging, foodborne pathogen. Hemolytic activity in E. coli has been attributed to hemolysin genes found in either uropathogenic or enterohemorrhagic E. coli. These E. coli hemolysins are classified as RTX toxins due to a repetitive toxin domain and similar gene organization, sequence homology, and mechanism of action and presence in animal and human E. coli isolates. Certain hemolytic animal isolates, however, lack these E. coli hemolysin genes. Recently, we identified a hemolysin from E. coli, isolated from poultry, with significant homology to the K12 "silent" hemolysin gene she. This gene was present only in one of four hemolytic, avian E. coli isolates examined, suggesting that the other three E. coli contain a gene distinct from the RTX toxin genes, hlyA and the she homolog, hlyE. A phagemid library was made from chicken E. coli isolate 963726, which was negative for hemolysin gene hlyA and hlyE. A hemolytic clone was identified from this library, which contained a 3.3-kb Sau3A DNA insert. The nucleotide sequences of this DNA insert revealed two, open reading frames (ORF). The first ORF encoded for a 40-Kdal protein with no significant homology to known hemolysins reported in the Gen- Bank DNA/Protein database. The second ORF specified a 26-Kdal protein with significant homology to a Salmonella regulatory gene mig-14 that had a broad distribution among the pathogenic, animal E. coli isolates. Deletion of the second orf did not abrogate hemolysis, indicating that the first ORF encoded the hemolysin. This new bacterial gene designated hlyF represents a new class of hemolysin.
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617. |
Valverde A,
Cantón R,
Galán JC,
Nordmann P,
Baquero F,
Coque TM,
( 2006 ) In117, an unusual In0-like class 1 integron containing CR1 and bla(CTX-M-2) and associated with a Tn21-like element. PMID : 16436750 : DOI : 10.1128/AAC.50.2.799-802.2006 PMC : PMC1366881 Abstract >>
An unusual In0-like class 1 integron containing a common region that includes the putative recombinase gene named orf513 (CR1) and bla(CTX-M-2) was characterized from Escherichia coli. The integron contained an unusual gene cassette array, estX-aadA1, embedded between the 5'-conserved segment (5'-CS) and 3'-CS1 regions and was flanked by mer-Tn21 sequences downstream of the tni truncated module. This element constitutes one of the few examples of CR1-bearing class 1 integrons that has been fully characterized.
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618. |
Turner AK,
Beavis JC,
Stephens JC,
Greenwood J,
Gewert C,
Thomas N,
Deary A,
Casula G,
Daley A,
Kelly P,
Randall R,
Darsley MJ,
( 2006 ) Construction and phase I clinical evaluation of the safety and immunogenicity of a candidate enterotoxigenic Escherichia coli vaccine strain expressing colonization factor antigen CFA/I. PMID : 16428753 : DOI : 10.1128/IAI.74.2.1062-1071.2006 PMC : PMC1360332 Abstract >>
Oral delivery of toxin-negative derivatives of enterotoxigenic Escherichia coli (ETEC) that express colonization factor antigens (CFA) with deletions of the aroC, ompC, ompF, and toxin genes may be an effective approach to vaccination against ETEC-associated diarrhea. We describe the creation and characterization of an attenuated CFA/I-expressing ETEC vaccine candidate, ACAM2010, from a virulent isolate in which the heat-stable enterotoxin (ST) and CFA/I genes were closely linked and on the same virulence plasmid as the enteroaggregative E. coli heat-stable toxin (EAST1) gene. A new suicide vector (pJCB12) was constructed and used to delete the ST and EAST1 genes and to introduce defined deletion mutations into the aroC, ompC, and ompF chromosomal genes. A phase I trial, consisting of an open-label dose escalation phase in 18 adult outpatient volunteers followed by a placebo-controlled double-blind phase in an additional 31 volunteers, was conducted. The vaccine was administered in two formulations, fresh culture and frozen suspension. These were both well tolerated, with no evidence of significant adverse events related to vaccination. Immunoglobulin A (IgA) and IgG antibody-secreting cells specific for CFA/I were assayed by ELISPOT. Positive responses (greater than twofold increase) were seen in 27 of 37 (73%) subjects who received the highest dose level of vaccine (nominally 5 x 10(9) CFU). Twenty-nine of these volunteers were secreting culturable vaccine organisms at day 3 following vaccination; five were still positive on day 7, with a single isolation on day 13. This live attenuated bacterial vaccine is safe and immunogenic in healthy adult volunteers.
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619. |
Vignoli R,
Varela G,
Mota MI,
Cordeiro NF,
Power P,
Ingold E,
Gadea P,
Sirok A,
Schelotto F,
Ayala JA,
Gutkind G,
( 2005 ) Enteropathogenic Escherichia coli strains carrying genes encoding the PER-2 and TEM-116 extended-spectrum beta-lactamases isolated from children with diarrhea in Uruguay. PMID : 15956426 : DOI : 10.1128/JCM.43.6.2940-2943.2005 PMC : PMC1151943 Abstract >>
We studied 13 extended-spectrum beta-lactamase (ESBL)-producing enteropathogenic Escherichia coli isolates from children suffering acute diarrhea in Uruguay. ESBL characterization in crude extracts showed a single band at pI 5.4. PCR amplification and sequencing data allowed identification of blaPER-2 and blaTEM-116. Retrospective analysis suggests that these strains were disseminated in the community, even if unnoticed, prior to their access to the hospital environment more than a decade ago.
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620. |
Delver EP,
Kotova VU,
Zavilgelsky GB,
Belogurov AA,
( 1991 ) Nucleotide sequence of the gene (ard) encoding the antirestriction protein of plasmid colIb-P9. PMID : 1653225 : DOI : 10.1128/jb.173.18.5887-5892.1991 PMC : PMC208323 Abstract >>
The IncI1 plasmid ColIb-P9 was found to encode an antirestriction function. The relevant gene, ard (alleviation of restriction of DNA), maps about 5 kb from the origin of transfer, in the region transferred early during bacterial conjugation. Ard inhibits both restriction and modification by each of the four type I systems of Escherichia coli tested, but it had no effect on restriction by either EcoRI, a type II system, or EcoP1, a type III system. The nucleotide sequence of the ColIb ard gene was determined; the predicted molecular weight of the Ard polypeptide is 19,193. The proposed polypeptide chain contains an excess of 25 negatively charged amino acids, suggesting that its overall character is very acidic. Deletion analysis of the gene revealed that the Ard protein contained a distinct functional domain located in the COOH-terminal half of the polypeptide. We suggest that the biological role of the ColIb Ard protein is associated with overcoming host-controlled restriction during bacterial conjugation.
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621. |
Gourmelon M,
Montet MP,
Lozach S,
Le Mennec C,
Pommepuy M,
Beutin L,
Vernozy-Rozand C,
( 2006 ) First isolation of Shiga toxin 1d producing Escherichia coli variant strains in shellfish from coastal areas in France. PMID : 16405688 : DOI : 10.1111/j.1365-2672.2005.02753.x Abstract >>
This study was carried out to evaluate the presence of Shiga toxin-producing Escherichia coli (STEC) and E. coli O157:H7 in shellfish from French coastal environments. Shellfish were collected in six growing areas or natural beds (B category) and nonfarming areas (D category) from July 2002 to August 2004. PCR detection of stx genes was performed on homogenized whole shellfish and digestive gland tissues enrichments. STEC strains were detected by colony DNA hybridization using a stx-specific gene probe and E. coli O157 strains were additionally searched by immunomagnetic separation with O157-specific magnetic beads. Stx genes were detected in 40 of 144 (27.8%) sample enrichments from mussels, oysters or cockles, 32 of 130 enrichments (24.6%) were from B-category areas and eight of 14 (57.1%) from the D-category area. Five strains carrying stx(1) or stx(1d) genes and one stx negative, eae and ehxA positive E. coli O157:H7 were isolated from six of 40 stx-positive enrichments. No relation was found between the total E. coli counts in shellfish and the presence of STEC strains in the samples. The STEC strains of different serotypes and stx types are present in shellfish from French coastal environments. It is the first isolation of STEC stx1d strains in France. Shellfish collected in coastal environments can serve as a vehicle for STEC transmission.
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622. |
Inamoto S,
Yoshioka Y,
Ohtsubo E,
( 1991 ) Site- and strand-specific nicking in vitro at oriT by the traY-traI endonuclease of plasmid R100. PMID : 1645338 : Abstract >>
We developed an in vitro system to reproduce a site- and strand-specific nicking at the oriT region of plasmid R100. The nicking reaction was dependent on the purified TraY protein and on the lysate, which was prepared from cells overproducing the TraI protein. This supports the idea that the protein products of two genes, traY and traI, constitute an endonuclease that introduces a specific nick in vivo in the oriT region of the conjugative plasmids related to R100. The products were the "complex" DNA molecules with a protein covalently linked with the 5'-end of the nick. The nick was introduced in the strand, which is supposed to be transferred to recipient cells during conjugation, and was located at the site 59 base pairs upstream of the TraY protein binding site, sbyA.
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623. |
Andreishcheva EN,
Vann WF,
( 2006 ) Gene products required for de novo synthesis of polysialic acid in Escherichia coli K1. PMID : 16484189 : DOI : 10.1128/JB.188.5.1786-1797.2006 PMC : PMC1426546 Abstract >>
Escherichia coli K1 is responsible for 80% of E. coli neonatal meningitis and is a common pathogen in urinary tract infections. Bacteria of this serotype are encapsulated with the alpha(2-8)-polysialic acid NeuNAc(alpha2-8), common to several bacterial pathogens. The gene cluster encoding the pathway for synthesis of this polymer is organized into three regions: (i) kpsSCUDEF, (ii) neuDBACES, and (iii) kpsMT. The K1 polysialyltransferase, NeuS, cannot synthesize polysialic acid de novo without other products of the gene cluster. Membranes isolated from strains having the entire K1 gene cluster can synthesize polysialic acid de novo. We designed a series of plasmid constructs containing fragments of regions 1 and 2 in two compatible vectors to determine the minimum number of gene products required for de novo synthesis of the polysialic acid from CMP-NeuNAc in K1 E. coli. We measured the ability of the various combinations of region 1 and 2 fragments to restore polysialyltransferase activity in vitro in the absence of exogenously added polysaccharide acceptor. The products of region 2 genes neuDBACES alone were not sufficient to support de novo synthesis of polysialic acid in vitro. Only membrane fractions harboring NeuES and KpsCS could form sialic polymer in the absence of exogenous acceptor at the concentrations formed by wild-type E. coli K1 membranes. Membrane fractions harboring NeuES and KpsC together could form small quantities of the sialic polymer de novo.
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624. |
Yip CK,
Kimbrough TG,
Felise HB,
Vuckovic M,
Thomas NA,
Pfuetzner RA,
Frey EA,
Finlay BB,
Miller SI,
Strynadka NC,
( 2005 ) Structural characterization of the molecular platform for type III secretion system assembly. PMID : 15931226 : DOI : 10.1038/nature03554 Abstract >>
Type III secretion systems (TTSSs) are multi-protein macromolecular 'machines' that have a central function in the virulence of many Gram-negative pathogens by directly mediating the secretion and translocation of bacterial proteins (termed effectors) into the cytoplasm of eukaryotic cells. Most of the 20 unique structural components constituting this secretion apparatus are highly conserved among animal and plant pathogens and are also evolutionarily related to proteins in the flagellar-specific export system. Recent electron microscopy experiments have revealed the gross 'needle-shaped' morphology of the TTSS, yet a detailed understanding of the structural characteristics and organization of these protein components within the bacterial membranes is lacking. Here we report the 1.8-A crystal structure of EscJ from enteropathogenic Escherichia coli (EPEC), a member of the YscJ/PrgK family whose oligomerization represents one of the earliest events in TTSS assembly. Crystal packing analysis and molecular modelling indicate that EscJ could form a large 24-subunit 'ring' superstructure with extensive grooves, ridges and electrostatic features. Electron microscopy, labelling and mass spectrometry studies on the orthologous Salmonella typhimurium PrgK within the context of the assembled TTSS support the stoichiometry, membrane association and surface accessibility of the modelled ring. We propose that the YscJ/PrgK protein family functions as an essential molecular platform for TTSS assembly.
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625. |
Fennell-Fezzie R,
Gradia SD,
Akey D,
Berger JM,
( 2005 ) The MukF subunit of Escherichia coli condensin: architecture and functional relationship to kleisins. PMID : 15902272 : DOI : 10.1038/sj.emboj.7600680 PMC : PMC1142612 Abstract >>
The Escherichia coli MukB, MukE, and MukF proteins form a bacterial condensin (MukBEF) that contributes to chromosome management by compacting DNA. MukB is an ATPase and DNA-binding protein of the SMC superfamily; however, the structure and function of non-SMC components, such as MukF, have been less forthcoming. Here, we report the crystal structure of the N-terminal 287 amino acids of MukF at 2.9 A resolution. This region folds into a winged-helix domain and an extended coiled-coil domain that self-associate to form a stable, doubly domain-swapped dimer. Protein dissection and affinity purification data demonstrate that the region of MukF C-terminal to this fragment binds to MukE and MukB. Our findings, together with sequence analyses, indicate that MukF is a kleisin subunit for E. coli condensin and suggest a means by which it may organize the MukBEF assembly.
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626. |
Gilmour MW,
Tracz DM,
Andrysiak AK,
Clark CG,
Tyson S,
Severini A,
Ng LK,
( 2006 ) Use of the espZ gene encoded in the locus of enterocyte effacement for molecular typing of shiga toxin-producing Escherichia coli. PMID : 16455898 : DOI : 10.1128/JCM.44.2.449-458.2006 PMC : PMC1392676 Abstract >>
Infections with Shiga toxin-producing Escherichia coli (STEC) result in frequent cases of sporadic and outbreak-associated enteric bacterial disease in humans. Classification of STEC is by stx genotype (encoding the Shiga toxins), O and H antigen serotype, and seropathotype (subgroupings based upon the clinical relevance and virulence-related genotypes of individual serotypes). The espZ gene is encoded in the locus of enterocyte effacement (LEE) pathogenicity island responsible for the attaching and effacing (A/E) lesions caused by various E. coli pathogens (but not limited to STEC), and this individual gene (approximately 300 bp) has previously been identified as hypervariable among these A/E pathogens. Sequence analysis of the espZ locus encoded by additional STEC serotypes and strains (including O26:H11, O121:H19, O111:NM, O145:NM, O165:H25, O121:NM, O157:NM, O157:H7, and O5:NM) indicated that distinct sequence variants exist which correlate to subgroups among these serotypes. Allelic discrimination at the espZ locus was achieved using Light Upon eXtension real-time PCR and by liquid microsphere suspension arrays. The allele subtype of espZ did not correlate with STEC seropathotype classification; however, a correlation with the allele type of the LEE-encoded intimin (eae) gene was supported, and these sequence variations were conserved among individual serotypes. The study focused on the characterization of three clinically significant seropathotypes of LEE-positive STEC, and we have used the observed genetic variation at a pathogen-specific locus for detection and subtyping of STEC.
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627. |
González-Zorn B,
Teshager T,
Casas M,
Porrero MC,
Moreno MA,
Courvalin P,
Domínguez L,
( 2005 ) armA and aminoglycoside resistance in Escherichia coli. PMID : 15963296 : DOI : 10.3201/eid1106.040553 PMC : PMC3367600 Abstract >>
We report armA in an Escherichia coli pig isolate from Spain. The resistance gene was borne by self-transferable IncN plasmid pMUR050. Molecular analysis of the plasmid and of the armA locus confirmed the spread of this resistance determinant.
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628. |
Poirel L,
Van De Loo M,
Mammeri H,
Nordmann P,
( 2005 ) Association of plasmid-mediated quinolone resistance with extended-spectrum beta-lactamase VEB-1. PMID : 15980408 : DOI : 10.1128/AAC.49.7.3091-3094.2005 PMC : PMC1168703 Abstract >>
Association of the plasmid-mediated quinolone resistance determinant QnrA and the bla(VEB-1) gene was identified in a single Enterobacter cloacae isolate from K.-Bic?tre, France, and in 11 out of 23 bla(VEB-1)-positive enterobacterial isolates from Bangkok, Thailand. This result may explain in part the association between quinolone and extended-spectrum beta-lactam resistance.
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629. |
Stepánek V,
Valesová R,
Kyslík P,
( 2005 ) Cryptic plasmid pRK2 from Escherichia coli W: sequence analysis and segregational stability. PMID : 15907542 : DOI : 10.1016/j.plasmid.2004.12.006 Abstract >>
Cryptic plasmid pRK2 of the strain Escherichia coli W (ATCC 9637), an ancestor of production strains for penicillin G acylase, was sequenced and characterized. Based on the data on replication region and origin (ori sequence AAC, 924-926nt), the plasmid was classified as ColE1-like plasmid. DNA sequence analysis revealed five orfs hypothetical products of which shared a significant sequence similarity with putative proteins encoded by DNA of plasmid pColE1. orf1 codes for protein Rom involved in the control of plasmid replication, orfs 2-5 code for putative mobilization proteins (Mob A-D) that show a high level of similarity with the ones encoded by DNA of plasmids pColE1 and pLG13 (E. coli), pECL18 and pEC01 (Enterobacter cloacae), pSFD10 (Salmonella choleraesuis), and pScol7 (Shigella sonnei). Recombinant plasmids pRS11 (4.91kbp), pRS12 (4.91kbp), pRS2 (2.996kbp), and pRS3 (2.623kbp) that bear the Spectinomycin resistance determinant (Spc(R)) were prepared on the basis of nucleotide sequence of pRK2. These constructs are stably maintained in the population of E. coli cells grown in the absence of the selection pressure for 63 generations. The copy number of Spc(R) constructs in E. coli host grown in antibiotic-free LB medium ranges from 25 to 40 molecules per chromosomal equivalent.
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630. |
Jeong JY,
Yoon HJ,
Kim ES,
Lee Y,
Choi SH,
Kim NJ,
Woo JH,
Kim YS,
( 2005 ) Detection of qnr in clinical isolates of Escherichia coli from Korea. PMID : 15917562 : DOI : 10.1128/AAC.49.6.2522-2524.2005 PMC : PMC1140518 Abstract >>
qnr was detected in 2 of 260 Escherichia coli clinical isolates collected from a Korean hospital during the period 2001 to 2003. The two strains were not clonally related. qnr was located in In4 family class 1 integrons of original structure, downstream of orf513 and upstream from another resistance gene (dfrA3b) and a gene of unknown function (orf105). Transfer of the qnr determinant by conjugation could be detected from only one strain.
|
631. |
Sunde M,
( 2005 ) Class I integron with a group II intron detected in an Escherichia coli strain from a free-range reindeer. PMID : 15917559 : DOI : 10.1128/AAC.49.6.2512-2514.2005 PMC : PMC1140521 Abstract >>
An Escherichia coli strain, isolated from wild reindeer in a remote mountain area, contained a class 1 integron with two unusual features: a group II intron and a cassette with homology to a superintegron cassette. Alignments indicate that attC sites of gene cassettes may be insertion sites for introns.
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632. |
Thielking V,
Selent U,
Köhler E,
Wolfes H,
Pieper U,
Geiger R,
Urbanke C,
Winkler FK,
Pingoud A,
( 1991 ) Site-directed mutagenesis studies with EcoRV restriction endonuclease to identify regions involved in recognition and catalysis. PMID : 1647200 : DOI : 10.1021/bi00240a011 Abstract >>
Guided by the X-ray structure analysis of a crystalline EcoRV-d(GGGATATCCC) complex (Winkler, in preparation), we have begun to identify functionally important amino acid residues of EcoRV. We show here that Asn70, Asp74, Ser183, Asn185, Thr186, and Asn188 are most likely involved in the binding and/or cleavage of the DNA, because their conservative substitution leads to mutants of no or strongly reduced activity. In addition, C-terminal amino acid residues of EcoRV seem to be important for its activity, since their deletion inactivates the enzyme. Following the identification of three functionally important regions, we have inspected the sequences of other restriction and modification enzymes for homologous regions. It was found that two restriction enzymes that recognize similar sequences as EcoRV (DpnII and HincII), as well as two modification enzymes (M.DpnII and, in a less apparent form, M.EcoRV), have the sequence motif -SerGlyXXXAsnIleXSer- in common, which in EcoRV contains the essential Ser183 and Asn188 residues. Furthermore, the C-terminal region, shown to be essential for EcoRV, is highly homologous to a similar region in the restriction endonuclease SmaI. On the basis of these findings we propose that these restriction enzymes and to a certain extent also some of their corresponding modification enzymes interact with DNA in a similar manner.
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633. |
Chouikha I,
Germon P,
Brée A,
Gilot P,
Moulin-Schouleur M,
Schouler C,
( 2006 ) A selC-associated genomic island of the extraintestinal avian pathogenic Escherichia coli strain BEN2908 is involved in carbohydrate uptake and virulence. PMID : 16428402 : DOI : 10.1128/JB.188.3.977-987.2006 PMC : PMC1347334 Abstract >>
The complete nucleotide sequence and genetic organization of a new genomic island (AGI-3) isolated from the extraintestinal avian pathogenic Escherichia coli strain BEN2908 is reported. This 49,600-bp island is inserted at the selC locus and contains putative mobile genetic elements such as a phage-related integrase gene, transposase genes, and direct repeats. AGI-3 shows a mosaic structure of five modules. Some of these modules are present in other E. coli strains and in other pathogenic bacterial species. The gene cluster aec-35 to aec-37 of module 1 encodes proteins associated with carbohydrates assimilation such as a major facilitator superfamily transporter (Aec-36), a glycosidase (Aec-37), and a putative transcriptional regulator of the LacI family (Aec-35). The aec-35 to aec-37 cluster was found in 11.6% of the tested pathogenic and nonpathogenic E. coli strains. When present, the aec-35 to aec-37 cluster is strongly associated with the selC locus (97%). Deletion of the aec-35-aec-37 region affects the assimilation of seven carbohydrates, decreases the growth rate of the strain in minimal medium containing galacturonate or trehalose, and attenuates the virulence of E. coli BEN2908 for chickens.
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634. |
Klemm P,
Roos V,
Ulett GC,
Svanborg C,
Schembri MA,
( 2006 ) Molecular characterization of the Escherichia coli asymptomatic bacteriuria strain 83972: the taming of a pathogen. PMID : 16369040 : DOI : 10.1128/IAI.74.1.781-785.2006 PMC : PMC1346676 Abstract >>
Escherichia coli 83972 is a clinical asymptomatia bacteriuric isolate that is able to colonize the human urinary bladder without inducing an immune response. Here we demonstrate that one of the mechanisms by which this strain has become attenuated is through the mutation of its genes encoding type 1 and P fimbriae.
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635. |
Kariyawasam S,
Johnson TJ,
Nolan LK,
( 2006 ) The pap operon of avian pathogenic Escherichia coli strain O1:K1 is located on a novel pathogenicity island. PMID : 16369033 : DOI : 10.1128/IAI.74.1.744-749.2006 PMC : PMC1346673 Abstract >>
We have identified a 56-kb pathogenicity island (PAI) in avian pathogenic Escherichia coli strain O1:K1 (APEC-O1). This PAI, termed PAI I(APEC-O1), is integrated adjacent to the 3' end of the pheV tRNA gene. It carries putative virulence genes of APEC (pap operon), other E. coli genes (tia and ireA), and a 1.5-kb region unique to APEC-O1. The kps gene cluster required for the biosynthesis of polysialic acid capsule was mapped to a location immediately downstream of this PAI.
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636. |
Yong D,
Lim Y,
Song W,
Choi YS,
Park DY,
Lee H,
Yum JH,
Lee K,
Kim JM,
Chong Y,
( 2005 ) Plasmid-mediated, inducible AmpC beta-lactamase (DHA-1)-producing Enterobacteriaceae at a Korean hospital: wide dissemination in Klebsiella pneumoniae and Klebsiella oxytoca and emergence in Proteus mirabilis. PMID : 15936167 : DOI : 10.1016/j.diagmicrobio.2005.03.008 Abstract >>
The aim of the study was to investigate the phenotypic and genetic characteristics of recently emerging cefoxitin-resistant and induction-positive isolates of Escherichia coli, Klebsiella species, and Proteus mirabilis. Strains of Enterobacteriaceae were isolated at a Korean tertiary care hospital between June and December 2002. Induction was tested using cefoxitin and aztreonam disks, the blaDHA allele was detected by PCR, and pulsed-field gel electrophoresis (PFGE) patterns were also analyzed. Among the cefoxitin-resistant isolates, 2.7% of E. coli, 21.1% of Klebsiella pneumoniae, 32.0% of Klebsiella oxytoca, and 8.3% of P. mirabilis isolates showed induction, and were blaDHA-1 allele positive. To the best of our knowledge, this is the first report of blaDHA-1 in P. mirabilis. The MICs of ceftazidime, cefotaxime, and aztreonam increased significantly by higher inoculum, suggesting that their clinical usefulness is limited. Presence of multiple PFGE patterns and identical patterns in some isolates suggest that the widely disseminated blaDHA-1 in Klebsiella species was because of both horizontal and clonal spread.
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637. |
Su J,
Shi L,
Yang L,
Xiao Z,
Li X,
Yamasaki S,
( 2006 ) Analysis of integrons in clinical isolates of Escherichia coli in China during the last six years. PMID : 16451182 : DOI : 10.1111/j.1574-6968.2005.00025.x Abstract >>
A multiple PCR for the detection of the integrase genes of the three classes of integrons was carried out, and their gene cassettes were characterized in 111 clinical strains of Escherichia coli isolated in Guangzhou City, China during the last 6 years. IntI1 and intI2 genes were detected in 95 isolates (85.6%) and four isolates (3.6%), respectively. No intI3 gene was detected. Six different gene cassettes were found in these strains, and a high prevalence of dfr and aad genes was observed. The E. coli isolates that contained a 1664-bp amplicon of dfrA17-aadA5 in class 1 integron were found to be phylogenetically unrelated to each other by using the enterobacterial repetitive intergenic consensus PCR, as the cassette could be transferred to recipient strains, indicating that the gene cassettes might be disseminated in the clinical strains by a horizontal gene transfer. Therefore, it is important that guidelines for the prudent use of antimicrobial agents are adopted and surveillance programs are established.
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638. |
Vimr ER,
Bergstrom R,
Steenbergen SM,
Boulnois G,
Roberts I,
( 1992 ) Homology among Escherichia coli K1 and K92 polysialytransferases. PMID : 1629170 : DOI : 10.1128/jb.174.15.5127-5131.1992 PMC : PMC206331 Abstract >>
The neuS-encoded polysialytransferase (polyST) in Escherichia coli K1 catalyzes synthesis of polysialic acid homopolymers composed of unbranched sialyl alpha 2,8 linkages. Subcloning and complementation experiments showed that the K1 neuS was functionally interchangeable with the neuS from E. coli K92 (S. M. Steenbergen, T. J. Wrona, and E. R. Vimr, J. Bacteriol. 174:1099-1108, 1992), which synthesizes polysialic acid capsules with alternating sialyl alpha 2,8-2,9 linkages. To better understand the relationship between these polySTs, the complete K92 neuS sequence was determined. The results demonstrated that K1 and K92 neuS genes are homologous and indicated that the K92 copy may have evolved from its K1 homolog. Both K1 and K92 structural genes comprised 1,227 bp. There were 156 (12.7%) differences between the two sequences; among these mutations, 55 did not affect the derived primary structure of K92 polyST and hence were synonymous with the K1 sequence. Assuming maximum parsimony, another estimated 17 synonymous mutations plus 84 nonsynonymous mutations could account for the 70 amino acid replacements in K92 polyST; 36 of these replacements were judged to be conservative when compared with those of K1 polyST. There were no changes detected in the first 146 5' or last 129 3' bp of either gene, suggesting, in addition to the observed mutational differences, the possibility of a past recombination event between neuS loci of two different kps clusters. The results indicate that relatively few amino acid changes can account for the evolution of a glycosyltransferase with novel linkage specificity.
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639. |
Fang Y,
Ngeleka M,
Middleton DM,
Simko E,
( 2005 ) Characterization and immuno-detection of AIDA-I adhesin isolated from porcine Escherichia coli. PMID : 15950405 : DOI : 10.1016/j.vetmic.2005.04.018 Abstract >>
A relatively high percentage of porcine Escherichia coli isolates from cases associated with neonatal and post-weaning diarrhea are positive for the gene encoding adhesin involved in diffuse adhesion I (AIDA-I). This gene and its corresponding protein were first identified and characterized in E. coli strain 2787 isolated from human infantile diarrhea. Little is known about the properties of the AIDA-I protein and its immuno-detection on surface of AIDA-I positive porcine E. coli isolates. In this study, we demonstrated that the AIDA-I adhesin isolated from porcine AIDA-I positive E. coli is an acidic protein consisting of five isoforms. It has a similar molecular weight (100 kDa) and relatively high amino acid homology (78-87%) with the AIDA-I adhesin expressed by human AIDA-I positive E. coli strain 2787. Based on limited comparison, it appears that there is a very high homology among AIDA-I proteins expressed by porcine AIDA-I positive E. coli isolates. Sensitivity of detection of surface AIDA-I adhesin of PCR-positive AIDA-I E. coli by immuno-dot-blot and coagglutination tests was 76 and 71%, respectively, whereas specificity was 89 and 84%, respectively. These tests are unlikely to be used for diagnostic detection of AIDA-I positive E. coli due to the relatively low sensitivity; however, they may be potentially useful for identification of false positive reactions generated by other diagnostic tests.
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640. |
Wang L,
Liu B,
Kong Q,
Steinrück H,
Krause G,
Beutin L,
Feng L,
( 2005 ) Molecular markers for detection of pathogenic Escherichia coli strains belonging to serogroups O 138 and O 139. PMID : 16280204 : DOI : 10.1016/j.vetmic.2005.10.006 Abstract >>
Escherichia coli strains belonging to O-serogroup 138 and 139 are important as disease agents in pigs causing post-weaning diarrhea and edema disease. Several types of shiga toxin-producing O 138 and O 139 strains were isolated from diarrheic humans and from cattle and food of bovine origin. Serotyping is the current method for detection of O 138 and O 139 strains but its applicability can be limited due to the presence of capsules and capsular-like bacterial surface antigens and in the case of rough LPS. To overcome these difficulties for diagnosis, we have developed a specific PCR method suitable for detection of different types of O 138 and O 139 strains. The O-antigen gene clusters of E. coli O 138 and O 139 type strains were sequenced, and the genes were identified on the basis of homology. By screening against 186 E. coli and Shigella type strains, two genes specific to each of E. coli O 138 and O 139 were identified, respectively, and were tested on 15 clinical and environmental isolates of those two serogroups in a double-blind test. The sensitivity of the PCR assays was determined, and the detection limits were 2 pg per mul of chromosomal DNA and 2 CFU per 10 g of water or pork samples. PCR-based detection of O-antigen specific genes of E. coli O 138 and O 139 was shown to be accurate, highly sensitive and rapid, and is suggested as a new diagnostic tool for investigations of infections and outbreaks with these strains in animals and humans and for control of food.
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641. |
Magalhaes ML,
Blanchard JS,
( 2005 ) The kinetic mechanism of AAC3-IV aminoglycoside acetyltransferase from Escherichia coli. PMID : 16331988 : DOI : 10.1021/bi051777d PMC : PMC2593831 Abstract >>
The aminoglycoside 3-N-acetyltransferase AAC(3)-IV from Escherichia coli exhibits a very broad aminoglycoside specificity, causing resistance to a large number of aminoglycosides, including the atypical veterinary antibiotic, apramycin. We report here on the characterization of the substrate specificity and kinetic mechanism of the acetyl transfer reaction catalyzed by AAC(3)-IV. The steady-state kinetic parameters revealed a narrow specificity for the acyl-donor and broad range of activity for aminoglycosides. AAC(3)-IV has the broadest substrate specificity of all AAC(3)'s studied to date. Dead-end inhibition and ITC experiments revealed that AAC(3)-IV follows a sequential, random bi-bi kinetic mechanism. The analysis of the pH dependence of the kinetic parameters revealed acid- and base-assisted catalysis and the existence of three additional ionizable groups involved in substrate binding. The magnitude of the solvent kinetic isotope effects suggests that a chemical step is at least partially rate limiting in the overall reaction.
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642. |
Doublet B,
Schwarz S,
Kehrenberg C,
Cloeckaert A,
( 2005 ) Florfenicol resistance gene floR is part of a novel transposon. PMID : 15855539 : DOI : 10.1128/AAC.49.5.2106-2108.2005 PMC : PMC1087673 Abstract >>
The florfenicol/chloramphenicol resistance gene floR was found to be part of the novel 4,284-bp transposon TnfloR from Escherichia coli. TnfloR consists of the gene floR, a putative regulatory gene, and the transposase gene tnpA. A circular form of TnfloR was detected and suggested the potential mobility of this transposon.
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643. |
Barak Y,
Thorne SH,
Ackerley DF,
Lynch SV,
Contag CH,
Matin A,
( 2006 ) New enzyme for reductive cancer chemotherapy, YieF, and its improvement by directed evolution. PMID : 16432167 : DOI : 10.1158/1535-7163.MCT-05-0365 Abstract >>
Reductive prodrugs, mitomycin C and 5-aziridinyl-2,4-dinitrobenzamide (CB 1954), are nontoxic in their native form but become highly toxic upon reduction. Their effectiveness in cancer chemotherapy can be enhanced by delivering to tumors enzymes with improved prodrug reduction kinetics. We report the discovery of a new prodrug-reducing enzyme, YieF, from Escherichia coli, and the improvement of its kinetics for reducing mitomycin C and CB 1954. A YieF-derived enzyme, Y6, killed HeLa spinner cells with >or=5-fold efficiency than the wild-type enzymes, YieF and NfsA, at a variety of drug and enzyme concentrations and incubation times. With adhered HeLa cells and Salmonella typhimurium SL 7838 bacteria as enzyme delivery vehicle, at least an order of magnitude less of Y6-producing bacteria were required to kill >90% of tumor cells compared with bacteria expressing the wild-type enzymes, which at a comparable level killed < 5% of the cells. Thus, Y6 is a promising enzyme for use in cancer chemotherapy, and Salmonella strain SL 7838, which specifically targets tumors, may be used to deliver the prodrug-activating enzymes to tumors.
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644. |
Robicsek A,
Strahilevitz J,
Jacoby GA,
Macielag M,
Abbanat D,
Park CH,
Bush K,
Hooper DC,
( 2006 ) Fluoroquinolone-modifying enzyme: a new adaptation of a common aminoglycoside acetyltransferase. PMID : 16369542 : DOI : 10.1038/nm1347 Abstract >>
Antimicrobial-modifying resistance enzymes have traditionally been class specific, having coevolved with the antibiotics they inactivate. Fluoroquinolones, antimicrobial agents used extensively in medicine and agriculture, are synthetic and have been considered safe from naturally occurring antimicrobial-modifying enzymes. We describe reduced susceptibility to ciprofloxacin in clinical bacterial isolates conferred by a variant of the gene encoding aminoglycoside acetyltransferase AAC(6')-Ib. This enzyme reduces the activity of ciprofloxacin by N-acetylation at the amino nitrogen on its piperazinyl substituent. Although approximately 30 variants of this gene have been reported since 1986, the two base-pair changes responsible for the ciprofloxacin modification phenotype are unique to this variant, first reported in 2003 and now widely disseminated. An intense increase in the medical use of ciprofloxacin seems to have been accompanied by a notable development: a single-function resistance enzyme has crossed class boundaries, and is now capable of enzymatically undermining two unrelated antimicrobial agents, one of them fully synthetic.
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645. |
Ueta M,
Yoshida H,
Wada C,
Baba T,
Mori H,
Wada A,
( 2005 ) Ribosome binding proteins YhbH and YfiA have opposite functions during 100S formation in the stationary phase of Escherichia coli. PMID : 16324148 : DOI : 10.1111/j.1365-2443.2005.00903.x Abstract >>
During the stationary phase of Escherichia coli growth, ribosomal structure changes drastically. Proteins RMF, YhbH, YfiA and SRA are expressed and bind to ribosome particles. In a process named 'ribosomal hibernation,' RMF binding induces the dimerization and subsequent inactivation of 70S ribosomes. Here, we examined the functions of YhbH and YfiA in the formation of 70S dimers using deletion mutants of YhbH and YfiA. The yfiA deletion mutant expressed YhbH and RMF in the stationary phase and formed a greater number of 100S particles than the wild-type, showing that YhbH promotes and stabilizes 100S formation. In contrast, the yhbH deletion mutant expressed YfiA and RMF and produced no 70S dimers, suggesting that YfiA prevents 70S dimer formation. Thus, YhbH and YfiA have opposite functions in 70S dimer formation. YhbH and YfiA share 40% sequence homology, suggesting that their binding sites overlap and they compete for a region proximal to the P- and A-sites on 30S subunits. In the yhbH and yfiA double deletion mutant, which expresses only RMF, 70S dimers were observed as 90S particles. Since 100S particles were seen in the yfiA deletion mutant containing RMF and YhbH, YhbH probably converts immature 90S ribosomes into mature 100S particles.
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646. |
Delgado MA,
Salomón RA,
( 2005 ) Molecular characterization of a DNA fragment carrying the basic replicon of pTUC100, the natural plasmid encoding the peptide antibiotic microcin J25 system. PMID : 15848229 : DOI : 10.1016/j.plasmid.2004.09.003 Abstract >>
The Escherichia coli plasmid pTUC100 encodes production of, and immunity to, the peptide antibiotic microcin J25. In the present study, an approximately 8-kb fragment immediately adjacent to the previously sequenced microcin region was isolated and its DNA sequence was determined. The main features of the newly characterized region are: (i) a basic replicon which is almost identical to that of the RepFIIA plasmid R100; (ii) two ORFs with 96% identity to two ORFs of unknown function on pO157, a large plasmid harbored by enterohemorragic E. coli, and a large ORF which does not show significant homology to any other reported nucleotide or protein sequence; and (iii) two intact insertion sequences, IS1294 and IS1. Sequence analysis, as well as that of the G+C content of both the 8-kb fragment and the previously sequenced microcin locus, lead us to propose that plasmid pTUC100 is a composite structure assembled from DNA elements from various sources.
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647. |
Isaac DD,
Pinkner JS,
Hultgren SJ,
Silhavy TJ,
( 2005 ) The extracytoplasmic adaptor protein CpxP is degraded with substrate by DegP. PMID : 16303867 : DOI : 10.1073/pnas.0508936102 PMC : PMC1308919 Abstract >>
In Escherichia coli, the CpxR/A two-component system senses various types of extracytoplasmic stresses and responds by activating the expression of genes encoding periplasmic protein folding and trafficking factors that clear such stresses to ensure the organism's survival. The cpxP gene encodes a small, stress-combative periplasmic protein and is the most strongly induced member of the Cpx regulon. We demonstrate that the Cpx stress response suppresses the toxicity associated with two misfolded proteins derived from the P pilus of uropathogenic E. coli and that mutations in either cpxP or the gene for the periplasmic protease DegP prevent suppression by preventing the degradation of these proteins. Strikingly, the presence of a periplasmic misfolded protein substrate significantly enhances the proteolysis of CpxP by DegP. Our data suggest that CpxP functions as a periplasmic adaptor protein that is required for the effective proteolysis of a subset of misfolded substrates by the DegP protease.
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648. |
Eckert C,
Gautier V,
Arlet G,
( 2006 ) DNA sequence analysis of the genetic environment of various blaCTX-M genes. PMID : 16291869 : DOI : 10.1093/jac/dki398 Abstract >>
Over a 3 year period (2000-2003) 21 Escherichia coli, 5 Klebsiella pneumoniae, 1 Serratia marcescens and 1 Proteus mirabilis producing CTX-M-type beta-lactamase were collected from five different hospitals in Paris, France. This study was conducted to analyse the genetic environment of these 28 bla(CTX-M) genes. Antimicrobial susceptibility testing was performed by the disc diffusion method and MICs of various beta-lactams were determined by an agar dilution method. PCR was used to detect and sequence alleles encoding CTX-M, TEM, SHV and CMY enzymes. The genetic environment was analysed by amplification and direct sequencing using various set of PCR primers or cloning in pBK-CMV. Sequence analysis revealed that these isolates contained seven different bla(CTX-M) genes: bla(CTX-M-1) (4 strains), bla(CTX-M-2) (2 strains), bla(CTX-M-3) (4 strains), bla(CTX-M-9) (1 strain), bla(CTX-M-14) (5 strains), bla(CTX-M-15) (11 strains), bla(CTX-M-24) (1 strain). TEM-1 was associated with CTX-M-type enzymes in 15 isolates. Two strains produced both CTX-M-15 and SHV-2 or CTX-M-14 and CMY-2. In 25 strains the insertion sequence ISEcp1 was located upstream of the 5' end of the bla(CTX-M) gene. Among these strains, in five isolates, ISEcp1 was disrupted by insertion sequences such as IS26 (in three of them) or IS1 or IS10. Insertion sequence IS903 was found downstream of bla(CTX-M-14) or bla(CTX-M-24). Examination of the other three bla(CTX-M) genes (two bla(CTX-M-2) and one bla(CTX-M-9)) by cloning, sequencing and PCR analysis revealed the presence of complex Class 1 integrons, In35, an integron similar to In60 and a novel integron. This work further confirmed the predominant role of ISEcp1 in the mobilization of bla(CTX-M) genes of the CTX-M-1 cluster and the presence of In35, of an integron similar to In60 and a novel complex Class 1 integron.
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649. |
Baraniak A,
Fiett J,
Mrówka A,
Walory J,
Hryniewicz W,
Gniadkowski M,
( 2005 ) Evolution of TEM-type extended-spectrum beta-lactamases in clinical Enterobacteriaceae strains in Poland. PMID : 15855509 : DOI : 10.1128/AAC.49.5.1872-1880.2005 PMC : PMC1087658 Abstract >>
Seventeen extended-spectrum beta-lactamase (ESBL)-producing isolates of the family Enterobacteriaceae recovered from 1998 to 2000 in hospitals of five different cities in Poland were analyzed. They expressed several TEM-type ESBLs, TEM-4, TEM-29, TEM-85, TEM-86, TEM-93, and TEM-94. TEM-85 (L21F, R164S, E240K, T265M), TEM-86 (L21F, R164S, A237T, E240K, T265M), TEM-93 (M182T, G238S, E240K), and TEM-94 (L21F, E104K, M182T, G238S, T265M) were identified for the first time. Including the enzymes described earlier, TEM-47, TEM-48, TEM-49, and TEM-68, the group of known ESBLs of the TEM family produced by enterobacteria in Polish hospitals has increased to 10 variants. Comparative sequence analysis of the genes coding for all these beta-lactamases revealed a view of their possible evolution, which, apart from the gradual acquisition of various mutations, could also have involved recombination events. Two different bla(TEM-1) gene alleles were precursors of the ESBL genes: bla(TEM-1A), which was the ancestor of bla(TEM-93), and bla(TEM-1F), from which all the remaining genes originated. The evolution of the bla(TEM-1F)-related genes most probably consisted of three major separate lineages, one of which, including bla(TEM-4), bla(TEM-47), bla(TEM-48), bla(TEM-49), bla(TEM-68), and bla(TEM-94), was highly structured itself and could have been initiated by the bla(TEM-25) gene, identified exclusively in France so far. Plasmid fingerprinting analysis revealed a high degree of diversity of plasmids carrying related bla(TEM) genes, which suggested either the intense diversification or transposition of bla(TEM) genes between different plasmids or some contribution of convergent evolution. The results of this study clearly demonstrate that the environment of Polish hospitals has been highly favorable for the rapid evolution of ESBLs.
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650. |
Guo H,
Yi W,
Shao J,
Lu Y,
Zhang W,
Song J,
Wang PG,
( 2005 ) Molecular analysis of the O-antigen gene cluster of Escherichia coli O86:B7 and characterization of the chain length determinant gene (wzz). PMID : 16332778 : DOI : 10.1128/AEM.71.12.7995-8001.2005 PMC : PMC1317457 Abstract >>
Escherichia coli O86:B7 has long been used as a model bacterial strain to study the generation of natural blood group antibody in humans, and it has been shown to possess high human blood B activity. The O-antigen structure of O86:B7 was solved recently in our laboratory. Comparison with the published structure of O86:H2 showed that both O86 subtypes shared the same O unit, yet each of the O antigens is polymerized from a different terminal sugar in a different glycosidic linkage. To determine the genetic basis for the O-antigen differences between the two O86 strains, we report the complete sequence of O86:B7 O-antigen gene cluster between galF and hisI, each gene was identified based on homology to other genes in the GenBank databases. Comparison of the two O86 O-antigen gene clusters revealed that the encoding regions between galF and gnd are identical, including wzy genes. However, deletion of the two wzy genes revealed that wzy in O86:B7 is responsible for the polymerization of the O antigen, while the deletion of wzy in O86:H2 has no effect on O-antigen biosynthesis. Therefore, we proposed that there must be another functional wzy gene outside the O86:H2 O-antigen gene cluster. Wzz proteins determine the degree of polymerization of the O antigen. When separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the lipopolysaccharide (LPS) of O86:B7 exhibited a modal distribution of LPS bands with relatively short O units attached to lipid A-core, which differs from the LPS pattern of O86:H2. We proved that the wzz genes are responsible for the different LPS patterns found in the two O86 subtypes, and we also showed that the very short type of LPS is responsible for the serum sensitivity of the O86:B7 strain.
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651. |
Ideses D,
Gophna U,
Paitan Y,
Chaudhuri RR,
Pallen MJ,
Ron EZ,
( 2005 ) A degenerate type III secretion system from septicemic Escherichia coli contributes to pathogenesis. PMID : 16291689 : DOI : 10.1128/JB.187.23.8164-8171.2005 PMC : PMC1291271 Abstract >>
The type III secretion system (T3SS) is an important virulence factor used by several gram-negative bacteria to deliver effector proteins which subvert host cellular processes. Enterohemorrhagic Escherichia coli O157 has a well-defined T3SS involved in attachment and effacement (ETT1) and critical for virulence. A gene cluster potentially encoding an additional T3SS (ETT2), which resembles the SPI-1 system in Salmonella enterica, was found in its genome sequence. The ETT2 gene cluster has since been found in many E. coli strains, but its in vivo role is not known. Many of the ETT2 gene clusters carry mutations and deletions, raising the possibility that they are not functional. Here we show the existence in septicemic E. coli strains of an ETT2 gene cluster, ETT2(sepsis), which, although degenerate, contributes to pathogenesis. ETT2(sepsis) has several premature stop codons and a large (5 kb) deletion, which is conserved in 11 E. coli strains from cases of septicemia and newborn meningitis. A null mutant constructed to remove genes coding for the putative inner membrane ring of the secretion complex exhibited significantly reduced virulence. These results are the first demonstration of the importance of ETT2 for pathogenesis.
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652. |
Barthélémy M,
Péduzzi J,
Bernard H,
Tancrède C,
Labia R,
( 1992 ) Close amino acid sequence relationship between the new plasmid-mediated extended-spectrum beta-lactamase MEN-1 and chromosomally encoded enzymes of Klebsiella oxytoca. PMID : 1633193 : DOI : 10.1016/0167-4838(92)90121-s Abstract >>
Isolated from an Escherichia coli strain MEN-1 is a plasmid-mediated beta-lactamase that confers resistance to methoxy imino third-generation cephalosporins. The protein purified to homogeneity was digested by trypsin, chymotrypsin and endoproteinase Asp-N. Amino acid sequence determinations of the resulting peptides gave rise to the alignment of the 263 residues of the beta-lactamase. From amino acid sequence comparison MEN-1 was found to share more than 72% identity with the chromosomally mediated beta-lactamases of Klebsiella oxytoca. Therefore, MEN-1 is the first transferable extended-spectrum beta-lactamase which is not directly derived from the widespread TEMs or SHV-1 penicillinases with which it presents less than 39% identity.
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653. |
Sota M,
Yano H,
Nagata Y,
Ohtsubo Y,
Genka H,
Anbutsu H,
Kawasaki H,
Tsuda M,
( 2006 ) Functional analysis of unique class II insertion sequence IS1071. PMID : 16391056 : DOI : 10.1128/AEM.72.1.291-297.2006 PMC : PMC1352228 Abstract >>
Various xenobiotic-degrading genes on many catabolic plasmids are often flanked by two copies of an insertion sequence, IS1071. This 3.2-kb IS element has long (110-bp) terminal inverted repeats (IRs) and a transposase gene that are phylogenetically related to those of the class II transposons. However, the transposition mechanism of IS1071 has remained unclear. Our study revealed that IS1071 was only able to transpose at high frequencies in two environmental beta-proteobacterial strains, Comamonas testosteroni and Delftia acidovorans, and not in any of the bacteria examined which belong to the alpha- and gamma-proteobacteria. IS1071 was found to have the functional features of the class II transposons in that (i) the final product of the IS1071 transposition was a cointegrate of its donor and target DNA molecules connected by two directly repeated copies of IS1071, one at each junction; (ii) a 5-bp duplication of the target sequence was observed at the insertion site; and (iii) a tnpA mutation of IS1071 was efficiently complemented by supplying the wild-type tnpA gene in trans. Deletion analysis of the IS1071 IR sequences indicated that nearly the entire region of the IRs was required for its transposition, suggesting that the interaction between the transposase and IRs of IS1071 might be different from that of the other well-characterized class II transposons.
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654. |
Sunde M,
( 2005 ) Prevalence and characterization of class 1 and class 2 integrons in Escherichia coli isolated from meat and meat products of Norwegian origin. PMID : 16239286 : DOI : 10.1093/jac/dki377 Abstract >>
The aim of the study was to investigate the prevalence of integrons and to characterize inserted gene cassettes in Escherichia coli isolated from meat and meat products of Norwegian origin. The strains investigated (n = 241 resistant out of 944 investigated) were collected within the frame of the Norwegian monitoring programme for antimicrobial resistance in bacteria from feed, food and animals (NORM-VET) during the years 2000-2003. PCR and DNA sequencing were used for detection of the integrase genes and gene cassettes within the integrons. Integrons were detected in 43 (18%) of the 241 resistant isolates. Class 1 integrons were detected in 29 (12%) strains and class 2 integrons were detected in 14 (6%) strains. Ten different gene cassettes were detected: dfrA1, dfr2a, dfrA12, aadA1, aadA2, catB2, oxa-30, sat, sat1 and orfF. The dfrA1 + aadA1 combination was the most prevalent cassette combination, detected in 12 of 29 class 1 integrons. Twelve (of 14) class 2 integrons contained a cassette area consistent with that on Tn7, the remaining two contained the cassettes sat + sat1 + aadA1. Nearly one-third of the class 1 integrons (9 of 29) lacked the sul1 gene. Ten gene cassettes (one dfr2a, two catB2 and seven aadA1) were expressed at levels below breakpoint values normally used to classify strains as resistant. Integrons of class 1 or 2 were present in approximately 18% of the resistant E. coli strains investigated. Certain cassette combinations in class 1 integrons seem to be more widespread than others, like the dfrA1 + aadA1. Low-level expression of antimicrobial resistance, caused by the expression of certain gene cassettes in some integrons represents an obstacle in classifying strains as susceptible or resistant.
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655. |
Johnson TJ,
Siek KE,
Johnson SJ,
Nolan LK,
( 2005 ) DNA sequence and comparative genomics of pAPEC-O2-R, an avian pathogenic Escherichia coli transmissible R plasmid. PMID : 16251312 : DOI : 10.1128/AAC.49.11.4681-4688.2005 PMC : PMC1280136 Abstract >>
In this study, a 101-kb IncF plasmid from an avian pathogenic Escherichia coli (APEC) strain (APEC O2) was sequenced and analyzed, providing the first completed APEC plasmid sequence. This plasmid, pAPEC-O2-R, has functional transfer and antimicrobial resistance-encoding regions. The resistance-encoding region encodes resistance to eight groups of antimicrobial agents, including silver and other heavy metals, quaternary ammonium compounds, tetracycline, sulfonamides, aminoglycosides, trimethoprim, and beta-lactam antimicrobial agents. This region of the plasmid is unique among previously described IncF plasmids in that it possesses a class 1 integron that harbors three gene cassettes and a heavy metal resistance operon. This region spans 33 kb and is flanked by the RepFII plasmid replicon and an assortment of plasmid maintenance genes. pAPEC-O2-R also contains a 32-kb transfer region that is nearly identical to that found in the E. coli F plasmid, rendering it transferable by conjugation to plasmid-less strains of bacteria, including an APEC strain, a fecal E. coli strain from an apparently healthy bird, a Salmonella enterica serovar Typhimurium strain, and a uropathogenic E. coli strain from humans. Differences in the G+C contents of individual open reading frames suggest that various regions of pAPEC-O2-R had dissimilar origins. The presence of pAPEC-O2-R-like plasmids that encode resistance to multiple antimicrobial agents and that are readily transmissible from APEC to other bacteria suggests the possibility that such plasmids may serve as a reservoir of resistance genes for other bacteria of animal and human health significance.
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656. |
Mukherjee S,
Chakraborty R,
( 2006 ) Incidence of class 1 integrons in multiple antibiotic-resistant Gram-negative copiotrophic bacteria from the River Torsa in India. PMID : 16239097 : DOI : 10.1016/j.resmic.2005.08.003 Abstract >>
The presence of class 1 integrons in multiple-antibiotic-resistant (MAR) Gram-negative copiotrophic bacteria from the River Torsa in India was detected using a polymerase chain reaction (PCR)-based screening method. Among 100 isolates that were resistant to at least five of the twelve antibiotics tested, 40 carried class 1 integrons, with inserted DNA regions of 0.7-3.2 kb. Carriage of integrons in strains of higher MAR index was found to be statistically significant. DNA sequencing was used to identify the genetic content of the integron-variable regions. In addition to the identification of gene cassettes dfrA1, dfrA5, dfrA7, dfrA17 and a variant of dfrA12 for trimethoprim, aac(6')-Ib for amikacin and tobramycin and aadA1 and aadA6 for streptomycin and spectinomycin resistance, a novel ORF predicted from a sequence of Morganella sp. TR 90 bearing homology with the Vibrio cholerae dfrA1 gene cassette was characterized. To our knowledge, this is the first report of the incidence and abundance of class 1 integrons in copiotrophic river water bacteria from India.
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657. |
Blanco M,
Schumacher S,
Tasara T,
Zweifel C,
Blanco JE,
Dahbi G,
Blanco J,
Stephan R,
( 2005 ) Serotypes, intimin variants and other virulence factors of eae positive Escherichia coli strains isolated from healthy cattle in Switzerland. Identification of a new intimin variant gene (eae-eta2). PMID : 15882459 : DOI : 10.1186/1471-2180-5-23 PMC : PMC1142320 Abstract >>
Enteropathogenic Escherichia coli (EPEC) and Shigatoxin-producing Escherichia coli (STEC) share the ability to introduce attaching-and-effacing (A/E) lesions on intestinal cells. The genetic determinants for the production of A/E lesions are located on the locus of enterocyte effacement (LEE), a pathogenicity island that also contains the genes encoding intimin (eae). This study reports information on the occurrence of eae positive E. coli carried by healthy cattle at the point of slaughter, and on serotypes, intimin variants, and further virulence factors of isolated EPEC and STEC strains. Of 51 eae positive bovine E. coli strains, 59% were classified as EPEC and 41% as STEC. EPEC strains belonged to 18 O:H serotypes, six strains to typical EPEC serogroups. EPEC strains harbored a variety of intimin variants with eae-beta1 being most frequently found. Moreover, nine EPEC strains harbored astA (EAST1), seven bfpA (bundlin), and only one strain was positive for the EAF plasmid. We have identified a new intimin gene (eta2) in three bovine bfpA and astA-positive EPEC strains of serotype ONT:H45. STEC strains belonged to seven O:H serotypes with one serotype (O103:H2) accounting for 48% of the strains. The majority of bovine STEC strains (90%) belonged to five serotypes previously reported in association with hemolytic uremic syndrom (HUS), including one O157:H7 STEC strain. STEC strains harbored four intimin variants with eae-epsilon1 and eae-gamma1 being most frequently found. Moreover, the majority of STEC strains carried only stx1 genes (13 strains), and was positive for ehxA (18 strains) encoding for Enterohemolysin. Four STEC strains showed a virulence pattern characteristic of highly virulent human strains (stx2 and eae positive). Our data confirm that ruminants are an important source of serologically and genetically diverse intimin-harboring E. coli strains. Moreover, cattle have not only to be considered as important asymptomatic carriers of O157 STEC but can also be a reservoir of EPEC and eae positive non-O157 STEC, which are described in association with human diseases.
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658. |
Power ML,
Littlefield-Wyer J,
Gordon DM,
Veal DA,
Slade MB,
( 2005 ) Phenotypic and genotypic characterization of encapsulated Escherichia coli isolated from blooms in two Australian lakes. PMID : 15819845 : DOI : 10.1111/j.1462-2920.2005.00729.x Abstract >>
Escherichia coli has long been used as an indicator organism for water quality assessment. Recently there has been an accumulation of evidence that suggests some strains of this organism are able to proliferate in the environment, a characteristic that would detract from its utility as an indicator of faecal pollution. Phenotypic and genotypic characterization of E. coli isolated from blooms in two Australian lakes, separated by a distance of approximately 200 km, identified that the blooms were dominated by three E. coli strains. A major phenotypic similarity among the three bloom strains was the presence of a group 1 capsule. Genetic characterization of a conserved region of the cps gene cluster, which encodes group 1 capsules, identified a high degree of genetic variation within the bloom isolates. This differs from previously described encapsulated E. coli strains which are highly conserved at the cps locus. The phenotypic or genotypic profiles of the bloom strains were not identified in 435 E. coli strains isolated from vertebrates. The occurrence of these encapsulated strains suggests that some E. coli have evolved a free-living lifestyle and do not require a host in order to proliferate. The presence of the same three strains in bloom events in different geographical regions of a temperate climate, and at different times, indicates that free-living E. coli strains are able to persist in these water reservoirs. This study provides further evidence of circumstances where caution is required in using E. coli as an indicator organism for water quality.
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659. |
Higgins JA,
Belt KT,
Karns JS,
Russell-Anelli J,
Shelton DR,
( 2005 ) tir- and stx-positive Escherichia coli in stream waters in a metropolitan area. PMID : 15870341 : DOI : 10.1128/AEM.71.5.2511-2519.2005 PMC : PMC1087540 Abstract >>
Diarrheagenic Escherichia coli, which may include the enteropathogenic E. coli and the enterohemorrhagic E. coli, are a significant cause of diarrheal disease among infants and children in both developing and developed areas. Disease outbreaks related to freshwater exposure have been documented, but the presence of these organisms in the urban aquatic environment is not well characterized. From April 2002 through April 2004 we conducted weekly surveys of streams in the metropolitan Baltimore, Md., area for the prevalence of potentially pathogenic E. coli by using PCR assays targeting the tir and stx(1) and stx(2) genes. Coliforms testing positive for the presence of the tir gene were cultured from 653 of 1,218 samples (53%), with a greater prevalence associated with urban, polluted streams than in suburban and forested watershed streams. Polluted urban streams were also more likely to test positive for the presence of one of the stx genes. Sequence analysis of the tir amplicon, as well as the entire tir gene from three isolates, indicated that the pathogenic E. coli present in the stream waters has a high degree of sequence homology with the E. coli O157:H7 serotype. Our data indicate that pathogenic E. coli are continually deposited into a variety of stream habitats and suggest that this organism may be a permanent member of the gastrointestinal microflora of humans and animals in the metropolitan Baltimore area.
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660. |
Robin F,
Delmas J,
Chanal C,
Sirot D,
Sirot J,
Bonnet R,
( 2005 ) TEM-109 (CMT-5), a natural complex mutant of TEM-1 beta-lactamase combining the amino acid substitutions of TEM-6 and TEM-33 (IRT-5). PMID : 16251281 : DOI : 10.1128/AAC.49.11.4443-4447.2005 PMC : PMC1280126 Abstract >>
Escherichia coli CF349 exhibited a complex beta-lactam resistance phenotype, including resistance to amoxicillin and ticarcillin alone and in combination with clavulanate and to some extended-spectrum cephalosporins. The double-disk synergy test was positive. CF349 harbored an 85-kb conjugative plasmid which encoded a beta-lactamase of pI 5.9. The corresponding bla gene was identified by PCR and sequencing as a bla(TEM) gene. The deduced protein sequence revealed a new complex mutant of TEM-1 beta-lactamase designated TEM-109 (CMT-5). TEM-109 contained both the substitutions Glu104Lys and Arg164His of the expanded-spectrum beta-lactamase (ESBL) TEM-6 and Met69Leu of the inhibitor-resistant TEM-33 (IRT-5). TEM-109 exhibited hydrolytic activity against ceftazidime similar to that of TEM-6 (k(cat), 56 s(-1) and 105 s(-1), respectively; K(m) values, 226 and 247 microM, respectively). The 50% inhibitory concentrations of clavulanate and tazobactam (0.13 microM and 0.27 microM, respectively) were 5- to 10-fold higher for TEM-109 than for TEM-6 (0.01 and 0.06 microM, respectively) but were almost 10-fold lower than those for TEM-33. The characterization of this novel CMT, which exhibits a low level of resistance to inhibitors, highlights the emergence of this new ESBL type.
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661. |
Beutin L,
Kaulfuss S,
Herold S,
Oswald E,
Schmidt H,
( 2005 ) Genetic analysis of enteropathogenic and enterohemorrhagic Escherichia coli serogroup O103 strains by molecular typing of virulence and housekeeping genes and pulsed-field gel electrophoresis. PMID : 15814965 : DOI : 10.1128/JCM.43.4.1552-1563.2005 PMC : PMC1081317 Abstract >>
We investigated the genetic relationships of 54 Escherichia coli O103 strains from humans, animals, and meat by molecular typing of housekeeping and virulence genes and by pulsed-field gel electrophoresis (PFGE). Multilocus sequence typing (MLST) of seven housekeeping genes revealed seven profiles, I through VII. MLST profiles I plus III cover 45 Shiga toxin-producing E. coli (STEC) O103:H2 strains from Australia, Canada, France, Germany, and Northern Ireland that are characterized by the intimin (eae) epsilon gene and carry enterohemorrhagic E. coli (EHEC) virulence plasmids. MLST profile II groups five human and animal enteropathogenic E. coli (EPEC) O103:H2 strains that were positive for intimin (eae) beta. Although strains belonging to MLST groups II and I plus III are closely related to each other (92.6% identity), major differences were found in the housekeeping icdA gene and in the virulence-associated genes eae and escD. E. coli O103 strains with MLST patterns IV to VII are genetically distant from MLST I, II, and III strains, as are the non-O103 E. coli strains EDL933 (O157), MG1655 (K-12), and CFT073 (O6). Comparison of MLST results with those of PFGE and virulence typing demonstrated that E. coli O103 STEC and EPEC have recently acquired different virulence genes and DNA rearrangements, causing alterations in their PFGE patterns. PFGE typing was very useful for identification of genetically closely related subgroups among MLST I strains, such as Stx2-producing STEC O103 strains from patients with hemolytic uremic syndrome. Analysis of virulence genes contributed to grouping of E. coli O103 strains into EPEC and STEC. Novel virulence markers, such as efa (EHEC factor for adherence), paa (porcine adherence factor), and cif (cell cycle-inhibiting factor), were found widely associated with E. coli O103 EPEC and STEC strains.
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662. |
Du X,
Shen Z,
Wu B,
Xia S,
Shen J,
( 2005 ) Characterization of class 1 integrons-mediated antibiotic resistance among calf pathogenic Escherichia coli. PMID : 15837385 : DOI : 10.1016/j.femsle.2005.03.021 Abstract >>
Escherichia coli isolates from calf diarrhea cases (n=22) in the Beijing surrounding region in China were characterized for disease serotype, virulence factors, antimicrobial susceptibility pattern and class 1 integrons. 59% (n=13) of the isolates were positive for the int I1 gene. The presence and genetic content of class 1 integrons in 13 E. coli isolates were examined by PCR and sequencing. Sequencing analysis revealed six gene cassettes, which encoded resistance to trimethoprim (dfrA1, dfrA17), aminoglycosides (aadB, aadA1 and aadA5) and chloramphenicol (cmlA). The gene cassette arrays dfrA1-orf (45%) and aadB-orf-cmlA (32%) were most prevalent among these isolates. These data revealed the high prevalence of class 1 integrons among calf pathogenic E. coli isolates in the Beijing surrounding region in China, which may provide important and useful surveillance information reflecting specific antibiotic selective pressure.
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663. |
Kashiwagi K,
Miyamoto S,
Suzuki F,
Kobayashi H,
Igarashi K,
( 1992 ) Excretion of putrescine by the putrescine-ornithine antiporter encoded by the potE gene of Escherichia coli. PMID : 1584788 : DOI : 10.1073/pnas.89.10.4529 PMC : PMC49116 Abstract >>
Excretion of putrescine from Escherichia coli was assessed by measuring its uptake into inside-out membrane vesicles. The vesicles were prepared from wild-type E. coli or E. coli transformed with plasmids containing one of the three polyamine transport systems. The results indicate that excretion of putrescine is catalyzed by the putrescine transport protein, encoded by the potE gene located at 16 min on the E. coli chromosome. Loading of ornithine (or lysine) inside the vesicles was essential for the uptake of putrescine, indicating that the protein exchanges putrescine and ornithine (or lysine) by an antiport mechanism. The Km and Vmax values for the putrescine uptake by inside-out membrane vesicles were 73 microM and 0.82 nmol/min per mg of protein, respectively. The antiport protein (potE protein) also catalyzed putrescine-putrescine and ornithine-ornithine exchange. The transport activity was not disturbed by inhibitors of energy production such as KCN and carbonyl cyanide m-chlorophenylhydrazone. When intact E. coli was used instead of the inside-out membrane vesicles, excretion of putrescine was also catalyzed by the antiport protein in the presence of ornithine in the medium.
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664. |
Chmelnitsky I,
Carmeli Y,
Leavitt A,
Schwaber MJ,
Navon-Venezia S,
( 2005 ) CTX-M-2 and a new CTX-M-39 enzyme are the major extended-spectrum beta-lactamases in multiple Escherichia coli clones isolated in Tel Aviv, Israel. PMID : 16251320 : DOI : 10.1128/AAC.49.11.4745-4750.2005 PMC : PMC1280129 Abstract >>
The rate of occurrence of the extended-spectrum beta-lactamase (ESBL)-producing phenotype among Escherichia coli isolates in Tel Aviv is 12% (22). The aim of this study was to understand the molecular epidemiology of E. coli ESBL producers and to identify the ESBL genes carried by them. We studied 20 single-patient ESBL-producing E. coli clinical isolates. They comprised 11 distinct nonrelated pulsed-field gel electrophoresis (PFGE) genotypes: six isolates belonged to the same PFGE clone, four other clones included two isolates each, and six unrelated clones included only one isolate. All isolates produced various beta-lactamases with pIs ranging from 5.2 to 8.2, varying within similar PFGE clones. The most prevalent ESBL gene was bla(CTX-M); 16 isolates carried bla(CTX-M-2) and three carried a new ESBL gene designated bla(CTX-M-39). Three strains carried bla(SHV) (two bla(SHV-12) and one bla(SHV-5)), and two strains carried inhibitor-resistant ESBL genes, bla(TEM-33) and bla(TEM-30); 18 strains carried bla(TEM-1) and eight strains carried bla(OXA-2). Plasmid mapping and Southern blot analysis with a CTX-M-2 probe demonstrated that bla(CTX-M-2) is plasmid borne. The wide dissemination of ESBLs among E. coli isolates in our institution is partly related to clonal spread, but more notably to various plasmid-associated ESBL genes, occurring in multiple clones, wherein the CTX-M gene family appears almost uniformly. We report here a new CTX-M gene, designated bla(CTX-M-39), which revealed 99% homology with bla(CTX-M-26), with a substitution of arginine for glutamine at position 225.
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665. |
Chen Y,
Delmas J,
Sirot J,
Shoichet B,
Bonnet R,
( 2005 ) Atomic resolution structures of CTX-M beta-lactamases: extended spectrum activities from increased mobility and decreased stability. PMID : 15811373 : DOI : 10.1016/j.jmb.2005.02.010 Abstract >>
Extended spectrum beta-lactamases (ESBLs) confer bacterial resistance to third-generation cephalosporins, such as cefotaxime and ceftazidime, increasing hospital mortality rates. Whereas these antibiotics are almost impervious to classic beta-lactamases, such as TEM-1, ESBLs have one to four orders greater activity against them. The origins of this activity have been widely studied for the TEM and SHV-type ESBLs, but have received less attention for the CTX-M beta-lactamases, an emerging family that is now the dominant ESBL in several regions. To understand how CTX-M beta-lactamases achieve their remarkable activity, biophysical and structural studies were undertaken. Using reversible, two-state thermal denaturation, it was found that as these enzymes evolve a broader substrate range, they sacrifice stability. Thus, the mutant enzyme CTX-M-16 is eightfold more active against ceftazidime than the pseudo-wild-type CTX-M-14 but is 1.9 kcal/mol less stable. This is consistent with a "stability-activity tradeoff," similar to that observed in the evolution of other resistance enzymes. To investigate the structural basis of enzyme activity and stability, the structures of four CTX-M enzymes were determined by X-ray crystallography. The structures of CTX-M-14, CTX-M-27, CTX-M-9 and CTX-M-16 were determined to 1.10 Angstroms, 1.20 Angstroms, 0.98 Angstroms and 1.74 Angstroms resolution, respectively. The enzyme active sites resemble those of the narrow-spectrum TEM-1 and SHV-1, and not the enlarged sites typical of ESBL mutants such as TEM-52 and TEM-64. Instead, point substitutions leading to specific interactions may be responsible for the improved activity against ceftazidime and cefotaxime, consistent with observations first made for the related Toho-1 enzyme. The broadened substrate range of CTX-M-16 may result from coupled defects in the enzyme's B3 strand, which lines the active site. Substitutions Val231-->Ala and Asp240-->Gly, which convert CTX-M-14 into CTX-M-16, occur at either end of this strand. These defects appear to increase the mobility of B3 based on anisotropic B-factor analyses at ultrahigh resolution, consistent with stability loss and activity gain. The unusually high resolution of these structures that makes such analyses possible also makes them good templates for inhibitor discovery.
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666. |
Miró E,
Mirelis B,
Navarro F,
Rivera A,
Mesa RJ,
Roig MC,
Gómez L,
Coll P,
( 2005 ) Surveillance of extended-spectrum beta-lactamases from clinical samples and faecal carriers in Barcelona, Spain. PMID : 16244084 : DOI : 10.1093/jac/dki395 Abstract >>
The aim of the present study was to characterize and compare the extended-spectrum beta-lactamase (ESBL)-producing organisms isolated from clinical samples and faecal carriers in 2001 and 2002. A total of 5251 Enterobacteriaceae isolated from clinical samples and 1321 stool samples were evaluated for the presence of ESBLs. The stool samples were spread onto plates of MacConkey agar containing 2 mg/L cefotaxime for selection of ESBL-producing strains. These strains were defined as those showing synergism between amoxicillin/clavulanic acid and third-generation cephalosporins. The beta-lactamases involved were characterized by isoelectric focusing, PCR assays and DNA sequencing. The prevalence of ESBL-producing strains among clinical Enterobacteriaceae was 1.7%. Of these, 87.6% produced CTX-M, 25.8% produced SHV and 2.2% were TEM-type-producing strains. All clinical ESBL-producing strains were Escherichia coli, with the exception of four Klebsiella pneumoniae and one Citrobacter freundii. The prevalence of faecal carriage of ESBL-producing organisms was 3.3%. Of these, 75% produced CTX-M-type enzymes followed by 22.7% SHV-producing strains. All faecal ESBL-producing strains were E. coli except for one Enterobacter cloacae and one Proteus mirabilis. This latter strain produced the PER-1 enzyme reported for the first time in Spain. The prevalence of ESBL-producing strains in stool samples was higher than that observed in clinical samples from the same period. The different types of ESBLs found were similar in both contexts. The most prevalent ESBLs were the CTX-M-related enzymes, with nine different types, followed by SHV-12.
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667. |
Boerlin P,
Travis R,
Gyles CL,
Reid-Smith R,
Janecko N,
Lim H,
Nicholson V,
McEwen SA,
Friendship R,
Archambault M,
( 2005 ) Antimicrobial resistance and virulence genes of Escherichia coli isolates from swine in Ontario. PMID : 16269706 : DOI : 10.1128/AEM.71.11.6753-6761.2005 PMC : PMC1287655 Abstract >>
A total of 318 Escherichia coli isolates obtained from diarrheic and healthy pigs in Ontario from 2001 to 2003 were examined for their susceptibility to 19 antimicrobial agents. They were tested by PCR for the presence of resistance genes for tetracycline, streptomycin, sulfonamides, and apramycin and of 12 common virulence genes of porcine E. coli. Antimicrobial resistance frequency among E. coli isolates from swine in Ontario was moderate in comparison with other countries and was higher in isolates from pigs with diarrhea than in isolates from healthy finisher pigs. Resistance profiles suggest that cephamycinases may be produced by > or = 8% of enterotoxigenic E. coli (ETEC). Resistance to quinolones was detected only in enterotoxigenic E. coli (< or = 3%). The presence of sul3 was demonstrated for the first time in Canada in porcine E. coli isolates. Associations were observed among tetA, sul1, aadA, and aac(3)IV and among tetB, sul2, and strA/strB, with a strong negative association between tetA and tetB. The paa and sepA genes were detected in 92% of porcine ETEC, and strong statistical associations due to colocation on a large plasmid were observed between tetA, estA, paa, and sepA. Due at least in part to gene linkages, the distribution of resistance genes was very different between ETEC isolates and other porcine E. coli isolates. This demonstrates that antimicrobial resistance epidemiology differs significantly between pathogenic and commensal E. coli isolates. These results may have important implications with regards to the spread and persistence of resistance and virulence genes in bacterial populations and to the prudent use of antimicrobial agents.
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668. |
Benz I,
Schmidt MA,
( 1992 ) AIDA-I, the adhesin involved in diffuse adherence of the diarrhoeagenic Escherichia coli strain 2787 (O126:H27), is synthesized via a precursor molecule. PMID : 1625582 : DOI : 10.1111/j.1365-2958.1992.tb00875.x Abstract >>
The adherence mechanisms of enteropathogenic Escherichia coli (EPEC) to epithelial cells are still not understood. To study the molecular basis of the diffuse adherence (DA) phenotype exhibited by diarrhoeagenic E. coli expressing classical EPEC serotypes we investigated strain 2787 (O126:H27) isolated from a case of infantile diarrhoea. A 6.0 kb plasmid-derived DNA fragment mediates the DA phenotype and encodes the 100 kDa adhesin protein AIDA-I (adhesin involved in diffuse adherence). Sequencing of the entire fragment revealed two open reading frames which encoded proteins of 45 kDa and 132 kDa, respectively. The 132 kDa protein has been identified as an AIDA-I precursor protein. After cleavage of the signal sequence further processing at the C-terminus of the 132 kDa precursor leads to the mature approximately 100 kDa AIDA-I. While the exact function of the cytoplasmic 45 kDa protein is not known, preliminary evidence indicates that it is necessary for the correct maturation of AIDA-I. The AIDA-I precursor exhibits significant homology with the virG(icsA) protein of Shigella flexneri which seems to be involved in the intercellular spread of invasive Shigella organisms.
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669. |
Murtaza I,
Dutt A,
Mushtaq D,
Ali A,
( 2005 ) Molecular cloning and genetic analysis of functional merB gene from indian isolates of Escherichia coli. PMID : 16211434 : DOI : 10.1007/s00284-005-0013-2 Abstract >>
Studies were carried out to characterize organomercurial lyase genes from wild type mercury-resistant Escherichia coli isolates, previously collected from five geographically distinct regions of the Indian subcontinent. PCR amplification followed by DNA sequencing of amplified fragments showed three merB identical to the previously characterized mer B from E. coli pR831b that were thus considered as the same gene. The remaining two genes derived from E. coli isolates of an almost mercury-free site (Dal lake, Kashmir) and designated as pIAAD3 merB and pIAAD14 merB showed slight variation (2%) at base. However, this variation in pIAAD3 due to the absence of base "T" at 479 position results in complete frame shift and the predicted MerB-like polypeptide derived from it showed 21.53% divergent at its C terminal end from the previously characterized pR831b MerB. The expression profile of pIAAD3 merB in pQE30 and pUC18 vectors each demonstrated 22.2 kDa proteins. The induced DH5alpha E. coli cells possessing pIAAD3 merB cloned in pUC18 vector split phenyl mercuric acetate (PMA) into benzene and inorganic mercury efficiently, thus giving a clue that the expressed gene product is biologically active. The current study suggests that such genetic changes may take place in the continued absence of mercury pressure, and with such modifications, they finally break down to act as vestigial remnants. Further work is going on in our lab to exploit pIAAD3 merB for the bioremediation of mercury-polluted sites.
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670. |
Olasz F,
Fekete PZ,
Blum-Oehler G,
Boldogkoi Z,
Nagy B,
( 2005 ) Characterization of an F18+ enterotoxigenic Escherichia coli strain from post weaning diarrhoea of swine, and of its conjugative virulence plasmid pTC. PMID : 15766780 : DOI : 10.1016/j.femsle.2005.01.057 Abstract >>
The enterotoxigenic Escherichia coli (ETEC) strain Ec2173, causing post weaning diarrhoea in swine, harbours six plasmids ranging from 13 to 200 kb in size. The heat stable toxin genes sta, stb and a tetracycline resistance gene were located on a self conjugative 120-kb plasmid, called pTC. In the cloned ColE1 type origin of replication of pTC a deletion was detected compared to other ColE1 replicons affecting the replication modulator gene rom. Epidemiological studies on ETEC isolates showed that pTC-like plasmids are widely distributed among porcine ETEC strains; thus representing an example of co-evolution of antibacterial resistance and virulence in pathogenic E. coli.
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671. |
García A,
Navarro F,
Miró E,
Mirelis B,
Campoy S,
Coll P,
( 2005 ) Characterization of the highly variable region surrounding the bla(CTX-M-9) gene in non-related Escherichia coli from Barcelona. PMID : 16188915 : DOI : 10.1093/jac/dki345 Abstract >>
The dispersion of a clone, a plasmid or a mobile element carrying the bla(CTX-M-9) gene was evaluated in 30 Escherichia coli strains isolated in Barcelona between 1996 and 1999. The presence of the previously described orf513-bearing class 1 integron, In60, carrying the bla(CTX-M-9) gene, was also studied. The clonality was analysed by pulsed-field gel electrophoresis. Plasmid analysis was performed by S1 digestion and hybridization with the CTX-M-9 probe. PCR mapping using specific designed primers was used to study the presence of In60 and In60-like structures. The clonality between the 30 strains was minor. The size of bla(CTX-M-9) carrying plasmids ranged between approximately 80 and 430 kb. One strain produced only a chromosome-encoded CTX-M-9 beta-lactamase. Thirty-six per cent of the strains showed differences with respect to the In60 structure due to an insertion or deletion events. These findings suggest that the bla(CTX-M-9) gene may be carried by a mobile element that disperses it between plasmids. The fast dispersion of the CTX-M-9 enzyme could therefore be due to both diffusion of plasmids and mobile elements.
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672. |
Germon P,
Chen YH,
He L,
Blanco JE,
Brée A,
Schouler C,
Huang SH,
Moulin-Schouleur M,
( 2005 ) ibeA, a virulence factor of avian pathogenic Escherichia coli. PMID : 15817785 : DOI : 10.1099/mic.0.27809-0 Abstract >>
The presence of ibeA, a gene encoding a known virulence factor of Escherichia coli strains responsible for neonatal meningitis in humans, was investigated in the genome of 213 avian pathogenic E. coli (APEC) strains and 55 non-pathogenic E. coli strains of avian origin. Fifty-three strains were found to be ibeA(+), all of which belonged to the APEC group. The ibeA gene is therefore positively linked to the pathogenicity of strains (P<0.0001). Analysis of the serogroup of strains revealed a positive association of ibeA with serogroups O18, O88 and O2. On the contrary, only 1/59 O78 strains are ibeA(+), indicating a negative association of ibeA with this serogroup (P<0.0001). The role of ibeA in the virulence of the APEC strain BEN 2908 was investigated by constructing an ibeA mutant. Challenge assays on 3-week-old chickens showed a reduced virulence for the ibeA mutant. Furthermore, the APEC strain BEN 2908 was able to invade brain microvascular epithelial cells, this invasion being significantly reduced upon inactivation of ibeA. Altogether, these results suggest a role of ibeA in the pathogenicity of some APEC strains and confirm the close relationship between APEC and other human extraintestinal pathogenic E. coli isolates.
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673. |
Kusumoto M,
Nishiya Y,
Kawamura Y,
Shinagawa K,
( 1999 ) Identification of an insertion sequence, IS1203 variant, in a Shiga toxin 2 gene of Escherichia coli O157:H7. PMID : 16232431 : Abstract >>
An insertion sequence composed of 1310 bp was found in Shiga toxin 2 genes of some isolates of Escherichia coli O157:H7. This insertion sequence showed extremely high homology with IS1203 of E. coli O111:H(-). This IS1203 variant was inserted in the region encoding the amino-terminus of the B subunit with a duplication of 3 bp at the target site, resulting in inactivation of the Shiga toxin 2 gene.
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674. |
Garmendia J,
Ren Z,
Tennant S,
Midolli Viera MA,
Chong Y,
Whale A,
Azzopardi K,
Dahan S,
Sircili MP,
Franzolin MR,
Trabulsi LR,
Phillips A,
Gomes TA,
Xu J,
Robins-Browne R,
Frankel G,
( 2005 ) Distribution of tccP in clinical enterohemorrhagic and enteropathogenic Escherichia coli isolates. PMID : 16272509 : DOI : 10.1128/JCM.43.11.5715-5720.2005 PMC : PMC1287796 Abstract >>
Enterohemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC) are diarrheagenic pathogens that colonize the gut through the formation of attaching and effacing lesions, which depend on the translocation of effector proteins via a locus of enterocyte effacement-encoded type III secretion system. Recently, two effector proteins, EspJ and TccP, which are encoded by adjacent genes on prophage CP-933U in EHEC O157:H7, have been identified. TccP consists of a unique N-terminus region and several proline-rich domains. In this project we determined the distribution of tccP in O157:H7, in non-O157 EHEC, and in typical and atypical EPEC isolates. All the EHEC O157:H7 strains tested were tccP(+). Unexpectedly, tccP was also found in non-O157 EHEC, and in typical and atypical EPEC isolates, particularly in strains belonging to serogroups O26 (EHEC), O119 (typical EPEC), and O55 (atypical EPEC). We recorded some variation in the length of tccP, which reflects diversity in the number of the proline-rich repeats. These results show the existence of a class of "attaching and effacing" pathogens which express a combination of EPEC and EHEC virulence determinants.
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675. |
Pertzev AV,
Ruban NM,
Zakharova MV,
Beletzkaja IV,
Petrov SI,
Kravetz AN,
Solonin AS,
( 1992 ) Eco29kI, a novel plasmid encoded restriction endonuclease from Escherichia coli. PMID : 1579502 : DOI : 10.1093/nar/20.8.1991 PMC : PMC312317 Abstract >>
N/A
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676. |
Chen Y,
Shoichet B,
Bonnet R,
( 2005 ) Structure, function, and inhibition along the reaction coordinate of CTX-M beta-lactamases. PMID : 15826180 : DOI : 10.1021/ja042850a PMC : PMC1360657 Abstract >>
CTX-M enzymes are an emerging group of extended spectrum beta-lactamases (ESBLs) that hydrolyze not only the penicillins but also the first-, second-, and third-generation cephalosporins. Although they have become the most frequently observed ESBLs in certain areas, there are few effective inhibitors and relatively little is known about their detailed mechanism. Here we describe the X-ray crystal structures of CTX-M enzymes in complex with different transition-state analogues and beta-lactam inhibitors, representing the enzyme as it progresses from its acylation transition state to its acyl enzyme complex to the deacylation transition state. As the enzyme moves along this reaction coordinate, two key catalytic residues, Lys73 and Glu166, change conformations, tracking the state of the reaction. Unexpectedly, the acyl enzyme complex with the beta-lactam inhibitor cefoxitin still has the catalytic water bound; this water had been predicted to be displaced by the unusual 7alpha-methoxy of the inhibitor. Instead, the 7alpha-group appears to inhibit by preventing the formation of the deacylation transition state through steric hindrance. From an inhibitor design standpoint, we note that the best of the reversible inhibitors, a ceftazidime-like boronic acid compound, binds to CTX-M-16 with a K(i) value of 4 nM. When used together in cell culture, this inhibitor reversed cefotaxime resistance in CTX-M-producing bacteria. The structure of its complex with CTX-M enzyme and the structural view of the reaction coordinate described here provide templates for inhibitor design and intervention to combat this family of antibiotic resistance enzymes.
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677. |
Pimenta AL,
Racher K,
Jamieson L,
Blight MA,
Holland IB,
( 2005 ) Mutations in HlyD, part of the type 1 translocator for hemolysin secretion, affect the folding of the secreted toxin. PMID : 16237030 : DOI : 10.1128/JB.187.21.7471-7480.2005 PMC : PMC1272971 Abstract >>
HlyD, a member of the membrane fusion protein family, is essential for the secretion of the RTX hemolytic toxin HlyA from Escherichia coli. Random point mutations affecting HlyA secretion were obtained, distributed in most periplasmic regions of the HlyD molecule. Analysis of the secretion phenotypes of different mutants allowed the identification of regions in HlyD involved in different steps of HlyA translocation. Four mutants, V349-I, T85-I, V334-I and L165-Q, were conditionally defective, a phenotype shown to be linked to the presence of inhibitory concentrations of Ca2+ in extracellular medium. Hly mutant T85-I was defective at an early stage in secretion, while mutants V334-I and L165-Q appeared to accumulate HlyA in the cell envelope, indicating a block at an intermediate step. Mutants V349-I, V334-I, and L165-Q were only partially defective in secretion, allowing significant levels of HlyA to be transported, but in the case of V349-I and L165-Q the HlyA molecules secreted showed greatly reduced hemolytic activity. Hemolysin molecules secreted from V349-I and V334-I are defective in normal folding and can be reactivated in vitro to the same levels as HlyA secreted from the wild-type translocator. Both V349-I and V334-I mutations mapped to the C-terminal lipoyl repeat motif, involved in the switching from the helical hairpin to the extended form of HlyD during assembly of the functional transport channel. These results suggest that HlyD is an integral component of the transport pathway, whose integrity is essential for the final folding of secreted HlyA into its active form.
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678. |
Nemoy LL,
Kotetishvili M,
Tigno J,
Keefer-Norris A,
Harris AD,
Perencevich EN,
Johnson JA,
Torpey D,
Sulakvelidze A,
Morris JG,
Stine OC,
( 2005 ) Multilocus sequence typing versus pulsed-field gel electrophoresis for characterization of extended-spectrum beta-lactamase-producing Escherichia coli isolates. PMID : 15814998 : DOI : 10.1128/JCM.43.4.1776-1781.2005 PMC : PMC1081380 Abstract >>
Extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli strains are emerging pathogens. Molecular typing of ESBL-producing E. coli is useful for surveillance purposes, to monitor outbreaks and track nosocomial spread. Although pulsed-field gel electrophoresis (PFGE) is the current "gold standard" for bacterial molecular typing, multilocus sequence typing (MLST) may offer advantages. Forty ESBL-producing E. coli isolates were selected at random from a cohort of intensive care unit patients who had active surveillance perirectal cultures done. PFGE identified 19 unique PFGE types (PT) among the 40 isolates; MLST identified 22 unique sequence types. MLST had greater discriminatory ability than PFGE for ESBL-producing E. coli. Simpson's indices of diversity for PFGE and MLST were 0.895 and 0.956, respectively. There were five clonal complexes (CCs) (isolates with differences of no more than two loci) that each contained multiple PT, but each PT was found in only one CC, indicating genetic consistency within a CC. MLST has clear utility in studies of ESBL-producing E. coli, based on a greater discriminatory ability and reproducibility than PFGE and the ability to a priori define genetically related bacterial strains.
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679. |
Delmas J,
Robin F,
Bittar F,
Chanal C,
Bonnet R,
( 2005 ) Unexpected enzyme TEM-126: role of mutation Asp179Glu. PMID : 16189109 : DOI : 10.1128/AAC.49.10.4280-4287.2005 PMC : PMC1251537 Abstract >>
The clinical isolate Escherichia coli CF884 exhibited low-level resistance to ceftazidime (4 mug/ml) by a positive double-disk synergy test and apparent susceptibility to cefuroxime, cefotaxime, cefepime, cefpirome, and aztreonam. The enzyme implicated in this phenotype was a novel 180-kb plasmid-encoded TEM-type extended-spectrum beta-lactamase designated TEM-126 which harbors the mutations Asp179Glu and Met182Thr. TEM-126 exhibited significant hydrolytic activity (k(cat), 2 s(-1)) and a K(m) value of 82 muM against ceftazidime. Molecular dynamics simulations suggested that the substitution Asp179Glu induces subtle conformational changes to the omega loop which may favor the insertion of ceftazidime in the binding site and the correct positioning of the crucial residue Glu166. Overall, these results highlight the remarkable plasticity of TEM enzymes, which can expand their activity against ceftazidime by the addition of one carbon atom in the side chain of residue 179.
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680. |
Beutin L,
Kong Q,
Feng L,
Wang Q,
Krause G,
Leomil L,
Jin Q,
Wang L,
( 2005 ) Development of PCR assays targeting the genes involved in synthesis and assembly of the new Escherichia coli O 174 and O 177 O antigens. PMID : 16207976 : DOI : 10.1128/JCM.43.10.5143-5149.2005 PMC : PMC1248525 Abstract >>
Escherichia coli O 174 and O 177 are newly described O serogroups which were reported as human pathogens. Identification of these strains by serotyping has been restricted, as the required sera are not commercially available. In this study, a collection of 13 E. coli O 174 strains and 12 E. coli O 177 strains was studied on the O:H serotypes and virulence markers. The O-antigen gene clusters of E. coli O 174 and O 177 were sequenced, and associated genes were assigned functions on the basis of homology. Two genes, each specific for E. coli O 174 and O 177, were identified. PCR assays based on the O-antigen-specific genes were developed and tested on 25 clinical and environmental isolates of those two serogroups as well as 26 isolates of other O serogroups. As little as 1 pg per mul of chromosomal DNA and as few as 0.1 CFU per g of pork and water samples were detected for either strain. The PCR assays established in this study were shown to be highly sensitive and reliable and could be the method of choice for detection of these two human pathogens from clinical, food, and other environmental samples.
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681. |
Tracz DM,
Boyd DA,
Bryden L,
Hizon R,
Giercke S,
Van Caeseele P,
Mulvey MR,
( 2005 ) Increase in ampC promoter strength due to mutations and deletion of the attenuator in a clinical isolate of cefoxitin-resistant Escherichia coli as determined by RT-PCR. PMID : 15761065 : DOI : 10.1093/jac/dki074 Abstract >>
To characterize the mechanism of cefoxitin resistance in clinical isolate Escherichia coli N99-0001. Plasmid analysis, PCR for beta-lactamases, and sequencing of the ampC genes was carried out. An RT-PCR method was developed to determine relative ampC expression. Analysis of the ampC promoter region of E. coli N99-0001 revealed a T-->A mutation at -32, a C-->A mutation at -11, an insertion of a T between -20 and -21, and a 28 bp deletion including the entire attenuator. RT-PCR showed that ampC was expressed 140-fold higher in E. coli N99-0001 than in E. coli ATCC 25922. Cefoxitin resistance in E. coli N99-0001 was due to overexpression of ampC caused by an increase in promoter strength.
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682. |
Batchelor M,
Clifton-Hadley FA,
Stallwood AD,
Paiba GA,
Davies RH,
Liebana E,
( 2005 ) Detection of multiple cephalosporin-resistant Escherichia coli from a cattle fecal sample in Great Britain. PMID : 15770096 : DOI : 10.1089/mdr.2005.11.58 Abstract >>
We describe the isolation of multiple cephalosporin-resistant Escherichia coli from cattle feces collected from animals at slaughter in Great Britain. Six E. coli strains were isolated with distinct XbaI pulsed-field gel electrophoresis (PFGE) profiles and different mechanisms of cephalosporin resistance from a single fecal sample. Two of these strains were found to contain conjugative plasmids conferring resistance to extended-spectrum cephalosporins that were indistinguishable from each other by restriction endonuclease digestion. Sequence analysis of the plasmid-encoded ampC showed that they were identical to bla(CMY-2), previously described in multiple-drug-resistant Salmonella and E. coli from animals in other parts of the world. DNA sequence analysis of the chromosomal ampC promoter regions for three cephalosporin-resistant strains lacking CMY-2 was determined. Several mutations were detected in the isolates tested including changes at positions -42 and -32, which are known to increase promoter strength. This report represents the first isolation of E. coli containing bla(CMY-2) from cattle in Great Britain, and, also to our knowledge, the first demonstration of multiple cephalosporin-resistant strains in a single animal.
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683. |
Feng L,
Han W,
Wang Q,
Bastin DA,
Wang L,
( 2005 ) Characterization of Escherichia coli O86 O-antigen gene cluster and identification of O86-specific genes. PMID : 15778030 : DOI : 10.1016/j.vetmic.2004.12.021 Abstract >>
Escherichia coli O86 belongs to the enteropathogenic E. coli (EPEC) group, some strains of which are pathogens of humans, wild birds and farm animals. The O-antigen gene cluster of E. coli O86 was amplified by long-range PCR using primers based on the housekeeping genes galF and gnd, and then sequenced. Genes involved in GDP-Fuc and N-acetyl-galactosamine (GalNAc) synthesis and genes encoding glycosyltransferases, O-unit flippase and O-antigen polymerase were identified on the basis of homology. By screening against 186 E. coli and Shigella-type strains, two genes specific to E. coli O86 were identified. A polymerase chain reaction (PCR) assay, based on the specific O-antigen genes identified here, could be used for the rapid detection of E. coli O86 in environmental and clinical samples. The relationship between E. coli O86 and O127 was also determined by comparing the two O-antigen gene clusters.
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684. |
Gilmour MW,
Cote T,
Munro J,
Chui L,
Wylie J,
Isaac-Renton J,
Horsman G,
Tracz DM,
Andrysiak A,
Ng LK,
( 2005 ) Multilocus sequence typing of Escherichia coli O26:H11 isolates carrying stx in canada does not identify genetic diversity. PMID : 16208008 : DOI : 10.1128/JCM.43.10.5319-5323.2005 PMC : PMC1248460 Abstract >>
Multilocus sequence typing of 31 stx-carrying Escherichia coli O26:H11 strains isolated in Canada between 1999 and 2003 revealed a high degree of genetic relatedness at 10 loci, suggesting either that this is a clonal serotype (similar to O157:H7) or that additional genetic loci need to be examined.
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685. |
Otto BR,
Sijbrandi R,
Luirink J,
Oudega B,
Heddle JG,
Mizutani K,
Park SY,
Tame JR,
( 2005 ) Crystal structure of hemoglobin protease, a heme binding autotransporter protein from pathogenic Escherichia coli. PMID : 15728184 : DOI : 10.1074/jbc.M412885200 Abstract >>
The acquisition of iron is essential for the survival of pathogenic bacteria, which have consequently evolved a wide variety of uptake systems to extract iron and heme from host proteins such as hemoglobin. Hemoglobin protease (Hbp) was discovered as a factor involved in the symbiosis of pathogenic Escherichia coli and Bacteroides fragilis, which cause intra-abdominal abscesses. Released from E. coli, this serine protease autotransporter degrades hemoglobin and delivers heme to both bacterial species. The crystal structure of the complete passenger domain of Hbp (110 kDa) is presented, which is the first structure from this class of serine proteases and the largest parallel beta-helical structure yet solved.
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686. |
Toma C,
Higa N,
Iyoda S,
Rivas M,
Iwanaga M,
( 2006 ) The long polar fimbriae genes identified in Shiga toxin-producing Escherichia coli are present in other diarrheagenic E. coli and in the standard E. coli collection of reference (ECOR) strains. PMID : 16125910 : DOI : 10.1016/j.resmic.2005.06.009 Abstract >>
Long polar fimbriae (LPF) are related to type I fimbriae in genetic organization and were first identified in Salmonella enterica serovar Typhimurium. Four lpfA genetic variants designated lpfA(O157/OI-141), lpfA(O157/OI-154), lpfA(O26) and lpfA(O113) have been identified in Shiga toxin-producing Escherichia coli (STEC). In this study, PCR was employed to determine the distribution of STEC-lpfAs in enteropathogenic, enteroaggregative, enterotoxigenic and enteroinvasive E. coli (EPEC, EAEC, ETEC and EIEC) and in the standard E. coli collection of reference (ECOR). Among the 97 diarrheagenic strains from our collection, only 2 EPEC strains of serotypes O55:H7 and O119:NM were positive for both lpfA(O157/OI-141) and lpfA(O157/OI-154). lpfA(O157/OI-141) was also positive in 1 of 25 ETEC strains. lpfA(O113) was present in 51 of 97 strains and lpfA(O26) in 13 of 97 strains of diverse diarrheagenic categories. STEC-lpfAs were also present in non-pathogenic ECOR strains of all phylogenetic groups. This study showed that the lpfA genes identified in the genome of STEC strains are not specific to this category. Our results suggest that there is a relationship between the lpfA variant and the phylogenetic group.
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687. |
Froehlich B,
Parkhill J,
Sanders M,
Quail MA,
Scott JR,
( 2005 ) The pCoo plasmid of enterotoxigenic Escherichia coli is a mosaic cointegrate. PMID : 16159784 : DOI : 10.1128/JB.187.18.6509-6516.2005 PMC : PMC1236633 Abstract >>
CS1 is the prototype of a class of pili of enterotoxigenic Escherichia coli (ETEC) associated with diarrheal disease in humans. The genes encoding this pilus are carried on a large plasmid, pCoo. We report the sequence of the complete 98,396-bp plasmid. Like many other virulence plasmids, pCoo is a mosaic consisting of regions derived from multiple sources. Complete and fragmented insertion sequences (IS) make up 24% of the total DNA and are scattered throughout the plasmid. The pCoo DNA between these IS elements has a wide range of G+C content (35 to 57%), suggesting that these regions have different ancestries. We find that the pCoo plasmid is a cointegrate of two functional replicons, related to R64 and R100, which are joined at a 1,953-bp direct repeat of IS100. Recombination between these repeats in the cointegrate generates the two smaller replicons which coexist with the cointegrate in the culture. Both of the smaller replicons have plasmid stability genes as well as genes that may be important in pathogenesis. Examination by PCR of 17 other unrelated CS1 ETEC strains with a variety of serotypes demonstrated that all contained at least parts of both replicons of pCoo and that strains of the O6 genotype appear to contain a cointegrate very similar to pCoo. The results suggest that this family of CS1-encoding plasmids is evolving rapidly.
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688. |
Ishii Y,
Kimura S,
Alba J,
Shiroto K,
Otsuka M,
Hashizume N,
Tamura K,
Yamaguchi K,
( 2005 ) Extended-spectrum beta-lactamase-producing Shiga toxin gene (Stx1)-positive Escherichia coli O26:H11: a new concern. PMID : 15750063 : PMC : PMC1081271 DOI : 10.1128/JCM.43.3.1072-1075.2005 Abstract >>
Escherichia coli strain TUM2139 was isolated from a stool sample from a 9-year-old girl on 16 June 2004. This strain was categorized as Shiga toxin-producing Escherichia coli (STEC) because the Shiga-like toxin gene stx(1) was detected by immunochromatography and PCR assay. The strain was highly resistant to cefotaxime (256 microg/ml) and was also resistant to cefepime, cefpodoxime, ceftriaxone, and aztreonam. In the presence of 4 microg of clavulanic acid per ml, the MIC of cefotaxime decreased to < or =0.12 microg/ml, indicating that this strain was an extended-spectrum beta-lactamase (ESBL) producer. Cefotaxime resistance was transferred to E. coli C600 by conjugation at a frequency of 3.0 x 10(-6). A PCR assay was performed with primer sets specific for TEM-type and SHV-type ESBLs and for the CTX-M-2 (Toho-1), CTX-M-3, and CTX-M-9 groups of ESBLs. A specific signal was observed with the primer set specific for the CTX-M-9 group of beta-lactamases. This beta-lactamase was confirmed to be the ESBL CTX-M-18 by DNA sequencing. This is the first report of an ESBL-producing STEC isolate.
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689. |
Fratamico PM,
DebRoy C,
Strobaugh TP,
Chen CY,
( 2005 ) DNA sequence of the Escherichia coli O103 O antigen gene cluster and detection of enterohemorrhagic E. coli O103 by PCR amplification of the wzx and wzy genes. PMID : 16121232 : DOI : 10.1139/w05-049 Abstract >>
Escherichia coli serogroup O103 has been associated with gastrointestinal illness and hemolytic uremic syndrome. To develop PCR-based methods for detection and identification of this serogroup, the DNA sequence of the 12,033-bp region containing the O antigen gene cluster of Escherichia coli O103 was determined. Of the 12 open reading frames identified, the E. coli O103 wzx (O antigen flippase) and wzy (O antigen polymerase) genes were selected as targets for development of both conventional and real-time PCR assays specific for this serogroup. In addition, a multiplex PCR targeting the Shiga toxin (Stx) 1 (stx1), Shiga toxin 2 (stx2), wzx, and wzy genes was developed to differentiate Stx-producing E. coli O103 from non-toxigenic strains. The PCR assays can be employed to identify E. coli serogroup O103, replacing antigen-based serotyping, and to potentially detect the organism in food, fecal, or environmental samples.
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690. |
Aoki SK,
Pamma R,
Hernday AD,
Bickham JE,
Braaten BA,
Low DA,
( 2005 ) Contact-dependent inhibition of growth in Escherichia coli. PMID : 16109881 : DOI : 10.1126/science.1115109 Abstract >>
Bacteria have developed mechanisms to communicate and compete with each other for limited environmental resources. We found that certain Escherichia coli, including uropathogenic strains, contained a bacterial growth-inhibition system that uses direct cell-to-cell contact. Inhibition was conditional, dependent upon the growth state of the inhibitory cell and the pili expression state of the target cell. Both a large cell-surface protein designated Contact-dependent inhibitor A (CdiA) and two-partner secretion family member CdiB were required for growth inhibition. The CdiAB system may function to regulate the growth of specific cells within a differentiated bacterial population.
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691. |
Ideses D,
Biran D,
Gophna U,
Levy-Nissenbaum O,
Ron EZ,
( 2005 ) The lpf operon of invasive Escherichia coli. PMID : 16128397 : DOI : 10.1016/j.ijmm.2005.04.009 Abstract >>
Extraintestinal pathogenic Escherichia coli (ExPEC) strains have been shown to code for several virulence factors involved in adherence to host tissues. Here we show the existence of an additional adherence gene cluster, coding for long polar fimbriae--LPF--in several strains of serotype O78 from septicemia and newborn meningitis. The complete gene cluster was sequenced in strain 789 (lpf789), where it is located between the genes glmS and pstS, and contains four ORFs, lpfA to lpfD. The lpf operon is expressed and is important for adherence to epithelial cells. The lpf operon was found only in four of the ExPEC strains tested and is likely to have been acquired by horizontal gene transfer.
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692. |
Grape M,
Farra A,
Kronvall G,
Sundström L,
( 2005 ) Integrons and gene cassettes in clinical isolates of co-trimoxazole-resistant Gram-negative bacteria. PMID : 15715715 : DOI : 10.1111/j.1469-0691.2004.01059.x Abstract >>
Despite a trend of declining consumption, resistance to co-trimoxazole has increased during a 12-year period in Stockholm. The molecular background to this surprising development was investigated by using PCR to screen for integrons and specific resistance genes, followed by sequence analysis of selected integrons, in 105 clinical urinary isolates of Gram-negative bacteria selected partly for trimethoprim resistance. Sixty-five integrons of class 1 or 2 were detected in a subset of 59 isolates, and of these positive isolates, all but one were resistant to trimethoprim. However, 11 isolates were resistant to trimethoprim, but negative for integrons. Isolates positive for integrons were resistant to an average of 4.2 antibiotics, compared with 1.9 antibiotics for integron-negative isolates. Despite this, the only gene cassettes identified in 19 class 1 integrons analysed were dfr and aadA cassettes. Thus, only resistance to trimethoprim, streptomycin, spectinomycin and sulphonamides could be explained by the presence of integrons in these isolates. A new dfr gene, named dfrA22, was discovered as a single gene cassette in a class 1 integron. In addition, sulphonamide resistance in many isolates was caused by carriage of sul2, which has no known association with integrons. Resistance to co-trimoxazole and many other antibiotics was thus not accounted for fully by the presence of integrons in these isolates.
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693. |
Demarre G,
Guérout AM,
Matsumoto-Mashimo C,
Rowe-Magnus DA,
Marlière P,
Mazel D,
( 2005 ) A new family of mobilizable suicide plasmids based on broad host range R388 plasmid (IncW) and RP4 plasmid (IncPalpha) conjugative machineries and their cognate Escherichia coli host strains. PMID : 15748991 : DOI : 10.1016/j.resmic.2004.09.007 Abstract >>
We describe the construction of the pSW family of conditionally replicating plasmids which are based on the IncX oriV origin (oriV(R6Kgamma)) of replication that is dependent on the pir-encoded protein. We constructed several Escherichia coli derivatives expressing pir from different chromosomal loci, and the pir gene could be transduced by phage P1 to any E. coli strain. These chromosomal constructions generate dapA and thyA knockouts, which lead to diaminopimelate or thymidine auxotrophies, respectively, and they serve to provide absolute counterselection even in rich media. These strains can be easily counterselected if used in plasmid transfer experiments into markerless recipients, and they have been demonstrated to work efficiently in E. coli xVibrio or E. coli xBartonella matings. We constructed different pSW plasmids carrying either the oriT(RP4) or the oriT(R388), and we demonstrated that these derivatives can be efficiently transferred using RP4 and R388 conjugation machineries, respectively. We also constructed two plasmids expressing the R388 conjugation machinery, but lacking the oriT(R388). We demonstrated that these plasmids enabled efficient and exclusive transfer of a pSW-oriT(R388) derivative from E. coli to V. cholerae, and we offer an alternative to the popular RP4-based delivery system.
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694. |
Duncan MJ,
Mann EL,
Cohen MS,
Ofek I,
Sharon N,
Abraham SN,
( 2005 ) The distinct binding specificities exhibited by enterobacterial type 1 fimbriae are determined by their fimbrial shafts. PMID : 16118220 : DOI : 10.1074/jbc.M501249200 Abstract >>
Type 1 fimbriae of enterobacteria are heteropolymeric organelles of adhesion composed of FimH, a mannose-binding lectin, and a shaft composed primarily of FimA. We compared the binding activities of recombinant clones expressing type 1 fimbriae from Escherichia coli, Klebsiella pneumoniae, and Salmonella typhimurium for gut and uroepithelial cells and for various soluble mannosylated proteins. Each fimbria was characterized by its capacity to bind particular epithelial cells and to aggregate mannoproteins. However, when each respective FimH subunit was cloned and expressed in the absence of its shaft as a fusion protein with MalE, each FimH bound a wide range of mannose-containing compounds. In addition, we found that expression of FimH on a heterologous fimbrial shaft, e.g. K. pneumoniae FimH on the E. coli fimbrial shaft or vice versa, altered the binding specificity of FimH such that it closely resembled that of the native heterologous type 1 fimbriae. Furthermore, attachment to and invasion of bladder epithelial cells, which were mediated much better by native E. coli type 1 fimbriae compared with native K. pneumoniae type 1 fimbriae, were found to be dependent on the background of the fimbrial shaft (E. coli versus K. pneumoniae) rather than the background of the FimH expressed. Thus, the distinct binding specificities of different enterobacterial type 1 fimbriae cannot be ascribed solely to the primary structure of their respective FimH subunits, but are also modulated by the fimbrial shaft on which each FimH subunit is presented, possibly through conformational constraints imposed on FimH by the fimbrial shaft. The capacity of type 1 fimbrial shafts to modulate the tissue tropism of different enterobacterial species represents a novel function for these highly organized structures.
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695. |
Brisse S,
Duijkeren Ev,
( 2005 ) Identification and antimicrobial susceptibility of 100 Klebsiella animal clinical isolates. PMID : 15708829 : DOI : 10.1016/j.vetmic.2004.11.010 Abstract >>
The objectives of this study were to determine the distribution of Klebsiella species and phylogenetic groups in animal clinical samples and to determine the levels of antimicrobial resistance of animal Klebsiella clinical isolates. One hundred Klebsiella veterinary clinical isolates were identified using gyrA PCR-RFLP and rpoB gene sequencing as a confirmatory method. Klebsiella pneumoniae phylogenetic group KpI was dominant (78 isolates), but KpII, KpIII (K. variicola), K. oxytoca, K. planticola and K. terrigena were also represented. The relative frequencies in animal infections of Klebsiella species and phylogenetic groups were similar to those observed in human nosocomial infections, suggesting that similar ecological and molecular factors cause Klebsiella infections in both situations. Resistance was common against ampicillin (99%) and cephalexin (43%) but not against ceftazidime, ceftiofur, tetracycline, enrofloxacin, gentamicin and trimethoprim-sulfamethoxazole. Thirteen isolates resistant to three or more antimicrobials or combinations thereof were found, but acquired antimicrobial resistance remains lower among animal isolates than among human nosocomial isolates.
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696. |
Zhu C,
Feng S,
Thate TE,
Kaper JB,
Boedeker EC,
( 2006 ) Towards a vaccine for attaching/effacing Escherichia coli: a LEE encoded regulator (ler) mutant of rabbit enteropathogenic Escherichia coli is attenuated, immunogenic, and protects rabbits from lethal challenge with the wild-type virulent strain. PMID : 16112258 : DOI : 10.1016/j.vaccine.2005.07.019 Abstract >>
The ler (LEE encoded regulator) gene product is a central regulator for the genes encoded on the locus of enterocyte effacement (LEE) pathogenicity island of attaching/effacing (A/E) pathogens, including human enteropathogenic E. coli (EPEC) and enterohemorrhagic E. coli (EHEC) as well as animal isolates. Although an in vivo role for Ler in bacterial virulence has not been documented, we hypothesized that a Ler deletion mutant should be attenuated for virulence but might retain immunogenicity. The goals of this study were to genetically characterize ler of a rabbit EPEC (rEPEC) strain (O103:H2), to examine the effect of ler on in vivo virulence, and to determine if intragastric inoculation of an attenuated rEPEC ler mutant was immunogenic and could protect rabbits against subsequent challenge with the wild-type virulent parent strain. The predicted ler gene product of rEPEC strain O103:H2 shares high homology (over 95% amino acid identity) with the Lers of another rEPEC strain RDEC-1 (O15:H-) and human EPEC and EHEC. A defined internal ler deletion mutant of rEPEC O103:H2 showed reduced production of secreted proteins. Although orogastric inoculation of rabbits with the virulent parent O103:H2 strain induced severe diarrhea, significant weight loss and early mortality with adherent mucosal bacteria found at sacrifice, the isogeneic ler mutant strain was well tolerated. Animals gained weight and showed no clinical signs of disease. Examination of histological sections of intestinal segments revealed the absence of mucosal bacterial adherence. This result demonstrates an essential role for Ler in in vivo pathogenicity of A/E E. coli. Single dose orogastric immunization with the rEPEC ler mutant induced serum IgG antibody to whole bacteria (but not to intimin). Immunized animals were protected against enteric infection with the WT virulent parent strain exhibiting normal weight gain, absence of diarrhea and absence of mucosally adherent bacteria at sacrifice. Such attenuated ler mutant strains may have potential for use as oral vaccines, or as vaccine vectors for delivery of foreign antigens. It remains to be determined whether such regulatory mutants can protect against infection with A/E bacteria of differing serotypes affecting different hosts.
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697. |
Yi W,
Shao J,
Zhu L,
Li M,
Singh M,
Lu Y,
Lin S,
Li H,
Ryu K,
Shen J,
Guo H,
Yao Q,
Bush CA,
Wang PG,
( 2005 ) Escherichia coli O86 O-antigen biosynthetic gene cluster and stepwise enzymatic synthesis of human blood group B antigen tetrasaccharide. PMID : 15713070 : DOI : 10.1021/ja045021y Abstract >>
Previous study showed that some Gram-negative bacteria possess human blood group activity. Among them, Escherichia coli O86 has high blood group B activity and weak blood group A activity. This is due to the cell surface O-antigen structure, which resembles that of human blood group B antigen. In this study, we sequenced the entire E. coli O86 antigen gene cluster and identified all the genes responsible for O-antigen biosynthesis by sequence comparative analysis. The blood group B-like antigen in E. coli O86 O-polysaccharide was synthesized by sequentially employing three glycosyltransferases identified in the gene cluster. More importantly, we identified a new bacterial glycosyltransferase (WbnI) equivalent to human blood group transferase B (GTB). The enzyme substrate specificity and stepwise enzymatic synthesis of blood group B-like antigen revealed that the biosynthetic pathway of B antigen is essentially the same in E. coli O86 as in humans. This new finding provides a model to study the specificity and structure relationship of blood group transferases and supports the hypothesis of anti-blood group antibody production by bacterial stimulation.
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698. |
Yajima S,
Muto Y,
Yokoyama S,
Masaki H,
Uozumi T,
( 1992 ) The secondary structure of the colicin E3 immunity protein as studied by 1H-1H and 1H-15N two-dimensional NMR spectroscopy. PMID : 1610804 : DOI : 10.1021/bi00139a022 Abstract >>
By performing 1H-1H and 1H-15N two-dimensional (2D) nuclear magnetic resonance (NMR) experiments, the complete sequence-specific resonance assignment was determined for the colicin E3 immunity protein (84 residues; ImmE3), which binds to colicin E3 and inhibits its RNase activity. First, the fingerprint region of the spectrum was analyzed by homonuclear 1H-1H HOHAHA and NOESY methods. For the identification of overlapping resonances, heteronuclear 1H-15N (HMQC-HOHAHA, HMQC-NOESY) experiments were performed, so that the complete 1H and 15N resonance assignments were provided. Then the secondary structure of ImmE3 was determined by examination of characteristic patterns of sequential backbone proton NOEs in combination with measurement of exchange rates of amide protons and 3JHN alpha coupling constants. From these results, it was concluded that ImmE3 contains a four-stranded antiparallel beta-sheet (residues 2-10, 19-22, 47-49, and 71-79) and a short alpha-helix (residues 31-36).
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699. |
D'Souza JM,
Samuel GN,
Reeves PR,
( 2005 ) Evolutionary origins and sequence of the Escherichia coli O4 O-antigen gene cluster. PMID : 15727817 : DOI : 10.1016/j.femsle.2005.01.012 Abstract >>
Escherichia coli express many types of O antigen, present in the outer membrane of the Gram-negative bacterial cell wall. O-Antigen biosynthesis genes are clustered together and differences seen in O-antigen types are due to genetic variation within this gene cluster. Sequencing of the E. coli O4 O-antigen gene cluster revealed a similar gene order and high levels of similarity to that of E. coli O26; indicating a common ancestor. These lateral transfer events observed within O-antigen gene clusters may occur as part of the evolution of the pathogenic clones.
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700. |
Bouzari S,
Oloomi M,
Oswald E,
( 2005 ) Detection of the cytolethal distending toxin locus cdtB among diarrheagenic Escherichia coli isolates from humans in Iran. PMID : 15748977 : DOI : 10.1016/j.resmic.2004.09.011 Abstract >>
Cytolethal distending toxin (CDT) represents an emerging family of newly described bacterial products that are produced by a number of pathogens. Genes encoding this toxin were identified as a cluster of three adjacent genes, cdt-A, cdt-B and cdt-C. Five cdt genetic variants, designated cdt-I, cdt-II, cdt-III, cdt-IV and cdt-V, have been identified so far. To determine the presence of cdt genes among Escherichia coli isolates, a PCR assay was employed. Using multiplex primers designed for detection of E. coli cdt genes, PCR analysis indicated the presence of cdt genes in 37.5% of these isolates. While specific primers were located in the cdt-B locus for detection of cdt-I, cdt-II, cdt-III and cdt-IV, in multiplex PCR positive isolates indicated that a cdt-I-like gene was present in 45.3% of these isolates. However, in 52% of the isolates, the cdt-III-like gene could be detected. It should be mentioned that in 30.8% of these isolates, cdt-II and -III were detected simultaneously. In 2.6% of the isolates, the cdt-IV gene was present. The cnf-1 gene was detected in 29.4% of strains carrying the cdt-I gene; however, the cnf-2 gene was detected only in 23.1% of the cdt-III-like strains. Furthermore, the data obtained by PCR analysis indicated the presence of cdt-like genes among our E. coli isolates, although in the CHO cell assay, all isolates showed a cytopathic effect characteristic of CDT.
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701. |
Balding C,
Bromley SA,
Pickup RW,
Saunders JR,
( 2005 ) Diversity of phage integrases in Enterobacteriaceae: development of markers for environmental analysis of temperate phages. PMID : 16156729 : DOI : 10.1111/j.1462-2920.2005.00845.x Abstract >>
Viruses are the most abundant biological entities in aquatic systems. Temperate bacteriophages have enormous influences on microbial diversity, genetic exchange and bacterial population dynamics. However, development of molecular tools for their detection in the environment has been problematic. The integrase gene is used here as a molecular marker to analyse the diversity of temperate bacteriophages in a population of freshwater bacteria. Interrogation of the GenBank database revealed 32 non-cryptic enteric phage integrase sequences, leading to the development of a suite of 11 degenerate primer sets specific to the extant sequences elucidated. Application of these primer sets to enterobacterial isolates recovered from a freshwater pond and the temperate phages induced from them revealed a number of diverse integrase genes, including novel integrase-like sequences not represented in the databases. This highlights the potential of utilizing the integrase gene family as a marker for phage diversity.
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702. |
Hengen PN,
Denicourt D,
Iyer VN,
( 1992 ) Isolation and characterization of kikA, a region on IncN group plasmids that determines killing of Klebsiella oxytoca. PMID : 1569033 : DOI : 10.1128/jb.174.9.3070-3077.1992 PMC : PMC205963 Abstract >>
Transfer of the IncN group plasmid pCU1 from Escherichia coli to Klebsiella oxytoca by conjugation kills a large proportion (90 to 95%) of the recipients of plasmid DNA, whereas transfer to E. coli or even to the closely related Enterobacter aerogenes does not. Two regions, kikA and kikB, have been identified on pCU1 that contribute to the Kik (killing in klebsiellas) phenotype. We have localized the kikA region to 500 bp by deletion analysis and show by DNA-DNA hybridization that kikA is highly conserved among the plasmids of incompatibility group N. The expression in K. oxytoca of kikA under the control of the strong inducible E. coli tac promoter results in loss of cell viability. The nucleotide sequence showed two overlapping open reading frames (ORFs) within the kikA region. The first ORF codes for a putative polypeptide of 104 amino acids (ORF104). The second ORF codes for a 70-amino-acid polypeptide (ORF70). The properties of the putative protein encoded by ORF104 and gene fusions of kikA to alkaline phosphatase by using TnphoA suggest that killing may involve an association with the bacterial membrane; however, we could not rule out the possibility that ORF70 plays a role in the Kik phenotype.
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703. |
Leomil L,
Pestana de Castro AF,
Krause G,
Schmidt H,
Beutin L,
( 2005 ) Characterization of two major groups of diarrheagenic Escherichia coli O26 strains which are globally spread in human patients and domestic animals of different species. PMID : 16046083 : DOI : 10.1016/j.femsle.2005.06.030 Abstract >>
Twenty-three Escherichia coli O26 strains from humans, cattle, sheep, pigs and chicken were investigated for virulence markers and for genetic similarity by pulsed field gel electrophoresis and multi locus sequence typing. Two groups of genetically closely related O26 strains were defined. One group is formed by enteropathogenic (EPEC) and enterohemorrhagic (EHEC) E. coli strains, which do not ferment rhamnose and dulcitol and most of these carry a plasmid encoding enterohemolysin. The other group consists of rhamnose and dulcitol fermenting EPEC strains, which carry plasmids encoding alpha-hemolysin. Multiple species of domestic animals were shown to serve as a reservoir for human pathogenic O26 EPEC and EHEC strains.
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704. |
Bischoff KM,
White DG,
Hume ME,
Poole TL,
Nisbet DJ,
( 2005 ) The chloramphenicol resistance gene cmlA is disseminated on transferable plasmids that confer multiple-drug resistance in swine Escherichia coli. PMID : 15668031 : DOI : 10.1016/j.femsle.2004.12.017 Abstract >>
A recent study of beta-hemolytic Escherichia coli isolated from diarrheic swine found that 53% were resistant to chloramphenicol, a drug that has been prohibited from use in food animals in the US since the mid-1980s. To identify the factors governing the persistence of chloramphenicol resistance in the absence of specific selection pressure, the location of the chloramphenicol resistance gene cmlA and its linkage to other resistance determinants were investigated. Southern blot analysis of plasmid DNA from 46 swine E. coli isolates indicated that cmlA was present on large plasmids greater than 100 kbp. Fifty-two percent of the isolates were able to transfer chloramphenicol resistance to an E. coli recipient at conjugation frequencies ranging from 10(-3) to 10(-8) per recipient. Antimicrobial susceptibility tests on transconjugant strains demonstrated that resistance to sulfamethoxazole, tetracycline, and kanamycin frequently transferred along with chloramphenicol resistance. The transconjugant strains possessed at least two distinct class 1 integrons that linked cmlA to both aminoglycoside resistance genes aadA1 and aadA2 and either to sul1 or to sul3 sulphonamide resistance genes. These results suggest that in the absence of specific chloramphenicol selection pressure, the cmlA gene is maintained by virtue of gene linkage to genes encoding resistance to antimicrobials that are currently approved for use in food animals.
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705. |
Buts L,
Wellens A,
Van Molle I,
Wyns L,
Loris R,
Lahmann M,
Oscarson S,
De Greve H,
Bouckaert J,
( 2005 ) Impact of natural variation in bacterial F17G adhesins on crystallization behaviour. PMID : 16041081 : DOI : 10.1107/S0907444905017038 DOI : 10.1107/S0907444905017038 Abstract >>
Since the introduction of structural genomics, the protein has been recognized as the most important variable in crystallization. Recent strategies to modify a protein to improve crystal quality have included rationally engineered point mutations, truncations, deletions and fusions. Five naturally occurring variants, differing in 1-18 amino acids, of the 177-residue lectin domain of the F17G fimbrial adhesin were expressed and purified in identical ways. For four out of the five variants crystals were obtained, mostly in non-isomorphous space groups, with diffraction limits ranging between 2.4 and 1.1 A resolution. A comparative analysis of the crystal-packing contacts revealed that the variable amino acids are often involved in lattice contacts and a single amino-acid substitution can suffice to radically change crystal packing. A statistical approach proved reliable to estimate the compatibilities of the variant sequences with the observed crystal forms. In conclusion, natural variation, universally present within prokaryotic species, is a valuable genetic resource that can be favourably employed to enhance the crystallization success rate with considerably less effort than other strategies.
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706. |
LaFayette PR,
Kane PM,
Phan BH,
Parrott WA,
( 2005 ) Arabitol dehydrogenase as a selectable marker for rice. PMID : 16151815 : DOI : 10.1007/s00299-005-0015-3 Abstract >>
Arabitol dehydrogenase has been adapted for use as a plant selectable marker. Arabitol is a five-carbon sugar alcohol that can be used by E. coli strain C, but not by the laboratory K12 strains. The enzyme converts the non-plant-metabolizable sugar arabitol into xylulose, which is metabolized by plant cells. Rice was transformed with a plant-expression-optimized synthetic gene using Biolistic-mediated transformation. Selection on 2.75% arabitol and 0.25% sucrose yielded a transformation efficiency (9.3%) equal to that obtained with hygromycin (9.2%). Molecular analyses showed that the atlD gene was integrated into the rice genome of selected plants and was inherited in a Mendelian manner. This study indicates that arabitol could serve as an effective means of plant selection.
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707. |
Bhagwat AA,
Chan L,
Han R,
Tan J,
Kothary M,
Jean-Gilles J,
Tall BD,
( 2005 ) Characterization of enterohemorrhagic Escherichia coli strains based on acid resistance phenotypes. PMID : 16041014 : DOI : 10.1128/IAI.73.8.4993-5003.2005 PMC : PMC1201262 Abstract >>
Acid resistance is perceived to be an important property of enterohemorrhagic Escherichia coli strains, enabling the organisms to survive passage through the acidic environment of the stomach so that they may colonize the mammalian gastrointestinal tract and cause disease. Accordingly, the organism has developed at least three genetically and physiologically distinct acid resistance systems which provide different levels of protection. The glutamate-dependent acid resistance (GDAR) system utilizes extracellular glutamate to protect cells during extreme acid challenges and is believed to provide the highest protection from stomach acidity. In this study, the GDAR system of 82 pathogenic E. coli isolates from 34 countries and 23 states within the United States was examined. Twenty-nine isolates were found to be defective in inducing GDAR under aerobic growth conditions, while five other isolates were defective in GDAR under aerobic, as well as fermentative, growth conditions. We introduced rpoS on a low-copy-number plasmid into 26 isolates and were able to restore GDAR in 20 acid-sensitive isolates under aerobic growth conditions. Four isolates were found to be defective in the newly discovered LuxR-like regulator GadE (formerly YhiE). Defects in other isolates could be due to a mutation(s) in a gene(s) with an as yet undefined role in acid resistance since GadE and/or RpoS could not restore acid resistance. These results show that in addition to mutant alleles of rpoS, mutations in gadE exist in natural populations of pathogenic E. coli. Such mutations most likely alter the infectivity of individual isolates and may play a significant role in determining the infective dose of enterohemorrhagic E. coli.
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708. |
Soong BW,
Lu FM,
Chak KF,
( 1992 ) Characterization of the cea gene of the ColE7 plasmid. PMID : 1603061 : DOI : 10.1007/bf00587577 Abstract >>
The complete nucleotide sequence (1731 nucleotides) of the gene encoding colicin E7 (cea) of plasmid ColE7-K317 was determined. This sequence encoded a deduced polypeptide of 576 amino acids of molecular weight 61349 Da. Comparison of the nucleotide and amino acid sequences of cea E7 with those of other E-group colicins revealed that colicin E7 was closely related to colicin E2, both in gene sequence and in predicted secondary structure of the deduced protein. Judging from the results of cross-immunity tests, we postulated that ColE7 is probably a proximate ancestor of ColE2 and ColE8. Based on results from colicin production tests on cells harboring a 5' end deleted form of the cea E7 gene, we propose that a previously unknown, non-inducible promoter may be involved in regulation of the constitutive expression of the cea E7 gene.
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709. |
Scaletsky IC,
Michalski J,
Torres AG,
Dulguer MV,
Kaper JB,
( 2005 ) Identification and characterization of the locus for diffuse adherence, which encodes a novel afimbrial adhesin found in atypical enteropathogenic Escherichia coli. PMID : 16040988 : DOI : 10.1128/IAI.73.8.4753-4765.2005 PMC : PMC1201181 Abstract >>
The O26 serogroup of enteropathogenic Escherichia coli (EPEC) is one of the serogroups most frequently implicated in infant diarrhea and is also common among enterohemorrhagic E. coli (EHEC) strains. The most common O26 strains belong to EPEC/EHEC serotype O26:H11 and are generally Shiga toxin (Stx) positive. Stx-negative E. coli strains that are negative for the EPEC EAF plasmid and bundle-forming pilus (Bfp) are classified as atypical EPEC. Here, we report a novel adhesin present in an stx-negative bfpA-negative atypical EPEC O26:H11 strain isolated from an infant with diarrhea. A cloned 15-kb genomic region from this strain, designated the locus for diffuse adherence (lda), confers diffuse adherence on HEp-2 cells when expressed in E. coli K-12. Sequence analysis of lda revealed a G+C content of 46.8% and 15 open reading frames sharing homology with the E. coli K88 fae and CS31A clp fimbrial operons. The lda region is part of a putative 26-kb genomic island inserted into the proP gene of the E. coli chromosome. Hybridization studies have demonstrated the prevalence of the minor structural subunit gene, ldaH, across E. coli serogroups O5, O26, O111, and O145. A second plasmid-encoded factor that contributed to the Hep-2 adherence of this strain was also identified but was not characterized. Null mutations that abolish adherence to HEp-2 cells can be restored by plasmid complementation. Antiserum raised against the major structural subunit, LdaG, recognizes a 25-kDa protein from crude heat-extracted protein preparations and inhibits the adherence of the E. coli DH5alpha lda(+) clone to HEp-2 cells. Electron microscopy revealed a nonfimbrial structure surrounding the bacterial cell.
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710. |
Gromek K,
Kaczorowski T,
( 2005 ) DNA sequencing by indexer walking. PMID : 16037413 : DOI : 10.1373/clinchem.2004.046599 Abstract >>
There is a need for DNA sequencing methods that are faster, more accurate, and less expensive than existing techniques. Here we present a new method for DNA analysis by means of indexer walking. For DNA sequencing by indexer walking, we ligated double-stranded synthetic oligonucleotides (indexers) to DNA fragments that were produced by type IIS restriction endonucleases, which generate nonidentical 4-nucleotide 5' overhangs. The subsequent amplification (30 thermal cycles) of indexed DNA provided a template for automated DNA sequencing with fluorescent dideoxy terminators. The data gathered in the first sequencing reaction permitted further movement into the unknown nucleotide sequence by digestion of analyzed DNA with selected type IIS restriction endonuclease followed by ligation of the next indexer. A library of presynthesized indexers consisting of 256 oligonucleotides was used for bidirectional analysis of DNA molecules and provided universal primers for sequencing. The proposed protocol was successfully applied to sequencing of cryptic plasmids isolated from pathogenic strains of Escherichia coli. The overall error rate for base-calling was 0.5%, with a mean read length of 550 nucleotides. Approximately 1000 nucleotides of high-quality sequence could be obtained per day from a single clone. Indexer walking can be used as a low-cost procedure for nucleotide sequence determination of DNA molecules, such as natural plasmids, cDNA clones, and longer DNA fragments. It can also serve as an alternative method for gap filling at the final stage of genome sequencing projects.
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711. |
Jores J,
Wagner S,
Rumer L,
Eichberg J,
Laturnus C,
Kirsch P,
Schierack P,
Tschäpe H,
Wieler LH,
( 2005 ) Description of a 111-kb pathogenicity island (PAI) encoding various virulence features in the enterohemorrhagic E. coli (EHEC) strain RW1374 (O103:H2) and detection of a similar PAI in other EHEC strains of serotype 0103:H2. PMID : 15715170 : Abstract >>
Human infections with enterohemorrhagic E. coli (EHEC) strains of serotype O103:H2 are of increasing importance in Germany. As bovines are the principal EHEC reservoir behind the occurrence of human infections, we analyzed a pathogenicity island (PAI I(RW1374)) of bovine O103:H2 strain RW1374 to identify putative virulence features. This PAI I(RW1374) harbors a functional 34-kb locus of enterocyte effacement (LEE) core region and has a total length of 111 kb. About 43 kb upstream of the LEE core a gene cassette consisting of efa1/lifA gene and flanking IS elements suggests another putative transposon within the PAI(IRW1374). In addition, the ent gene, encoding a Shigella ShET-2 enterotoxin homologue, is present about 57 kb upstream of the LEE core. This PAI is therefore a complex assembly of various virulence determinants including the efa1/lifA and the ent gene resembling O157:H7 PAI OI-122/SpLE3 as well as the LEE core region. An integrase gene on the very left end of PAI I(Rw1374) is disrupted by an IS629 homologue. In an attempt to mobilize the LEE core we performed conjugation, transformation and transduction experiments. We were, however, unable to mobilize the whole or even single regions of PAI I(RW1374). Comparative studies with other strains of serotype O103:H2 isolated from humans, bovines and food showed that they all harbored a similar phe V-inserted PAI including the virulence genes ent and lifA/efa1 as well as the large virulence-associated plasmid encoding the EHEC hemolysin. This combination of several virulence factors confirms the complex virulence of O103:H2 EHEC and may at least partly explain the high virulence of this EHEC serotype in humans.
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712. |
Ahmed AM,
Miyoshi S,
Shinoda S,
Shimamoto T,
( 2005 ) Molecular characterization of a multidrug-resistant strain of enteroinvasive Escherichia coli O164 isolated in Japan. PMID : 15713611 : DOI : 10.1099/jmm.0.45908-0 Abstract >>
Enteroinvasive Escherichia coli (EIEC) O164 strain RIMD05091045 was isolated from a travelling patient suffering from diarrhoea at the Osaka airport quarantine facility in Japan. The strain showed multidrug resistance against streptomycin, spectinomycin, co-trimoxazole (trimethoprim/sulfamethoxazole) and ampicillin, and reduced susceptibility to ciprofloxacin. Molecular characterization of the multidrug-resistance phenotype revealed the presence of a class 1 integron containing three genes, a dihydrofolate reductase type XII gene, dfrXII, which confers resistance to trimethoprim, an aminoglycoside adenyltransferase gene, aadA2, which confers resistance to streptomycin and spectinomycin, and an ORF of unknown function. Southern blot hybridization and conjugation experiments showed that the class 1 integron was located on a transferable plasmid that was less than 90 kb in size. The resistance of EIEC O164 to ampicillin was found to be due to the presence of TEM-1 beta-lactamase. On the other hand, a single mutation that has not previously been described, P158-to-S, was detected downstream of the quinolone-resistance-determining region of parC of topoisomerase IV and may be responsible for the reduced susceptibility to ciprofloxacin in this strain.
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713. |
Enne VI,
Delsol AA,
Davis GR,
Hayward SL,
Roe JM,
Bennett PM,
( 2005 ) Assessment of the fitness impacts on Escherichia coli of acquisition of antibiotic resistance genes encoded by different types of genetic element. PMID : 16040624 : DOI : 10.1093/jac/dki255 Abstract >>
Little is known of the fitness cost that antibiotic resistance exerts on wild-type bacteria, especially in their natural environments. We therefore examined the fitness costs that several antibiotic resistance elements imposed on a wild-type Escherichia coli isolate, both in the laboratory and in a pig gut colonization model. Plasmid R46, Tn1 and Tn7 and a K42R RpsL substitution were separately introduced into E. coli 345-2 RifC, a rifampicin-resistant derivative of a recent porcine isolate. The insertion site of Tn1 was determined by DNA sequencing. The fitness cost of each resistance element was assessed in vitro by pairwise growth competition and in vivo by regularly monitoring the recovery of strains from faeces for 21 days following oral inoculation of organic piglets. Each derivative of 345-2 RifC carrying a resistance element was grown in antibiotic-free broth for 200 generations and the experiments to assess fitness were repeated. RpsL K42R was found to impose a small fitness cost on E. coli 345-2 RifC in vitro but did not compromise survival in vivo. R46 imposed a cost both before and after laboratory passage in vitro, but only the pre-passage strain was at a disadvantage in vivo. The post-passage isolate had an advantage in pigs. Acquisition of Tn7 had no impact on the fitness of E. coli 345-2 RifC. Two derivatives containing Tn1 were isolated and, in both cases, the transposon inserted into the same cryptic chromosomal sequence. Acquisition of Tn1 improved fitness of E. coli 345-2 RifC in vitro and in vivo in the case of the first derivative, but in the case of a second, independent derivative, Tn1 had a neutral effect on fitness. The fitness impact imposed on E. coli 345-2 RifC by carriage of antibiotic resistance elements was generally low or non-existent, suggesting that once established, resistance may be difficult to eliminate through reduction in prescribing alone.
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714. |
Ren CP,
Beatson SA,
Parkhill J,
Pallen MJ,
( 2005 ) The Flag-2 locus, an ancestral gene cluster, is potentially associated with a novel flagellar system from Escherichia coli. PMID : 15687208 : DOI : 10.1128/JB.187.4.1430-1440.2005 PMC : PMC545627 Abstract >>
Escherichia coli K-12 possesses two adjacent, divergent, promoterless flagellar genes, fhiA-mbhA, that are absent from Salmonella enterica. Through bioinformatics analysis, we found that these genes are remnants of an ancestral 44-gene cluster and are capable of encoding a novel flagellar system, Flag-2. In enteroaggregative E. coli strain 042, there is a frameshift in lfgC that is likely to have inactivated the system in this strain. Tiling path PCR studies showed that the Flag-2 cluster is present in 15 of 72 of the well-characterized ECOR strains. The Flag-2 system resembles the lateral flagellar systems of Aeromonas and Vibrio, particularly in its apparent dependence on RpoN. Unlike the conventional Flag-1 flagellin, the Flag-2 flagellin shows a remarkable lack of sequence polymorphism. The Flag-2 gene cluster encodes a flagellar type III secretion system (including a dedicated flagellar sigma-antisigma combination), thus raising the number of distinct type III secretion systems in Escherichia/Shigella to five. The presence of the Flag-2 cluster at identical sites in E. coli and its close relative Citrobacter rodentium, combined with its absence from S. enterica, suggests that it was acquired by horizontal gene transfer after the former two species diverged from Salmonella. The presence of Flag-2-like gene clusters in Yersinia pestis, Yersinia pseudotuberculosis, and Chromobacterium violaceum suggests that coexistence of two flagellar systems within the same species is more common than previously suspected. The fact that the Flag-2 gene cluster was not discovered in the first 10 Escherichia/Shigella genome sequences studied emphasizes the importance of maintaining an energetic program of genome sequencing for this important taxonomic group.
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715. |
Tórtola MT,
Lavilla S,
Miró E,
González JJ,
Larrosa N,
Sabaté M,
Navarro F,
Prats G,
( 2005 ) First detection of a carbapenem-hydrolyzing metalloenzyme in two enterobacteriaceae isolates in Spain. PMID : 16048967 : DOI : 10.1128/AAC.49.8.3492-3494.2005 PMC : PMC1196258 Abstract >>
Two strains of Enterobacteriaceae, Escherichia coli and Klebsiella pneumoniae, producing VIM-1 were isolated for the first time in Spain. In both strains, bla(VIM-1) was found to be carried on a gene cassette inserted into a class 1 integron. The bla(VIM-1)-containing integron was located on a transferable plasmid.
|
716. |
Mammeri H,
Van De Loo M,
Poirel L,
Martinez-Martinez L,
Nordmann P,
( 2005 ) Emergence of plasmid-mediated quinolone resistance in Escherichia coli in Europe. PMID : 15616277 : DOI : 10.1128/AAC.49.1.71-76.2005 PMC : PMC538905 Abstract >>
Although quinolone resistance results mostly from chromosomal mutations, it may also be mediated by a plasmid-encoded qnr gene in members of the family Enterobacteriaceae. Thus, 297 nalidixic-acid resistant strains of 2,700 Escherichia coli strains that had been isolated at the Bicetre Hospital (Le Kremlin-Bicetre, France) in 2003 were screened for qnr by PCR. A single E. coli isolate that carried a ca. 180-kb conjugative plasmid encoding a qnr determinant was identified. It conferred low-level resistance to quinolones and was associated with a chromosomal mutation in subunit A of the topoisomerase II gene. The qnr gene was located on a sul1-type class 1 integron just downstream of a conserved region (CR) element (CR1) comprising the Orf513 recombinase. Promoter sequences for qnr expression overlapped the extremity of CR1, indicating the role of CR1 in the expression of antibiotic resistance genes. This integron was different from other qnr-positive sul1-type integrons identified in American and Chinese enterobacterial isolates. In addition, plasmid pQR1 carried another class 1 integron that was identical to In53 from E. coli. The latter integron possessed a series of gene cassettes, including those coding for the extended-spectrum beta-lactamase VEB-1, the rifampin ADP ribosyltransferase ARR-2, and several aminoglycoside resistance markers. This is the first report of plasmid-mediated quinolone resistance in Europe associated with an unknown level of plasmid-mediated multidrug resistance in Enterobacteriaceae.
|
717. |
Ma L,
Alba J,
Chang FY,
Ishiguro M,
Yamaguchi K,
Siu LK,
Ishii Y,
( 2005 ) Novel SHV-derived extended-spectrum beta-lactamase, SHV-57, that confers resistance to ceftazidime but not cefazolin. PMID : 15673739 : DOI : 10.1128/AAC.49.2.600-605.2005 PMC : PMC547208 Abstract >>
A new SHV-derived extended-spectrum beta-lactamase, SHV-57, that confers high-level resistance to ceftazidime but not cefotaxime or cefazolin was identified from a national surveillance study conducted in Taiwan in 1998. An Escherichia coli isolate resistant to ampicillin, cephalothin, and ceftazidime but sensitive to cefoxitin, ceftriaxone, cefotaxime, imipenem, and a narrow-spectrum cephem (cefazolin) was isolated from the urine of a patient treated with beta-lactam antibiotics. Resistance to beta-lactams was conjugatively transferred with a plasmid of about 50 kbp. The pI of this enzyme was 8.3. The sequence of the gene was determined, and the open reading frame of the gene was found to consist of 861 bases (GenBank accession number AY223863). Kinetic parameters showed that SHV-57 had a poor affinity to cefazolin. The K(m) value toward cefazolin (5.57 x 10(3) muM) was extremely high in comparison to those toward ceftazidime (30.9 muM) and penicillin G (67 muM), indicating its low affinity to cefazolin. Although the K(m) value of the beta-lactamase inhibitor was too high for the study of catalytic activity (k(cat)), indicating the low k(cat) of SHV-57, the SHV-57 carrier was highly susceptible to a beta-lactam-beta-lactamase inhibitor combination. Comparison of the three-dimensional molecular model of SHV-57 with that of the SHV-1 beta-lactamase suggests that the substitution of arginine for leucine-169 in the Omega loop is important for the substrate specificity.
|
718. |
Yip CK,
Finlay BB,
Strynadka NC,
( 2005 ) Structural characterization of a type III secretion system filament protein in complex with its chaperone. PMID : 15619638 : DOI : 10.1038/nsmb879 Abstract >>
The type III secretion system (TTSS) mediates the specific translocation of bacterial proteins into the cytoplasm of eukaryotic cells, a process essential for the virulence of many Gram-negative pathogens. The enteropathogenic Escherichia coli TTSS protein EspA forms a hollow extracellular filament believed to be a molecular conduit for type III protein translocation. Structural analysis of EspA has been hampered by its polymeric nature. We show that EspA alone is sufficient to form filamentous structures in the absence of other pathogenicity island-encoded proteins. CesA is the recently proposed chaperone of EspA, and we demonstrate that CesA traps EspA in a monomeric state and inhibits its polymerization. Crystallographic analysis of the heterodimeric CesA-EspA complex at a resolution of 2.8 A reveals that EspA contains two long a-helices, which are involved in extensive coiled-coil interactions with CesA.
|
719. |
Durso LM,
Bono JL,
Keen JE,
( 2005 ) Molecular serotyping of Escherichia coli O26:H11. PMID : 16085902 : DOI : 10.1128/AEM.71.8.4941-4944.2005 PMC : PMC1183322 Abstract >>
Serotyping is the foundation of pathogenic Escherichia coli diagnostics; however, few laboratories have this capacity. We developed a molecular serotyping protocol that targets, genetically, the same somatic and flagellar antigens of E. coli O26:H11 used in traditional serotyping. It correctly serotypes strains untypeable by traditional methods, affording primary laboratories serotyping capabilities.
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720. |
Janka A,
Becker G,
Sonntag AK,
Bielaszewska M,
Dobrindt U,
Karch H,
( 2005 ) Presence and characterization of a mosaic genomic island which distinguishes sorbitol-fermenting enterohemorrhagic Escherichia coli O157:H- from E. coli O157:H7. PMID : 16085887 : DOI : 10.1128/AEM.71.8.4875-4878.2005 PMC : PMC1183287 Abstract >>
A mosaic genomic island comprising Shigella resistance locus (SRL) sequences flanked by segments of Escherichia coli O157:H7 strain EDL933 O islands 43, 81, and 82 was identified in sorbitol-fermenting (SF) enterohemorrhagic Escherichia coli (EHEC) O157:H(-) strain 493/89. This mosaic island is absent from strain EDL933. PCR targeting the SRL-related sequence is a useful tool to distinguish SF EHEC O157:H(-) from EHEC O157:H7.
|
721. |
Handal T,
Olsen I,
Walker CB,
Caugant DA,
( 2005 ) Detection and characterization of beta-lactamase genes in subgingival bacteria from patients with refractory periodontitis. PMID : 15621454 : DOI : 10.1016/j.femsle.2004.11.023 Abstract >>
Fifty-three beta-lactamase-producing strains of oral bacteria isolated from patients with refractory periodontitis in Norway and USA were screened for the presence of the bla(TEM), bla(SHV), bla(OXA), bla(ampC), bla(cfxA), and bla(cepA/cblA) genes by the polymerase chain reaction (PCR). The PCR products were characterized by direct sequencing of the amplified DNA. Thirty-four of the 53 enzyme-producing strains (64%) were positive in one of the PCR assays. All beta-lactamase-producing Prevotella and Capnocytophaga spp. were CfxA positive. TEM-type beta-lactamases were identified in one strain each of Escherichia coli and Neisseria sp., and one strain of Citrobacter freundii possessed an AmpC-type beta-lactamase. Screening for gene cassettes and genes known to be associated with integrons did not reveal the presence of integrons in these oral bacteria. Sequence analyses showed that most CfxA positive Prevotella and Capnocytophaga isolates from patients with refractory periodontitis harboured variants of the CfxA2 and CfxA3 enzyme. The present study also showed that many different genetic determinants of beta-lactamase production are found in bacteria isolated from refractory periodontitis, many of which remain to be characterized.
|
722. |
Gangoue-Pieboji J,
Miriagou V,
Vourli S,
Tzelepi E,
Ngassam P,
Tzouvelekis LS,
( 2005 ) Emergence of CTX-M-15-producing enterobacteria in Cameroon and characterization of a blaCTX-M-15-carrying element. PMID : 15616331 : DOI : 10.1128/AAC.49.1.441-443.2005 PMC : PMC538870 Abstract >>
CTX-M-15-producing Klebsiella pneumoniae and Escherichia coli emerged recently in Cameroon. CTX-M-15 was encoded by two different multiresistance plasmids, of which one carried an ISEcp1-bla(CTX-M-15) element flanked by a 5-bp target site duplication and inserted within a Tn2-derived sequence. A truncated form of this element in the second plasmid was identified.
|
723. |
DebRoy C,
Fratamico PM,
Roberts E,
Davis MA,
Liu Y,
( 2005 ) Development of PCR assays targeting genes in O-antigen gene clusters for detection and identification of Escherichia coli O45 and O55 serogroups. PMID : 16085897 : DOI : 10.1128/AEM.71.8.4919-4924.2005 PMC : PMC1183307 Abstract >>
The Escherichia coli O45 O-antigen gene cluster of strain O45:H2 96-3285 was sequenced, and conventional (singleplex), multiplex, and real-time PCR assays were designed to amplify regions in the wzx (O-antigen flippase) and wzy (O-antigen polymerase) genes. In addition, PCR assays targeting the E. coli O55 wzx and wzy genes were designed based on previously published sequences. PCR assays targeting E. coli O45 showed 100% specificity for this serogroup, whereas by PCR assays specific for E. coli O55, 97/102 strains serotyped as E. coli O55 were positive for wzx and 98/102 for wzy. Multiplex PCR assays targeting the E. coli O45 and the E. coli O55 wzx and wzy genes were used to detect the organisms in fecal samples spiked at levels of 10(6) and 10(8) CFU/0.2 g feces. Thus, the PCR assays can be used to detect and identify E. coli serogroups O45 and O55.
|
724. |
Miriagou V,
Carattoli A,
Tzelepi E,
Villa L,
Tzouvelekis LS,
( 2005 ) IS26-associated In4-type integrons forming multiresistance loci in enterobacterial plasmids. PMID : 16048979 : DOI : 10.1128/AAC.49.8.3541-3543.2005 PMC : PMC1196216 Abstract >>
Three distinct multiresistant loci from enterobacterial plasmids each comprised an integron and an IS26-associated sequence. Sequence comparison suggested a common ancestral structure that derived from an IS26 insertion into the 5' conserved segment of an In4-type integron and evolved through acquisition of gene cassettes and IS26-mediated recruitment of additional resistance genes of diverse origin.
|
725. |
González-Zorn B,
Catalan A,
Escudero JA,
Domínguez L,
Teshager T,
Porrero C,
Moreno MA,
( 2005 ) Genetic basis for dissemination of armA. PMID : 16027145 : DOI : 10.1093/jac/dki246 Abstract >>
armA is a novel plasmid-borne 16S rRNA methyltransferase that confers high-level resistance to 4,6-disubstituted deoxystreptamines. Recently, we have isolated from a high-level broad-spectrum aminoglycoside-resistant Escherichia coli animal isolate a plasmid, pMUR050, that bore the armA gene. In order to elucidate the genetic basis for the spread of armA, we have determined the complete nucleotide sequence of pMUR050. armA was borne by a complex transposon composite flanked by two direct repeats of IS26. The transposon composite included a class one integron with sul1 for resistance to sulphonamides and ant3''9 conferring resistance to spectinomycin-streptomycin, and a macrolide efflux pump and mefE/mel conferring high-level resistance to erythromycin. We identified in GenBank that another plasmid, pCTX-M3, from a Polish Citrobacter freundii human isolate, bore the same genetic structure, including armA. armA is present in human and animal isolates within a novel transposon composite. Further spread of armA between bacteria of diverse origin is to be expected.
|
726. |
Beutin L,
Tao J,
Feng L,
Krause G,
Zimmermann S,
Gleier K,
Xia Q,
Wang L,
( 2005 ) Sequence analysis of the Escherichia coli O15 antigen gene cluster and development of a PCR assay for rapid detection of intestinal and extraintestinal pathogenic E. coli O15 strains. PMID : 15695667 : DOI : 10.1128/JCM.43.2.703-710.2005 PMC : PMC548065 Abstract >>
A collection of 33 Escherichia coli serogroup O15 strains was studied with regard to O:H serotypes and virulence markers and for detection of the O-antigen-specific genes wzx and wzy. The strains were from nine different countries, originated from healthy or diseased humans and animals and from food, and were isolated between 1941 and 2003. On the basis of virulence markers and clinical data the strains could be split into different pathogroups, such as uropathogenic E. coli, enteropathogenic E. coli, Shiga toxin-producing E. coli, and enteroaggregative E. coli. H serotyping and genotyping of the flagellin (fliC) gene revealed 11 different H types and a close association between certain H types, virulence markers, and pathogroups was found. Nucleotide sequence analysis of the O-antigen gene cluster revealed putative genes for biosynthesis of O15 antigen. PCR assays were developed for sensitive and specific detection of the O15-antigen-specific genes wzx and wzy. The high pathotype diversity found in the collection of 33 O15 strains contrasted with the high level of similarity found in the genes specific to the O15 antigen. This might indicate that the O15 determinant has been spread by horizontal gene transfer to a number of genetically unrelated strains of E. coli.
|
727. |
Riley JG,
Menggad M,
Montoya-Peleaz PJ,
Szarek WA,
Marolda CL,
Valvano MA,
Schutzbach JS,
Brockhausen I,
( 2005 ) The wbbD gene of E. coli strain VW187 (O7:K1) encodes a UDP-Gal: GlcNAc{alpha}-pyrophosphate-R {beta}1,3-galactosyltransferase involved in the biosynthesis of O7-specific lipopolysaccharide. PMID : 15625181 : DOI : 10.1093/glycob/cwi038 Abstract >>
In this work, we demonstrate that the wbbD gene of the O7 lipopolysaccharide (LPS) biosynthesis cluster in Escherichia coli strain VW187 (O7:K1) encodes a galactosyltransferase involved in the synthesis of the O7-polysaccharide repeating unit. The galactosyltransferase catalyzed the transfer of Gal from UDP-Gal to the GlcNAc residue of a GlcNAc-pyrophosphate-lipid acceptor. A mutant strain with a defective wbbD gene was unable to form O7 LPS and lacked this specific galactosyltransferase activity. The normal phenotype was restored by complementing the mutant with the cloned wbbD gene. To characterize the WbbD galactosyltransferase, we used a novel acceptor substrate containing GlcNAcalpha-pyrophosphate covalently bound to a hydrophobic phenoxyundecyl moiety (GlcNAc alpha-O-PO(3)-PO(3)-(CH(2))(11)-O-phenyl). The WbbD galactosyltransferase had optimal activity at pH 7 in the presence of 2.5 mM MnCl(2). Detergents in the assay did not increase glycosyl transfer. Digestion of enzyme product by highly purified bovine testicular beta-galactosidase demonstrated a beta-linkage. Cleavage of product by pyrophosphatase and phosphatase, followed by HPLC and NMR analyses, revealed a disaccharide with the structure Gal beta1-3GlcNAc. Our results conclusively demonstrate that WbbD is a UDP-Gal: GlcNAcalpha-pyrophosphate-R beta1,3-galactosyltransferase and suggest that the novel synthetic glycolipid acceptor may be generally applicable to characterize other bacterial glycosyltransferases.
|
728. |
Frey J,
Bagdasarian MM,
Bagdasarian M,
( 1992 ) Replication and copy number control of the broad-host-range plasmid RSF1010. PMID : 1563624 : DOI : 10.1016/0378-1119(92)90675-f Abstract >>
Initiation of replication of the broad-host-range plasmid RSF1010, is accurately controlled by the plasmid-encoded proteins, RepB (MobA), RepB', RepA and RepC [Haring et al., Proc. Natl. Acad. Sci. USA 82 (1985) 6090-6094; Scherzinger et al., Nucleic Acids Res. 19 (1991) 1203-1211]. The genes encoding these proteins which are essential for replication and conjugative mobilization are transcribed from a cluster of promoters, P1/P3 and P2, which partly overlap with the origin of conjugal transfer, oriT. Three regions were found where deletion mutations affect the mobilization of RSF1010 and increase its copy number in Escherichia coli. A deletion in the mobC gene increased the copy number of RSF1010 four-fold. Another deletion, that removed oriT and part of the promoter believed to be responsible for the expression of mobC, results in a three-fold increase in copy number. The third type of deletions affect the N-terminal part of RepB (MobA). A deletion that created a frame-shift results in a three-fold increase in copy number. A smaller, in-frame deletion of this region only affected the mobilization of RSF1010, but not its copy number. The extent by which RSF1010 or its deletion derivatives could repress the P1/P3 and P2 promoters has indicated that these promoters are negatively regulated by MobC and RepB (MobA), presumably by their attachment to the oriT region of RSF1010. Both MobC and RepB are required for the maximal repression of the rep operon. Optimal function of RSF1010 thus involves not only overlapping genes, but also proteins that exert multiple functions, mobilization, replication and regulation.
|
729. |
Piatek R,
Zalewska B,
Kolaj O,
Ferens M,
Nowicki B,
Kur J,
( 2005 ) Molecular aspects of biogenesis of Escherichia coli Dr Fimbriae: characterization of DraB-DraE complexes. PMID : 15618148 : DOI : 10.1128/IAI.73.1.135-145.2005 PMC : PMC538934 Abstract >>
The Dr hemagglutinin of uropathogenic Escherichia coli is a fimbrial homopolymer of DraE subunits encoded by the dra operon. The dra operon includes the draB and draC genes, whose products exhibit homology to chaperone-usher proteins involved in the biogenesis of surface-located polymeric structures. DraB is one of the periplasmic proteins belonging to the superfamily of PapD-like chaperones. It possesses two conserved cysteine residues characteristic of the FGL subfamily of Caf1M-like chaperones. In this study we obtained evidence that DraB cysteines form a disulfide bond in a mature chaperone and have the crucial function of forming the DraB-DraE binary complex. Expression experiments showed that the DraB protein is indispensable in the folding of the DraE subunit to a form capable of polymerization. Accumulation of DraB-DraE(n) oligomers, composed of head-to-tail subunits and the chaperone DraB, was observed in the periplasm of a recombinant E. coli strain which expressed DraB and DraE (but not DraC). To investigate the donor strand exchange mechanism during the formation of DraE oligomers, we constructed a series of DraE N-terminal deletion mutants. Deletion of the first three N-terminal residues of a potential donor strand resulted in a DraE protein lacking an oligomerization function. In vitro data showed that the DraE disulfide bond was not needed to form a binary complex with the DraB chaperone but was essential in the polymerization process. Our data suggest that assembly of Dr fimbriae requires a chaperone-usher pathway and the donor strand exchange mechanism.
|
730. |
Sauer B,
McDermott J,
( 2004 ) DNA recombination with a heterospecific Cre homolog identified from comparison of the pac-c1 regions of P1-related phages. PMID : 15550568 : DOI : 10.1093/nar/gkh941 PMC : PMC534624 Abstract >>
Sequencing of the 7 kb immC region from four P1-related phages identified a novel DNA recombinase that exhibits many Cre-like characteristics, including recombination in mammalian cells, but which has a distinctly different DNA specificity. DNA sequence comparison to the P1 immC region showed that all phages had related DNA terminase, C1 repressor and DNA recombinase genes. Although these genes from phages P7, phi(w39) and p15B were highly similar to those from P1, those of phage D6 showed significant divergence. Moreover, the D6 sequence showed evidence of DNA deletion and substitution in this region relative to the other phages. Characterization of the D6 site-specific DNA recombinase (Dre) showed that it was a tyrosine recombinase closely related to the P1 Cre recombinase, but that it had a distinct DNA specificity for a 32 bp DNA site (rox). Cre and Dre are heterospecific: Cre did not catalyze recombination at rox sites and Dre did not catalyze recombination at lox sites. Like Cre, Dre catalyzed both integrative and excisive recombination and required no other phage-encoded proteins for recombination. Dre-mediated recombination in mammalian cells showed that, like Cre, no host bacterial proteins are required for efficient Dre-mediated site-specific DNA recombination.
|
731. |
Toma C,
Martínez Espinosa E,
Song T,
Miliwebsky E,
Chinen I,
Iyoda S,
Iwanaga M,
Rivas M,
( 2004 ) Distribution of putative adhesins in different seropathotypes of Shiga toxin-producing Escherichia coli. PMID : 15528677 : DOI : 10.1128/JCM.42.11.4937-4946.2004 PMC : PMC525252 Abstract >>
The distribution of eight putative adhesins that are not encoded in the locus for enterocyte effacement (LEE) in 139 Shiga toxin-producing Escherichia coli (STEC) of different serotypes was investigated by PCR. Five of the adhesins (Iha, Efa1, LPF(O157/OI-141), LPF(O157/OI-154), and LPF(O113)) are encoded in regions corresponding to genomic O islands of E. coli EDL933, while the other three adhesins have been reported to be encoded in the STEC megaplasmid of various serotypes (ToxB [O157:H7], Saa [O113:H21], and Sfp [O157:NM]). STEC strains were isolated from humans (n = 54), animals (n = 52), and food (n = 33). They were classified into five seropathotypes (A through E) based on the reported occurrence of STEC serotypes in human disease, in outbreaks, and in the hemolytic-uremic syndrome (M. A. Karmali, M. Mascarenhas, S. Shen, K. Ziebell, S. Johnson, R. Reid-Smith, J. Isaac-Renton, C. Clark, K. Rahn, and J. B. Kaper, J. Clin. Microbiol. 41:4930-4940, 2003). The most prevalent adhesin was that encoded by the iha gene (91%; 127 of 139 strains), which was distributed in all seropathotypes. toxB and efa1 were present mainly in strains of seropathotypes A and B, which were LEE positive. saa was present only in strains of seropathotypes C, D, and E, which were LEE negative. Two fimbrial genes, lpfA(O157/OI-141) and lpfA(O157/OI-154), were strongly associated with seropathotype A. The fimbrial gene lpfA(O113) was present in all seropathotypes except for seropathotype A, while sfpA was not present in any of the strains studied. The distribution of STEC adhesins depends mainly on serotypes and not on the source of isolation. Seropathotype A, which is associated with severe disease and frequently is involved in outbreaks, possesses a unique adhesin profile which is not present in the other seropathotypes. The wide distribution of iha in STEC strains suggested that it could be a candidate for vaccine development.
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732. |
Rezwan F,
Lan R,
Reeves PR,
( 2004 ) Molecular basis of the indole-negative reaction in Shigella strains: extensive damages to the tna operon by insertion sequences. PMID : 15489459 : DOI : 10.1128/JB.186.21.7460-7465.2004 PMC : PMC523188 Abstract >>
The molecular basis of the loss of tryptophan utilization (indole-negative phenotype) of Shigella strains, in effect clones of Escherichia coli, was investigated. Analysis of the tna operon of 23 Shigella strains representing each of the indole-negative serotypes revealed that insertion sequence-mediated insertion and/or deletions damaged the tna operon, leading to inability to convert tryptophan to indole. These events differ for cluster 1, cluster 3, and the outlier Shigella strains, confirming our previous observation of independent origins of these lineages from within E. coli. Parallel loss of the trait and prevalence of indole-negative strains suggest that the trait is deleterious in Shigella strains and advantages those without it.
|
733. |
Feng L,
Senchenkova SN,
Tao J,
Shashkov AS,
Liu B,
Shevelev SD,
Reeves PR,
Xu J,
Knirel YA,
Wang L,
( 2005 ) Structural and genetic characterization of enterohemorrhagic Escherichia coli O145 O antigen and development of an O145 serogroup-specific PCR assay. PMID : 15629947 : DOI : 10.1128/JB.187.2.758-764.2005 PMC : PMC543545 Abstract >>
Enterohemorrhagic Escherichia coli O145 strains are emerging as causes of hemorrhagic colitis and hemolytic uremic syndrome. In this study, we present the structure of the E. coli O145 O antigen and the sequence of its gene cluster. The O145 antigen has repeat units containing three monosaccharide residues: 2-acetamido-2-deoxy-D-glucose (GlcNAc), 2-acetamidoylamino-2,6-dideoxy-L-galactose, and N-acetylneuraminic acid. It is very closely related to Salmonella enterica serovar Touera and S. enterica subsp. arizonae O21 antigen. The E. coli O145 gene cluster is located between the JUMPStart sequence and the gnd gene and consists of 15 open reading frames. Putative genes for the synthesis of the O-antigen constituents, for sugar transferase, and for O-antigen processing were annotated based on sequence similarities and the presence of conserved regions. The putative genes located in the E. coli O145 O-antigen gene cluster accounted for all functions expected for synthesis of the structure. An E. coli O145 serogroup-specific PCR assay based on the genes wzx and wzy was also developed by screening E. coli and Shigella isolates of different serotypes.
|
734. |
Wang W,
Seah SY,
( 2005 ) Purification and biochemical characterization of a pyruvate-specific class II aldolase, HpaI. PMID : 15996099 : DOI : 10.1021/bi050607y Abstract >>
HpaI, a class II pyruvate-specific aldolase involved in the catabolic pathway of hydroxyphenylacetate, is overexpressed and purified. A previous suggestion that phosphate is involved in proton transfer of pyruvate, based on the crystal structure of the homologous 2-dehydro-3-deoxygalactarate aldolase, is not substantiated from biochemical studies with HpaI. Thus, specific activities of the enzyme for the substrate 4-hydroxy-2-ketopentanoate in sodium HEPES and Tris-acetate buffers are higher than in sodium phosphate buffer. The enzyme also catalyzed the partial reaction of pyruvate proton exchange with an initial rate of 0.77 mmol min(-)(1) mg(-)(1) in phosphate-free buffer, as monitored by nuclear magnetic resonance. Steady-state kinetic analysis shows that the enzyme is also able to catalyze the aldol cleavage of 4-hydroxy-2-ketohexanoate and 3-deoxy-d-manno-oct-2-ulosonic acid (KDO). The enzyme exhibits significant oxaloacetate decarboxylase activity, with a k(cat) value 2.4-fold higher than the corresponding value for the aldol cleavage of 4-hydroxy-2-ketopentanoate. Sodium oxalate, an analogue of the enolate intermediate of the enzyme-catalyzed reaction, is a competitive inhibitor of the enzyme, with a K(i) value of 5.5 microM. Replacement of an active site arginine residue (R70) with alanine by site-specific mutagenesis resulted in an enzyme that lacks both aldolase and decarboxylase activities. The mutant enzyme is also unable to catalyze pyruvate proton exchange. The dissociation constant for pyruvate in the R70A mutant, determined by fluorescence titration, is similar to that of the wild-type enzyme, indicating that pyruvate binding is not affected by this mutation. Together, the results show that R70 influences catalysis in HpaI, particularly at the pyruvate proton exchange step.
|
735. |
Anantha RP,
McVeigh AL,
Lee LH,
Agnew MK,
Cassels FJ,
Scott DA,
Whittam TS,
Savarino SJ,
( 2004 ) Evolutionary and functional relationships of colonization factor antigen i and other class 5 adhesive fimbriae of enterotoxigenic Escherichia coli. PMID : 15557644 : DOI : 10.1128/IAI.72.12.7190-7201.2004 PMC : PMC529125 Abstract >>
Colonization factor antigen I (CFA/I) is the archetype of eight genetically related fimbriae of enterotoxigenic Escherichia coli (ETEC) designated class 5 fimbriae. Assembled by the alternate chaperone pathway, these organelles comprise a rigid stalk of polymerized major subunits and an apparently tip-localized minor adhesive subunit. We examined the evolutionary relationships of class 5-specific structural proteins and correlated these with functional properties. We sequenced the gene clusters encoding coli surface antigen 4 (CS4), CS14, CS17, CS19, and putative colonization factor antigen O71 (PCFO71) and analyzed the deduced proteins and the published homologs of CFA/I, CS1, and CS2. Multiple alignment and phylogenetic analysis of the proteins encoded by each operon define three subclasses, 5a (CFA/I, CS4, and CS14), 5b (CS1, CS17, CS19, and PCFO71), and 5c (CS2). These share distant evolutionary relatedness to fimbrial systems of three other genera. Subclass divisions generally correlate with distinguishing in vitro adherence phenotypes of strains bearing the ETEC fimbriae. Phylogenetic comparisons of the individual structural proteins demonstrated greater intrasubclass conservation among the minor subunits than the major subunits. To correlate this with functional attributes, we made antibodies against CFA/I and CS17 whole fimbriae and maltose-binding protein fusions with the amino-terminal half of the corresponding minor subunits. Anti-minor subunit Fab preparations showed hemagglutination inhibition (HAI) of ETEC expressing homologous and intrasubclass heterologous colonization factors while anti-fimbrial Fab fractions showed HAI activity limited to colonization factor-homologous ETEC. These results were corroborated with similar results from the Caco-2 cell adherence assay. Our findings suggest that the minor subunits of class 5 fimbriae may be superior to whole fimbriae in inducing antiadhesive immunity.
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736. |
Zalewska B,
Piatek R,
Bury K,
Samet A,
Nowicki B,
Nowicki S,
Kur J,
( 2005 ) A surface-exposed DraD protein of uropathogenic Escherichia coli bearing Dr fimbriae may be expressed and secreted independently from DraC usher and DraE adhesin. PMID : 16000738 : DOI : 10.1099/mic.0.28083-0 Abstract >>
The dra gene cluster, expressed by uropathogenic Escherichia coli strains, determines bacterial attachment and invasion. The Dr fimbrial structures formed at the bacterial cell surface are composed of DraE subunits. The Dr fimbriae-coding cluster contains six open reading frames--draA, draB, draC, draD, draP and draE--among which the draE gene encodes the structural fimbrial subunit DraE. Very little is known about E. coli surface expression of the draD gene product. The expression of DraD and its role in the biogenesis of Dr fimbriae were determined by constructing mutants in the dra operon and by immunoblot and immunofluorescence experiments. In this study, DraD was found to be a surface-exposed protein. The expression of DraD was independent of the DraC usher and DraE fimbrial subunits. Polymerization of DraE fimbrial subunits into fimbrial structures did not require expression of the DraD protein.
|
737. |
Hashimoto H,
Shimizu T,
Imasaki T,
Kato M,
Shichijo N,
Kita K,
Sato M,
( 2005 ) Crystal structures of type II restriction endonuclease EcoO109I and its complex with cognate DNA. PMID : 15590682 : DOI : 10.1074/jbc.M411684200 Abstract >>
EcoO109I is a type II restriction endonuclease that recognizes the DNA sequence of RGGNCCY. Here we describe the crystal structures of EcoO109I and its complex with DNA. A comparison of the two structures shows that the catalytic domain moves drastically to capture the DNA. One metal ion and two water molecules are observed near the active site of the DNA complex. The metal ion is a Lewis acid that stabilizes the pentavalent phosphorus atom in the transition state. One water molecule, activated by Lys-126, attacks the phosphorus atom in an S(N)2 mechanism, whereas the other water interacts with the 3'-leaving oxygen to donate a proton to the oxygen. EcoO109I is similar to EcoRI family enzymes in terms of its DNA cleavage pattern and folding topology of the common motif in the catalytic domain, but it differs in the manner of DNA recognition. Our findings propose a novel classification of the type II restriction endonucleases and lead to the suggestion that EcoO109I represents a new subclass of the EcoRI family.
|
738. |
Müller V,
Jones CJ,
Kawagishi I,
Aizawa S,
Macnab RM,
( 1992 ) Characterization of the fliE genes of Escherichia coli and Salmonella typhimurium and identification of the FliE protein as a component of the flagellar hook-basal body complex. PMID : 1551848 : DOI : 10.1128/jb.174.7.2298-2304.1992 PMC : PMC205851 Abstract >>
Within flagellar region III of Escherichia coli and Salmonella typhimurium, the genomic organization has been largely established. An exception is fliE, a gene whose exact location and product function are not well understood. We cloned the fliE gene, obtained its DNA sequence, and identified its product.fliE was found to be a monocistronic transcriptional unit, adjacent to and divergent from the large fliF operon. It is several kilobases distant from the nearest flagellar operon in the other direction, the fliD operon, and constitutes the first operon within the newly defined region IIIb, which contains the genes fliE through fliR.fliE encodes a small, moderately hydrophilic protein with a deduced molecular mass of 11,114 Da (E. coli) or 11,065 Da (S. typhimurium). We identified a protein within the isolated hook-basal body complex as the fliE gene product on the basis of its size and comparison of its N-terminal amino acid sequence with that deduced from the gene sequence. From gel electrophoresis and autoradiography of 35S-labeled S. typhimurium hook-basal body complexes (C.J. Jones, R.M. Macnab, H. Okino, and S.-I. Aizawa, J. Mol. Biol. 212:377-387, 1990) and the deduced number of sulfur-containing residues in FliE, we estimated the stoichiometry of the protein in the hook-basal body complex to be about nine subunits. FliE does not undergo cleavage of a signal peptide, nor does it show any sequence similarity to the axial components like the rod or hook proteins, which are believed to be exported by the flagellum-specific export pathway. On the basis of this and other evidence, we suggest that FliE may be in the vicinity of the MS ring, perhaps acting as an adaptor protein between the ring and rod substructures.
|
739. |
Olesen I,
Hasman H,
Aarestrup FM,
( 2004 ) Prevalence of beta-lactamases among ampicillin-resistant Escherichia coli and Salmonella isolated from food animals in Denmark. PMID : 15650379 : DOI : 10.1089/mdr.2004.10.334 Abstract >>
The genetic background for beta-lactamase-mediated resistance to beta-lactam antibiotics was examined by PCR and sequencing in 160 ampicillin-resistant isolates (109 Escherichia coli and 51 Salmonella) obtained from healthy and diseased food animals in Denmark. Sequencing revealed three different variants of bla (TEM-1), of which bla (TEM-1b) was the most frequently detected (80 E. coli and 47 Salmonella), followed by bla (TEM-1a) (eight E. coli, one Salmonella) and bla (TEM-1c) (seven E. coli). A few isolates were found to express OXA, TEM-30, or PSE beta-lactamases. Mutations in the ampC promoter leading to increased production of the AmpC beta-lactamase were demonstrated in 11 cefoxitin-resistant or intermediate E. coli isolates. Nine of these isolates did not contain any bla (TEM) genes, whereas the remaining two did. No genes encoding SHV or extended-spectrum beta-lactamases were detected. Two new variants of bla (TEM) were detected, which have been designated bla (TEM-127) and bla (TEM-128). In TEM-127, amino acid 158 is substituted from His to Asn, whereas a substitution from Asp to Glu is seen at amino acid 157 in TEM-128. According to MIC determinations, these novel enzymes do not possess activity against extended-spectrum beta-lactams.
|
740. |
Zhang W,
Bielaszewska M,
Friedrich AW,
Kuczius T,
Karch H,
( 2005 ) Transcriptional analysis of genes encoding Shiga toxin 2 and its variants in Escherichia coli. PMID : 15640236 : DOI : 10.1128/AEM.71.1.558-561.2005 PMC : PMC544208 Abstract >>
Six of 37 non-O157 Escherichia coli strains possessing Shiga toxin (Stx) 2 gene variant stx(2d) or stx(2e) secreted no detectable Stx. These isolates produced significantly less stx mRNA than Stx2d, Stx2e, Stx2c, or Stx2 secretors did. Standard screening procedures miss a significant subset of E. coli harboring stx(2) variants.
|
741. |
Kim JY,
Song JS,
Bak SH,
Cho YE,
Kim DW,
Jeong SH,
Park YM,
Lee KJ,
Lee SH,
( 2004 ) Dissemination of Escherichia coli producing AmpC-type beta-lactamase (CMY-11) in Korea. PMID : 15380255 : DOI : 10.1016/j.ijantimicag.2004.03.023 Abstract >>
Among the 51 clinical isolates collected from a university hospital in Korea, nine isolates were resistant to cephamycins. Nine isolates were shown to produce CMY-11 and these also included three isolates producing TEM-1. The results from ERIC-PCR revealed that dissemination of CMY-11 was due to outbreaks of resistant species and to the intra-species spread of resistance to cephamycins in Korea. CMY-11 beta-lactamase genes from nine clinical isolates that were responsible for resistance to cephamycins (cefoxitin and cefotetan), amoxicillin, cephalothin and amoxicillin-clavulanic acid, were cloned and characterised. A sequence identical to the common regions in In6, In7 and a novel integron from pSAL-1 was found upstream from bla(CMY-11) gene at nucleotide 1-71. Eighteen nucleotides between position 71 and 72 were inserted into the bla(CMY-11) gene.
|
742. |
Dezfulian H,
Tremblay D,
Harel J,
( 2004 ) Molecular characterization of extraintestinal pathogenic Escherichia coli (ExPEC) pathogenicity islands in F165-positive E. coli strain from a diseased animal. PMID : 15358417 : DOI : 10.1016/j.femsle.2004.07.052 Abstract >>
Septicemic Escherichia coli 4787 (O115: K-: H51: F165) of porcine origin possess gene clusters related to extraintestinal E. coli fimbrial adhesins. This strain produces two fimbriae: F165(1) and F165(2). F165(1) (Prs-like) belongs to the P fimbrial family, encoded by foo operon and F165(2) is a F1C-like encoded by fot operon. Data from this study suggest that these two operons are part of two PAIs. PAI I(4787) includes a region of 20 kb, which not only harbors the foo operon but also contains a potential P4 integrase gene and is located within the pheU tRNA gene, at 94 min of the E. coli chromosome. PAI II(4787) includes a region of over 35 kb, which harbors the fot operon, iroBCDEN gene clusters, as well as part of microcin M genes and nonfunctional mobility genes. PAI II(4787) is found between the proA and yagU at 6 min of the E. coli chromosome.
|
743. |
Gärtner JF,
Schmidt MA,
( 2004 ) Comparative analysis of locus of enterocyte effacement pathogenicity islands of atypical enteropathogenic Escherichia coli. PMID : 15501811 : DOI : 10.1128/IAI.72.11.6722-6728.2004 PMC : PMC523029 Abstract >>
The pathogenicity of enteropathogenic Escherichia coli (EPEC) is linked to the locus of enterocyte effacement, or LEE, encoding a type III secretion system (T3SS) that directly transfers bacterial effector proteins into eukaryotic cells. Atypical diffusely adhering EPEC (DA-EPEC) strains that harbor homologues of the LEE but lack the EPEC adherence factor plasmid have been increasingly associated with outbreaks of diarrhea. In this study, we have completely sequenced and functionally characterized LEE pathogenicity islands derived from the clinical DA-EPEC isolates 3431 (O8:H-) and 0181 (O119:H9:K61). LEE3431 and LEE0181 exhibit genetic organization analogous to that of the prototype LEE(E2348/69). Genes constituting the T3SS apparatus are highly conserved. However, LEE-encoded effector proteins exhibit major differences. Transfer and functional expression of LEE0181 in an E. coli XL1 blue MR background demonstrated that LEE0181 contains all the information for signal transduction and pedestal formation.
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744. |
Jiménez Gómez PA,
García de los Rios JE,
Rojas Mendoza A,
de Pedro Ramonet P,
García Albiach R,
Reche Sainz MP,
( 2004 ) Molecular basis of quinolone resistance in Escherichia coli from wild birds. PMID : 15352551 : PMC : PMC1142146 Abstract >>
Nine quinolone resistant (minimal inhibitory concentration [MIC] was > 32 microg/mL for nalidixic acid, > 1 microg/mL for ciprofloxacin) isolates of Escherichia coli have been found in wild birds with septicemia. All of the isolates were aerobactin positive. The mechanisms of resistance were characterised by sequencing the quinolone resistance-determining region (QRDR) of the gyrA, gyrB, parC, and parE genes. Sequence analysis of the gyrA gene in all isolates identified only 1 nucleotide substitution at codon Serine-83 for Leucine-83. Sequence analysis of the gyrB, parC, and parE QRDR genes revealed no mutations in any of the isolates. This study was conducted to determine the importance of these genes in the susceptibility of E. coli strains isolated from wild birds to quinolones.
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745. |
Meyer T,
Karch H,
Hacker J,
Bocklage H,
Heesemann J,
( 1992 ) Cloning and sequencing of a Shiga-like toxin II-related gene from Escherichia coli O157:H7 strain 7279. PMID : 1559006 : Abstract >>
Escherichia coli O157:H7 strains are newly recognized pathogens associated with haemorrhagic colitis and haemolytic-uremic syndrome. In addition to Shiga-like toxin types I and II known to be produced by E. coli O157 isolates, we have identified in E. coli O157:H7 strain 7279 a third toxin, designated SLT-IIvhc, that is neutralized neither by anti-SLT-I nor by anti-SLT-II antibodies. The genes for this toxin were isolated by using a PCR-mediated cell-free cloning technique. DNA sequence analysis revealed a high degree of homologies to SLT-II and several SLT-II variants. The predicted amino acid sequence of the A subunit of SLT-IIvhc differed from that of the O91:H7 toxin VT2ha and SLT-IIc from E. coli O157:H- strain E2511 by 2 and 3 amino acids, respectively. The amino acid sequence of its B subunit was identical to VT2ha and SLT-IIc but different from SLT-II and SLT-IIv. Immunological differences of SLT-IIvhc and SLT-II as well as their different toxicity to HeLa cells presumably resulted from the small deviations within the primary structure of the B subunit.
|
746. |
Kostakioti M,
Stathopoulos C,
( 2004 ) Functional analysis of the Tsh autotransporter from an avian pathogenic Escherichia coli strain. PMID : 15385451 : DOI : 10.1128/IAI.72.10.5548-5554.2004 PMC : PMC517524 Abstract >>
The temperature-sensitive hemagglutinin (Tsh) is an autotransporter protein secreted by avian-pathogenic Escherichia coli strains that colonize the respiratory tract and lead to airsacculitis, pericarditis, and colisepticemia. It is synthesized as a 140-kDa precursor protein, whose processing results in a 106-kDa passenger domain (Tshs) and a 33-kDa beta-domain (Tsh(beta)). The presence of a conserved 7-amino-acid serine protease motif within Tshs classifies the protein in a subfamily of autotransporters, known as serine protease autotransporters of the Enterobacteriaceae. In this study, we report that purified Tshs is capable of adhering to red blood cells, hemoglobin, and the extracellular matrix proteins fibronectin and collagen IV. We also demonstrate that Tshs exerts proteolytic activity against casein, and we provide experimental evidence demonstrating that serine 259 is essential for the protease function. However, this residue is not required for adherence to substrates, and its replacement by an alanine does not abolish binding activity. In summary, our results demonstrate that Tsh is a bifunctional protein with both adhesive and proteolytic properties.
|
747. |
Sherlock O,
Schembri MA,
Reisner A,
Klemm P,
( 2004 ) Novel roles for the AIDA adhesin from diarrheagenic Escherichia coli: cell aggregation and biofilm formation. PMID : 15547278 : DOI : 10.1128/JB.186.23.8058-8065.2004 PMC : PMC529095 Abstract >>
Diarrhea-causing Escherichia coli strains are responsible for numerous cases of gastrointestinal disease and constitute a serious health problem throughout the world. The ability to recognize and attach to host intestinal surfaces is an essential step in the pathogenesis of such strains. AIDA is a potent bacterial adhesin associated with some diarrheagenic E. coli strains. AIDA mediates bacterial attachment to a broad variety of human and other mammalian cells. It is a surface-displayed autotransporter protein and belongs to the selected group of bacterial glycoproteins; only the glycosylated form binds to mammalian cells. Here, we show that AIDA possesses self-association characteristics and can mediate autoaggregation of E. coli cells. We demonstrate that intercellular AIDA-AIDA interaction is responsible for bacterial autoaggregation. Interestingly, AIDA-expressing cells can interact with antigen 43 (Ag43)-expressing cells, which is indicative of an intercellular AIDA-Ag43 interaction. Additionally, AIDA expression dramatically enhances biofilm formation by E. coli on abiotic surfaces in flow chambers.
|
748. |
Timms AR,
Steingrimsdottir H,
Lehmann AR,
Bridges BA,
( 1992 ) Mutant sequences in the rpsL gene of Escherichia coli B/r: mechanistic implications for spontaneous and ultraviolet light mutagenesis. PMID : 1552908 : DOI : 10.1007/bf00299141 Abstract >>
Mutants able to grow in the presence of 1.2 mg/ml streptomycin were isolated from Escherichia coli WP2 after exposure to ultraviolet light (UV) or in the absence of any treatment (spontaneous), and from a umuC derivative after exposure to UV and delayed photoreversal. These mutants, characterized as streptomycin resistant (Smr) or dependent (Smd), carry mutations in the rpsL gene. This gene was amplified using the polymerase chain reaction and sequenced. Mutations induced by UV were largely (76%) of the Smr phenotype, all of which were changes at an A:T base pair at codons 42 or 87. Mutations induced by UV plus delayed photoreversal in the non-UV-mutable umuC122 derivative of WP2 were exclusively of the Smd phenotype and all occurred at G:C base pairs at codons 41, 90 or 91. These results are consistent with current understanding of the mechanism of mutagenesis by UV and delayed photoreversal. A broader spectrum of mutations was seen in the spontaneous series including three-base deletions leading to amino acid loss (2 of codon 93, 1 of codon 87). Of particular note was the number of intragenic second site mutations in the spontaneous series, most if not all of which appeared to be silent with respect to streptomycin phenotype. It is necessary to postulate a high rate of formation of such mutations at some stage during the experiment. One possibility is that spontaneous mutation may often occur in bursts when an error correction mechanism (eg., proofreading, mismatch correction) is temporarily inactive.(ABSTRACT TRUNCATED AT 250 WORDS)
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749. |
Gentschev I,
Goebel W,
( 1992 ) Topological and functional studies on HlyB of Escherichia coli. PMID : 1552901 : DOI : 10.1007/bf00299135 Abstract >>
The topology of HlyB, a protein located in the inner membrane of Escherichia coli and involved in the secretion of alpha-haemolysin (HlyA), was determined by the generation of HlyB-PhoA and HlyB-LacZ fusion proteins. The data obtained by this biochemical method together with computer predictions suggest that HlyB is inserted in the cytoplasmic membrane by six stable hydrophobic, alpha-helical transmembrane segments. These segments extend from amino acid positions 158 to 432 of HlyB. The cytoplasmic loops between these transmembrane segments are relatively large and carry an excess of positively charged amino acids, while the periplasmic loops are rather small. In addition to these six transmembrane segments, two additional regions in the 78 N-terminal amino acids of HlyB appear to be also inserted in the cytoplasmic membrane. However, the association of these two segments with the cytoplasmic membrane seems to be less tight, since active PhoA and LacZ fusions were obtained by insertion into the same positions of these segments. A LacZ-HlyAs, fusion protein carrying, at the C-terminus of LacZ, the 60-amino acid signal sequence of HlyA was not secreted in the presence of HlyB/HlyD. However, transport of this fusion protein into the cytoplasmic membrane appeared to be initiated, as suggested by the tight association of this protein with the inner membrane. A similar close association of LacZ-HlyAs with the inner membrane was also observed in the presence of HlyB alone but not in its absence. These data suggest that HlyB recognizes the HlyA signal sequence and initiates the transport of HlyA into the membrane.
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750. |
Ojo KK,
Tung D,
Luis H,
Bernardo M,
Leitao J,
Roberts MC,
( 2004 ) Gram-positive merA gene in gram-negative oral and urine bacteria. PMID : 15358427 : DOI : 10.1016/j.femsle.2004.08.004 Abstract >>
Clinical mercury resistant (Hg(r)) Gram-negative bacteria carrying Gram-positive mercury reductase (merA)-like genes were characterized using DNA-DNA hybridization, PCR and sequencing. A PCR assay was developed which discriminated between the merA genes related to Staphylococcus and those related to the Bacillus/Streptococcus merA genes by the difference in size of the PCR product. DNA sequence analysis correlated with the PCR assay. The merA genes from Acinetobacter junii, Enterobacter cloacae and Escherichia coli were sequenced and shared 98-99% identical nucleotide (nt) and 99.6-100% amino acid identity with the Staphylococcus aureus MerA protein. A fourth merA gene, from Pantoeae agglomerans, was partially sequenced (60%) and had 99% identical nt and 100% amino acid identity with the Streptococcus oralis MerA protein. All the Hg(r) Gram-negative bacteria transferred their Gram-positive merA genes to a Gram-positive Enterococcus faecalis recipient with the resulting transconjugants expressing mercury resistance. These Gram-positive merA genes join Gram-positive tetracycline resistance and Gram-positive macrolide resistance genes in their association with mobile elements which are able to transfer and express in Gram-negative bacteria.
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751. |
Borovok I,
Gorovitz B,
Yanku M,
Schreiber R,
Gust B,
Chater K,
Aharonowitz Y,
Cohen G,
( 2004 ) Alternative oxygen-dependent and oxygen-independent ribonucleotide reductases in Streptomyces: cross-regulation and physiological role in response to oxygen limitation. PMID : 15522084 : DOI : 10.1111/j.1365-2958.2004.04325.x Abstract >>
Ribonucleotide reductases (RNRs) catalyse the conversion of ribonucleotides to deoxyribonucleotides and are essential for de novo DNA synthesis and repair. Streptomyces spp. contain genes coding for two RNRs. We show here that the Streptomyces coelicolor M145 nrdAB genes encoding an oxygen-dependent class I RNR are co-transcribed with nrdS, which encodes an AraC-like regulatory protein. Likewise, the class II oxygen-independent RNR nrdJ gene forms an operon with a likely regulatory gene, nrdR, which encodes a protein possessing an ATP-cone domain like those present in the allosteric activity site of many class Ia RNRs. Deletions in nrdB and nrdJ had no discernible effect on growth individually, but abolition of both RNR systems, using hydroxyurea to inactivate the class Ia RNR (NrdAB) in the nrdJ deletion mutant, was lethal, establishing that S. coelicolor possesses just two functional RNR systems. The class II RNR (NrdJ) may function to provide a pool of deoxyribonucleotide precursors for DNA repair during oxygen limitation and/or for immediate growth after restoration of oxygen, as the nrdJ mutant was slower in growth recovery than the nrdB mutant or the parent strain. The class Ia and class II RNR genes show complex regulation. The nrdRJ genes were transcribed some five- to sixfold higher than the nrdABS genes in vegetative growth, but when nrdJ was deleted, nrdABS transcription was upregulated by 13-fold. In a reciprocal experiment, deletion of nrdB had little effect on nrdRJ transcription. Deletion of nrdR caused a dramatic increase in transcription of nrdJ and to a less extent nrdABS, whereas disruption of cobN, a gene required for synthesis of coenzyme B12 a cofactor for the class II RNR, caused similar upregulation of transcription of nrdRJ and nrdABS. In contrast, deletion of nrdS had no detectable effect on transcription of either set of RNR genes. These results establish the existence of control mechanisms that sense and regulate overall RNR gene expression.
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752. |
Bernier-Fébreau C,
du Merle L,
Turlin E,
Labas V,
Ordonez J,
Gilles AM,
Le Bouguénec C,
( 2004 ) Use of deoxyribose by intestinal and extraintestinal pathogenic Escherichia coli strains: a metabolic adaptation involved in competitiveness. PMID : 15385522 : DOI : 10.1128/IAI.72.10.6151-6156.2004 PMC : PMC517565 Abstract >>
We showed that the deoK operon, which confers the ability to use deoxyribose as a carbon source, is more common among pathogenic than commensal Escherichia coli strains. The expression of the deoK operon increases the competitiveness of clinical isolates, suggesting that this biochemical characteristic plays a role in host infectivity.
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753. |
Schneider G,
Dobrindt U,
Brüggemann H,
Nagy G,
Janke B,
Blum-Oehler G,
Buchrieser C,
Gottschalk G,
Emödy L,
Hacker J,
( 2004 ) The pathogenicity island-associated K15 capsule determinant exhibits a novel genetic structure and correlates with virulence in uropathogenic Escherichia coli strain 536. PMID : 15385503 : DOI : 10.1128/IAI.72.10.5993-6001.2004 PMC : PMC517556 Abstract >>
The K15 capsule determinant of uropathogenic Escherichia coli strain 536 (O6:K15:H31) is part of a novel 79.6-kb pathogenicity island (PAI) designated PAI V536 that is absent from the genome of nonpathogenic E. coli K-12 strain MG1655. PAI V536 shows typical characteristics of a composite PAI that is associated with the pheV tRNA gene and contains the pix fimbriae determinant as well as genes coding for a putative phosphoglycerate transport system, an autotransporter protein, and hypothetical open reading frames. A gene cluster coding for a putative general secretion pathway system, together with a kps(K15) determinant, is localized downstream of a truncated pheV gene ('pheV) also present in this chromosomal region. The distribution of genes present on PAI V536 was studied by PCR in different pathogenic and nonpathogenic E. coli isolates of various sources. Analysis of the 20-kb kps locus revealed a so far unknown genetic organization. Generally, the kps(K15) gene cluster resembles that of group 2 and 3 capsules, where two conserved regions (regions 1 and 3) are located up- or downstream of a highly variable serotype-specific region (region 2). Interestingly, recombination of a group 2 and 3 determinant may have been involved in the evolution of the K15 capsule-encoding gene cluster. Expression of the K15 capsule is important for virulence in a murine model of ascending urinary tract infection but not for serum resistance of E. coli strain 536.
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754. |
Munday CJ,
Boyd DA,
Brenwald N,
Miller M,
Andrews JM,
Wise R,
Mulvey MR,
Hawkey PM,
( 2004 ) Molecular and kinetic comparison of the novel extended-spectrum beta-lactamases CTX-M-25 and CTX-M-26. PMID : 15561863 : DOI : 10.1128/AAC.48.12.4829-4834.2004 PMC : PMC529179 Abstract >>
CTX-M-25 is a novel extended-spectrum beta-lactamase isolated from a single Canadian Escherichia coli isolate. Susceptibility testing demonstrated that this enzyme confers resistance to both cefotaxime and ceftazidime, but the level of resistance was reduced with the addition of beta-lactamase inhibitors. The bla(CTX-M-25) gene was detected on a 111-kb plasmid. It is a member of the CTX-M-8 group and has the closest amino acid identity (99%; three amino acid substitutions) with CTX-M-26. The bla(CTX-M-26) gene was detected on a 100-kb plasmid isolated from a Klebsiella pneumoniae strain from the United Kingdom, and plasmid profiling revealed that it showed some homology to the bla(CTX-M-25)-harboring plasmid. Both CTX-M genes were located downstream of ISEcp1, although the copy upstream of bla(CTX-M-25) was disrupted by IS50-A. Comparative kinetic studies of recombinant CTX-M-25 and CTX-M-26 enzymes showed that CTX-M-25 has a higher level of ceftazidime hydrolysis (kcat values, 33 and 0.005 s(-1) for CTX-M-25 and CTX-M-26, respectively).
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755. |
Fratamico PM,
Bagi LK,
Bush EJ,
Solow BT,
( 2004 ) Prevalence and characterization of shiga toxin-producing Escherichia coli in swine feces recovered in the National Animal Health Monitoring System's Swine 2000 study. PMID : 15574914 : DOI : 10.1128/AEM.70.12.7173-7178.2004 PMC : PMC535163 Abstract >>
A study was conducted to determine the prevalence of Shiga toxin-producing Escherichia coli (STEC) in swine feces in the United States as part of the National Animal Health Monitoring System's Swine 2000 study. Fecal samples collected from swine operations from 13 of the top 17 swine-producing states were tested for the presence of STEC. After enrichment of swine fecal samples in tryptic soy broth, the samples were tested for the presence of stx1 and stx2 by use of the TaqMan E. coli STX1 and STX2 PCR assays. Enrichments of samples positive for stx1 and/or stx2 were plated, and colony hybridization was performed using digoxigenin-labeled probes complementary to the stx1 and stx2 genes. Positive colonies were picked and confirmed by PCR for the presence of the stx1, stx2, or stx2e genes, and the isolates were serotyped. Out of 687 fecal samples tested using the TaqMan assays, 70% (484 of 687) were positive for Shiga toxin genes, and 54% (370 of 687), 64% (436 of 687), and 38% (261 of 687) were positive for stx1, stx2, and both toxin genes, respectively. Out of 219 isolates that were characterized, 29 (13%) produced stx1, 14 (6%) produced stx2, and 176 (80%) produced stx2e. Twenty-three fecal samples contained at least two STEC strains that had different serotypes but that had the same toxin genes or included a strain that possessed stx1 in addition to a strain that possessed stx2 or stx2e. The STEC isolates belonged to various serogroups, including O2, O5, O7, O8, O9, OX10, O11, O15, OX18, O20, O57, O65, O68, O69, O78, O91, O96, O100, O101, O120, O121, O152, O159, O160, O163, and O untypeable. It is noteworthy that no isolates of serogroup O157 were recovered. Results of this study indicate that swine in the United States harbor STEC that can potentially cause human illness.
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756. |
Boyd DA,
Tyler S,
Christianson S,
McGeer A,
Muller MP,
Willey BM,
Bryce E,
Gardam M,
Nordmann P,
Mulvey MR,
( 2004 ) Complete nucleotide sequence of a 92-kilobase plasmid harboring the CTX-M-15 extended-spectrum beta-lactamase involved in an outbreak in long-term-care facilities in Toronto, Canada. PMID : 15388431 : DOI : 10.1128/AAC.48.10.3758-3764.2004 PMC : PMC521865 Abstract >>
A major outbreak involving an Escherichia coli strain that was resistant to expanded-spectrum cephalosporins occurred in Toronto and surrounding regions in 2000 to 2002. We report the complete sequence of a plasmid, pC15-1a, that was found associated with the outbreak strain. Plasmid pC15-1a is a circular molecule of 92,353 bp consisting of two distinct regions. The first is a 64-kb region that is essentially homologous to the non-R-determinant region of plasmid R100 except for several point mutations, a few small insertions and deletions, and the absence of Tn10. The second is a 28.4-kb multidrug resistance region (MDR) that has replaced the R-determinant region of the R100 progenitor and consists mostly of transposons or partial transposons and five copies of the insertion element IS26. All drug resistance genes found in pC15-1a, including the beta-lactamase genes bla(CTX-M-15), bla(OXA-1), and bla(TEM-1), the tetracycline resistance gene tetA, and aminoglycoside resistance genes aac(6')-Ib and aac(3)-II, are located in the MDR. The bla(CTX-M-15) gene was found downstream of ISEcp1as part of a transposition unit, as determined from the surrounding sequence. Examination of the plasmids from CTX-M-15-harboring strains isolated from hospitals across Canada showed that pC15-1a was found in several strains isolated from a site in western Canada. Comparison of pC15-1a and pCTX15, found in an E. coli strain isolated in India in 1999, revealed that the plasmids had several features in common, including an R100 backbone and several of the resistance genes, including bla(CTX-M-15), bla(TEM-1), bla(OXA-1), tetA, and aac(6')-Ib.
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757. |
Reischl U,
Youssef MT,
Wolf H,
Hyytia-Trees E,
Strockbine NA,
( 2004 ) Real-time fluorescence PCR assays for detection and characterization of heat-labile I and heat-stable I enterotoxin genes from enterotoxigenic Escherichia coli. PMID : 15364995 : DOI : 10.1128/JCM.42.9.4092-4100.2004 PMC : PMC516355 Abstract >>
To facilitate the diagnosis of enterotoxigenic Escherichia coli (ETEC) infections in humans, we developed and evaluated real-time fluorescence PCR assays for the Roche LightCycler (LC) against the enterotoxin genes commonly present in strains associated with human illness. Separate LC-PCR assays with identical cycling conditions were designed for the type I heat-labile enterotoxin (LT I) and the type I heat-stable enterotoxin (ST I) genes, using the LC hybridization probe format. A duplex assay for ST I with two sets of amplification primers and three hybridization probes was required to detect the major nucleotide sequence variants of ST I, ST Ia and ST Ib. LC-PCR findings from the testing of 161 E. coli isolates of human origin (138 ETEC and 23 non-ETEC) were compared with those obtained by block cycler PCR analysis. The sensitivities and specificities of the LC-PCR assays were each 100% for the LT I and ST I genes. The LC-PCR and block cycler PCR assays were also compared for their abilities to detect LT I and ST I genes in spiked stool specimens with different methods of sample preparation. Findings from these experiments revealed that the limits of detection for the LC-PCR assays were the same or substantially lower than those observed for the block cycler PCR assay. Melting curve analysis of the amplified LT I and ST I genes revealed sequence variation within each gene, which for the ST I genes correlated with the presence of ST Ia and ST Ib. The rapidity, sensitivity, and specificity of the LC-PCR assays make them attractive alternatives to block cycler PCR assays for the detection and characterization of ETEC.
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758. |
Mammeri H,
Nazic H,
Naas T,
Poirel L,
Léotard S,
Nordmann P,
( 2004 ) AmpC beta-lactamase in an Escherichia coli clinical isolate confers resistance to expanded-spectrum cephalosporins. PMID : 15388478 : DOI : 10.1128/AAC.48.10.4050-4053.2004 PMC : PMC521871 Abstract >>
Cloning, sequencing, and biochemical analysis identified a novel AmpC-type beta-lactamase conferring resistance to extended-spectrum cephalosporins in an Escherichia coli clinical isolate. This enzyme, exhibiting 14 amino acid substitutions compared to a reference AmpC cephalosporinase of E. coli, hydrolyzed ceftazidime and cefepime significantly.
|
759. |
Lee JH,
Jung HI,
Jung JH,
Park JS,
Ahn JB,
Jeong SH,
Jeong BC,
Lee JH,
Lee SH,
( 2004 ) Dissemination of transferable AmpC-type beta-lactamase (CMY-10) in a Korean hospital. PMID : 15383166 : DOI : 10.1089/mdr.2004.10.224 Abstract >>
To determine dissemination and genotype of AmpC beta-lactamases and an extended-spectrum beta-lactamase among clinical isolates of Enterobacteriaceae, we performed antibiotic susceptibility testing, pI determination, induction test, plasmid profiles, transconjugation test, enterobacterial repetitive consensus (ERIC)-PCR, and DNA sequencing. Among the 51 clinical isolates collected from a university hospital in Korea, six isolates were resistant to cephamycins. All six isolates produced a plasmid-encoded AmpC-type beta-lactamase, CMY-10. Five strains also produced one or more other beta-lactamases: SHV-12, an extended-spectrum beta-lactamase (five isolates); TEM-1, a class A beta-lactamase (two isolates); and a chromosomal AmpC beta-lactamase (one isolate, a strain of Enterobacter aerogenes, which produced all four of the beta-lactamases that were identified). One of six isolates produced only CMY-10. ERIC-PCR analysis revealed that dissemination of CMY-10 and SHV-12 was due to a clonal outbreak of a resistant strain and to the interspecies spread of resistance to cephamycins and broad-spectrum beta-lactams in Korea. CMY-10 beta-lactamase genes that are responsible for the resistance to cephamycins (cefoxitin and cefotetan), amoxicillin, cephalothin, and amoxicillin-clavulanic acid were cloned and characterized from six clinical isolates. A sequence identical to the common regions in In6, In7, and a novel integron from pSAL-1 was found upstream from blaCMY-10 gene at nucleotides 1-71. A total of 15 nucleotides (I-15) or 18 nucleotides (I-18) between position 71 and 72 were inserted into the blaCMY-10 gene. The blaCMY-10 gene might be inserted into a sul1-type complex integron by I-15 or I-18.
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760. |
Woodford N,
Ward ME,
Kaufmann ME,
Turton J,
Fagan EJ,
James D,
Johnson AP,
Pike R,
Warner M,
Cheasty T,
Pearson A,
Harry S,
Leach JB,
Loughrey A,
Lowes JA,
Warren RE,
Livermore DM,
( 2004 ) Community and hospital spread of Escherichia coli producing CTX-M extended-spectrum beta-lactamases in the UK. PMID : 15347638 : DOI : 10.1093/jac/dkh424 Abstract >>
During 2003, the Health Protection Agency's Antibiotic Resistance Monitoring and Reference Laboratory began to receive isolates of Escherichia coli for confirmation of extended-spectrum beta-lactamase production with a phenotype implying a CTX-M-type beta-lactamase, i.e. MICs of cefotaxime > or = 8-fold higher than MICs of ceftazidime. Many were referred as being from community patients. We examined 291 CTX-M-producing isolates from the UK and investigated the genetic basis of their phenotype. PCR was used to detect alleles encoding CTX-M enzymes and to assign these to their blaCTX-M phylogenetic groups. Selected alleles were sequenced. Producers were compared by analysis of banding patterns generated by pulsed-field gel electrophoresis of XbaI-digested genomic DNA. MICs were determined by an agar dilution method or by Etest. Of 291 CTX-M-producing E. coli isolates studied from 42 UK centres, 70 (24%) were reportedly from community patients, many of whom had only limited recent hospital contact. Community isolates were referred by 12 centres. Two hundred and seventy-nine (95.9%) producers contained genes encoding group 1 CTX-M enzymes and 12 contained blaCTX-M-9-like alleles. An epidemic CTX-M-15-producing strain was identified, with 110 community and inpatient isolates referred from six centres. Representatives of four other major strains also produced CTX-M-15, as did several sporadic isolates examined. Most producers were multi-resistant to fluoroquinolones, trimethoprim, tetracycline and aminoglycosides as well as to non-carbapenem beta-lactams. CTX-M-producing E. coli are a rapidly developing problem in the UK, with CTX-M-15 particularly common. The diversity of producers and geographical scatter of referring laboratories indicates wide dissemination of blaCTX-M genes. Because of the public health implications, including for the treatment of community-acquired urinary tract infections, the spread of these strains--and CTX-M-15 beta-lactamase in particular--merits close monitoring.
|
761. |
Zhang H,
Shi L,
Li L,
Guo S,
Zhang X,
Yamasaki S,
Miyoshi S,
Shinoda S,
( 2004 ) Identification and characterization of class 1 integron resistance gene cassettes among Salmonella strains isolated from healthy humans in China. PMID : 15383699 : DOI : 10.1111/j.1348-0421.2004.tb03473.x Abstract >>
Twenty-three strains of Salmonella spp. isolated from healthy humans in Guangdong, China, were examined for their susceptibility to ten common antibiotics and the presence of antibiotic resistance integrons. All the strains were resistant to at least one antibiotic, and 4 strains were positive for the intI1 gene. Polymerase chain reaction using in-F and in-B primers showed the existence of amplicons of 1,009 bp in two, 1,664 bp in one, and 1,009 bp and 1,664 bp in one of the intI1 -positive isolates, respectively. Sequence analysis revealed that the 1,009-bp amplicon harbored gene cassette aadA2, conferring resistance to spectinomycin, and the 1,664-bp amplicon harbored genes aadA5 and dfr17, conferring resistance to spectinomycin, streptomycin and trimethoprim. Meanwhile the experiments of plasmid conjugation and Southern hybridization with intI1 as the DNA probe indicated that all the integrons found in these strains were chromosomal. Because the strains carrying class 1 integrons were isolated from healthy humans, it suggests the need for all-round surveillance of the antibiotic resistance of pathogens.
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762. |
De Baets L,
Van der Taelen I,
De Filette M,
Piérard D,
Allison L,
De Greve H,
Hernalsteens JP,
Imberechts H,
( 2004 ) Genetic typing of shiga toxin 2 variants of Escherichia coli by PCR-restriction fragment length polymorphism analysis. PMID : 15466582 : DOI : 10.1128/AEM.70.10.6309-6314.2004 PMC : PMC522131 Abstract >>
Shiga toxins Stx1 and Stx2 play a prominent role in the pathogenesis of Shiga toxin-producing Escherichia coli (STEC) infections. Several variants of the stx(2) gene, encoding Stx2, have been described. In this study, we developed a PCR-restriction fragment length polymorphism system for typing stx(2) genes of STEC strains. The typing system discriminates eight described variants and allows the identification of new stx(2) variants and STEC isolates carrying multiple stx(2) genes. A phylogenetic tree, based on the nucleotide sequences of the toxin-encoding genes, demonstrates that stx(2) sequences with the same PvuII HaeIII HincII AccI type generally cluster together.
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763. |
Möncke-Buchner E,
Mackeldanz P,
Krüger DH,
Reuter M,
( 2004 ) Overexpression and affinity chromatography purification of the Type III restriction endonuclease EcoP15I for use in transcriptome analysis. PMID : 15464603 : DOI : 10.1016/j.jbiotec.2004.06.014 Abstract >>
The Type III restriction endonuclease EcoP15I is a multifunctional hetero-oligomeric enzyme that recognizes the non-symmetric DNA sequence 5'-CAGCAG. For efficient cleavage, EcoP15I needs the interaction with two copies of the recognition sequence that have to be inversely oriented in the DNA double strand. The enzyme cuts the upper DNA strand 25-26 bp and the lower DNA strand 27-28 bp, respectively, downstream of the recognition sequence-a distinct feature that makes the enzyme particularly valuable for gene expression profiling methods relying on the SAGE procedure (Matsumura et al., PNAS 100, 15718, 2003). Because the broader use of this transcriptome analysis method requires the availability of larger amounts of restriction endonuclease EcoP15I and the enzyme is not commercially available, we have cloned the genes coding for the EcoP15I restriction endonuclease into pQE-16 plasmid vector that provides the enzyme with a C-terminal 6xHis-tag. After Ni-NTA affinity chromatography and ion exchange chromatography on heparin sepharose, we obtained 5mg homogeneous EcoP15I per gram cell pellet within 1-2 day(s). Moreover, the C-terminally 6xHis-tagged EcoP15I restriction endonuclease shows comparable enzymatic activity as the untagged enzyme.
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764. |
Meng SY,
Bennett GN,
( 1992 ) Nucleotide sequence of the Escherichia coli cad operon: a system for neutralization of low extracellular pH. PMID : 1556085 : DOI : 10.1128/jb.174.8.2659-2669.1992 PMC : PMC205906 Abstract >>
Lysine decarboxylase of Escherichia coli has been the subject of enzymological studies, and the gene encoding lysine decarboxylase (cadA) and a regulatory gene (cadR) have been mapped. This enzyme is induced at low pH in the presence of lysine and achieves maximal level under anaerobic conditions. The induction of lysine decarboxylase increases the pH of the extracellular medium and provides a distinctive marker in tests of clinical strains. We report the sequence of the cad operon encoding lysine decarboxylase, a protein of 715 amino acids, and another protein, CadB, of 444 amino acids. The amino acid sequence of lysine decarboxylase showed high homology to that of the lysine decarboxylase of Hafnia alvei with less homology to the sequence of speC, which encodes the biosynthetic ornithine decarboxylase of E. coli. The cadA and cadB genes were separately cloned and placed under the control of lac and tac promoters, respectively, to facilitate independent study of their physiological effects. The cadB gene product had a mobility characteristic of a smaller protein on protein gels, analogous to that found for some other membrane proteins. The CadB sequence showed homology to that of ArcD of Pseudomonas aeruginosa, encoding an arginine/ornithine antiporter. Excretion studies of various strains, the coinduction of cadB and cadA, and the attractive physiological role for an antiport system led to a model for the coupled action of cadA and cadB in uptake of lysine, the reduction of H+ concentration, and excretion of cadaverine.
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765. |
Maas R,
( 2004 ) Prereplicative purine methylation and postreplicative demethylation in each DNA duplication of the Escherichia coli replication cycle. PMID : 15448156 : DOI : 10.1074/jbc.M407394200 Abstract >>
Escherichia coli plasmid DNA activated for initiation of duplication is in a stable low linking number supercoiled conformation. Low linking number DNA is methylated at the internal purines of a frequent 5'-Pyr-Pyr-Pur-Pur tetramer with a 5'-Pyr-Pur-3' axis of symmetry and is cut at the axis of symmetry by pneumococcal restriction enzyme DpnI when methylated in both strands. Purine methylation is of adenine in one strand and guanine in the other. Methylation of one of the two purines is removed during the cell cycle, presumably before the reverse shift to the B-supercoiled conformation. The topological transition was reconstituted in vitro only with DNA unmethylated at purines. Methylation-restriction analyses coupled with the chemical properties of low-linking number DNA and B-DNA respectively, suggest that removal of guanine methylation is essential for the low-linking number to B-DNA transition and hence for the deactivation of replication. Demethylation of methylguanine could explain the presence in E. coli of the two-member inducible operon known as ada. Characteristics of ada suggest a cascade of chemical DNA modifications that reverse prereplicative guanine methylation. Guanine demethylation could provide a model for the pivotal role played by de novo methylation in replication and for the essential role of "repair" enzyme ExoIII in demethylation leading to the reversal of replicative DNA activation and other processes that affect DNA function.
|
766. |
Galani I,
Souli M,
Chryssouli Z,
Katsala D,
Giamarellou H,
( 2004 ) First identification of an Escherichia coli clinical isolate producing both metallo-beta-lactamase VIM-2 and extended-spectrum beta-lactamase IBC-1. PMID : 15301681 : DOI : 10.1111/j.1469-0691.2004.00913.x Abstract >>
An Escherichia coli strain with decreased susceptibility to carbapenems was isolated from a hospitalised patient in Athens, Greece. The strain was resistant to all beta-lactams, including aztreonam, whereas the MIC of imipenem and meropenem was 0.5 mg/L. A positive EDTA-disk synergy test suggested the production of a metallo-beta-lactamase. PCR experiments revealed the presence of the bla(VIM-2), bla(IBC-1), and bla(TEM-1) genes. Resistance to beta-lactams was not transferable by conjugation. This is the first report of a clinical isolate of E. coli producing VIM-2, and the first report of the coexistence of bla(VIM-2) and bla(IBC-1) in a single clinical isolate.
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767. |
Ludwig A,
von Rhein C,
Bauer S,
Hüttinger C,
Goebel W,
( 2004 ) Molecular analysis of cytolysin A (ClyA) in pathogenic Escherichia coli strains. PMID : 15292132 : DOI : 10.1128/JB.186.16.5311-5320.2004 PMC : PMC490944 Abstract >>
Cytolysin A (ClyA) of Escherichia coli is a pore-forming hemolytic protein encoded by the clyA (hlyE, sheA) gene that was first identified in E. coli K-12. In this study we examined various clinical E. coli isolates with regard to the presence and integrity of clyA. PCR and DNA sequence analyses demonstrated that 19 of 23 tested Shiga toxin-producing E. coli (STEC) strains, all 7 tested enteroinvasive E. coli (EIEC) strains, 6 of 8 enteroaggregative E. coli (EAEC) strains, and 4 of 7 tested enterotoxigenic E. coli (ETEC) strains possess a complete clyA gene. The remaining STEC, EAEC, and ETEC strains and 9 of the 17 tested enteropathogenic E. coli (EPEC) strains were shown to harbor mutant clyA derivatives containing 1-bp frameshift mutations that cause premature termination of the coding sequence. The other eight EPEC strains and all tested uropathogenic and new-born meningitis-associated E. coli strains (n = 14 and 3, respectively) carried only nonfunctional clyA fragments due to the deletion of two sequences of 493 bp and 204 or 217 bp at the clyA locus. Expression of clyA from clinical E. coli isolates proved to be positively controlled by the transcriptional regulator SlyA. Several tested E. coli strains harboring a functional clyA gene produced basal amounts of ClyA when grown under standard laboratory conditions, but most of them showed a clyA-dependent hemolytic phenotype only when SlyA was overexpressed. The presented data indicate that cytolysin A can play a role only for some of the pathogenic E. coli strains.
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768. |
Grozdanov L,
Raasch C,
Schulze J,
Sonnenborn U,
Gottschalk G,
Hacker J,
Dobrindt U,
( 2004 ) Analysis of the genome structure of the nonpathogenic probiotic Escherichia coli strain Nissle 1917. PMID : 15292145 : DOI : 10.1128/JB.186.16.5432-5441.2004 PMC : PMC490877 Abstract >>
Nonpathogenic Escherichia coli strain Nissle 1917 (O6:K5:H1) is used as a probiotic agent in medicine, mainly for the treatment of various gastroenterological diseases. To gain insight on the genetic level into its properties of colonization and commensalism, this strain's genome structure has been analyzed by three approaches: (i) sequence context screening of tRNA genes as a potential indication of chromosomal integration of horizontally acquired DNA, (ii) sequence analysis of 280 kb of genomic islands (GEIs) coding for important fitness factors, and (iii) comparison of Nissle 1917 genome content with that of other E. coli strains by DNA-DNA hybridization. PCR-based screening of 324 nonpathogenic and pathogenic E. coli isolates of different origins revealed that some chromosomal regions are frequently detectable in nonpathogenic E. coli and also among extraintestinal and intestinal pathogenic strains. Many known fitness factor determinants of strain Nissle 1917 are localized on four GEIs which have been partially sequenced and analyzed. Comparison of these data with the available knowledge of the genome structure of E. coli K-12 strain MG1655 and of uropathogenic E. coli O6 strains CFT073 and 536 revealed structural similarities on the genomic level, especially between the E. coli O6 strains. The lack of defined virulence factors (i.e., alpha-hemolysin, P-fimbrial adhesins, and the semirough lipopolysaccharide phenotype) combined with the expression of fitness factors such as microcins, different iron uptake systems, adhesins, and proteases, which may support its survival and successful colonization of the human gut, most likely contributes to the probiotic character of E. coli strain Nissle 1917.
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769. |
Vandemaele FJ,
Hensen SM,
Goddeeris BM,
( 2004 ) Conservation of deduced amino acid sequence of FimH among Escherichia coli of bovine, porcine and avian disease origin. PMID : 15172698 : DOI : 10.1016/j.vetmic.2004.03.013 Abstract >>
The FimH subunit of type 1 pili mediates adhesion of Escherichia coli to epithelium in different animal hosts. In this study, we sequenced and analyzed the fimH genes of 24 E. coli strains from bovine and porcine clinical cases. The obtained sequences were compared among each other and also with 24 known fimH sequences from avian E. coli strains. This comparison revealed a substantial homology (>99%) among strains from the different animal species origins. Moreover, specific mutations were found, some of which were present more frequently in avian strains or in bovine and porcine strains.
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770. |
Joensuu JJ,
Kotiaho M,
Riipi T,
Snoeck V,
Palva ET,
Teeri TH,
Lång H,
Cox E,
Goddeeris BM,
Niklander-Teeri V,
( 2004 ) Fimbrial subunit protein FaeG expressed in transgenic tobacco inhibits the binding of F4ac enterotoxigenic Escherichia coli to porcine enterocytes. PMID : 15359606 : Abstract >>
Plants offer a promising alternative for the production of foreign proteins for pharmaceutical purposes in tissues that are consumed as food and/or feed. Our long-term strategy is to develop edible vaccines against piglet diarrhoea caused by enterotoxigenic Escherichia coli (F4 ETEC) in feed plants. In this work, we isolated a gene, faeG, encoding for a major F4ac fimbrial subunit protein. Our goal was to test whether the FaeG protein, when isolated from its fimbrial background and produced in a plant cell, would retain the key properties of an oral vaccine, that is, stability in gastrointestinal conditions, binding to intestinal receptors and inhibition of the F4 ETEC attachment. For this purpose, tobacco was first transformed with a faeG construct that included a transit peptide encoding sequence to target the FaeG protein to the chloroplast. The best transgenic lines produced FaeG protein in amounts of 1% total soluble protein. The stability of the plant-produced FaeG was tested in fluids simulating piglet gastric (SGF) and intestinal (SIF) conditions. Plant-produced FaeG proved to be stable up to 2 h under these conditions. The binding and inhibition properties were tested with isolated piglet villi. These results showed that the plant-produced FaeG could bind to the receptors on the villi and subsequently inhibit F4 ETEC binding in a dose-dependent manner. Thus, the first two prerequisites for the development of an oral vaccine have been met.
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771. |
Ahmed AM,
Shimamoto T,
( 2004 ) A plasmid-encoded class 1 integron carrying sat, a putative phosphoserine phosphatase gene and aadA2 from enterotoxigenic Escherichia coli O159 isolated in Japan. PMID : 15183870 : DOI : 10.1016/j.femsle.2004.04.042 Abstract >>
A class 1 integron was detected in a single multidrug-resistant strain of enterotoxigenice Escherichia coli (ETEC) O159 after examination of 23 clinical E. coli isolates. This isolate was resistant to streptomycin, kanamycin, gentamicin, chloramphenicol and ampicillin. Sequencing of the class 1 integron identified three-gene cassettes. The first is the streptothricin acetyltransferase gene, sat, which confers resistance to streptothricin. The second is an ORF whose product is a putative phosphoserine phosphatase (PSP), and the last is an aminoglycoside adenyltransferase gene, aadA2, which confers resistance to streptomycin and spectinomycin. The putative PSP gene product was found to be 39%, 38%, 28%, and 27% identical to PSP gene products of Vibrio vulnificus CMCP6, V. vulnificus YJ016, Pseudomonas syringae, and P. aeruginosa, respectively. Southern-blot hybridization showed that this integron is located on a 90 kb plasmid. This is the first report identifying a putative PSP gene in an integron.
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772. |
Beranek A,
Zettl M,
Lorenzoni K,
Schauer A,
Manhart M,
Koraimann G,
( 2004 ) Thirty-eight C-terminal amino acids of the coupling protein TraD of the F-like conjugative resistance plasmid R1 are required and sufficient to confer binding to the substrate selector protein TraM. PMID : 15466052 : DOI : 10.1128/JB.186.20.6999-7006.2004 PMC : PMC522193 Abstract >>
Coupling proteins (CPs) are present in type IV secretion systems of plant, animal, and human pathogens and are essential for DNA transfer in bacterial conjugation systems. CPs connect the DNA-processing machinery to the mating pair-forming transfer apparatus. In this report we present in vitro and in vivo data that demonstrate specific binding of CP TraD of the IncFII R1 plasmid transfer system to relaxosomal protein TraM. With overlay assays and enzyme-linked immunosorbent assays we showed that a truncated version of TraD, termed TraD11 (DeltaN155), interacted strongly with TraM. The apparent TraD11-TraM association constant was determined to be 2.6 x 10(7) liters/mol. Electrophoretic mobility shift assays showed that this variant of TraD also strongly bound to TraM when it was in complex with its target DNA. When 38 amino acids were additionally removed from the C terminus of TraD, no binding to TraM was observed. TraD15, comprising the 38 amino-acid-long C terminus of TraD, bound to TraM, indicating that the main TraM interaction domain resides in these 38 amino acids of TraD. TraD15 exerted a dominant negative effect on DNA transfer but not on phage infection by pilus-specific phage R17, indicating that TraM-TraD interaction is important for conjugative DNA transfer but not for phage infection. We also observed that TraD encoded by the closely related F factor bound to TraM encoded by the R1 plasmid. Our results thus provide evidence that substrate selection within the IncF plasmid group is based on TraM's capability to select the correct DNA molecule for transport and not on substrate selection by the CP.
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773. |
Hansen LH,
Johannesen E,
Burmølle M,
Sørensen AH,
Sørensen SJ,
( 2004 ) Plasmid-encoded multidrug efflux pump conferring resistance to olaquindox in Escherichia coli. PMID : 15328093 : DOI : 10.1128/AAC.48.9.3332-3337.2004 PMC : PMC514751 Abstract >>
We report here the first gene-encoded resistance mechanism to the swine growth enhancer olaquindox. The genetic elements involved in resistance to olaquindox were subcloned and sequenced from a conjugative plasmid isolated from Escherichia coli. The subcloned fragment contained two open reading frames, oqxA and oqxB, that are homologous to several resistance-nodulation-cell-division family efflux systems from different species. The putative protein sequences were aligned to both experimentally verified and putative efflux pumps. We show that oqxA and oqxB are expressed in E. coli. Plasmids containing the oqxAB genes yielded high (>128 microg/ml) resistance to olaquindox in E. coli, whereas strains containing the control plasmid showed low resistance to the drug (8 microg/ml). The oqxAB-encoded pump also conferred high (>64 microg/ml) resistance to chloramphenicol. We demonstrate that the subcloned fragment conferred H(+)-dependent ethidium efflux abilities to E. coli strain N43. In addition, we show that the efflux system is dependent on the host TolC outer membrane protein when expressed in E. coli.
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774. |
Allard JD,
Bertrand KP,
( 1992 ) Membrane topology of the pBR322 tetracycline resistance protein. TetA-PhoA gene fusions and implications for the mechanism of TetA membrane insertion. PMID : 1517220 : Abstract >>
The tetracycline resistance gene of pBR322 encodes a 41-kDa inner membrane protein (TetA) that acts as a tetracycline/H+ antiporter. Based on hydrophobicity profiles, we identified 12 potential transmembrane segments in TetA. We used oligonucleotide deletion mutagenesis to fuse alkaline phosphatase (PhoA) to the C-terminal edge of each of the predicted periplasmic and cytoplasmic segments of TetA. In general, the PhoA activities of the TetA-PhoA fusions support a TetA topology model consisting of 12 transmembrane segments with the N and C termini in the cytoplasm. However, several TetA-PhoA fusions have unexpected properties. One PhoA fusion to a predicted cytoplasmic segment (C6) has high activity. However, previous protease accessibility studies on the related Tn10 TetA protein indicated that C6 is cytoplasmically localized as predicted (Eckert, B., and Beck, C. F. (1989) J. Biol. Chem. 264, 11663-11670). PhoA fusions to three predicted periplasmic segments (P1, P2, and P5) have low to intermediate activity. In each case, the preceding transmembrane segment (TM1, TM3, and TM9) contains an aspartate (Asp17, Asp86, and Asp287). We show that these aspartates act like signal sequence mutations for PhoA export: (i) Asp----Ala mutations increase the PhoA activity of fusions to P1, P2, and P5. (ii) The signal sequence mutation suppressor prlA402 increases the PhoA activity of these same fusions. We also show that the aspartates in TM1, TM3, and TM9 are critical for wild-type TetA function; they are conserved in related TetA proteins and Asp----Ala mutations reduce or eliminate tetracycline resistance. The properties of the anomalous TetA-PhoA fusions suggest that TetA sequences C-terminal to some cytoplasmic and periplasmic segments are required for the proper localization of those segments, i.e. long range interactions may be more important in determining the membrane topology of TetA than suggested in some general models.
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775. |
Lan R,
Alles MC,
Donohoe K,
Martinez MB,
Reeves PR,
( 2004 ) Molecular evolutionary relationships of enteroinvasive Escherichia coli and Shigella spp. PMID : 15322001 : DOI : 10.1128/IAI.72.9.5080-5088.2004 PMC : PMC517479 Abstract >>
Enteroinvasive Escherichia coli (EIEC), a distinctive pathogenic form of E. coli causing dysentery, is similar in many properties to bacteria placed in the four species of Shigella. Shigella has been separated as a genus but in fact comprises several clones of E. coli. The evolutionary relationships of 32 EIEC strains of 12 serotypes have been determined by sequencing of four housekeeping genes and two plasmid genes which were used previously to determine the relationships of Shigella strains. The EIEC strains were grouped in four clusters with one outlier strain, indicating independent derivation of EIEC several times. Three of the four clusters contain more than one O antigen type. One EIEC strain (an O112ac:H- strain) was found in Shigella cluster 3 but is not identical to the Shigella cluster 3 D2 and B15 strains with the same O antigen. Two forms of the virulence plasmid pINV have been identified in Shigella strains by using the sequences of ipgD and mxiA genes, and all but two of our EIEC strains have pINV A. The EIEC strains were grouped in two subclusters with a very low level of variation, generally not intermingled with Shigella pINV A strains. The EIEC clusters based on housekeeping genes were reflected in the plasmid gene sequences, with some exceptions. Two strains were found in the pINV B form by using the ipgD sequence, with one strain having an mxiA sequence similar to the divergent sequence of D1. Clearly, EIEC and Shigella spp. form a pathovar of E. coli.
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776. |
Agrawal RK,
Sharma MR,
Kiel MC,
Hirokawa G,
Booth TM,
Spahn CM,
Grassucci RA,
Kaji A,
Frank J,
( 2004 ) Visualization of ribosome-recycling factor on the Escherichia coli 70S ribosome: functional implications. PMID : 15178758 : DOI : 10.1073/pnas.0401904101 PMC : PMC428444 Abstract >>
After the termination step of protein synthesis, a deacylated tRNA and mRNA remain associated with the ribosome. The ribosome-recycling factor (RRF), together with elongation factor G (EF-G), disassembles this posttermination complex into mRNA, tRNA, and the ribosome. We have obtained a three-dimensional cryo-electron microscopic map of a complex of the Escherichia coli 70S ribosome and RRF. We find that RRF interacts mainly with the segments of the large ribosomal subunit's (50S) rRNA helices that are involved in the formation of two central intersubunit bridges, B2a and B3. The binding of RRF induces considerable conformational changes in some of the functional domains of the ribosome. As compared to its binding position derived previously by hydroxyl radical probing study, we find that RRF binds further inside the intersubunit space of the ribosome such that the tip of its domain I is shifted (by approximately 13 A) toward protein L5 within the central protuberance of the 50S subunit, and domain II is oriented more toward the small ribosomal subunit (30S). Overlapping binding sites of RRF, EF-G, and the P-site tRNA suggest that the binding of EF-G would trigger the removal of deacylated tRNA from the P site by moving RRF toward the ribosomal E site, and subsequent removal of mRNA may be induced by a shift in the position of 16S rRNA helix 44, which harbors part of the mRNA.
|
777. |
Valvatne H,
Steinsland H,
Grewal HM,
Mølbak K,
Vuust J,
Sommerfelt H,
( 2004 ) Identification and molecular characterization of the gene encoding coli surface antigen 20 of enterotoxigenic Escherichia coli. PMID : 15451111 : DOI : 10.1016/j.femsle.2004.08.028 Abstract >>
Enterotoxigenic Escherichia coli (ETEC) is a major cause of diarrhea among children living in developing countries and of travelers' diarrhea. Current ETEC vaccine designs aim to induce an anti-colonizing immunity by including the ETEC surface colonization factor antigens. We isolated and characterized the structural gene of the coli surface antigen 20 (CS20). CS20 has an N-terminal amino acid sequence similar to that of CS18. We therefore used a DNA fragment carrying the CS18 fotA gene as a probe in a hybridization assay to detect the corresponding gene in a CS20-positive strain isolated from an Indian child. Cross hybridizing DNA was isolated and found to contain an open reading frame encoding a polypeptide of 195 amino acids, including a 22 amino acid signal peptide. The gene, which we named csnA, shows a high degree of identity to the major fimbrial subunits of CS12, CS18 and F6 (also referred to as 987P), a CS of porcine ETEC. The coding region of csnA was inserted into an expression system to generate a polypeptide confirmed to be CS20 by Western blot. A CS20 colony hybridization assay using a DNA probe derived from csnA was developed.
|
778. |
Jelsch C,
Lenfant F,
Masson JM,
Samama JP,
( 1992 ) Beta-lactamase TEM1 of E. coli. Crystal structure determination at 2.5 A resolution. PMID : 1544485 : DOI : 10.1016/0014-5793(92)80232-6 Abstract >>
The crystal structure of beta-lactamase TEM1 from E. coli has been solved to 2.5 A resolution by X-ray diffraction methods and refined to a crystallographic R-factor of 22.7%. The structure was determined by multiple isomorphous replacement using four heavy atom derivatives. The solution from molecular replacement, using a polyalanine model constructed from the C alpha coordinates of S. Aureus PCl enzyme, provided a set of phases used for heavy atom derivatives analysis. The E. coli beta-lactamase TEM1 is made up of two domains whose topology is similar to that of the PCl enzyme. However, global superposition of the two proteins shows significant differences.
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779. |
Cartelle M,
del Mar Tomas M,
Molina F,
Moure R,
Villanueva R,
Bou G,
( 2004 ) High-level resistance to ceftazidime conferred by a novel enzyme, CTX-M-32, derived from CTX-M-1 through a single Asp240-Gly substitution. PMID : 15155242 : DOI : 10.1128/AAC.48.6.2308-2313.2004 PMC : PMC415568 Abstract >>
A clinical strain of Escherichia coli isolated from pleural liquid with high levels of resistance to cefotaxime, ceftazidime, and aztreonam harbors a novel CTX-M gene (bla(CTX-M-32)) whose amino acid sequence differs from that of CTX-M-1 by a single Asp240-Gly substitution. Moreover, by site-directed mutagenesis we demonstrated that this replacement is a key event in ceftazidime hydrolysis
|
780. |
Verdonck F,
Cox E,
Schepers E,
Imberechts H,
Joensuu J,
Goddeeris BM,
( 2004 ) Conserved regions in the sequence of the F4 (K88) fimbrial adhesin FaeG suggest a donor strand mechanism in F4 assembly. PMID : 15327796 : DOI : 10.1016/j.vetmic.2004.06.002 Abstract >>
Oral immunization of newly weaned piglets with recombinant F4 (K88) fimbrial adhesin FaeG induces a F4-specific immune response, significantly reducing F4+ Escherichia coli excretion following challenge. In order to use FaeG subunits in an oral vaccine against F4+ enterotoxigenic E. coli, it is necessary to determine the conservation of the adhesin subunit. Hereto, the faeG sequence was determined of 21 F4ac+ E. coli field isolates from piglets with diarrhoea and subsequently compared with these of the reference strain GIS26 and previously reported FaeG sequences from F4ab, F4ac and F4ad antigenic variant strains. The FaeG amino acid sequence was 96-100% homologous within each F4 serotype, but only 92 and 88% when the F4ab and F4ad antigenic variants were compared with the F4ac antigenic variant. Furthermore, the conserved regions of the adhesin suggest a donor strand mechanism in F4 fimbriae assembly as reported for type 1 and P pili. In conclusion, the results of the reported experiments support the usefulness FaeG in an oral subunit vaccine against F4+ E. coli infections or as a mucosal carrier since the adhesin is conserved among F4+ E. coli field isolates.
|
781. |
Nandiwada LS,
Schamberger GP,
Schafer HW,
Diez-Gonzalez F,
( 2004 ) Characterization of an E2-type colicin and its application to treat alfalfa seeds to reduce Escherichia coli O157:H7. PMID : 15163583 : DOI : 10.1016/j.ijfoodmicro.2003.11.009 Abstract >>
Several outbreaks of Escherichia coli O157:H7 infections have been associated with contaminated alfalfa seeds. A recently isolated E. coli strain Hu194 was capable of inhibiting 22 strains of E. coli O157:H7 and this inhibition was mediated by the production of a colicin named Hu194. The objectives of this study were to test the efficacy of treating alfalfa seeds with colicin Hu194 against E. coli O157:H7 strains, and to characterize this antimicrobial protein. Significant reductions (approximately 5 log CFU ml-1) in the viable cell counts of strains 43890 and 43895 were observed after 1-day incubation with semi-crude colicin, and after 2 days for strain 3081. Strain 43890 was successfully eliminated (5 log CFU g-1) from inoculated alfalfa seeds after soaking in a colicin suspension at a concentration of 10,000 AU/g. Treatment of alfalfa seeds inoculated with strains 43895 and 3081 required 20-fold higher concentrations of colicin Hu194 to achieve as much as 3 log CFU g-1 reductions. The genes encoding the colicin Hu194 operon were located on a 6 kb plasmid, and the sequence analysis revealed that this colicin was an E-type DNAse. From the sequence data, the estimated molecular masses of colicin Hu194, its immunity protein and lysis protein were 61.3, 10.0 and 4.8 kDa, respectively. Based on DNA and protein sequence comparisons with other E-type colicin, colicin Hu194 belonged to the type E2-colicin cluster. However, cross-immunity tests between E-group colicins suggested that Hu194 colicin was divergent from the previously characterized E2 colicins.
|
782. |
Jeong SH,
Bae IK,
Lee JH,
Sohn SG,
Kang GH,
Jeon GJ,
Kim YH,
Jeong BC,
Lee SH,
( 2004 ) Molecular characterization of extended-spectrum beta-lactamases produced by clinical isolates of Klebsiella pneumoniae and Escherichia coli from a Korean nationwide survey. PMID : 15243036 : DOI : 10.1128/JCM.42.7.2902-2906.2004 PMC : PMC446292 Abstract >>
To determine the prevalence and genotypes of extended-spectrum beta-lactamases (ESBLs) among clinical isolates of Klebsiella pneumoniae and Escherichia coli, we performed antibiotic susceptibility testing, pI determination, induction testing, transconjugation, and DNA sequencing analysis. Among the 509 isolates collected from 13 university hospitals in Korea, 39.2% produced ESBLs. ESBL-producing isolates were detected in every region in Korea. A total of 44.6% of the isolates produced both TEM- and SHV-type ESBLs, and 52% of ESBL-producing isolates transferred resistance to ceftazidime by transconjugation. The ESBLs were TEM-19, TEM-20, TEM-52, SHV-2a, SHV-12, and one new variant identified for the first time in Korea, namely, TEM-116. TEM-1 and SHV-12 were by far the most common variants. TEM-1, TEM-116, and SHV-12 showed a high prevalence in K. pneumoniae. Two isolates (E. coli SH16 and K. pneumoniae SV3) produced CMY-1-like beta-lactamases, which play a decisive role in resistance to cefoxitin and cefotetan, as well as TEM-type enzymes (TEM-20 and TEM-52, respectively). Using MIC patterns and DNA sequencing analysis, we postulated a possible evolution scheme among TEM-type beta-lactamases in Korea: from TEM-1 to TEM-19, from TEM-19 to TEM-20, and from TEM-20 to TEM-52.
|
783. |
Huerta-Saquero A,
Calderón-Flores A,
Díaz-Villaseñor A,
Du Pont G,
Durán S,
( 2004 ) Regulation of transcription and activity of Rhizobium etli glutaminase A. PMID : 15279892 : DOI : 10.1016/j.bbagen.2004.05.001 Abstract >>
The present study determines the regulatory mechanisms that operate on Rhizobium etli glutaminase A. glsA gene expression levels were evaluated under several metabolic conditions by fusions of the glsA gene promoter and the transcriptional reporter cassette uidA2-aad. glsA expression was directly correlated to the glutaminase A activity found under the tested growth conditions, reaching its maximum level in the presence of glutamine and during exponential growth phase. Glutamine induces glsA expression. The influence of allosteric metabolites on glutaminase A activity was also determined. The purified enzyme was inhibited by 2-oxoglutarate and pyruvate, whereas oxaloacetate and glyoxylate modulate it positively. Glutaminase A is not inhibited by glutamate and is activated by ammonium. Glutaminase A participates in an ATP-consuming cycle where glutamine is continually degraded and resynthesized by glutamine synthetase (GS). GS and glutaminase A activities appear simultaneously during bacterial growth under different metabolic conditions and their control mechanisms are not reciprocal. Slight overproduction in glutaminase A expression causes a reduction in growth yield and a dramatic decrease in bacterial growth. We propose a model for regulation of glutaminase A, and discuss its contribution to glutamine cycle regulation.
|
784. |
Vourli S,
Giakkoupi P,
Miriagou V,
Tzelepi E,
Vatopoulos AC,
Tzouvelekis LS,
( 2004 ) Novel GES/IBC extended-spectrum beta-lactamase variants with carbapenemase activity in clinical enterobacteria. PMID : 15135524 : DOI : 10.1016/j.femsle.2004.03.028 Abstract >>
Two clinical isolates, an Escherichia coli and a Klebsiella pneumoniae, with decreased susceptibility to carbapenems were studied. This phenotype was associated with production of novel GES/IBC variant beta-lactamases, designated GES-3 (from E. coli) and GES-4 (from K. pneumoniae), exhibiting carbapenemase activity. Both enzymes possessed Ser at Ambler's position 170 instead of Gly found in the beta-lactamases GES-1 and IBC-1 that lack carbapenemase activity. Additionally, position 104 in GES-4 was occupied by a Lys as in IBC-1. bla(GES-3) and bla(GES-4) occurred as gene cassettes in the variable regions of class 1 integrons carried by plasmids. The structure of the GES-4-encoding integron was similar to that of previously described IBC-1 integrons. The GES-3-encoding integron was, most likely, truncated at the 3' conserved segment.
|
785. |
Tamulaitiene G,
Grazulis S,
Janulaitis A,
Janowski R,
Bujacz G,
Jaskolski M,
( 2004 ) Crystallization and preliminary crystallographic studies of a bifunctional restriction endonuclease Eco57I. PMID : 15134658 : DOI : 10.1016/j.bbapap.2003.12.006 Abstract >>
Restriction endonuclease Eco57I from Escherichia coli recognizes asymmetric DNA sequence 5'-CTGAAG and has both restriction (DNA cleavage a short distance away from the recognition site) and modification (methylation) activities residing in a single polypeptide chain. Single crystals of wild-type Eco57I ternary complexes with double-stranded DNA and sinefungin, a stimulator of endonuclease activity, were obtained by the vapor diffusion technique and characterized crystallographically for different variants of the DNA component. The best data for the complex with 25-mer DNA were collected to 4.2-A resolution at 100 K using synchrotron radiation. The crystals are orthorhombic, space group P2(1)2(1)2, with a=164.3, b=293.0, c=71.1 A, and contain two to four copies of the protein in the asymmetric unit.
|
786. |
Poupart MC,
Villemain M,
Sirot J,
De Champs C,
Chanal C,
Sirot D,
Baraduc R,
Romaszko JP,
Bonnet R,
Plaidy A,
Boyer M,
Carroy E,
Gbadamassi MC,
Laluque S,
Oules O,
( 2004 ) Frequency and diversity of Class A extended-spectrum beta-lactamases in hospitals of the Auvergne, France: a 2 year prospective study. PMID : 15282240 : DOI : 10.1093/jac/dkh395 Abstract >>
To evaluate the frequency and diversity of extended-spectrum beta-lactamases (ESBLs) produced by Enterobacteriaceae and Pseudomonas aeruginosa in one French region. During 2001-2002, all the non-duplicate isolates of P. aeruginosa resistant to ceftazidime and of Enterobacteriaceae intermediate or resistant to ceftazidime and/or cefotaxime and/or aminoglycosides with an AAC(6') I phenotype were collected in nine hospitals of the area. ESBL isoelectric points were determined, bla genes were amplified and sequenced and epidemic isolates were genotyped with ERIC2-PCR. ESBLs were observed in 297 Enterobacteriaceae (0.8%). The most frequent were TEM-3 like (n=152; 51.2%) and TEM-24 (n=115; 38.7%). Four new enzymes were observed, TEM-112 (pI 5.4), TEM-113 (pI 6.3), TEM-114 (pI 5.9) and TEM-126 (pI 5.4). Other TEMs were TEM-8, TEM-12, TEM-16, TEM-19, TEM-20, TEM-21, TEM-29 and TEM-71. The other ESBLs were SHV-4, SHV-5 and SHV-12, CTX-M-1, CTX-M-3, CTX-M-14 and CTX-M-15. In 37 P. aeruginosa (0.7%) only one ESBL was observed, PER-1. Five epidemic strains were detected, Serratia marcescens TEM-3 and four observed in several hospitals, Enterobacter aerogenes TEM-24, Citrobacter koseri TEM-3, Proteus mirabilis TEM-3 and P. aeruginosa PER-1. ESBL frequency was lower than in 1998, and CTX-M-type frequency higher (2.1% of ESBLs in 2001, 4.9% in 2002). This long-term survey detected new sporadic enzymes (TEM-112, TEM-113, TEM-114 and TEM-126) and interhospital epidemic strains while avoiding any overestimation of ESBL frequency that may otherwise have occurred because of acute epidemics.
|
787. |
Du X,
Xia C,
Shen J,
Wu B,
Shen Z,
( 2004 ) Characterization of florfenicol resistance among calf pathogenic Escherichia coli. PMID : 15251195 : DOI : 10.1016/j.femsle.2004.05.013 Abstract >>
Three floR genes were cloned from calf pathogenic Escherichia coli strains, and the efflux-mediated accumulation of florfenicol in the floR gene-JM109 E. coli system was determined by HPLC. The floR genes resulted in a 1356-bp fragment covering the ORF in region 66-1280 coding for 404 amino acids. The common motifs of 12-transmembrane segments efflux pumps family were conserved in the deduced floR amino acid sequences. HPLC results indicated a significant difference in florfenicol accumulation between florfenicol-resistant strains and the susceptible strains, which was almost reversed by the addition of a proton motive force blocker. These results suggest that the florfenicol resistance mediated by the floR gene involves active efflux of florfenicol.
|
788. |
Miriagou V,
Tzouvelekis LS,
Villa L,
Lebessi E,
Vatopoulos AC,
Carattoli A,
Tzelepi E,
( 2004 ) CMY-13, a novel inducible cephalosporinase encoded by an Escherichia coli plasmid. PMID : 15273143 : DOI : 10.1128/AAC.48.8.3172-3174.2004 PMC : PMC478546 Abstract >>
An IncN plasmid (p541) from Escherichia coli carried a Citrobacter freundii-derived sequence of 4,252 bp which included an ampC-ampR region and was bound by two directly repeated IS26 elements. ampC encoded a novel cephalosporinase (CMY-13) with activity similar to that of CMY-2. AmpR was likely functional as indicated in induction experiments.
|
789. |
Froehlich B,
Holtzapple E,
Read TD,
Scott JR,
( 2004 ) Horizontal transfer of CS1 pilin genes of enterotoxigenic Escherichia coli. PMID : 15126486 : DOI : 10.1128/jb.186.10.3230-3237.2004 PMC : PMC400639 Abstract >>
CS1 is one of a limited number of serologically distinct pili found in enterotoxigenic Escherichia coli (ETEC) strains associated with disease in people. The genes for the CS1 pilus are on a large plasmid, pCoo. We show that pCoo is not self-transmissible, although our sequence determination for part of pCoo shows regions almost identical to those in the conjugative drug resistance plasmid R64. When we introduced R64 into a strain containing pCoo, we found that pCoo was transferred to a recipient strain in mating. Most of the transconjugant pCoo plasmids result from recombination with R64, leading to acquisition of functional copies of all of the R64 transfer genes. Temporary coresidence of the drug resistance plasmid R64 with pCoo leads to a permanent change in pCoo so that it is now self-transmissible. We conclude that when R64-like plasmids are transmitted to an ETEC strain containing pCoo, their recombination may allow for spread of the pCoo plasmid to other enteric bacteria.
|
790. |
Doi Y,
Wachino J,
Ishiguro M,
Kurokawa H,
Yamane K,
Shibata N,
Shibayama K,
Yokoyama K,
Kato H,
Yagi T,
Arakawa Y,
( 2004 ) Inhibitor-sensitive AmpC beta-lactamase variant produced by an Escherichia coli clinical isolate resistant to oxyiminocephalosporins and cephamycins. PMID : 15215122 : DOI : 10.1128/AAC.48.7.2652-2658.2004 PMC : PMC434168 Abstract >>
Escherichia coli HKY28, a ceftazidime-resistant strain isolated from a urine specimen in Japan, produced an inhibitor-sensitive AmpC beta-lactamase variant. The deduced amino acid sequence of the enzyme contained a number of substitutions and a tripeptide deletion (Gly286-Ser287-Asp288) compared with the sequence of native AmpC of E. coli. When the deletion was reverted by a 9-base insertion at the relevant site of ampC in the clone, the typical inhibitor-resistant phenotype of AmpC was restored, while at the same time the levels of resistance to ceftazidime, cefpirome, and cefepime were reduced eightfold or more. Molecular modeling studies indicated that a structural change took place in the H-10 helix as a result of the deletion, and this change caused an alteration of the substrate binding site, leading to a unique phenotype analogous to that of inhibitor-sensitive class A extended-spectrum beta-lactamases. The degree of inhibition was greater with sulbactam and tazobactam than with clavulanic acid. To our knowledge, this is the first report to have characterized an E. coli ampC that encodes chromosomal AmpC beta-lactamase sensitive to the available beta-lactamase inhibitors.
|
791. |
Enne VI,
Bennett PM,
Livermore DM,
Hall LM,
( 2004 ) Enhancement of host fitness by the sul2-coding plasmid p9123 in the absence of selective pressure. PMID : 15102746 : DOI : 10.1093/jac/dkh217 Abstract >>
Despite a 97% reduction in clinical sulphonamide usage, the prevalence of sulphonamide resistance among Escherichia coli has remained constant in the UK. Genetic linkage of sulphonamide resistance to other resistances is thought important for this maintenance, but the finding also implies that sulphonamide resistance exerts little fitness cost. To test this hypothesis, we examined the fitness impact of four naturally occurring sul2-coding plasmids upon their hosts. The fitness impact of the plasmids upon E. coli was determined by pairwise growth competition in a minimal medium. The DNA sequence of plasmid p9123 was obtained by primer walking and PCR. Three of the four sul2-coding plasmids studied imposed fitness costs on their hosts. The fourth plasmid, a 6.2 kb resistance element carrying sul2, strA and strB designated p9123, conferred a 4% fitness advantage upon its original clinical host and also on E. coli K12 JM109. The complete sequence of p9123 revealed eight open reading frames, including five of unknown function. There was no obvious gene to which the fitness advantage might be attributed. The novel finding that p9123 can improve host fitness may explain why this plasmid and its close relatives are so widespread among enteric bacteria. In addition to other factors such as co-selection of sulphonamide resistance by other agents, the fitness advantage conferred by plasmids such as p9123 may have contributed to the maintenance of sulphonamide resistance in the UK in the absence of clinical selection pressure. These data indicate that once antibiotic resistance has been established on mobile genetic elements, it may be difficult to eliminate.
|
792. |
McCool JD,
Ford CC,
Sandler SJ,
( 2004 ) A dnaT mutant with phenotypes similar to those of a priA2::kan mutant in Escherichia coli K-12. PMID : 15238512 : DOI : 10.1534/genetics.103.025296 PMC : PMC1470904 Abstract >>
The ability to repair damaged replication forks and restart them is important for cell survival. DnaT is essential for replication restart in vitro and yet no definite genetic analysis has been done in Escherichia coli K-12. To begin, dnaT822, an in-frame six-codon (87-92) deletion was constructed. DnaT822 mutants show colony size, cell morphology, inability to properly partition nucleoids, UV sensitivity, and basal SOS expression similar to priA2::kan mutants. DnaT822 priA2::kan double mutants had phenotypes similar to those of the single mutants. DnaT822 and dnaT822 priA2::kan mutant phenotypes were fully suppressed by dnaC809. Previously, a dominant temperature-sensitive lethal mutation, dnaT1, had been isolated in E. coli 15T(-). DnaT1 was found to have a base-pair change relative to the E. coli 15T(-) and E. coli K-12 dnaT genes that led to a single amino acid change: R152C. A plasmid-encoded E. coli K-12 mutant dnaT gene with the R152C amino acid substitution did not display a dominant temperature-sensitive lethal phenotype in a dnaT(+) strain of E. coli K-12. Instead, this mutant dnaT gene was found to complement the E. coli K-12 dnaT822 mutant phenotypes. The significance of these results is discussed in terms of models for replication restart.
|
793. |
Steiniger-White M,
Rayment I,
Reznikoff WS,
( 2004 ) Structure/function insights into Tn5 transposition. PMID : 15102449 : DOI : 10.1016/j.sbi.2004.01.008 Abstract >>
Prokaryotic transposon 5 (Tn5) serves as a model system for studying the molecular mechanism of DNA transposition. Elucidation of the X-ray co-crystal structure of Tn5 transposase complexed with a DNA recognition end sequence provided the first three-dimensional picture of an intermediate in a transposition/retroviral integration pathway. The many Tn5 transposase-DNA co-crystal structures now available complement biochemical and genetic studies, allowing a comprehensive and detailed understanding of transposition mechanisms. Specifically, the structures reveal two different types of protein-DNA contacts: cis contacts, required for initial DNA recognition, and trans contacts, required for catalysis. Protein-protein contacts required for synapsis are also seen. Finally, the two divalent metals in the active site of the transposase support a 'two-metal-ion' mechanism for Tn5 transposition.
|
794. |
Feng L,
Senchenkova SN,
Yang J,
Shashkov AS,
Tao J,
Guo H,
Cheng J,
Ren Y,
Knirel YA,
Reeves PR,
Wang L,
( 2004 ) Synthesis of the heteropolysaccharide O antigen of Escherichia coli O52 requires an ABC transporter: structural and genetic evidence. PMID : 15231783 : DOI : 10.1128/JB.186.14.4510-4519.2004 PMC : PMC438562 Abstract >>
The structural and genetic organization of the Escherichia coli O52 O antigen was studied. As identified by sugar and methylation analysis and nuclear magnetic resonance spectroscopy, the O antigen of E. coli O52 has a partially O-acetylated disaccharide repeating unit (O unit) containing D-fucofuranose and 6-deoxy-D-manno-heptopyranose, as well as a minor 6-deoxy-3-O-methylhexose (most likely, 3-O-methylfucose). The O-antigen gene cluster of E. coli O52, which is located between the galF and gnd genes, was found to contain putative genes for the synthesis of the O-antigen constituents, sugar transferase genes, and ABC-2 transporter genes. Further analysis confirmed that O52 employs an ATP-binding cassette (ABC) transporter-dependent pathway for translocation and polymerization of the O unit. This is the first report of an ABC transporter being involved in translocation of a heteropolysaccharide O antigen in E. coli. Genes specific for E. coli O52 were also identified.
|
795. |
Jagura-Burdzy G,
Khanim F,
Smith CA,
Thomas CM,
( 1992 ) Crosstalk between plasmid vegetative replication and conjugative transfer: repression of the trfA operon by trbA of broad host range plasmid RK2. PMID : 1508679 : DOI : 10.1093/nar/20.15.3939 PMC : PMC334070 Abstract >>
Previous deletion and complementation analysis has indicated that the region between trfA and kilBI (trbB) encodes trans-acting factor, designated trbA, required for conjugative transfer of broad host range plasmid RK2. In analysing the nucleotide sequence of this region we have discovered a gene encoding a 12 kDa polypeptide. The predicted amino acid sequence of this protein shows similarity at its C-terminal to KorA from the central control operon of RK2 and at its N-terminal to immunity repressor protein from phage phi 105 of Bacillus subtilis as well as the Sin protein of B. subtilis which regulates alternate developmental processes including sporulation, motility and competence. We show that TrbA represses transcription of both trfA (vegetative replication) and kilBI (trbB) (required for conjugative transfer and whose product has similarity to ComG, required for competence of B. subtilis) and may help to coordinate expression of both sets of functions. This region has similarities to some temperate bacteriophage immunity regions in modulating divergent transcription required for alternative means of propagation.
|
796. |
Chiang RC,
Cavicchioli R,
Gunsalus RP,
( 1992 ) Identification and characterization of narQ, a second nitrate sensor for nitrate-dependent gene regulation in Escherichia coli. PMID : 1508040 : DOI : 10.1111/j.1365-2958.1992.tb01364.x Abstract >>
In response to nitrate availability, Escherichia coli regulates the synthesis of a number of enzymes involved in anaerobic respiration and fermentation. When nitrate is present, nitrate reductase (narGHJI) gene expression is induced, while expression of the DMSO/TMAO reductase (dmsABC), fumarate reductase (frdABCD) and fermentation related genes are repressed. The narL and narX gene products are required for this nitrate-dependent control, and apparently function as members of a two-component regulatory system. NarX is a presumed sensor-transmitter for nitrate and possibly molybdenum detection. The presumed response-regulator, NarL, when activated by NarX then binds at the regulatory DNA sites of genes to modulate their expression. In this study a third nitrate regulatory gene, narQ, was identified that also participates in nitrate-dependent gene regulation. Strains defective in either narQ or narX alone exhibited no nitrate-dependent phenotype whereas mutants defective in both narQ and narX were fully inactive for nitrate-dependent repression or activation. In all conditions tested, this regulation required a functional narL gene product. These findings suggest that the narX and narQ products have complementary sensor-transmitter functions for nitrate detection, and can work independently to activate NarL, for eliciting nitrate-dependent regulation of anaerobic electron transport and fermentation functions. The narQ gene was cloned, sequenced, and compared with the narX gene. Both gene products are similar in size, hydrophobicity, and sequence, and contain a highly conserved histidine residue common to sensor-transmitter proteins.
|
797. |
Fröderberg L,
Houben EN,
Baars L,
Luirink J,
de Gier JW,
( 2004 ) Targeting and translocation of two lipoproteins in Escherichia coli via the SRP/Sec/YidC pathway. PMID : 15140892 : DOI : 10.1074/jbc.M403229200 Abstract >>
In Escherichia coli, two main protein targeting pathways to the inner membrane exist: the SecB pathway for the essentially posttranslational targeting of secretory proteins and the SRP pathway for cotranslational targeting of inner membrane proteins (IMPs). At the inner membrane both pathways converge at the Sec translocase, which is capable of both linear transport into the periplasm and lateral transport into the lipid bilayer. The Sec-associated YidC appears to assist the lateral transport of IMPs from the Sec translocase into the lipid bilayer. It should be noted that targeting and translocation of only a handful of secretory proteins and IMPs have been studied. These model proteins do not include lipoproteins. Here, we have studied the targeting and translocation of two secretory lipoproteins, the murein lipoprotein and the bacteriocin release protein, using a combined in vivo and in vitro approach. The data indicate that both murein lipoprotein and bacteriocin release protein require the SRP pathway for efficient targeting to the Sec translocase. Furthermore, we show that YidC plays an important role in the targeting/translocation of both lipoproteins.
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798. |
Liebana E,
Gibbs M,
Clouting C,
Barker L,
Clifton-Hadley FA,
Pleydell E,
Abdalhamid B,
Hanson ND,
Martin L,
Poppe C,
Davies RH,
( 2004 ) Characterization of beta-lactamases responsible for resistance to extended-spectrum cephalosporins in Escherichia coli and Salmonella enterica strains from food-producing animals in the United Kingdom. PMID : 15140388 : DOI : 10.1089/107662904323047745 Abstract >>
Nine epidemiologically unrelated isolates [1 Salmonella Bredeney from turkeys, and 8 Escherichia coli [3 environmental isolates (2 from chickens, 1 from pigs), and 5 isolates from cattle with neonatal diarrhea]] were examined both pheno- and genotypically for extended-spectrum beta-lactam (ESBL) resistance. Resistance phenotypes (ampicillin, aztreonam, cefotaxime, cefpodoxime, ceftazidime, and ceftriaxone) suggested the presence of an ESBL enzyme, but cefoxitin MICs (>/= 32 mg/L) suggested the presence of an AmpC-like enzyme. Synergism experiments with benzo(b)thiophene-2-boronic acid (BZBTH2B) and isoelectric focusing (IEF) revealed the presence of an AmpC beta-lactamase with a pI >/= 9. amp C multiplex PCR, sequence, and Southern analyses indicated that only the Salmonella isolate had a plasmid-encoded AmpC beta-lactamase CMY-2 on a nonconjugative 60-MDa plasmid. PCR and sequence analysis of the E. coli ampC promoter identified mutations at positions -88(T), -82(G), -42(T), -18(A), -1(T) and +58(T) in all the isolates. In addition one strain had two extra-mutations at positions +23(A) and +49(G), and another strain had one extra-mutation at position +32(A). DNA fingerprinting revealed that all the E. coli isolates were different clones. It also showed that the U.K. Salmonella isolate was indistinguisable from a Canadian Salmonella isolate from turkeys; both had identical resistance phenotypes and produced CMY-2. This is the first report of a CMY-2 Salmonella isolate in the United Kingdom. These data imply that beta-lactam resistance in animal isolates can be generated de novo as evidenced by the E. coli strains, or in the case of the Salmonella strains be the result of intercontinental transmission due to an acquired resistance mechanism.
|
799. |
Kaye KS,
Gold HS,
Schwaber MJ,
Venkataraman L,
Qi Y,
De Girolami PC,
Samore MH,
Anderson G,
Rasheed JK,
Tenover FC,
( 2004 ) Variety of beta-lactamases produced by amoxicillin-clavulanate-resistant Escherichia coli isolated in the northeastern United States. PMID : 15105100 : DOI : 10.1128/aac.48.5.1520-1525.2004 PMC : PMC400555 Abstract >>
This study analyzed the enzymatic basis and molecular epidemiology of amoxicillin-clavulanate-resistant Escherichia coli isolated by the microbiology laboratory of a United States tertiary care hospital. From October 1998 to December 1999, all E. coli isolates were screened for ampicillin-sulbactam resistance. Of 283 isolates that tested resistant to ampicillin-sulbactam, 69 unique patient isolates were also resistant to amoxicillin-clavulanate by disk diffusion testing (zone diameter /= 32 micro g/ml). Two isolates were susceptible to amoxicillin-clavulanic acid by agar dilution, although they were resistant by disk diffusion testing. The distribution of beta-lactamases was as follows: the TEM type alone was found in 52 isolates, the AmpC type was found in 4 isolates (2 identified as containing CMY-2), the TEM type and CMY-2 were found in 2 isolates, and the OXA type was found in 1 isolate. Also, there was one isolate with the TEM type and the SHV type and one with the TEM type and a second, unidentified enzyme. Among the isolates with TEM-type enzymes, two extended-spectrum beta-lactamase-producing isolates were identified but two isolates with inhibitor-resistant TEM (IRT) enzymes (one with TEM-34 [IRT-6] and the other with a novel enzyme [tentatively assigned the designation TEM-122]) were more interesting.
|
800. |
Paton AW,
Srimanote P,
Talbot UM,
Wang H,
Paton JC,
( 2004 ) A new family of potent AB(5) cytotoxins produced by Shiga toxigenic Escherichia coli. PMID : 15226357 : DOI : 10.1084/jem.20040392 PMC : PMC2213318 Abstract >>
The Shiga toxigenic Escherichia coli (STEC) O113:H21 strain 98NK2, which was responsible for an outbreak of hemolytic uremic syndrome, secretes a highly potent and lethal subtilase cytotoxin that is unrelated to any bacterial toxin described to date. It is the prototype of a new family of AB(5) toxins, comprising a single 35-kilodalton (kD) A subunit and a pentamer of 13-kD B subunits. The A subunit is a subtilase-like serine protease distantly related to the BA_2875 gene product of Bacillus anthracis. The B subunit is related to a putative exported protein from Yersinia pestis, and binds to a mimic of the ganglioside GM2. Subtilase cytotoxin is encoded by two closely linked, cotranscribed genes (subA and subB), which, in strain 98NK2, are located on a large, conjugative virulence plasmid. Homologues of the genes are present in 32 out of 68 other STEC strains tested. Intraperitoneal injection of purified subtilase cytotoxin was fatal for mice and resulted in extensive microvascular thrombosis, as well as necrosis in the brain, kidneys, and liver. Oral challenge of mice with E. coli K-12-expressing cloned subA and subB resulted in dramatic weight loss. These findings suggest that the toxin may contribute to the pathogenesis of human disease.
|
801. |
Koh TH,
Wang GC,
Sng LH,
Koh TY,
( 2004 ) CTX-M and plasmid-mediated AmpC-producing Enterobacteriaceae Singapore. PMID : 15224678 : DOI : 10.3201/eid1006.030726 PMC : PMC3323164 Abstract >>
N/A
|
802. |
Scoulica EV,
Neonakis IK,
Gikas AI,
Tselentis YJ,
( 2004 ) Spread of bla(VIM-1)-producing E. coli in a university hospital in Greece. Genetic analysis of the integron carrying the bla(VIM-1) metallo-beta-lactamase gene. PMID : 15023424 : DOI : 10.1016/j.diagmicrobio.2003.09.012 Abstract >>
Bla(VIM-1) gene was detected in four Escherichia coli clinical isolates with both reduced susceptibility to carbapenems and an ESBL phenotype. The VIM-1 determinant was located within the variable region of a Class I integron along with a 6'-N-aminoglycoside acetyltransferase gene (aac(6')-Ib) and it could be transferred by conjugation. In all four clinical isolates the VIM-1 gene cassette presented a characteristic duplication of the 3' end coding 153 nucleotides followed by the first 14 nucleotides of the 59 base element (59be) that however did not seem to affect either the integrity of the coding sequence or the 59be of the gene cassette. These clinical isolates not only harbored the same Class I integron, but they also shared the same discrete ribotype-pattern, indicative for their clonal origin. Spread of carbapenem resistance genes among Enterobacteriaceae in hospital is a matter of great concern.
|
803. |
Escobar-Páramo P,
Clermont O,
Blanc-Potard AB,
Bui H,
Le Bouguénec C,
Denamur E,
( 2004 ) A specific genetic background is required for acquisition and expression of virulence factors in Escherichia coli. PMID : 15014151 : DOI : 10.1093/molbev/msh118 Abstract >>
In bacteria, the evolution of pathogenicity seems to be the result of the constant arrival of virulence factors (VFs) into the bacterial genome. However, the integration, retention, and/or expression of these factors may be the result of the interaction between the new arriving genes and the bacterial genomic background. To test this hypothesis, a phylogenetic analysis was done on a collection of 98 Escherichia coli/Shigella strains representing the pathogenic and commensal diversity of the species. The distribution of 17 VFs associated to the different E. coli pathovars was superimposed on the phylogenetic tree. Three major types of VFs can be recognized: (1) VFs that arrive and are expressed in different genetic backgrounds (such as VFs associated with the pathovars of mild chronic diarrhea: enteroaggregative, enteropathogenic, and diffusely-adhering E. coli), (2) VFs that arrive in different genetic backgrounds but are preferentially found, associated with a specific pathology, in only one particular background (such as VFs associated with extraintestinal diseases), and (3) VFs that require a particular genetic background for the arrival and expression of their virulence potential (such as VFs associated with pathovars typical of severe acute diarrhea: enterohemorragic, enterotoxigenic, and enteroinvasive E. coli strains). The possibility of a single arrival of VFs by chance, followed by a vertical transmission, was ruled out by comparing the evolutionary histories of some of these VFs to the strain phylogeny. These evidences suggest that important changes in the genome of E. coli have occurred during the diversification of the species, allowing the virulence factors associated with severe acute diarrhea to arrive in the population. Thus, the E. coli genome seems to be formed by an "ancestral" and a "derived" background, each one responsible for the acquisition and expression of different virulence factors.
|
804. |
Nakano R,
Okamoto R,
Nakano Y,
Kaneko K,
Okitsu N,
Hosaka Y,
Inoue M,
( 2004 ) CFE-1, a novel plasmid-encoded AmpC beta-lactamase with an ampR gene originating from Citrobacter freundii. PMID : 15047515 : DOI : 10.1128/aac.48.4.1151-1158.2004 PMC : PMC375250 Abstract >>
A clinical isolate of Escherichia coli from a patient in Japan, isolate KU6400, was found to produce a plasmid-encoded beta-lactamase that conferred resistance to extended-spectrum cephalosporins and cephamycins. Resistance arising from production of a beta-lactamase could be transferred by either conjugation or transformation with plasmid pKU601 into E. coli ML4947. The substrate and inhibition profiles of this enzyme resembled those of the AmpC beta-lactamase. The resistance gene of pKU601, which was cloned and expressed in E. coli, proved to contain an open reading frame showing 99.8% DNA sequence identity with the ampC gene of Citrobacter freundii GC3. DNA sequence analysis also identified a gene upstream of ampC whose sequence was 99.0% identical to the ampR gene from C. freundii GC3. In addition, a fumarate operon (frdABCD) and an outer membrane lipoprotein (blc) surrounding the ampR-ampC genes in C. freundii were identified, and insertion sequence (IS26) elements were observed on both sides of the sequences identified (forming an IS26 composite transposon); these results confirm the evidence of the translocation of a beta-lactamase-associated gene region from the chromosome to a plasmid. Finally, we describe a novel plasmid-encoded AmpC beta-lactamase, CFE-1, with an ampR gene derived from C. freundii.
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805. |
Potenza L,
Ubaldi L,
De Sanctis R,
De Bellis R,
Cucchiarini L,
Dach? M,
( 2004 ) Effects of a static magnetic field on cell growth and gene expression in Escherichia coli. PMID : 15238230 : DOI : 10.1016/j.mrgentox.2004.03.009 Abstract >>
Escherichia coli cultures exposed to a 300mT static magnetic field (SMF) were studied in order to analyse possible induced changes in cellular growth and gene expression. Biomass was evaluated by visible-light spectrometry and gene expression analyses were carried out by use of RNA arbitrarily primed PCR. The bacterial strain XL-1Blue, cultivated in traditional and modified Luria-Bertani medium, was exposed to SMF generated by permanent neodymium magnetic disks. The results show alterations induced by SMF in terms of increased cell proliferation and changes in gene expression compared with control groups. Three cDNAs were found to be expressed only in the exposed cells, whereas one cDNA was more expressed in the controls. One clone, expressed only in the exposed cells, corresponds to a putative transposase. This is of particular interest in that it suggests that exposure to a magnetic field may stimulate transposition activity.
|
806. |
Doroshenko VG,
Livshits VA,
( 2004 ) Structure and mode of transposition of Tn2555 carrying sucrose utilization genes. PMID : 15063507 : DOI : 10.1016/j.femsle.2004.03.016 Abstract >>
The sucrose transposon Tn2555 from Escherichia coli, which has an unstable structure, was studied in more detail. Sequence analysis of one of the transposon variants, designated Tn2555.3, revealed the presence of two direct IS26 copies on its flanks, and a third inverted IS26 copy inside the transposon. The sucrose utilization genes of Tn2555.3 were found to be identical to those of the previously described pUR400 plasmid. It was demonstrated that Tn2555.3 translocation from pBR325 to RP4 occurs via a cointegrate formation, mediated by one of the three IS26 copies, followed by its resolution due to RecA-dependent recombination between two direct IS26 copies flanking the donor replicon.
|
807. |
McSweeney LA,
Dreyfus LA,
( 2004 ) Nuclear localization of the Escherichia coli cytolethal distending toxin CdtB subunit. PMID : 15056215 : DOI : 10.1111/j.1462-5822.2004.00373.x Abstract >>
Cytolethal distending toxin (CDT) is a heterotrimeric protein toxin produced by several bacterial pathogens. Cells exposed to CDT die from either activation of the mitotic checkpoint cascade or apoptosis. Introduction of the purified CdtB subunit, a homologue of mammalian type I DNase, into cells mimics the action of the CDT holotoxin. Mutant CdtBs lacking DNase activity are devoid of biological activity. Chromosomal DNA appears to be the CDT target; thus, nuclear translocation of CdtB must precede cytolethal activity. Examination of the CdtB sequence indicates the presence of putative candidate bipartite nuclear localization signals (NLS). Here, we examine the functionality of the two potential NLS sequences found in the Escherichia coli CdtB-II. Nuclear translocation of EcCdtB-II was examined by monitoring the localization of an EcCdtB-II-EGFP fusion in Cos-7 cells. Our results indicated that EGFP-EcCdtB-II localized to the nucleus. The candidate EcCdtB-II-II NLS sequences were modified by site-directed mutagenesis such that tandem arginine residues were changed to threonine and serine respectively. Mutation of both putative NLS sequences had no effect on EcCdtB-II-associated DNase activity; however, cell cycle arrest and nuclear localization were significantly impaired in cells that received CDT reconstituted from the EcCdtB-II-DeltaNLS mutants. When HeLa cells were electroporated with the EcCdtB-II-DeltaNLS1 and the EcCdtB-II-NLS double mutants, toxicity was not observed, whereas the activity of EcCdtB-II-DeltaNLS2 was similar to that of wild-type EcCdtB-II. These data indicate that the putative NLS sequences are important for CDT-mediated action arrest and that they are likely to function in the nuclear translocation of EcCdtB-II.
|
808. |
Ansaldi M,
Théraulaz L,
Méjean V,
( 2004 ) TorI, a response regulator inhibitor of phage origin in Escherichia coli. PMID : 15197250 : DOI : 10.1073/pnas.0401927101 PMC : PMC438992 Abstract >>
The torI gene has been identified by using a genetic multicopy approach as a negative regulator of the torCAD operon that encodes the trimethylamine N-oxide reductase respiratory system in Escherichia coli. The negative effect was due to a previously unidentified small ORF (66 aa) of phage origin that we called torI for Tor inhibition. Overexpression of torI led to an 8-fold decrease of the torCAD operon transcription. This operon is positively regulated, in the presence of trimethylamine N-oxide, by a four-step phosphorelay involving the TorS sensor and the TorR response regulator. Epistatic experiments showed that TorI acts downstream of TorS and needs the presence of TorR. In vitro experiments showed that it is neither a TorR phosphatase nor a histidine kinase inhibitor and that it binds to the effector domain of TorR. Unexpectedly, TorI did not impede TorR DNA binding, and we propose that it may prevent RNA polymerase recruitment to the torC promoter. This study thus reveals a previously uncharacterized class of response regulator inhibitors.
|
809. |
Clarke BR,
Cuthbertson L,
Whitfield C,
( 2004 ) Nonreducing terminal modifications determine the chain length of polymannose O antigens of Escherichia coli and couple chain termination to polymer export via an ATP-binding cassette transporter. PMID : 15184370 : DOI : 10.1074/jbc.M404738200 Abstract >>
The chain length of bacterial lipopolysaccharide O antigens is regulated to give a modal distribution that is critical for pathogenesis. This paper describes the process of chain length determination in the ATP-binding cassette (ABC) transporter-dependent pathway, a pathway that is widespread among Gram-negative bacteria. Escherichia coli O8 and O9/O9a polymannans are synthesized in the cytoplasm, and an ABC transporter exports the nascent polymer across the inner membrane prior to completion of the LPS molecule. The polymannan O antigens have nonreducing terminal methyl groups. The 3-O-methyl group in serotype O8 is transferred from S-adenosylmethionine by the WbdD(O8) enzyme, and this modification terminates polymerization. Methyl groups are added to the O9a polymannan in a reaction dependent on preceding phosphorylation. The bifunctional WbdD(O9a) catalyzes both reactions, but only the kinase activity controls chain length. Chain termination occurs in a mutant lacking the ABC transporter, indicating that it precedes export. An E. coli wbdD(O9a) mutant accumulated O9a polymannan in the cytoplasm, indicating that WbdD activity coordinates polymannan chain termination with export across the inner membrane.
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810. |
Escobar-Páramo P,
Sabbagh A,
Darlu P,
Pradillon O,
Vaury C,
Denamur E,
Lecointre G,
( 2004 ) Decreasing the effects of horizontal gene transfer on bacterial phylogeny: the Escherichia coli case study. PMID : 15022774 : Abstract >>
Phylogenetic reconstructions of bacterial species from DNA sequences are hampered by the existence of horizontal gene transfer. One possible way to overcome the confounding influence of such movement of genes is to identify and remove sequences which are responsible for significant character incongruence when compared to a reference dataset free of horizontal transfer (e.g., multilocus enzyme electrophoresis, restriction fragment length polymorphism, or random amplified polymorphic DNA) using the incongruence length difference (ILD) test of Farris et al. [Cladistics 10 (1995) 315]. As obtaining this "whole genome dataset" prior to the reconstruction of a phylogeny is clearly troublesome, we have tested alternative approaches allowing the release from such reference dataset, designed for a species with modest level of horizontal gene transfer, i.e., Escherichia coli. Eleven different genes available or sequenced in this work were studied in a set of 30 E. coli reference (ECOR) strains. Either using ILD to test incongruence between each gene against the all remaining (in this case 10) genes in order to remove sequences responsible for significant incongruence, or using just a simultaneous analysis without removals, gave robust phylogenies with slight topological differences. The use of the ILD test remains a suitable method for estimating the level of horizontal gene transfer in bacterial species. Supertrees also had suitable properties to extract the phylogeny of strains, because the way they summarize taxonomic congruence clearly limits the impact of individual gene transfers on the global topology. Furthermore, this work allowed a significant improvement of the accuracy of the phylogeny within E. coli.
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811. |
Tominaga A,
( 2004 ) Characterization of six flagellin genes in the H3, H53 and H54 standard strains of Escherichia coli. PMID : 15056931 : Abstract >>
Six flagellin genes in three H standard Escherichia coli strains for H3, H53 and H54 were characterized. Each strain has two flagellin genes, one of which is expressed as its standard H antigen. A pair of flagellin genes flkA3 (encoding for H3 antigen) and fliC16 (H16) was cloned from Bi7327-41, flkA53 (H53) and fliC-53 from E480-68, and flmA54 (H54) and fliC-54 from E223-69. Two fliC genes, fliC-53 and fliC-54, are nonfunctional owing to the insertions of IS1 and IS1222, respectively. The flkA and flmA regions are located in the 3' end of the rnpB gene and near the nlpA gene, respectively. Each of them is followed by a gene homologous to fljA, which is known to repress the expression of fliC(i) in Salmonella enterica serovar Typhimurium. These results suggest that they are derived from the same origin of the fljBA operon. However, these regions contain neither the hin gene nor the invertible H segment. The four flagellin genes, fliC16, flkA3, flkA53 and flmA54, share high homology in nucleotide and amino-acid sequences with one another and with the S. enterica serovar Typhimurium flagellin genes. The promoter sequence of fliC16 is homologous to that of fliC(i), whereas the promoter sequences of flkA and flmA are homologous to that of fljB. The terminator sequences of the fliC16, fliC-53 and fliC-54 genes are conserved among themselves and identical with that of the E. coli fliC48 gene. Three FljA repressors, FljA3, FljA53 and FljA54, are homologous highly with one another and moderately with FljA of Salmonella. These results indicate that six flagellin genes analyzed are markedly similar to the Salmonella flagellin genes, suggesting their lateral transfer from Salmonella.
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812. |
Stürenburg E,
Kühn A,
Mack D,
Laufs R,
( 2004 ) A novel extended-spectrum beta-lactamase CTX-M-23 with a P167T substitution in the active-site omega loop associated with ceftazidime resistance. PMID : 15201232 : DOI : 10.1093/jac/dkh334 Abstract >>
In recent years, cefotaximases of the CTX-M type have become a predominant cause of resistance to extended-spectrum cephalosporins in Gram-negative bacteria. Although most enzymes provide higher levels of resistance to cefotaxime than to ceftazidime, mutants with enhanced catalytic efficiency against ceftazidime have recently been described. This report identifies another ceftazidime-resistant mutant of the CTX-M class of enzymes. Two ceftazidime-resistant strains, Escherichia coli IFI-1 and Klebsiella pneumoniae IFI-2, were isolated from a 46-year-old man during treatment of postoperative peritonitis with ceftazidime. Susceptibility testing, mating-out assays, isoelectric focusing as well as PCR and sequencing techniques were carried out to investigate the underlying mechanism of resistance. E. coli IFI-1 and K. pneumoniae IFI-2 exhibited a clavulanic acid-inhibited substrate profile that included extended-spectrum cephalosporins. Notably, both strains had up to a 32-fold higher level of resistance to ceftazidime than to cefotaxime. Further characterization revealed that a novel bla(CTX-M) gene encoding a beta-lactamase with a pI of 8.9 was implicated in this resistance: CTX-M-23. Along with the substitutions D114N and S140A, CTX-M-23 differed from CTX-M-1, the most closely related enzyme, by a P167T replacement in the active-site omega loop, which has not previously been observed in other CTX-M enzymes. By analogy with what was observed with certain TEM/PSE/BPS-type beta-lactamases, the amino acid substitution in the omega loop may explain ceftazidime resistance, which has only rarely been reported for other CTX-M enzymes. The emergence of a new ceftazidime-resistant CTX-M-type mutant provides evidence that these enzymes are able to broaden their substrate spectrum towards ceftazidime, probably due to substitutions in the active-site omega loop.
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813. |
Azpiroz MF,
Laviña M,
( 2004 ) Involvement of enterobactin synthesis pathway in production of microcin H47. PMID : 15047525 : DOI : 10.1128/aac.48.4.1235-1241.2004 PMC : PMC375329 Abstract >>
Microcin H47 (MccH47) is a gene-encoded peptide antibiotic produced by an Escherichia coli clinical isolate which is active on strains of gram-negative bacteria. Its uptake by E. coli K-12-susceptible cells depends on the presence of any of the outer membrane proteins Cir, Fiu, and FepA, the three catechol receptors of this organism. The nucleotide sequence of a portion of the MccH47 genetic system that had not yet been studied was elucidated. Five open reading frames were identified, three of which corresponded to genes encoding functions related to catechol-type siderophores: mchA and mchS1 are iroB and iroD homologues, respectively, and mchS4 was found to promote the production of the catecholate siderophore enterobactin. The possible relationship between enterobactin synthesis and MccH47 production was studied. Enterobactin-deficient strains failed to produce MccH47 when transformed with the antibiotic genetic determinants and upon introduction of the ent genetic cluster, the production of both the siderophore and MccH47 was restored. Further studies demonstrated that at least the enterobactin nonribosomal peptide synthase EntF is necessary for MccH47 synthesis. The relationship found between MccH47 and catecholate siderophore production is discussed, and a model outlining MccH47 synthesis is proposed.
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814. |
Greune L,
Prager R,
Fruth A,
Tschäpe H,
Schmidt MA,
Karch H,
Bielaszewska M,
Fell M,
( 2004 ) Characterization of cytolethal distending toxin genes and expression in shiga toxin-producing Escherichia coli strains of non-O157 serogroups. PMID : 14977993 : DOI : 10.1128/iai.72.3.1812-1816.2004 PMC : PMC356029 Abstract >>
We identified cytolethal distending toxin and its gene (cdt) in 17 of 340 non-O157 Shiga toxin-producing Escherichia coli (STEC) strains (serotypes O73:H18, O91:H21, O113:H21, and O153:H18), all of which were eae negative. cdt is either chromosomal and homologous to cdt-V (serotypes O73:H18, O91:H21, and O113:H21) or plasmidborne and identical to cdt-III (serotype O153:H18). Among eae-negative STEC, cdt was associated with disease (P = 0.003).
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815. |
Patel SK,
Dotson J,
Allen KP,
Fleckenstein JM,
( 2004 ) Identification and molecular characterization of EatA, an autotransporter protein of enterotoxigenic Escherichia coli. PMID : 14977988 : DOI : 10.1128/iai.72.3.1786-1794.2004 PMC : PMC356008 Abstract >>
Enterotoxigenic Escherichia coli (ETEC) strains remain a formidable cause of diarrheal disease. To identify novel surface proteins of ETEC, we performed TnphoA mutagenesis of prototype ETEC strain H10407 and discovered a secreted protein not previously recognized in ETEC. DNA sequencing of the interrupted locus in mutant TnphoA.977 revealed a candidate 4,095-bp open reading frame without significant homology to commensal E. coli K-12 genomic DNA. Translation of this sequence revealed that it encoded a predicted peptide of 147.7 kDa that bears significant homology to members of the autotransporter family of bacterial virulence factors, particularly the serine protease autotransporters of the Enterobacteriaceae proteins. The gene identified in H10407, eatA (ETEC autotransporter A), encodes a potential serine protease motif (GDSGSP) in the secreted amino-terminal domain, and the predicted peptide shows more than 80% homology with SepA, a virulence protein secreted by Shigella flexneri. DNA hybridization and PCR demonstrated that eatA resides on the 92-kDa pCS1 virulence plasmid of H10407 and that it is present in multiple clinical ETEC strains. Immunoblots with antisera directed against a recombinant EatA passenger protein fragment identified a 110-kDa protein in supernatants purified from H10407 but not from the TnphoA.977 mutant or H10407-P, which lacks pCS1. EatA possesses serine protease activity that is abolished by mutations within a serine protease catalytic triad formed by residues H(134), D(162), and S(267). Finally, interruption of the eatA gene retarded fluid accumulation in the rabbit ileal loop model, suggesting that this autotransporter contributes to the virulence of ETEC.
|
816. |
Shen S,
Mascarenhas M,
Rahn K,
Kaper JB,
Karmali MA,
Karmal MA,
( 2004 ) Evidence for a hybrid genomic island in verocytotoxin-producing Escherichia coli CL3 (serotype O113:H21) containing segments of EDL933 (serotype O157:H7) O islands 122 and 48. PMID : 14977955 : DOI : 10.1128/iai.72.3.1496-1503.2004 PMC : PMC355994 Abstract >>
Genomic O island 122 (OI-122) of the verocytotoxin-producing Escherichia coli (VTEC) strain EDL933 contains four putative virulence genes, Z4321, Z4326, Z4332, and Z4333. However, strain CL3 (serotype O113:H21) contains only Z4321, not the other three genes. To determine whether Z4321 is part of a different genomic island in CL3, a region of 27,293 bp up- and downstream of Z4321 was sequenced and found to contain elements of two different EDL933 genomic islands (OI-48 and OI-122) and a Yersinia pestis-like hemolysin/adhesin gene cluster. The region contained OI-48 genes Z1635, Z1636, and Z1637 at the left terminus and Z1641, Z1642, Z1643, and Z1644 at the right. The middle portion consisted of OI-48 gene Z1640, which was separated into three fragments by genomic segments including the Y. pestis cluster and EDL933 OI-122 genes Z4322, Z4321, and Z4318. In a PCR investigation of 36 VTEC strains of different serotypes, intact Z1640 was present in strains of serotypes O157:H7, O26:H11, O103:H2, O111:NM, and O145:NM, which are associated with hemolytic uremic syndrome and outbreaks. In contrast, fragmented Z1640 was seen in strains of nonepidemic serotypes, such as O91:H21 and O113:H21, and in animal serotypes that have not been associated with human disease, indicating that Z1640 might be a virulence gene.
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817. |
Sonntag AK,
Prager R,
Bielaszewska M,
Zhang W,
Fruth A,
Tschäpe H,
Karch H,
( 2004 ) Phenotypic and genotypic analyses of enterohemorrhagic Escherichia coli O145 strains from patients in Germany. PMID : 15004038 : DOI : 10.1128/jcm.42.3.954-962.2004 PMC : PMC356868 Abstract >>
Enterohemorrhagic Escherichia coli (EHEC) strains of serogroup O145 are emerging as causes of diarrhea and the hemolytic-uremic syndrome. However, there have been few genetic analyses of this EHEC group. We investigated the serotypes, virulence genes, plasmid profiles, pulsed-field gel electrophoresis (PFGE) patterns, and genetic variability of the fliC and eae genes in 120 EHEC O145 strains isolated from cases of hemolytic-uremic syndrome (n = 24) or diarrhea (n = 96) in Germany between 1996 and 2002. Three isolates belonged to serotype O145:H28, one to serotype O145:H25, and 116 were nonmotile (O145:H(-)). One hundred fourteen of the nonmotile strains shared fliC restriction fragment length polymorphism (RFLP) patterns identical to that of the O145:H28 strains. The remaining two nonmotile strains displayed a fliC-RFLP pattern identical to that of the O145:H25 strain. Each of the 117 strains with the fliC-RFLP(H28) pattern harbored eae gamma, whereas the three strains with the fliC-RFLP(H25) pattern possessed eae beta. Five different stx genotypes, six combinations of plasmid-encoded putative virulence genes, 29 plasmid profiles, and 47 PFGE types were identified. Strains within some of the PFGE types could be further subtyped by means of distinct plasmid profiles. These data demonstrate that the EHEC O145 serogroup is comprised of two different serotypes that possess distinct eae types. The heterogeneity of EHEC O145 strains at the chromosomal and plasmid level, in particular the high diversity in PFGE patterns, provides a basis for molecular subtyping of these pathogens.
|
818. |
Ackerley DF,
Gonzalez CF,
Park CH,
Blake R,
Keyhan M,
Matin A,
( 2004 ) Chromate-reducing properties of soluble flavoproteins from Pseudomonas putida and Escherichia coli. PMID : 14766567 : DOI : 10.1128/aem.70.2.873-882.2004 PMC : PMC348923 Abstract >>
Cr(VI) (chromate) is a toxic, soluble environmental contaminant. Bacteria can reduce chromate to the insoluble and less toxic Cr(III), and thus chromate bioremediation is of interest. Genetic and protein engineering of suitable enzymes can improve bacterial bioremediation. Many bacterial enzymes catalyze one-electron reduction of chromate, generating Cr(V), which redox cycles, generating excessive reactive oxygen species (ROS). Such enzymes are not appropriate for bioremediation, as they harm the bacteria and their primary end product is not Cr(III). In this work, the chromate reductase activities of two electrophoretically pure soluble bacterial flavoproteins--ChrR (from Pseudomonas putida) and YieF (from Escherichia coli)-were examined. Both are dimers and reduce chromate efficiently to Cr(III) (kcat/Km = approximately 2 x 10(4) M(-1) x s(-1)). The ChrR dimer generated a flavin semiquinone during chromate reduction and transferred >25% of the NADH electrons to ROS. However, the semiquinone was formed transiently and ROS diminished with time. Thus, ChrR probably generates Cr(V), but only transiently. Studies with mutants showed that ChrR protects against chromate toxicity; this is possibly because it preempts chromate reduction by the cellular one-electron reducers, thereby minimizing ROS generation. ChrR is thus a suitable enzyme for further studies. During chromate reduction by YieF, no flavin semiquinone was generated and only 25% of the NADH electrons were transferred to ROS. The YieF dimer may therefore be an obligatory four-electron chromate reducer which in one step transfers three electrons to chromate and one to molecular oxygen. As a mutant lacking this enzyme could not be obtained, the role of YieF in chromate protection could not be directly explored. The results nevertheless suggest that YieF may be an even more suitable candidate for further studies than ChrR.
|
819. |
Horne SM,
Goplin JL,
Giddings CW,
Dyer NW,
Nolan LK,
( 2004 ) Cloning and sequencing of cnf1 from Escherichia coli incriminated in mink and bovine colibacillosis. PMID : 14992240 : Abstract >>
Colibacillosis is responsible for significant losses to the mink and cattle industries. Previous work in our laboratory and by others has suggested that possession of cnf1, the gene encoding cytotoxic necrotizing factor (CNF1), may contribute to the virulence of isolates of E. coli from mink and cattle. The cnf1 gene from E. coli isolated from a mink with colisepticaemia and a bovid with scours was amplified and cloned as a 3.5 kb fragment, and the fragment was sequenced. The cnf1 sequences from the mink and bovine isolates of E. coli were compared to each other and to cnf1 sequences of E. coli from urinary tract and diarrhoea-associated infections of humans. The difference was only 7 nucleotides between the cnf1 sequences of the mink and bovine isolates of E. coli, which translated into 7 differences in amino acids. The cnf1 sequence of the mink isolate of E. coli had 15 nucleotide differences from the cnf1 sequences of the human isolate of E. coli (GenBank X70670), which translated into 11 differences in amino acids between these proteins. The cnf1 sequence of the bovine isolate of E. coli had 14 nucleotide differences from the cnf1 sequence of the human isolate of E. coli (GenBank X70670), which translated into 10 differences in amino acids between these proteins. The highly conserved sequences of the amino acids of CNF1 proteins make them a promising target for detection and control of the CNF1-producing E. coli involved in disease among various host species.
|
820. |
Newton HJ,
Sloan J,
Bennett-Wood V,
Adams LM,
Robins-Browne RM,
Hartland EL,
( 2004 ) Contribution of long polar fimbriae to the virulence of rabbit-specific enteropathogenic Escherichia coli. PMID : 14977923 : DOI : 10.1128/iai.72.3.1230-1239.2004 PMC : PMC356030 Abstract >>
Enteropathogenic Escherichia coli (EPEC) is a major of cause of diarrhea among children in developing countries. Although EPEC is a human specific pathogen, some related strains are natural pathogens of animals, including laboratory-bred rabbits. We have identified two chromosomal loci in rabbit-specific EPEC (REPEC) O15:H- strain 83/39, which are predicted to encode long polar fimbriae (LPF). lpf(R154) was identical to a fimbrial gene cluster, lpf(O113), identified previously in enterohemorrhagic E. coli (EHEC) O113:H21. The second locus, lpf(R141), comprised a novel sequence with five predicted open reading frames, lpfA to lpfE, that encoded long fine fimbriae in nonfimbriated E. coli ORN103. The predicted products of lpf(R141) shared identity with components of the lpfABCC'DE gene cluster from EHEC O157:H7, and the fimbriae were similar in morphology and length to LPF from EHEC O157:H7. Interruption of lpf(R141) resulted in significant attenuation of REPEC 83/39 for rabbits with respect to the early stages of colonization and severity of diarrhea. However, there was no significant difference in the number of bacteria shed at later time points or in overall body weight and mortality rate of rabbits infected with lpf(R141) mutant strains or wild-type REPEC 83/39. Although rabbits infected with the lpf(R141) mutants did not develop severe diarrhea, there was evidence of attaching and effacing histopathology, which was indistinguishable in morphology, location, and extent compared to rabbits infected with wild-type REPEC 83/39. The results suggested that lpf(R141) contributes to the early stages of REPEC-mediated disease and that this is important for the development of severe diarrhea in susceptible animals.
|
821. |
Schülein R,
Gentschev I,
Mollenkopf HJ,
Goebel W,
( 1992 ) A topological model for the haemolysin translocator protein HlyD. PMID : 1495479 : DOI : 10.1007/bf00272357 Abstract >>
A topological model for HlyD is proposed that is based on results obtained with gene fusions of lacZ and phoA to hlyD. Active HlyD-LacZ fusion proteins were only generated when lacZ was fused to hlyD within the first 180 bp (60 amino acids). HlyD-PhoA proteins exhibiting alkaline phosphatase (AP) activity were obtained when phoA was inserted into hlyD between nucleotides 262 (behind amino acid position 87) and 1405 (behind amino acid position 468, only 10 amino acids away from the C-terminus of HlyD). Active insertions of phoA into the middle region of hlyD were not observed on in vivo transposition but such fusions exhibiting AP activity could be constructed by in vitro techniques. A fusion protein that carried the PhoA part close to the C-terminal end of HlyD proved to be the most stable HlyD-PhoA fusion protein. In contrast to the other, rather unstable, HlyD-PhoA+ fusions, no proteolytic degradation product of this HlyD-PhoA protein was observed and nearly all the alkaline phosphatase activity was membrane bound. Protease accessibility and cell fractionation experiments indicated that the alkaline phosphatase moiety of this fusion protein was located in the periplasm as for all other HlyD-PhoA+ proteins. These data and computer-assisted predictions suggest a topological model for HlyD with the N-terminal 60 amino acids located in the cytoplasm, a single transmembrane segment from amino acids 60 to 80 and a large periplasmic region extending from amino acid 80 to the C-terminus.(ABSTRACT TRUNCATED AT 250 WORDS)
|
822. |
Guo H,
Feng L,
Tao J,
Zhang C,
Wang L,
( 2004 ) Identification of Escherichia coli O172 O-antigen gene cluster and development of a serogroup-specific PCR assay. PMID : 15186455 : DOI : 10.1111/j.1365-2672.2004.02305.x Abstract >>
To characterize the locus for O-antigen biosynthesis from Escherichia coli O172 type strain and to develop a rapid, specific and sensitive PCR-based method for identification and detection of E. coli O172. DNA of O-antigen gene cluster of E. coli O172 was amplified by long-range PCR method using primers based on housekeeping genes galF and gnd Shot gun bank was constructed and high quality sequencing was performed. The putative genes for synthesis of UDP-FucNAc, O-unit flippase, O-antigen polymerase and glycosyltransferases were assigned by the homology search. The evolutionary relationship between O-antigen gene clusters of E. coli O172 and E. coli O26 is shown by sequence comparison. Genes specific to E. coli O172 strains were identified by PCR assays using primers based on genes for O-unit flippase, O-antigen polymerase and glycosyltransferases. The specificity of PCR assays was tested using all E. coli and Shigella O-antigen type strains, as well as 24 clinical E. coli isolates. The sensitivity of PCR assays was determined, and the detection limits were 1 pg microl(-1) chromosomal DNA, 0.2 CFU g(-1) pork and 0.2 CFU ml(-1) water. The total time required from beginning to end of the procedure was within 16 h. The O-antigen gene cluster of E. coli O172 was identified and PCR assays based on O-antigen specific genes showed high specificity and sensitivity. An O-antigen gene cluster was identified by sequencing. The specific genes were determined for E. coli O172. The sensitivity of O-antigen specific PCR assay was tested. Although Shiga toxin-producing O172 strains were not yet isolated from clinical specimens, they may emerge as pathogens.
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823. |
Pickett CL,
Lee RB,
Eyigor A,
Elitzur B,
Fox EM,
Strockbine NA,
( 2004 ) Patterns of variations in Escherichia coli strains that produce cytolethal distending toxin. PMID : 14742509 : DOI : 10.1128/iai.72.2.684-690.2004 PMC : PMC321568 Abstract >>
A collection of 20 Escherichia coli strains that produce cytolethal distending toxin (CDT) were analyzed for their virulence-associated genes. All of these strains were serotyped, and multiplex PCR analysis was used to ascertain the presence of genes encoding other virulence factors, including Shiga toxin, intimin, enterohemolysin, cytotoxic necrotizing factor type 1 (CNF1) and CNF2, heat-stable toxin, and heat-labile toxin. These CDT-producing strains possessed various combinations of known virulence genes, some of which have not been noted before. Partial cdtB sequences were obtained from 10 of these strains, and their predicted CdtB sequences were compared to known E. coli CdtB sequences; some of the sequences were identical to known CdtB sequences, but two were not. PCR primers based on sequence differences between the known cdt sequences were tested for their ability to detect CDT producers and to determine CDT type. Correlations between the type of CDT produced, the presence of other virulence properties, and overall strain relatedness revealed that the CDT producers studied here can be divided into three general groups, with distinct differences in CDT type and in their complement of virulence-associated genes.
|
824. |
Wren BW,
Colby SM,
Cubberley RR,
Pallen MJ,
( 1992 ) Degenerate PCR primers for the amplification of fragments from genes encoding response regulators from a range of pathogenic bacteria. PMID : 1490612 : DOI : 10.1016/0378-1097(92)90042-m Abstract >>
Many bacterial responses to environmental stimuli are mediated by response regulators which coordinately regulate genes involved in particular adaptive responses. Degenerate oligonucleotide primers were used to amplify by the polymerase chain reaction (PCR), fragments from genes encoding eleven novel response regulators. Sequence and phylogenetic analysis revealed that phoB, phoP and creB gene fragments had been amplified from Yersinia enterocolitica and Yersinia pseudotuberculosis, and that a creB sequence had been amplified from Campylobacter jejuni. Four amplified fragments from C. jejuni, Listeria monocytogenes, Mycobacterium tuberculosis and Escherichia coli clearly came from response regulator genes, but were not closely related to any of the known genes. Mutagenesis of the newly identified genes should allow us to determine their function and the genes under their control.
|
825. |
Ly A,
Henderson J,
Lu A,
Culham DE,
Wood JM,
( 2004 ) Osmoregulatory systems of Escherichia coli: identification of betaine-carnitine-choline transporter family member BetU and distributions of betU and trkG among pathogenic and nonpathogenic isolates. PMID : 14702297 : DOI : 10.1128/jb.186.2.296-306.2004 PMC : PMC305767 Abstract >>
Multiple transporters mediate osmoregulatory solute accumulation in Escherichia coli K-12. The larger genomes of naturally occurring strains such as pyelonephritis isolates CFT073 and HU734 may encode additional osmoregulatory systems. CFT073 is more osmotolerant than HU734 in the absence of organic osmoprotectants, yet both strains grew in high osmolality medium at low K(+) (micromolar concentrations) and retained locus trkH, which encodes an osmoregulatory K(+) transporter. Both lacked the trkH homologue trkG. Transporters ProP and ProU account for all glycine-betaine uptake activity in E. coli K-12 and CFT073, but not in HU734, yet elimination of ProP and ProU impairs the growth of HU734, but not CFT073, in high osmolality human urine. No known osmoprotectant stimulated the growth of CFT073 in high osmolality minimal medium, but putative transporters YhjE, YiaMNO, and YehWXYZ may mediate uptake of additional osmoprotectants. Gene betU was isolated from HU734 by functional complementation and shown to encode a betaine uptake system that belongs to the betaine-choline-carnitine transporter family. The incidence of trkG and betU within the ECOR collection, representatives of the E. coli pathotypes (PATH), and additional strains associated with urinary tract infection (UTI) were determined. Gene trkG was present in 66% of the ECOR collection but only in 16% of the PATH and UTI collections. Gene betU was more frequently detected in ECOR groups B2 and D (50% of isolates) than in groups A, B1, and E (20%), but it was similar in overall incidence in the ECOR collection and in the combined UTI and PATH collections (32 and 34%, respectively). Genes trkG and betU may have been acquired by lateral gene transfer, since trkG is part of the rac prophage and betU is flanked by putative insertion sequences. Thus, BetU and TrkG contribute, with other systems, to the osmoregulatory capacity of the species E. coli, but they are not characteristic of a particular phylogenetic group or pathotype.
|
826. |
Schubert S,
Dufke S,
Sorsa J,
Heesemann J,
( 2004 ) A novel integrative and conjugative element (ICE) of Escherichia coli: the putative progenitor of the Yersinia high-pathogenicity island. PMID : 14731283 : DOI : 10.1046/j.1365-2958.2003.03870.x Abstract >>
Diversification of bacterial species and pathotypes is largely caused by horizontal transfer of diverse DNA elements such as plasmids, phages and genomic islands (e.g. pathogenicity islands, PAIs). A PAI called high-pathogenicity island (HPI) carrying genes involved in siderophore-mediated iron acquisition (yersiniabactin system) has previously been identified in Yersinia pestis, Y. pseudotuberculosis and Y. enterocolitica IB strains, and has been characterized as an essential virulence factor in these species. Strikingly, an orthologous HPI is a widely distributed virulence determinant among Escherichia coli and other Enterobacteriaceae which cause extraintestinal infections. Here we report on the HPI of E. coli strain ECOR31 which is distinct from all other HPIs described to date because the ECOR31 HPI comprises an additional 35 kb fragment at the right border compared to the HPI of other E. coli and Yersinia species. This part encodes for both a functional mating pair formation system and a DNA-processing region related to plasmid CloDF13 of Enterobacter cloacae. Upon induction of the P4-like integrase, the entire HPI of ECOR31 is precisely excised and circularised. The HPI of ECOR31 presented here resembles integrative and conjugative elements termed ICE. It may represent the progenitor of the HPI found in Y. pestis and E. coli, revealing a missing link in the horizontal transfer of an element that contributes to microbial pathogenicity upon acquisition.
|
827. |
Sibley MH,
Raleigh EA,
( 2004 ) Cassette-like variation of restriction enzyme genes in Escherichia coli C and relatives. PMID : 14744977 : DOI : 10.1093/nar/gkh194 PMC : PMC373321 Abstract >>
A surprising result of comparative bacterial genomics has been the large amount of DNA found to be present in one strain but not in another of the same species. We examine in detail one location where gene content varies extensively, the restriction cluster in Escherichia coli. This region is designated the Immigration Control Region (ICR) for the density and variability of restriction functions found there. To better define the boundaries of this variable locus, we determined the sequence of the region from a restrictionless strain, E.coli C. Here we compare the 13.7 kb E.coli C sequence spanning the site of the ICR with corresponding sequences from five E.coli strains and Salmonella typhimurium LT2. To discuss this variation, we adopt the term 'framework' to refer to genes that are stable components of genomes within related lineages, while 'migratory' genes are transient inhabitants of the genome. Strikingly, seven different migratory DNA segments, encoding different sets of genes and gene fragments, alternatively occupy a single well-defined location in the seven strains examined. The flanking framework genes, yjiS and yjiA, display approximately normal patterns of conservation. The patterns observed are consistent with the action of a site-specific recombinase. Since no nearby gene codes for a likely recombinase of known families, such a recombinase must be of a new family or unlinked.
|
828. |
Herbeck JT,
Funk DJ,
Degnan PH,
Wernegreen JJ,
( 2003 ) A conservative test of genetic drift in the endosymbiotic bacterium Buchnera: slightly deleterious mutations in the chaperonin groEL. PMID : 14704156 : PMC : PMC1462895 Abstract >>
The obligate endosymbiotic bacterium Buchnera aphidicola shows elevated rates of sequence evolution compared to free-living relatives, particularly at nonsynonymous sites. Because Buchnera experiences population bottlenecks during transmission to the offspring of its aphid host, it is hypothesized that genetic drift and the accumulation of slightly deleterious mutations can explain this rate increase. Recent studies of intraspecific variation in Buchnera reveal patterns consistent with this hypothesis. In this study, we examine inter- and intraspecific nucleotide variation in groEL, a highly conserved chaperonin gene that is constitutively overexpressed in Buchnera. Maximum-likelihood estimates of nonsynonymous substitution rates across Buchnera species are strikingly low at groEL compared to other loci. Despite this evidence for strong purifying selection on groEL, our intraspecific analysis of this gene documents reduced synonymous polymorphism, elevated nonsynonymous polymorphism, and an excess of rare alleles relative to the neutral expectation, as found in recent studies of other Buchnera loci. Comparisons with Escherichia coli generally show patterns predicted by their differences in N(e). The sum of these observations is not expected under relaxed or balancing selection, selective sweeps, or increased mutation rate. Rather, they further support the hypothesis that drift is an important force driving accelerated protein evolution in this obligate mutualist.
|
829. |
Bertin Y,
Boukhors K,
Livrelli V,
Martin C,
( 2004 ) Localization of the insertion site and pathotype determination of the locus of enterocyte effacement of shiga toxin-producing Escherichia coli strains. PMID : 14711626 : DOI : 10.1128/aem.70.1.61-68.2004 PMC : PMC321293 Abstract >>
Of 220 Shiga toxin-producing Escherichia coli (STEC) strains collected in central France from healthy cattle, food samples, and asymptomatic children, 12 possessed the eae gene included in the locus of enterocyte effacement (LEE) pathogenicity island. Based on gene typing, we observed 7 different eae espA espB tir pathotypes among the 12 STEC strains and described the new espAbetav variant. As previously observed, the O157 serogroup is associated with eaegamma, O26 is associated with eaebeta, and O103 is associated with eaeepsilon. However, the unexpected eaezeta allele was detected in 5 of the 12 isolates. PCR amplification and pulsed-field gel electrophoresis using the I-CeuI endonuclease followed by Southern hybridization indicated that the LEE was inserted in the vicinity of the selC (three isolates), pheU (two isolates), or pheV (six isolates) tRNA gene. Six isolates harbored two or three of these tRNA loci altered by the insertion of integrase genes (CP4-int and/or int-phe), suggesting the insertion of additional foreign DNA fragments at these sites. In spite of great genetic diversity of LEE pathotypes and LEE insertion sites, bovine strains harbor alleles of LEE genes that are frequently found in clinical STEC strains isolated from outbreaks and sporadic cases around the world, underscoring the potential risk of the bovine strains on human health.
|
830. |
Blanco M,
Blanco JE,
Mora A,
Dahbi G,
Alonso MP,
González EA,
Bernárdez MI,
Blanco J,
( 2004 ) Serotypes, virulence genes, and intimin types of Shiga toxin (verotoxin)-producing Escherichia coli isolates from cattle in Spain and identification of a new intimin variant gene (eae-xi). PMID : 14766831 : DOI : 10.1128/jcm.42.2.645-651.2004 PMC : PMC344521 Abstract >>
A total of 514 Shiga toxin-producing Escherichia coli (STEC) isolates from diarrheic and healthy cattle in Spain were characterized in this study. PCR showed that 101 (20%) isolates carried stx(1) genes, 278 (54%) possessed stx(2) genes, and 135 (26%) possessed both stx(1) and stx(2). Enterohemolysin (ehxA) and intimin (eae) virulence genes were detected in 326 (63%) and in 151 (29%) of the isolates, respectively. STEC isolates belonged to 66 O serogroups and 113 O:H serotypes (including 23 new serotypes). However, 67% were of one of these 15 serogroups (O2, O4, O8, O20, O22, O26, O77, O91, O105, O113, O116, O157, O171, O174, and OX177) and 52% of the isolates belonged to only 10 serotypes (O4:H4, O20:H19, O22:H8, O26:H11, O77:H41, O105:H18, O113:H21, O157:H7, O171:H2, and ONT:H19). Although the 514 STEC isolates belonged to 164 different seropathotypes (associations between serotypes and virulence genes), only 12 accounted for 43% of isolates. Seropathotype O157:H7 stx(2) eae-gamma1 ehxA (46 isolates) was the most common, followed by O157:H7 stx(1) stx(2) eae-gamma1 ehxA (34 isolates), O113:H21 stx(2) (25 isolates), O22:H8 stx(1) stx(2) ehxA (15 isolates), O26:H11 stx(1) eae-beta1 ehxA (14 isolates), and O77:H41 stx(2) ehxA (14 isolates). Forty-one (22 of serotype O26:H11) isolates had intimin beta1, 82 O157:H7 isolates possessed intimin gamma1, three O111:H- isolates had intimin type gamma2, one O49:H- strain showed intimin type delta, 13 (six of serotype O103:H2) isolates had intimin type epsilon and eight (four of serotype O156:H-) isolates had intimin zeta. We have identified a new variant of the eae intimin gene designated xi (xi) in two isolates of serotype O80:H-. The majority (85%) of bovine STEC isolates belonged to serotypes previously found for human STEC organisms and 54% to serotypes associated with STEC organisms isolated from patients with hemolytic uremic syndrome. Thus, this study confirms that cattle are a major reservoir of STEC strains pathogenic for humans.
|
831. |
Wertz JE,
Goldstone C,
Gordon DM,
Riley MA,
( 2003 ) A molecular phylogeny of enteric bacteria and implications for a bacterial species concept. PMID : 14640415 : Abstract >>
A molecular phylogeny for seven taxa of enteric bacteria (Citrobacter freundii, Enterobacter cloacae, Escherichia coli, Hafnia alvei, Klebsiella oxytoca, Klebsiella pneumoniae, and Serratia plymuthica) was made from multiple isolates per taxa taken from a collection of environmental enteric bacteria. Sequences from five housekeeping genes (gapA, groEL, gyrA, ompA, and pgi) and the 16S rRNA gene were used to infer individual gene trees and were concatenated to infer a composite molecular phylogeny for the species. The isolates from each taxa formed tight species clusters in the individual gene trees, suggesting the existence of 'genotypic' clusters that correspond to traditional species designations. These sequence data and the resulting gene trees and consensus tree provide the first data set with which to assess the utility of the recently proposed core genome hypothesis (CGH). The CGH provides a genetically based approach to applying the biological species concept to bacteria.
|
832. |
Blickwede M,
Schwarz S,
( 2004 ) Molecular analysis of florfenicol-resistant Escherichia coli isolates from pigs. PMID : 14645321 : DOI : 10.1093/jac/dkh007 Abstract >>
The aim of this study was to analyse florfenicol-resistant Escherichia coli isolates from pigs for the genetic basis of florfenicol resistance, and to compare these data with those previously determined for E. coli isolates from cattle and poultry. Fourteen porcine E. coli isolates were included in this study and subjected to serotyping, plasmid profiling and macrorestriction analysis. MICs of florfenicol were determined by broth microdilution. The presence of the gene floR was confirmed by hybridization and PCR analysis. Transformation experiments were conducted to isolate florfenicol resistance plasmids. The floR region of a florfenicol resistance plasmid was cloned and sequenced. All florfenicol-resistant E. coli isolates exhibited MICs of florfenicol >128 mg/L and carried the floR gene. A single isolate had a floR-carrying plasmid of approximately 35 kb, designated pMBSF1. Sequence analysis identified the floR gene flanked by truncated transposase genes. Moreover, a truncated copy of Tn5393 with complete streptomycin resistance genes strA and strB was found upstream of the floR gene of pMBSF1. Chromosomally resistant E. coli isolates, which shared the same BlnI macrorestriction pattern, differed in their floR hybridization patterns. The plasmid pMBSF1 is the smallest floR-carrying plasmid reported to date. Its floR region differed from those previously found in E. coli isolates from cattle. Variations in the RFLPs of chromosomal EcoRI fragments carrying floR in isolates that had the same macrorestriction pattern might suggest variable chromosomal integration sites.
|
833. |
Pons AM,
Delalande F,
Duarte M,
Benoit S,
Lanneluc I,
Sablé S,
Van Dorsselaer A,
Cottenceau G,
( 2004 ) Genetic analysis and complete primary structure of microcin L. PMID : 14742202 : DOI : 10.1128/aac.48.2.505-513.2004 PMC : PMC321509 Abstract >>
Escherichia coli LR05, in addition to producing MccB17, J25, and D93, secretes microcin L, a newly discovered microcin that exhibits strong antibacterial activity against related Enterobacteriaceae, including Salmonella enterica serovars Typhimurium and Enteritidis. Microcin L was purified using a two-step procedure including solid-phase extraction and reverse-phase C(18) high-performance liquid chromatography. A 4,901-bp region of the DNA plasmid of E. coli LR05 was sequenced revealing that the microcin L cluster consists of four genes, mclC, mclI, mclA, and mclB. The structural gene mclC encoded a 105-amino-acid precursor with a 15-amino-acid N-terminal extension ending with a Gly-Ala motif upstream of the cleavage site. This motif is typical of the class II microcins and other gram-positive bacteriocins exported by ABC transporters. The mclI immunity gene was identified upstream of the mclC gene and encodes a 51-amino-acid protein with two potential transmembrane domains. Located on the reverse strand, two genes, mclA and mclB, encoded the proteins MclA and MclB, respectively. They bear strong relatedness with the ABC transporter proteins and accessory factors involved in the secretion of microcins H47, V, E492, and 24. The microcin L genetic system resembles the genetic organization of MccV. Furthermore the MccL primary structure has been determined. It is a 90-amino-acid peptide of 8,884 Da with two disulfide bridges. The N-terminal region has significant homologies with several gram-positive bacteriocins. The C-terminal 32-amino-acid sequence is 87.5% identical to that of MccV. Together, these results strongly indicate that microcin L is a gram-negative class II microcin.
|
834. |
Marchès O,
Ledger TN,
Boury M,
Ohara M,
Tu X,
Goffaux F,
Mainil J,
Rosenshine I,
Sugai M,
De Rycke J,
Oswald E,
( 2003 ) Enteropathogenic and enterohaemorrhagic Escherichia coli deliver a novel effector called Cif, which blocks cell cycle G2/M transition. PMID : 14651638 : DOI : 10.1046/j.1365-2958.2003.03821.x Abstract >>
Enteropathogenic Escherichia coli (EPEC) and enterohaemorrhagic E. coli (EHEC) are closely related pathogens. Both use a type III secretion system (TTSS) encoded by the 'locus of enterocyte effacement' (LEE) to subvert and attach to epithelial cells through the injection of a repertoire of effector molecules. Here, we report the identification of a new TTSS translocated effector molecule called Cif, which blocks cell cycle G2/M transition and induces the formation of stress fibres through the recruitment of focal adhesions. Cif is not encoded by the LEE but by a lambdoid prophage present in EPEC and EHEC. A cif mutant causes localized effacement of microvilli and intimately attaches to the host cell surface, but is defective in the ability to block mitosis. When expressed in TTSS competent LEE-positive pathogens, Cif is injected into the infected epithelial cells. These cells arrested at the G2/M phase displayed accumulation of inactive phosphorylated Cdk1. In conclusion, Cif is a new member of a growing family of bacterial cyclomodulins that subvert the host eukaryotic cell cycle.
|
835. |
Ojo KK,
Kehrenberg C,
Odelola HA,
Schwarz S,
( 2003 ) Structural analysis of the tetracycline resistance gene region of a small multiresistance plasmid from uropathogenic Escherichia coli isolated in Nigeria. PMID : 14613948 : DOI : 10.1093/jac/dkh005 Abstract >>
N/A
|
836. |
Yeo HJ,
Yuan Q,
Beck MR,
Baron C,
Waksman G,
( 2003 ) Structural and functional characterization of the VirB5 protein from the type IV secretion system encoded by the conjugative plasmid pKM101. PMID : 14673074 : DOI : 10.1073/pnas.2535211100 PMC : PMC307673 Abstract >>
Type IV secretion systems mediate intercellular transfer of macro-molecules via a mechanism ancestrally related to that of bacterial conjugation machineries. TraC of the IncN plasmid pKM101 belongs to the VirB5 family of proteins, an essential component of most type IV secretion systems. Here, we present the structure of TraC. VirB5/TraC is a single domain protein, which consists of a three helix bundle and a loose globular appendage. Structure-based site-directed mutagenesis followed by functional studies indicates that VirB5 proteins participate in protein-protein interactions important for pilus assembly and function.
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837. |
Vandemaele FJ,
Mugasa JP,
Vandekerchove D,
Goddeeris BM,
( 2003 ) Predominance of the papGII allele with high sequence homology to that of human isolates among avian pathogenic Escherichia coli (APEC). PMID : 14654294 : DOI : 10.1016/j.vetmic.2003.09.017 Abstract >>
Avian pathogenic Escherichia coli (APEC) are often found in poultry and are responsible for a set of diseases, commonly referred to as avian colibacillosis. One of the important virulence factors is adhesion to different epithelial surfaces, which is mediated by pili. P pili are thought to play a role by means of their PapG adhesin, which occurs in three molecular variants: PapGI, PapGII and PapGIII. This study is the first to determine and analyse the distribution of the different papG alleles in APEC. Our results show a significant predominance of the papGII allele above all other alleles or allele combinations. No statistically significant associations could be found between papG allele distribution and the type of bird, organ of isolation and O serogroup. Finally, the papGII and papGIII sequences showed high homology with mammalian (including human) source papG sequences.
|
838. |
Heritage J,
Chambers PA,
Tyndall C,
Buescher ES,
( 2003 ) SHV-34: an extended-spectrum beta-lactamase encoded by an epidemic plasmid. PMID : 14613945 : DOI : 10.1093/jac/dkh017 Abstract >>
To elucidate the causes for treatment failure in children given extended-spectrum cephalosporins. During April 1998-March 2000, 18 isolates of members of the family Enterobacteriaceae, fulfilling microbiological criteria for carriage of extended-spectrum beta-lactamases (ESBLs) and carrying blaSHV, were isolated from paediatric inpatients. The collection was subjected to a retrospective molecular analysis. Three species were represented in the collection: Citrobacter koseri (one isolate), Escherichia coli (one isolate) and Klebsiella pneumoniae (16 isolates). A common plasmid was found in these bacteria, as judged by restriction endonuclease digestion. This was able to transfer an ESBL phenotype from donors to a laboratory strain of E. coli. Nucleotide sequence analysis revealed that this phenotype was associated with a new variant in blaSHV encoding SHV-34. Analysis reveals the presence of an epidemic plasmid in this collection of bacteria. This carries a gene encoding the SHV-34 ESBL, described for the first time in this report. Nucleotide sequence analysis shows that there is a mutation from A-->G affecting the codon at amino acid position 64 (GAA-->GGA), changing the glutamic acid typically seen in this position to glycine.
|
839. |
Leung PH,
Peiris JS,
Ng WW,
Robins-Browne RM,
Bettelheim KA,
Yam WC,
( 2003 ) A newly discovered verotoxin variant, VT2g, produced by bovine verocytotoxigenic Escherichia coli. PMID : 14660413 : DOI : 10.1128/aem.69.12.7549-7553.2003 PMC : PMC309948 Abstract >>
A new verotoxin (VT) variant, designated vt2g, was identified from a bovine strain of verocytotoxigenic Escherichia coli (VTEC) serotype O2:H25. When vt2g was aligned with published sequences of vt2 and vt variants, it exhibited the highest DNA sequence homology with vt2 and vt2c. However, vt2g was not detected by vt2-specific primers and probes, although it was partially neutralized by an antiserum to the VT2A subunit. VT2g was cytotoxic for Vero and HeLa cells and was not activated by mouse intestinal mucus. The vt2g gene was detected in 3 of 409 (0.7%) bovine VTEC strains, including serotypes O2:H25, O2:H45 and Ont:H-.
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840. |
Gueneau de Novoa P,
Williams KP,
( 2004 ) The tmRNA website: reductive evolution of tmRNA in plastids and other endosymbionts. PMID : 14681369 : DOI : 10.1093/nar/gkh102 PMC : PMC308836 Abstract >>
tmRNA combines tRNA- and mRNA-like properties and ameliorates problems arising from stalled ribosomes. Research on the mechanism, structure and biology of tmRNA is served by the tmRNA website (http://www.indiana.edu/~ tmrna), a collection of sequences, alignments, secondary structures and other information. Because many of these sequences are not in GenBank, a BLAST server has been added; another new feature is an abbreviated alignment for the tRNA-like domain only. Many tmRNA sequences from plastids have been added, five found in public sequence data and another 10 generated by direct sequencing; detection in early-branching members of the green plastid lineage brings coverage to all three primary plastid lineages. The new sequences include the shortest known tmRNA sequence. While bacterial tmRNAs usually have a lone pseudoknot upstream of the mRNA segment and a string of three or four pseudoknots downstream, plastid tmRNAs collectively show loss of pseudoknots at both postions. The pseudoknot-string region is also too short to contain the usual pseudoknot number in another new entry, the tmRNA sequence from a bacterial endosymbiont of insect cells, Tremblaya princeps. Pseudoknots may optimize tmRNA function in free-living bacteria, yet become dispensible when the endosymbiotic lifestyle relaxes selective pressure for fast growth.
|
841. |
Ohmura-Hoshino M,
Ho ST,
Kurazono H,
Igarashi K,
Yamasaki S,
Takeda Y,
( 2003 ) Genetic and immunological analysis of a novel variant of Shiga toxin 1 from bovine Escherichia coli strains and development of bead-ELISA to detect the variant toxin. PMID : 14605438 : DOI : 10.1111/j.1348-0421.2003.tb03441.x Abstract >>
A novel variant of Shiga toxin 1 (Stx1) was identified from bovine Escherichia coli strains. The stx1 variant genes designated as stx1v51 and stx1v52 were cloned and sequenced. The two variant genes differed each other by 2 bp, but the deduced amino acid sequences of the two Stx1 variant toxins were the same and had 94% and 92% homology to that of prototype A and B subunits of Stx1, respectively. The variant toxin designated as Stx1v52 was purified to homogeneity. Although inhibition of protein synthesis in vitro by purified Stx1v52 was almost equal to that of purified Stx1, Vero cell cytotoxicity and mouse lethality of Stx1v52 were several folds lower than those of prototype Stx1. In Ouchterlony double gel diffusion test, the precipitin line between Stx1v52 and Stx1 formed a spur against anti-Stx1 serum but was fused against anti-Stx1v52 serum. Stx1v52 and Stx1v52-specific-bead-ELISA was developed, and both Stx1 and Stx1v52 could be detected with high sensitivity using Stx1v52 conjugate. However, Stx1v52 but not Stx1 could be detected with Stx1v52-specific bead-ELISA.
|
842. |
Pandey M,
Khan A,
Das SC,
Sarkar B,
Kahali S,
Chakraborty S,
Chattopadhyay S,
Yamasaki S,
Takeda Y,
Nair GB,
Ramamurthy T,
( 2003 ) Association of cytolethal distending toxin locus cdtB with enteropathogenic Escherichia coli isolated from patients with acute diarrhea in Calcutta, India. PMID : 14605183 : DOI : 10.1128/jcm.41.11.5277-5281.2003 PMC : PMC262502 Abstract >>
Among Escherichia coli strains isolated from stool specimens from patients with acute diarrhea, 1.4% were found to harbor cdtB by use of enrichment cytolethal distending toxin (CDT) PCR. These isolates were identified as being enteropathogenic E. coli (EPEC). In a retrospective study using a probe hybridization assay, 6 of 138 EPEC strains were found to harbor the cdtB locus. cdtB-positive isolates mostly belong to the O86a and O127a serogroups, with the former being associated with higher expression of CDT. Pulsed-field gel electrophoresis profiles showed that the EPEC strains harboring cdtB strains are genetically diverse.
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843. |
Ramachandran V,
Brett K,
Hornitzky MA,
Dowton M,
Bettelheim KA,
Walker MJ,
Djordjevic SP,
( 2003 ) Distribution of intimin subtypes among Escherichia coli isolates from ruminant and human sources. PMID : 14605134 : DOI : 10.1128/jcm.41.11.5022-5032.2003 PMC : PMC262460 Abstract >>
The intimin gene eae, located within the locus of enterocyte effacement pathogenicity island, distinguishes enteropathogenic Escherichia coli (EPEC) and some Shiga toxin-producing E. coli (STEC) strains from all other pathotypes of diarrheagenic E. coli. EPEC is a leading cause of infantile diarrhea in developing countries, and intimin-positive STEC isolates are typically associated with life-threatening diseases such as hemolytic-uremic syndrome and hemorrhagic colitis. Here we describe the development of a PCR-restriction fragment length polymorphism (RFLP) assay that reliably differentiates all 11 known intimin types (alpha1, alpha2, beta, gamma, kappa, epsilon, eta, iota, lambda, theta, and zeta) and three new intimin genes that show less than 95% nucleotide sequence identity with existing intimin types. We designated these new intimin genes Int- micro, Int-nu, and Int-xi. The PCR-RFLP assay was used to screen 213 eae-positive E. coli isolates derived from ovine, bovine, and human sources comprising 60 serotypes. Of these, 82 were STEC isolates, 89 were stx-negative (stx(-)) and ehxA-positive (ehxA(+)) isolates, and 42 were stx(-) and ehxA-negative isolates. Int-beta, the most commonly identified eae subtype (82 of 213 [38.5%] isolates), was associated with 21 serotypes, followed by Int-zeta (39 of 213 [18.3%] isolates; 11 serotypes), Int-theta (25 of 213 [11.7%] isolates; 15 serotypes), Int-gamma (19 of 213 [8.9%] isolates; 9 serotypes), and Int-epsilon (21 of 213 [9.9%] isolates; 5 serotypes). Intimin subtypes alpha1, alpha2, kappa, lambda, xi, micro, nu, and iota were infrequently identified; and Int-eta was not detected. Phylogenetic analyses with the Phylip package of programs clustered the intimin subtypes into nine distinct families (alpha, beta-xi, gamma, kappa, epsilon-eta-nu, iota- micro, lambda, theta, and zeta). Our data confirm that ruminants are an important source of serologically and genetically diverse intimin-containing E. coli strains.
|
844. |
Roberts RC,
Helinski DR,
( 1992 ) Definition of a minimal plasmid stabilization system from the broad-host-range plasmid RK2. PMID : 1459960 : DOI : 10.1128/jb.174.24.8119-8132.1992 PMC : PMC207551 Abstract >>
The stable inheritance of the broad-host-range plasmid RK2 is due at least in part to functions within a region located at coordinates 32.8 to 35.9 kb, termed the RK2 par locus. This locus encodes four previously identified genes in two operons (parCBA and parD; M. Gerlitz, O. Hrabak, and H. Schwab, J. Bacteriol. 172:6194-6203, 1990, and R. C. Roberts, R. Burioni, and D. R. Helinski, J. Bacteriol. 172:6204-6216, 1990). The parCBA operon is functional in resolving plasmid multimers to monomers. Analysis of the plasmid stabilization capacity of deletions within this region, however, indicates that this multimer resolution operon is required for stabilization only in certain Escherichia coli strains and under specific growth conditions. The deletion analysis further allowed a redefinition of the minimal functional region as 790 bp in length, consisting of the parD gene (243 bp) and its promoter as well as sequences downstream of parD. This minimal region stabilizes an RK2-derived minireplicon in several different gram-negative bacteria and, at least in E. coli, in a vector-independent manner. By insertional mutagenesis, both the parD gene and downstream (3') regions were found to be required for plasmid stabilization. The downstream DNA sequence contained an open reading frame which was subsequently shown by transcriptional and translational fusions to encode a protein with a predicted size of 11,698 Da, designated ParE. Since the parDE operon requires the presence of the parCBA operon for efficient stabilization under certain growth conditions, the potential role of multimer resolution in plasmid stabilization was tested by substituting the ColE1 cer site for the parCBA operon. While the cer site did function to resolve plasmid multimers, it was not sufficient to restore stabilization activity to the parDE operon under growth conditions that require the parCBA operon for plasmid stability. This suggests that plasmid stabilization by the RK2 par locus relies on a complex mechanism, representing a multifaceted stabilization system of which multimer resolution is a conditionally dispensable component, and that the function(s) encoded by the parDE operon is essential.
|
845. |
Subbarayan PR,
Sarkar M,
( 2004 ) A comparative study of variation in codon 33 of the rpoS gene in Escherichia coli K12 stocks: implications for the synthesis of sigma(s). PMID : 14618393 : DOI : 10.1007/s00438-003-0944-x Abstract >>
The Escherichia coli rpoS gene encodes an RNA polymerase sigma factor (sigma S or sigma(S)) required for the expression of stationary-phase genes. In the first published rpoS sequence from E. coli K-12 codon 33 is given as CAG. However, several subsequent independent studies found the amber codon TAG at this position (rpoSAm). Besides this amber codon, other codons such as TAT have also been found at this location in rpoS. Comparative genome analysis now leads us to propose TAG as the parental codon 33 in rpoS in E. coli K-12. Five different stocks of the strain W3110, which differ in the levels of sigma(S) protein they express, were investigated. We sequenced the rpoS gene from these, and found a T at nucleotide position 97 in four out of the five stocks and a G at position 99 in three out of the five. W1485, a parental strain of W3110, and W3350, a derivative of W3110, are also rpoSAm mutants. Such rpoSAm mutants would be expected to show no RpoS activity. The retention of partial or intermediate sigma(S) activity by suppressor-free rpoSAm mutants is therefore puzzling. We propose that a functional, N-terminally truncated, sigma(S) (Delta1-53sigma(S)) can be translated from a Secondary Translation Initiation Region (STIR) located downstream of the amber codon 33. It has recently been reported that a fragment of RpoS (Delta1-53sigma(S)) that lacks the first 53 amino acids is functional when synthesized in vivo. Taken together, our results support the hypothesis that the original codon 33 of the rpoS gene in E. coli K-12 strains is the amber codon TAG.
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846. |
Aoshima M,
Ishii M,
Yamagishi A,
Oshima T,
Igarashi Y,
( 2003 ) Metabolic characteristics of an isocitrate dehydrogenase defective derivative of escherichia coli BL21(DE3). PMID : 14595786 : DOI : 10.1002/bit.10832 Abstract >>
A 7.8 kb fragment containing isocitrate dehydrogenase (ICDH) gene and its flanking regions was cloned from Escherichia coli BL21(DE3) and sequenced. Unlike the case of the K-12 strain, the e14 element was not found. The nucleotide divergence between these two strains was about 2%. Using the cloned fragment, ICDH defective mutant strain, MA1935, was generated from BL21(DE3). Although MA1935 accumulated citrate, citrate synthase activity was not repressed but was rather high. In addition, isocitrate lyase was not highly induced at the stationary phase. MA1935 was shown to be a good host strain for ICDH gene expression.
|
847. |
Leslie AG,
( 1990 ) Refined crystal structure of type III chloramphenicol acetyltransferase at 1.75 A resolution. PMID : 2187098 : DOI : 10.1016/S0022-2836(05)80129-9 Abstract >>
High level bacterial resistance to chloramphenicol is generally due to O-acetylation of the antibiotic in a reaction catalysed by chloramphenicol acetyltransferase (CAT, EC 2.3.1.28) in which acetyl-coenzyme A is the acyl donor. The crystal structure of the type III enzyme from Escherichia coli with chloramphenicol bound has been determined and refined at 1.75 A resolution, using a restrained parameter reciprocal space least squares procedure. The refined model, which includes chloramphenicol, 204 solvent molecules and two cobalt ions has a crystallographic R-factor of 18.3% for 27,300 reflections between 6 and 1.75 A resolution. The root-mean-square deviation in bond lengths from ideal values is 0.02 A. The cobalt ions play a crucial role in stabilizing the packing of the molecule in the crystal lattice. CAT is a trimer of identical subunits (monomer Mr 25,000) and the trimeric structure is stabilized by a number of hydrogen bonds, some of which result in the extension of a beta-sheet across the subunit interface. Chloramphenicol binds in a deep pocket located at the boundary between adjacent subunits of the trimer, such that the majority of residues forming the binding pocket belong to one subunit while the catalytically essential histidine belongs to the adjacent subunit. His195 is appropriately positioned to act as a general base catalyst in the reaction, and the required tautomeric stabilization is provided by an unusual interaction with a main-chain carbonyl oxygen.
|
848. |
Alvarez P,
Buscaglia CA,
Campetella O,
( 2004 ) Improving protein pharmacokinetics by genetic fusion to simple amino acid sequences. PMID : 14612434 : DOI : 10.1074/jbc.M311356200 Abstract >>
The role of primary amino acid sequences in protein pharmacokinetics, an issue of relevance in both basic knowledge and biotechnology, was addressed here using as a starting point two repetitive antigens from the hemoflagellate Trypanosoma cruzi that are known to stabilize their associated proteins in the bloodstream. A major drawback to their pharmacological application is that these repetitive sequences are highly immunogenic, being therefore the deletion of this characteristic desirable. Based on sequence homology and epitope mapping analyses, an artificial repetitive sequence (PSTAD) was engineered. This motif was tested by genetic fusion to the C terminus of both the trypanosomal trans-sialidase and the rat tyrosine aminotransferase and found to produce a 4.5-6-fold increase in the half-life of the associated proteins in blood while displaying significantly lower immunogenicity. Residues involved in the stabilizing properties of the novel peptide were mapped by a site-directed mutagenesis approach, allowing us to successfully identify another two motifs. Searching databases for sequences displaying some homology, embedded in proline frameworks and associated to shed virulence factors from unrelated microorganisms, resulted in the identification of four other protein extensions. Remarkably, three of them (from Streptococcus pneumoniae, Actinomyces viscosus, and Escherichia coli) revealed similar pharmacokinetic features, suggesting therefore an analogous evolutionarily acquired mechanism to ensure the biodistribution of their corresponding proteins. Our findings indicate that the insertion of defined motifs into a proline-rich framework constitutes a suitable alternative to construct a chimeric protein with extended half-life in blood.
|
849. |
Guasch A,
Lucas M,
Moncalián G,
Cabezas M,
Pérez-Luque R,
Gomis-Rüth FX,
de la Cruz F,
Coll M,
( 2003 ) Recognition and processing of the origin of transfer DNA by conjugative relaxase TrwC. PMID : 14625590 : DOI : 10.1038/nsb1017 Abstract >>
Relaxases are DNA strand transferases that catalyze the initial and final stages of DNA processing during conjugative cell-to-cell DNA transfer. Upon binding to the origin of transfer (oriT) DNA, relaxase TrwC melts the double helix. The three-dimensional structure of the relaxase domain of TrwC in complex with its cognate DNA at oriT shows a fold built on a two-layer alpha/beta sandwich, with a deep narrow cleft that houses the active site. The DNA includes one arm of an extruded cruciform, an essential feature for specific recognition. This arm is firmly embraced by the protein through a beta-ribbon positioned in the DNA major groove and a loop occupying the minor groove. It is followed by a single-stranded DNA segment that enters the active site, after a sharp U-turn forming a hydrophobic cage that traps the N-terminal methionine. Structural analysis combined with site-directed mutagenesis defines the architecture of the active site.
|
850. |
Flores C,
Qadri MI,
Lichtenstein C,
( 1990 ) DNA sequence analysis of five genes; tnsA, B, C, D and E, required for Tn7 transposition. PMID : 2156235 : DOI : 10.1093/nar/18.4.901 PMC : PMC330344 Abstract >>
A region of DNA sequence of the bacterial transposon Tn7, which is required for transposition, has been determined. This DNA sequence completes an 8351 base pair (bp) region containing five long open reading frames (ORF's) that correspond to the genetically defined genes, tnsA, B, C, D and E, required for Tn7 transposition. All of the ORF's are oriented in the same direction, ie. inward from the element's right end. The genes are in a very compact arrangement with the presumed initiation codons never more than two bases beyond the preceding termination codon. Domains with similarity to the helix-turn-helix genre of Cro-like, sequence specific DNA binding sites occur within the deduced amino acid (a.a.) sequence of the TnsA, TnsB, TnsD and TnsE proteins. Translation of the tnsC ORF reveals strong homology to a consensus sequence for nucleotide binding sites as well as a region of similarity to a transcriptional activator (MalT). No striking a.a. sequence similarity to other DNA recombinases is observed. The possible roles of these proteins in Tn7 transposition is discussed in light of the analysis presented.
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851. |
Drieux L,
Bourgeois-Nicolaos N,
Cremniter J,
Lawrence C,
Jarlier V,
Doucet-Populaire F,
Sougakoff W,
( 2011 ) Accumulation of carbapenemase-producing Gram-negative bacteria in a single patient linked to the acquisition of multiple carbapenemase producers and to the in vivo transfer of a plasmid encoding VIM-1. PMID : 21570257 : DOI : 10.1016/j.ijantimicag.2011.03.017 Abstract >>
N/A
|
852. |
Zhou X,
Shen LP,
Chi CW,
( 1990 ) Isolation and nucleotide sequence determination of a gene encoding a heat-stable enterotoxin of Escherichia coli. PMID : 2190361 : DOI : 10.1016/0041-0101(90)90085-l Abstract >>
An enterotoxin-producing strain of E. coli has been isolated from an infant patient in Shanghai Children Hospital and the gene of its heat-stable enterotoxin has been cloned and sequenced. The pre-pro-STI was composed of 72 amino acid residues corresponding to the encoding of 216 base pairs. There was only one nucleotide difference in the pro-part between this STI gene and the STIb gene reported in the literature. A guanosine base in our STI gene was substituted for a cytosine base in STIb gene resulting in a replacement of proline by alanine. Hence, the STI genes from different human sources are highly conserved though mutagenesis still occurs.
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853. |
Tietze E,
Brevet J,
( 1990 ) Nucleotide sequence of the streptothricin-acetyl-transferase gene sat-2. PMID : 2157196 : DOI : 10.1093/nar/18.5.1283 PMC : PMC330445 Abstract >>
N/A
|
854. |
Wong JJ,
Lu J,
Edwards RA,
Frost LS,
Glover JN,
( 2011 ) Structural basis of cooperative DNA recognition by the plasmid conjugation factor, TraM. PMID : 21565799 : DOI : 10.1093/nar/gkr296 PMC : PMC3159463 Abstract >>
The conjugative transfer of F-like plasmids such as F, R1, R100 and pED208, between bacterial cells requires TraM, a plasmid-encoded DNA-binding protein. TraM tetramers bridge the origin of transfer (oriT) to a key component of the conjugative pore, the coupling protein TraD. Here we show that TraM recognizes a high-affinity DNA-binding site, sbmA, as a cooperative dimer of tetramers. The crystal structure of the TraM-sbmA complex from the plasmid pED208 shows that binding cooperativity is mediated by DNA kinking and unwinding, without any direct contact between tetramers. Sequence-specific DNA recognition is carried out by TraM's N-terminal ribbon-helix-helix (RHH) domains, which bind DNA in a staggered arrangement. We demonstrate that both DNA-binding specificity, as well as selective interactions between TraM and the C-terminal tail of its cognate TraD mediate conjugation specificity within the F-like family of plasmids. The ability of TraM to cooperatively bind DNA without interaction between tetramers leaves the C-terminal TraM tetramerization domains free to make multiple interactions with TraD, driving recruitment of the plasmid to the conjugative pore.
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855. |
Metlitskaya A,
Severinov K,
Nair SK,
Agarwal V,
( 2011 ) Structural basis for microcin C7 inactivation by the MccE acetyltransferase. PMID : 21507941 : DOI : 10.1074/jbc.M111.226282 PMC : PMC3122189 Abstract >>
The antibiotic microcin C7 (McC) acts as a bacteriocide by inhibiting aspartyl-tRNA synthetase and stalling the protein translation machinery. McC is synthesized as a heptapeptide-nucleotide conjugate, which is processed by cellular peptidases within target strains to yield the biologically active compound. As unwanted processing of intact McC can result in self-toxicity, producing strains utilize multiple mechanisms for autoimmunity against processed McC. We have shown previously that the mccE gene within the biosynthetic cluster can inactivate processed McC by acetylating the antibiotic. Here, we present the characterization of this acetylation mechanism through biochemical and structural biological studies of the MccE acetyltransferase domain (MccE(AcTase)). We have also determined five crystal structures of the MccE-acetyl-CoA complex with bound substrates, inhibitor, and reaction product. The structural data reveal an unexpected mode of substrate recognition through �k-stacking interactions similar to those found in cap-binding proteins and nucleotidyltransferases. These studies provide a rationale for the observation that MccE(AcTase) can detoxify a range of aminoacylnucleotides, including those that are structurally distinct from microcin C7.
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856. |
Obeng AS,
Rickard H,
Ndi O,
Sexton M,
Barton M,
( 2012 ) Antibiotic resistance, phylogenetic grouping and virulence potential of Escherichia coli isolated from the faeces of intensively farmed and free range poultry. PMID : 21856098 : DOI : 10.1016/j.vetmic.2011.07.010 Abstract >>
Antibiotic use in poultry production is a risk factor for promoting the emergence of resistant Escherichia coli. To ascertain differences in different classes of chickens, the resistance profile, some virulence genes and phylogenetic grouping on 251 E. coli isolates from intensive meat (free range and indoor commercial) and free range egg layer chickens collected between December 2008 and June 2009 in South Australia were performed. Among the 251 strains, 102 (40.6%) and 67 (26.7%) were found to be resistant to tetracycline and ampicillin respectively. Resistance was also observed to trimethoprim-sulfamethoxazole (12.4%), streptomycin (10.8%), spectinomycin (9.6%), neomycin (6.0%) and florfenicol (2.0%) but no resistance was found to ceftiofur, ciprofloxacin or gentamicin. Amplification of DNA of the isolates by polymerase chain reaction revealed the presence of genes that code for resistant determinants: tetracycline (tet(A), tet(B) and tet(C)), ampicillin (bla(TEM) and bla(SHV)), trimethoprim (dhfrV and dhfrXIII), sulphonamide (sulI and sulII), neomycin (aph(3)-Ia(aphA1)), and spectinomycin-streptinomycin (aadA2). In addition, 32.3-39.4% of the isolates were found to belong to commensal groups (A and B1) and 11.2-17.1% belonged to the virulent groups (B2 and D). Among the 251 E. coli isolates, 25 (10.0%) carried two or more virulence genes typical of Extraintestinal pathogenic E. coli (ExPEC). Furthermore, 17 of the isolates with multi-resistance were identified to be groups B2 and D. Although no significant difference was observed between isolates from free range and indoor commercial meat chickens (P>0.05), significant differences was observed between the different classes of meat chickens (free range and indoor commercial) and egg layers (P<0.05). While this study assessed the presence of a limited number of virulence genes, our study re emphasises the zoonotic potential of poultry E. coli isolates.
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857. |
Davis MA,
Besser TE,
Orfe LH,
Baker KN,
Lanier AS,
Broschat SL,
New D,
Call DR,
( 2011 ) Genotypic-phenotypic discrepancies between antibiotic resistance characteristics of Escherichia coli isolates from calves in management settings with high and low antibiotic use. PMID : 21421795 : DOI : 10.1128/AEM.02588-10 PMC : PMC3126435 Abstract >>
We hypothesized that bacterial populations growing in the absence of antibiotics will accumulate more resistance gene mutations than bacterial populations growing in the presence of antibiotics. If this is so, the prevalence of dysfunctional resistance genes (resistance pseudogenes) could provide a measure of the level of antibiotic exposure present in a given environment. As a proof-of-concept test, we assayed field strains of Escherichia coli for their resistance genotypes using a resistance gene microarray and further characterized isolates that had resistance phenotype-genotype discrepancies. We found a small but significant association between the prevalence of isolates with resistance pseudogenes and the lower antibiotic use environment of a beef cow-calf operation versus a higher antibiotic use dairy calf ranch (Fisher's exact test, P = 0.044). Other significant findings include a very strong association between the dairy calf ranch isolates and phenotypes unexplained by well-known resistance genes (Fisher's exact test, P < 0.0001). Two novel resistance genes were discovered in E. coli isolates from the dairy calf ranch, one associated with resistance to aminoglycosides and one associated with resistance to trimethoprim. In addition, isolates resistant to expanded-spectrum cephalosporins but negative for bla(CMY-2) had mutations in the promoter regions of the chromosomal E. coli ampC gene consistent with reported overexpression of native AmpC beta-lactamase. Similar mutations in hospital E. coli isolates have been reported worldwide. Prevalence or rates of E. coli ampC promoter mutations may be used as a marker for high expanded-spectrum cephalosporin use environments.
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858. |
Guillon H,
Tande D,
Mammeri H,
( 2011 ) Emergence of ertapenem resistance in an Escherichia coli clinical isolate producing extended-spectrum beta-lactamase AmpC. PMID : 21746958 : DOI : 10.1128/AAC.01513-10 PMC : PMC3165280 Abstract >>
Escherichia coli isolate MEV, responsible for a bloodstream infection, was resistant to penicillins, cephalosporins, and ertapenem. Molecular and biochemical characterization revealed the production of a novel, chromosome-borne, extended-spectrum AmpC (ESAC) �]-lactamase with a Ser-282 duplication and increased carbapenemase activity. This study demonstrates for the first time that chromosome-borne ESAC �]-lactamases can contribute to the emergence of ertapenem resistance in E. coli clinical isolates.
|
859. |
Fernández-Alarcón C,
Singer RS,
Johnson TJ,
( 2011 ) Comparative genomics of multidrug resistance-encoding IncA/C plasmids from commensal and pathogenic Escherichia coli from multiple animal sources. PMID : 21858108 : DOI : 10.1371/journal.pone.0023415 PMC : PMC3155540 Abstract >>
Incompatibility group A/C (IncA/C) plasmids have received recent attention for their broad host range and ability to confer resistance to multiple antimicrobial agents. Due to the potential spread of multidrug resistance (MDR) phenotypes from foodborne pathogens to human pathogens, the dissemination of these plasmids represents a public health risk. In this study, four animal-source IncA/C plasmids isolated from Escherichia coli were sequenced and analyzed, including isolates from commercial dairy cows, pigs and turkeys in the U.S. and Chile. These plasmids were initially selected because they either contained the floR and tetA genes encoding for florfenicol and tetracycline resistance, respectively, and/or the bla(CMY-2) gene encoding for extended spectrum �]-lactamase resistance. Overall, sequence analysis revealed that each of the four plasmids retained a remarkably stable and conserved backbone sequence, with differences observed primarily within their accessory regions, which presumably have evolved via horizontal gene transfer events involving multiple modules. Comparison of these plasmids with other available IncA/C plasmid sequences further defined the core and accessory elements of these plasmids in E. coli and Salmonella. Our results suggest that the bla(CMY-2) plasmid lineage appears to have derived from an ancestral IncA/C plasmid type harboring floR-tetAR-strAB and Tn21-like accessory modules. Evidence is mounting that IncA/C plasmids are widespread among enteric bacteria of production animals and these emergent plasmids have flexibility in their acquisition of MDR-encoding modules, necessitating further study to understand the evolutionary mechanisms involved in their dissemination and stability in bacterial populations.
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860. |
Poirel L,
Bonnin RA,
Nordmann P,
( 2011 ) Analysis of the resistome of a multidrug-resistant NDM-1-producing Escherichia coli strain by high-throughput genome sequencing. PMID : 21746951 : DOI : 10.1128/AAC.00165-11 PMC : PMC3165296 Abstract >>
The resistome of the multidrug-resistant Escherichia coli strain 271 carrying the plasmid-mediated bla(NDM-1) carbapenemase gene was analyzed by high-throughput genome sequencing. The p271A plasmid carrying the bla(NDM-1) gene was 35.9 kb in size and possessed an IncN-type backbone that harbored a novel replicase gene. Acquisition of the bla(NDM-1) gene on plasmid p271A had been likely the result of a cointegration event involving the transposase of Tn5403. The expression of bla(NDM-1) was associated with the insertion sequence ISAba125 likely originating from Acinetobacter baumannii. E. coli 271 accumulated multiple resistance determinants, including five �]-lactamase genes (comprising the extended-spectrum �]-lactamase CTX-M-15), two 16S RNA methylase ArmA- and RmtB-encoding genes, and the qepA gene encoding an efflux pump involved in resistance to fluoroquinolones. These resistance genes were located on three additional plasmids, of 160 kb (IncA/C), 130 kb (IncF), and 110 kb (IncI1). In addition, several chromosomally encoded resistance determinants were identified, such as topoisomerase mutations, porin modifications and truncations, and the intrinsic ampC gene of E. coli that was weakly expressed. The multidrug resistance pattern observed for E. coli 271 was therefore the result of combined chromosome- and plasmid-encoded mechanisms.
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861. |
Szmolka A,
Fortini D,
Villa L,
Carattoli A,
Anjum MF,
Nagy B,
( 2011 ) First report on IncN plasmid-mediated quinolone resistance gene qnrS1 in porcine Escherichia coli in Europe. PMID : 21834664 : DOI : 10.1089/mdr.2011.0068 Abstract >>
Plasmid-mediated quinolone resistance (PMQR) of enterobacteria encoded by qnr genes is an emerging concern in human and veterinary medicine. Here we aimed to study PMQR of porcine Escherichia coli in two large piggeries in Romania and Hungary. The studies identified PMQR E. coli strains in 34% of piglets in the Romanian farm. Clonality of six qnrS1 E. coli strains representing the Romanian pig farm was established by multilocus sequence typing (MLST), and the qnrS1 plasmids were characterized by plasmid transfer and PCR-based replicon typing. The six tested strains were assigned to three different MLST types. All proved to carry IncN plasmids, representing the first IncN-borne qnrS1 gene to be identified in E. coli from food-producing animals. DNA sequences flanking the qnrS1 gene showed ?99% homology with the corresponding resistance region of the pINF5 plasmid from Salmonella Infantis isolated from chicken carcass and of IncN plasmids from human clinical E. coli strains. Thus, our data suggest that transfer of qnrS1 plasmids occurs between Salmonella and E. coli of animal and human origin, with pigs representing one of the potential reservoirs. Further, we report on identification and characterization of the qnrS1 gene in porcine E. coli for the first time in Europe.
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862. |
Chen HD,
Jewett MW,
Groisman EA,
( 2011 ) Ancestral genes can control the ability of horizontally acquired loci to confer new traits. PMID : 21811415 : DOI : 10.1371/journal.pgen.1002184 PMC : PMC3140997 Abstract >>
Horizontally acquired genes typically function as autonomous units conferring new abilities when introduced into different species. However, we reasoned that proteins preexisting in an organism might constrain the functionality of a horizontally acquired gene product if it operates on an ancestral pathway. Here, we determine how the horizontally acquired pmrD gene product activates the ancestral PmrA/PmrB two-component system in Salmonella enterica but not in the closely related bacterium Escherichia coli. The Salmonella PmrD protein binds to the phosphorylated PmrA protein (PmrA-P), protecting it from dephosphorylation by the PmrB protein. This results in transcription of PmrA-dependent genes, including those conferring polymyxin B resistance. We now report that the E. coli PmrD protein can activate the PmrA/PmrB system in Salmonella even though it cannot do it in E. coli, suggesting that these two species differ in an additional component controlling PmrA-P levels. We establish that the E. coli PmrB displays higher phosphatase activity towards PmrA-P than the Salmonella PmrB, and we identified a PmrB subdomain responsible for this property. Replacement of the E. coli pmrB gene with the Salmonella homolog was sufficient to render E. coli resistant to polymyxin B under PmrD-inducing conditions. Our findings provide a singular example whereby quantitative differences in the biochemical activities of orthologous ancestral proteins dictate the ability of a horizontally acquired gene product to confer species-specific traits. And they suggest that horizontally acquired genes can potentiate selection at ancestral loci.
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863. |
Bielak E,
Bergenholtz RD,
Jørgensen MS,
Sørensen SJ,
Hansen LH,
Hasman H,
( 2011 ) Investigation of diversity of plasmids carrying the blaTEM-52 gene. PMID : 21831988 : DOI : 10.1093/jac/dkr331 Abstract >>
To investigate the diversity of plasmids that carry bla(TEM-52) genes among Escherichia coli and Salmonella enterica originating from animals, meat products and humans. A collection of 22 bla(TEM-52)-encoding plasmids was characterized by restriction fragment length polymorphism (RFLP), replicon typing (by PCR or replicon sequencing), susceptibility testing, assessment of plasmid ability to self-transfer by conjugation and typing of the genetic environment of the bla(TEM-52) gene. Detected IncI1 plasmids underwent further plasmid multilocus sequence typing. RFLP profiles demonstrated dissemination of bla(TEM-52) in Denmark (imported meat from Germany), France, Belgium and the Netherlands from 2000 to 2006 by mainly two different plasmids, one encoding bla(TEM-52b) (IncX1A, 45 kb) and the other bla(TEM-52c) (IncI1, 80 kb). In addition, bla(TEM-52b) was also found to be located on various other plasmids belonging to IncA/C and IncL/M, while bla(TEM-52c) was found on IncN-like as well as on IncR plasmids. In the majority of cases (n = 21) the bla(TEM-52) gene was located on a Tn3 transposon. Seven out of 10 bla(TEM-52) plasmids tested in conjugation experiments were shown to be capable of self-transfer to a plasmid-free E. coli recipient. The bla(TEM-52) gene found in humans could have been transmitted on transferable plasmids originating from animal sources. Some of the bla(TEM-52) plasmids carry replicons that differ from the classical ones. Two novel replicons were detected, IncX1A and IncN-like. Unlike its predecessor bla(TEM-1), the bla(TEM-52) gene was not detected on F-type replicons suggesting that this gene evolved on other types of plasmid scaffolds.
|
864. |
Ben Slama K,
Ben Sallem R,
Jouini A,
Rachid S,
Moussa L,
Sáenz Y,
Estepa V,
Somalo S,
Boudabous A,
Torres C,
( 2011 ) Diversity of genetic lineages among CTX-M-15 and CTX-M-14 producing Escherichia coli strains in a Tunisian hospital. PMID : 21479796 : DOI : 10.1007/s00284-011-9930-4 Abstract >>
Fourteen broad-spectrum-cephalosporin-resistant Escherichia coli isolates were recovered between June and December 2007 in a Tunisian hospital. Genes encoding extended-spectrum-beta-lactamases (ESBL) and other resistance genes were characterized by PCR and sequencing. The following ESBL genes were identified: bla (CTX-M-15) (12 isolates), bla (CTX-M-14a) (one isolate), and bla (CTX-M-14b) (one isolate). The bla (OXA-1) gene was detected in 13 bla (CTX-M)-producing strains and a bla (TEM-1) gene in 6 of them. The ISEcp1 sequence was found upstream of bla (CTX-M) genes in 8 of 14 strains, and orf477 or IS903 downstream of this gene in 13 strains. Nine of the strains carried class 1 integrons and five different gene cassette arrangements were detected, dfrA17-aadA5 being the most common. One of the strains (bla (CTX-M-14a)-positive) harbored three class 1 integrons, and one of them was non-previously described containing as gene cassettes new variants of aac(6')-Ib and cmlA1 genes and it was linked to the bla (CTX-M-14a) gene flanked by a truncated ISEcp1 sequence (included in GenBank with accession number JF701188). CTX-M-15-producing strains were ascribed to phylogroup B2 (six isolates) and D (six isolates). Multilocus-sequence-typing revealed ten different sequence-types (STs) among ESBL-positive E. coli strains with prevalence of ST405 (four strains of phylogroup D) and ST131 types (two strains of phylogroup B2 and serogroup O25b). A high clonal diversity was also observed among studied strains by pulsed-field-gel-electrophoresis (11 unrelated profiles). CTX-M-15 is an emergent mechanism of resistance in the studied hospital and the world-disseminated 0:25b-ST131-B2 and ST405-D clones have been identified among CTX-M-15-producing isolates.
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865. |
Mata C,
Miró E,
Toleman M,
Rivera MA,
Walsh TR,
Navarro F,
( 2011 ) Association of bla(DHA-1) and qnrB genes carried by broad-host-range plasmids among isolates of Enterobacteriaceae at a Spanish hospital. PMID : 21781207 : DOI : 10.1111/j.1469-0691.2011.03539.x Abstract >>
A collection of 30 DHA-1-Enterobacteriaceae producers was examined for the presence of qnr genes. PCR-based replicon typing, plasmid profile and Southern hybridisation analyses revealed that all isolates co-harboured bla(DHA-1) and qnrB genes on the same plasmid. All but one of these plasmids belonged to the L/M group. Genetic organization analyses of a randomly selected isolate revealed the co-localization of both genes on an IS26-composite transposon. As plasmids carrying both genes seem to have a high prevalence and a worldwide distribution, care should be taken when quinolones are used to treat infections caused by DHA-1 producers.
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866. |
Damborg P,
Marskar P,
Baptiste KE,
Guardabassi L,
( 2012 ) Faecal shedding of CTX-M-producing Escherichia coli in horses receiving broad-spectrum antimicrobial prophylaxis after hospital admission. PMID : 21820821 : DOI : 10.1016/j.vetmic.2011.07.005 Abstract >>
The objective of this longitudinal study was to investigate the occurrence and genetic background of faecal Escherichia coli resistant to cefotaxime (CTX) in horses receiving broad-spectrum antimicrobial prophylaxis after admission to a veterinary teaching hospital. The ten horses enrolled in the study were treated with cefquinome either alone (n=4) or in combination with metronidazole (n=3) or other antimicrobial agents (n=3). CTX-resistant coliforms in faeces collected before, during and after treatment were quantified on selective MacConkey agar supplemented with CTX, and a colony isolated randomly from each positive sample was characterized by pulsed-field gel electrophoresis, and by PCR detection and sequencing of bla(TEM), bla(SHV), bla(CTX-M) and bla(CMY). All horses were negative for CTX-resistant coliforms at admission but became positive within the first three days of treatment. The average faecal densities of CTX-resistant coliforms increased significantly following antimicrobial prophylaxis (P<0.001). Genetic characterization of 29 faecal isolates revealed that this effect was due to proliferation of E. coli producing either CTX-M-1 (n=28) or CTX-M-14 (n=1). Five CTX-M-1 isolates produced additional �]-lactamases (TEM-1, CMY-34 and the novel variant CMY-53). Shedding of CTX-M-producing E. coli appeared intermittent in four horses and persisted two weeks after antimicrobial treatments in five of six patients tested after discharge from hospital. Nosocomial transmission was suggested by finding five identical CTX-M-1-producing E. coli pulsotypes in multiple horses. The originality of the study lies in the unanticipated high frequency and genetic diversity of CTX-M-producing E. coli observed in the faecal flora of hospitalized patients receiving broad-spectrum antimicrobial prophylaxis.
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867. |
Partridge SR,
Zong Z,
Iredell JR,
( 2011 ) Recombination in IS26 and Tn2 in the evolution of multiresistance regions carrying blaCTX-M-15 on conjugative IncF plasmids from Escherichia coli. PMID : 21859935 : DOI : 10.1128/AAC.00025-11 PMC : PMC3195058 Abstract >>
CTX-M-15 now appears to be the dominant extended-spectrum �]-lactamase worldwide, and a number of different factors may contribute to this success. These include associations between bla(CTX-M-15) and particular plasmids (IncF) and/or strains, such as Escherichia coli ST131, as well as the genetic contexts in which this gene is found. We previously identified bla(CTX-M-15) as the dominant ESBL gene in the western Sydney area, Australia, and found that it was carried mainly on IncF or IncI1 plasmids. Here, we have mapped the multiresistance regions of the 11 conjugative plasmids with one or more IncF replicons obtained from that survey and conducted a limited comparison of plasmid backbones. Two plasmids with only an IncFII replicon appear to be very similar to the published plasmids pC15-1a and pEK516. The remaining nine plasmids, with multiple IncF replicons, have multiresistance regions related to those of pC15-1a and pEK516, but eight contain additional modules previously found in resistance plasmids from different geographic locations that carry a variety of different resistance genes. Differences between the multiresistance regions are largely due to IS26-mediated deletions, insertions, and/or rearrangements, which can explain the observed variable associations between bla(CTX-M-15) and certain other resistance genes. We found no evidence of independent movement of bla(CTX-M-15) or of a large multiresistance region between different plasmid backbones. Instead, homologous recombination between common components, such as IS26 and Tn2, appeared to be more important in creating new multiresistance regions, and this may be coupled with recombination in plasmid backbones to reassort multiple IncF replicons as well as components of multiresistance regions.
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868. |
Mancini J,
Weckselblatt B,
Chung YK,
Durante JC,
Andelman S,
Glaubman J,
Dorff JD,
Bhargava S,
Lijek RS,
Unger KP,
Okeke IN,
( 2011 ) The heat-resistant agglutinin family includes a novel adhesin from enteroaggregative Escherichia coli strain 60A. PMID : 21764925 : DOI : 10.1128/JB.05142-11 PMC : PMC3165684 Abstract >>
Heat-resistant agglutinin 1 (Hra1) is an accessory colonization factor of enteroaggregative Escherichia coli (EAEC) strain 042. Tia, a close homolog of Hra1, is an invasin and adhesin that has been described in enterotoxigenic E. coli. We devised a PCR-restriction fragment length polymorphism screen for the associated genes and found that they occur among 55 (36.7%) of the enteroaggregative E. coli isolates screened, as well as lower proportions of enterotoxigenic, enteropathogenic, enterohemorrhagic, and commensal E. coli isolates. Overall, 25%, 8%, and 3% of 150 EAEC strains harbored hra1 alone, tia alone, or both genes, respectively. One EAEC isolate, 60A, produced an amplicon with a unique restriction profile, distinct from those of hra1 and tia. We cloned and sequenced the full-length agglutinin gene from strain 60A and have designated it hra2. The hra2 gene was not detected in any of 257 diarrheagenic E. coli isolates in our collection but is present in the genome of Salmonella enterica serovar Heidelberg strain SL476. The cloned hra2 gene from strain 60A, which encodes a predicted amino acid sequence that is 64% identical to that of Hra1 and 68% identical to that of Tia, was sufficient to confer adherence on E. coli K-12. We constructed an hra2 deletion mutant of EAEC strain 60A. The mutant was deficient in adherence but not autoaggregation or invasion, pointing to a functional distinction from the autoagglutinin Hra1 and the Tia invasin. Hra1, Tia, and the novel accessory adhesin Hra2 are members of a family of integral outer membrane proteins that confer different colonization-associated phenotypes.
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869. |
Dolejska M,
Duskova E,
Rybarikova J,
Janoszowska D,
Roubalova E,
Dibdakova K,
Maceckova G,
Kohoutova L,
Literak I,
Smola J,
Cizek A,
( 2011 ) Plasmids carrying blaCTX-M-1 and qnr genes in Escherichia coli isolates from an equine clinic and a horseback riding centre. PMID : 21393204 : DOI : 10.1093/jac/dkq500 Abstract >>
The aim of this study was to determine the occurrence of extended-spectrum �]-lactamase (ESBL)-producing Escherichia coli at an equine clinic and a horseback riding centre, and to discuss the impact of antimicrobial treatment on resistance selection. Faeces from horses, environmental smears and flies were sampled at both the clinic and riding centre. Staff at the equine clinic were also examined. The samples were cultivated on MacConkey agar with cefotaxime (2 mg/L) to isolate ESBL-producing E. coli. The presence of bla and qnr genes was tested by PCR, and transferability was determined by conjugation. Replicon typing and restriction analysis of plasmids harbouring ESBL and qnr genes were performed. E. coli with the blaCTX-M-1 gene were isolated from horses, staff, environmental smears and flies at the two sites. E. coli isolates from the equine clinic harboured an IncHI1 conjugative 235-285 kb plasmid containing blaCTX-M-1, catA1, strA, sul2 and tet(B) genes. Some of these were positive for qnrS1 and/or qnrB19, and were located on 40 or 45 kb IncN or IncX1 conjugative plasmids. The gene blaCTX-M-1 in isolates from the riding centre was carried by IncN (30 kb) and IncI1 (85 kb) conjugative plasmids. Horizontal gene transfer seems to be involved in disseminating E. coli with ESBL and qnr genes at the clinic and riding centre. The study illustrates that ESBL-producing E. coli, as well as plasmids carrying ESBL genes of clinical interest, can be easily transferred among horses, humans and flies living in close contact.
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870. |
Kim J,
Bae IK,
Jeong SH,
Chang CL,
Lee CH,
Lee K,
( 2011 ) Characterization of IncF plasmids carrying the blaCTX-M-14 gene in clinical isolates of Escherichia coli from Korea. PMID : 21415040 : DOI : 10.1093/jac/dkr106 Abstract >>
The purpose of this study was to investigate the molecular epidemiology of CTX-M-14-producing Escherichia coli clinical isolates from Korea. A total of 138 non-duplicate E. coli clinical isolates showing reduced susceptibility or resistance to ceftazidime and/or cefotaxime were included in the study. Resistance genes, genetic environment, R plasmid size and replicon type, sequence type (ST) and XbaI-macrorestriction patterns were determined. Among 138 isolates, 35 were found to carry the bla(CTX-M-14) gene. The ISEcp1 element was identified in the upstream region of the bla(CTX-M-14) gene in 32 isolates. The bla(CTX-M-14) gene was located on an IncF plasmid in 21 isolates, on an IncA/C plasmid in 1 isolate, on the chromosome in 8 isolates and on both the chromosome and an IncF plasmid in 5 isolates. The most prevalent ST was ST405 (n = 8), followed by ST354 (n = 4), ST38 (n = 3), ST69 (n = 3) and the intercontinental ST, ST131 (n = 3). PFGE and multilocus sequence typing experiments demonstrated no major clonal relationship among the CTX-M-14-producing isolates. The bla(CTX-M-14) gene was probably mobilized by IncF plasmids, which can readily spread in E. coli, causing horizontal dissemination of the resistance gene in Korea.
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871. |
Bogaerts P,
Bouchahrouf W,
de Castro RR,
Deplano A,
Berhin C,
Piérard D,
Denis O,
Glupczynski Y,
( 2011 ) Emergence of NDM-1-producing Enterobacteriaceae in Belgium. PMID : 21444697 : DOI : 10.1128/AAC.00049-11 PMC : PMC3101394 Abstract >>
Five multidrug-resistant nonclonally related Enterobacteriaceae isolates were recovered in Belgium in 2010 from three patients who had been hospitalized in Pakistan, Montenegro, and Serbia/Kosovo. New Delhi metallo-�]-lactamase (NDM-1) was detected in each of the isolates in addition to several extended-spectrum �]-lactamases (CTX-M-15, SHV-12), plasmidic cephalosporinases (CMY-16, CMY-58), rRNA methylases (ArmA, RmtB), and Qnr genes (qnrA6, qnrB1, qnrB2). One patient died from uncontrolled sepsis, while the two others recovered. No secondary cases occurred in any of the hospitals.
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872. |
Gomez SA,
Pasteran FG,
Faccone D,
Tijet N,
Rapoport M,
Lucero C,
Lastovetska O,
Albornoz E,
Galas M,
N/A N/A,
Melano RG,
Corso A,
Petroni A,
( 2011 ) Clonal dissemination of Klebsiella pneumoniae ST258 harbouring KPC-2 in Argentina. PMID : 21851480 : DOI : 10.1111/j.1469-0691.2011.03600.x Abstract >>
The present work describes the abrupt emergence of Klebsiella pneumoniae carbapenemase (KPC) and characterizes the first 79 KPC-producing enterobacteria from Argentina (isolated from 2006 to 2010). The emergence of bla(KPC-2) was characterized by two patterns of dispersion: the first was the sporadic occurrence in diverse enterobacteria from distant geographical regions, harbouring plasmids of different incompatibility groups and bla(KPC-2) in an unusual genetic environment flanked by ISKpn8-�Gbla(TEM-1) and ISKpn6-like. bla(KPC-2) was associated with IncL/M transferable plasmids; the second was the abrupt clonal spread of K. pneumoniae ST258 harbouring bla(KPC-2) in Tn4401a.
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873. |
Aslanidis C,
Schmitt R,
( 1990 ) Regulatory elements of the raffinose operon: nucleotide sequences of operator and repressor genes. PMID : 2180920 : DOI : 10.1128/jb.172.4.2178-2180.1990 PMC : PMC208720 Abstract >>
The raffinose (raf) operon is negatively controlled by the specific binding of raf repressor (rafR gene) to raf operator (rafO) DNA. Both rafR and rafO have been sequenced. The 1,011-base-pair rafR gene encodes a 336-amino-acid polypeptide containing an N-terminal helix-turn-helix motif. rafO, as defined by in vivo titration of raf repressor, consists of two nearly identical 18-base-pair palindromes that flank the -35 box of the raf promoter.
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874. |
Ho PL,
Lo WU,
Wong RC,
Yeung MK,
Chow KH,
Que TL,
Tong AH,
Bao JY,
Lok S,
Wong SS,
( 2011 ) Complete sequencing of the FII plasmid pHK01, encoding CTX-M-14, and molecular analysis of its variants among Escherichia coli from Hong Kong. PMID : 21393220 : DOI : 10.1093/jac/dkr010 Abstract >>
We characterized plasmids encoding CTX-M-14 �]-lactamase originating from Escherichia coli isolates recovered from patients with uncomplicated cystitis or individuals with faecal colonization in Hong Kong from 2002 to 2004. Plasmids carrying CTX-M-14 were studied by conjugation, replicon typing, S1 nuclease-PFGE and plasmid PCR-restriction fragment length polymorphism (RFLP). The complete sequence of pHK01, a 70 kb plasmid encoding CTX-M-14 from an E. coli strain, was determined and the results compared with reference plasmids and aligned with GenBank data. The blaCTX-M-14 plasmids could be transferred in 23 of 44 E. coli strains tested. Among the 23 transconjugants, the replicon types of the CTX-M-14-encoding plasmid were FII (n=13), I1-I�^ (n=4), F1B (n=2), FII and I1-I�^ (n=1), K (80 kb, n=1) and undetermined (n=2). Plasmid pHK01 (FII replicon) shares a high degree of homology with R100 except mainly for a 11 kb variable region containing blaCTX-M-14 (with an upstream ISEcp1 and a downstream truncated IS903), an iron transport system, an outer membrane protein (malB, maltoporin) and a putative toxin-antitoxin plasmid stability system (yacABC). It was highly related to blaCTX-M-14 (pKF3-70) and blaCTX-M-24 (pEG356) plasmids reported from mainland China in 2006 and Vietnam in 2007, respectively. Subtyping by a plasmid PCR-RFLP scheme showed that 10 of the 13 FII plasmids originating from isolates collected by multiple laboratories exhibited either identical or highly similar profiles. This study showed that narrow host-range FII plasmids play important roles in the dissemination of CTX-M-14. FII plasmids closely related to pHK01 have disseminated widely in the Hong Kong community.
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875. |
Ho PL,
Lo WU,
Yeung MK,
Lin CH,
Chow KH,
Ang I,
Tong AH,
Bao JY,
Lok S,
Lo JY,
( 2011 ) Complete sequencing of pNDM-HK encoding NDM-1 carbapenemase from a multidrug-resistant Escherichia coli strain isolated in Hong Kong. PMID : 21445317 : DOI : 10.1371/journal.pone.0017989 PMC : PMC3061923 Abstract >>
The emergence of plasmid-mediated carbapenemases, such as NDM-1 in Enterobacteriaceae is a major public health issue. Since they mediate resistance to virtually all �]-lactam antibiotics and there is often co-resistance to other antibiotic classes, the therapeutic options for infections caused by these organisms are very limited. We characterized the first NDM-1 producing E. coli isolate recovered in Hong Kong. The plasmid encoding the metallo-�]-lactamase gene was sequenced. The plasmid, pNDM-HK readily transferred to E. coli J53 at high frequencies. It belongs to the broad host range IncL/M incompatibility group and is 88803 bp in size. Sequence alignment showed that pNDM-HK has a 55 kb backbone which shared 97% homology with pEL60 originating from the plant pathogen, Erwina amylovora in Lebanon and a 28.9 kb variable region. The plasmid backbone includes the mucAB genes mediating ultraviolet light resistance. The 28.9 kb region has a composite transposon-like structure which includes intact or truncated genes associated with resistance to �]-lactams (bla(TEM-1), bla(NDM-1), �Gbla(DHA-1)), aminoglycosides (aacC2, armA), sulphonamides (sul1) and macrolides (mel, mph2). It also harbors the following mobile elements: IS26, ISCR1, tnpU, tnpAcp2, tnpD, �GtnpATn1 and insL. Certain blocks within the 28.9 kb variable region had homology with the corresponding sequences in the widely disseminated plasmids, pCTX-M3, pMUR050 and pKP048 originating from bacteria in Poland in 1996, in Spain in 2002 and in China in 2006, respectively. The genetic support of NDM-1 gene suggests that it has evolved through complex pathways. The association with broad host range plasmid and multiple mobile genetic elements explain its observed horizontal mobility in multiple bacterial taxa.
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876. |
Bailey JK,
Pinyon JL,
Anantham S,
Hall RM,
( 2011 ) Distribution of the blaTEM gene and blaTEM-containing transposons in commensal Escherichia coli. PMID : 21393132 : DOI : 10.1093/jac/dkq529 Abstract >>
The context of antibiotic resistance genes can provide valuable information about the epidemiology of mobile genetic elements. This study examined the distribution of the closely related blaTEM transposons Tn1, Tn2 and Tn3, or blaTEM-containing fragments of them, in ampicillin-resistant human commensal Escherichia coli isolates. A PCR mapping protocol was used to detect different segments of the transposons or to link partial copies to the insertion sequence IS26. Restriction digestion of one amplicon was used to assign transposons to Tn1, Tn2 or Tn3 groups and sequencing validated this approach. Restriction digestion and sequencing were used to determine how much of the transposon remained when blaTEM was linked to IS26. Sequences were compared with those in GenBank. Of 25 ampicillin-resistant E. coli strains recovered from the faecal flora of healthy humans that carried the blaTEM gene, 15 carried a complete copy of Tn2 or a Tn2 variant; one was interrupted by IS4. A further isolate carried Tn3. Tn2 was also most abundant in sequences available in GenBank. Two isolates carried Tn2 and an IS26-blaTEM fragment. The remaining 10 isolates carried only the blaTEM end of the transposon and 9 of these partial copies were flanked by IS26 at varying distances upstream of blaTEM. One configuration corresponded to that in Tn6029B and the complete transposon was shown to be present. Tn1, Tn2 and Tn3 can be simply and rapidly identified. Tn2 appears to be the most widely distributed. However, the blaTEM-containing end associated with an IS26 is also widely distributed.
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877. |
Nada RA,
Shaheen HI,
Khalil SB,
Mansour A,
El-Sayed N,
Touni I,
Weiner M,
Armstrong AW,
Klena JD,
( 2011 ) Discovery and phylogenetic analysis of novel members of class b enterotoxigenic Escherichia coli adhesive fimbriae. PMID : 21289147 : DOI : 10.1128/JCM.02006-10 PMC : PMC3122862 Abstract >>
Enterotoxigenic Escherichia coli (ETEC) is recognized to be a common cause of acute watery diarrhea in children from developing countries. Colonization factors (CFAs) have been identified predominantly in ETEC isolates secreting heat-stable enterotoxin (ST) or cosecreting ST with a heat-labile toxin (LT). We hypothesized that LT-only-secreting ETEC produces unique colonization factors not previously described in ST and LTST-secreting ETEC. A set of degenerate primers based on nucleotide sequence similarities between the major structural genes of CS20 (csnA), CS18 (fotA), CS12 (cswA), and porcine antigen 987 (fasA) was developed and used to screen a collection of 266 LT-secreting ETEC isolates in which no known CFA was detected. PCR-amplified products of different molecular masses were obtained from 49 (18.4%) isolates. Nucleotide sequence analysis of the PCR amplicons followed by GenBank nucleotide BLASTn analysis revealed five novel DNA sequences; translated amino acid BLASTx analysis confirmed sequence similarity to class 1b major structural proteins encoded by csnA, fotA, and fasA. Strains expressing the novel CFAs were phylotyped and analyzed using multilocus sequence typing (MLST; Achtman scheme), and the types detected were compared to those of a collection of archived global E. coli strains. In conclusion, application of the degenerate primer sets to ETEC isolates from surveillance studies increased the total number of ETEC isolates with detectable CFAs by almost 20%. Additionally, MLST analysis suggests that for many CFAs, there may be a requirement for certain genetic backgrounds to acquire and maintain plasmids carrying genes encoding CFAs.
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878. |
Deng Y,
Zeng Z,
Chen S,
He L,
Liu Y,
Wu C,
Chen Z,
Yao Q,
Hou J,
Yang T,
Liu JH,
( 2011 ) Dissemination of IncFII plasmids carrying rmtB and qepA in Escherichia coli from pigs, farm workers and the environment. PMID : 21375663 : DOI : 10.1111/j.1469-0691.2011.03472.x Abstract >>
Fifty-one fluoroquinolone-resistant Escherichia coli isolates recovered from pigs, workers and environmental samples in one pig farm were screened for 16S rRNA methylase genes and qepA, a fluoroquinolone efflux pump gene, by PCR. Clonal relatedness of the E. coli isolates was examined by pulsed-field gel electrophoresis (PFGE), multilocus sequence typing and phylogenetic analysis. Plasmids from the E. coli isolates were characterized by incompatibility group, restriction enzyme digestion and Southern hybridization analysis. The genetic environment of rmtB and qepA was also determined by PCR mapping. Eleven isolates that were highly resistant to amikacin and fluoroquinolones were positive for rmtB and qepA. All of these isolates belonged to phylogenetic group A, but most of them had different PFGE patterns or belonged to different sequence types (STs). Four isolates from different sources (two from pigs, one from a farm worker and one from an environmental sample) belonged to the same ST (ST160). Both rmtB and qepA were located on approximately 75-kb IncFII conjugative plasmids with nearly the same EcoRI digestion pattern. Tn3, IS26 and ISCR3 were found to be associated with rmtB and qepA. This study has found, for the first time, the transmission of rmtB and qepA among E. coli isolates from pigs, farm workers and the environment. Both horizontal transfer of IncFII plasmids and clonal dissemination have occurred and been seen to contribute to the dissemination of these resistance genes in a pig farm.
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879. |
Hentschke M,
Kotsakis SD,
Wolters M,
Heisig P,
Miriagou V,
Aepfelbacher M,
( 2011 ) CMY-42, a novel plasmid-mediated CMY-2 variant AmpC beta-lactamase. PMID : 21388298 : DOI : 10.1089/mdr.2010.0137 Abstract >>
We isolated a clinical Escherichia coli strain with an antimicrobial resistance phenotype characteristic for the expression of an AmpC beta-lactamase. Molecular methods revealed a novel, plasmid-localized variant of CMY-2 with a substitution of valine 231 for serine (V231S), which was designated CMY-42. Like the CMY-2-like AmpC beta-lactamase CMY-30, carrying the substitution V231G, CMY-42 displayed increased activity toward expanded spectrum cephalosporins. This finding supports the hypothesis that a bulky side chain at position 231 (Ambler's position 211) may pose a steric clash with certain cephalosporins hindering the access of the AmpC beta-lactamase; however, additional phenomena may account for the observed hydrolytic properties.
|
880. |
Duda KA,
Lindner B,
Brade H,
Leimbach A,
Brzuszkiewicz E,
Dobrindt U,
Holst O,
( 2011 ) The lipopolysaccharide of the mastitis isolate Escherichia coli strain 1303 comprises a novel O-antigen and the rare K-12 core type. PMID : 21372091 : DOI : 10.1099/mic.0.046912-0 Abstract >>
Mastitis represents one of the most significant health problems of dairy herds. The two major causative agents of this disease are Escherichia coli and Staphylococcus aureus. Of the first, its lipopolysaccharide (LPS) is thought to play a prominent role during infection. Here, we report the O-antigen (OPS, O-specific polysaccharide) structure of the LPS from bovine mastitis isolate E. coli 1303. The structure was determined utilizing chemical analyses, mass spectrometry, and 1D and 2D NMR spectroscopy methods. The O-repeating unit was characterized as -[��4)-�]-D-Quip3NAc-(1��3)-�\-L-Fucp2OAc-(1��4)-�]-D-Galp-(1��3)-�\-D-GalpNAc-(1��]- in which the O-acetyl substitution was non-stoichiometric. The nucleotide sequence of the O-antigen gene cluster of E. coli 1303 was also determined. This cluster, located between the gnd and galF genes, contains 13 putative open reading frames, most of which represent unknown nucleotide sequences that have not been described before. The O-antigen of E. coli 1303 was shown to substitute O-7 of the terminal LD-heptose of the K-12 core oligosaccharide. Interestingly, the non-OPS-substituted core oligosaccharide represented a truncated version of the K-12 outer core - namely terminal LD-heptose and glucose were missing; however, it possessed a third Kdo residue in the inner core. On the basis of structural and genetic data we show that the mastitis isolate E. coli 1303 represents a new serotype and possesses the K-12 core type, which is rather uncommon among human and bovine isolates.
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881. |
Zhu J,
Yin X,
Yu H,
Zhao L,
Sabour P,
Gong J,
( 2011 ) Involvement of quorum sensing and heat-stable enterotoxin a in cell damage caused by a porcine enterotoxigenic Escherichia coli strain. PMID : 21300771 : DOI : 10.1128/IAI.01281-10 PMC : PMC3067552 Abstract >>
Enterotoxigenic Escherichia coli (ETEC) strains with K88 fimbriae are often associated with the outbreaks of diarrhea in newborn and weaned piglets worldwide. In the present study, we observed that 10? CFU/ml of K88(+) ETEC strain JG280 caused more death of pig intestinal IPEC-J2 cells than did 10? CFU/ml, suggesting that ETEC-induced cell death was cell density dependent and that quorum sensing (QS) may play a role in pathogenesis. Subsequent investigations demonstrated a positive correlation between autoinducer 2 (AI-2) activity of JG280 and death of IPEC-J2 cells during the infection for up to 3 h. However, there was a negative correlation between AI-2 activity and expression of the JG280 enterotoxin genes estA and estB when IPEC-J2 cells were exposed to the pathogen at 10? CFU/ml. We therefore cloned the luxS gene (responsible for AI-2 production) from JG280 and overexpressed it in E. coli DH5�\, because deletion of the luxS gene was retarded by the lack of suitable antibiotic selection markers and the resistance of this pathogen to a wide range of antibiotics. The addition of culture fluid from E. coli DH5�\ with the overexpressed luxS reduced cell death of IPEC-J2 cells by 10? CFU/ml JG280. The addition also reduced the estA expression by JG280. Nonpathogenic K88(+) strain JFF4, which lacks the enterotoxin genes, caused no death of IPEC-J2 cells, although it produced AI-2 activity comparable to that produced by JG280. These results suggest the involvement of AI-2-mediated quorum sensing in K88(+) ETEC pathogenesis, possibly through a negative regulation of STa production.
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882. |
Gerlitz M,
Hrabak O,
Schwab H,
( 1990 ) Partitioning of broad-host-range plasmid RP4 is a complex system involving site-specific recombination. PMID : 2172207 : DOI : 10.1128/jb.172.11.6194-6203.1990 PMC : PMC526800 Abstract >>
The broad-host-range plasmid RP4 encodes a highly efficient partitioning system (par) that was previously mapped within the 6.2-kb PstI C fragment. The essential functions were assigned to a region of 2.2 kb between fiwA and IS21 (IS8). On the basis of the nucleotide sequence data of the entire par locus and of in vitro and in vivo expression studies, three distinct loci encoding polypeptides of 9, 18, and 24 kDa were identified. Evidence for the expression of another polypeptide was found. A putative divergent promoter was localized in an intergenic region and is suggested to be responsible for transcription of these genes. It was found that the RP4 par region includes a function resolving plasmid dimers. The 24-kDa polypeptide is considered to function as a resolvase, since its predicted amino acid sequence shows homology to sequences of resolvases of the Tn3 family. Furthermore, palindromes present in the intergenic region containing the divergent promoter resemble repeat structures specific for res sites of Tn3-related transposons. However, it was found that dimer resolution itself was not sufficient for stabilization; additional functions, including the other two polypeptides, seemed to play an important role. These results suggested that RP4 contains a complex stabilization system involving resolution of plasmid dimers during cell division, thus ensuring the delivery of at least one copy to each daughter cell.
|
883. |
Spehr V,
Warrass R,
Höcherl K,
Ilg T,
( 2011 ) Large-scale production of the immunomodulator c-di-GMP from GMP and ATP by an enzymatic cascade. PMID : 21710212 : DOI : 10.1007/s12010-011-9294-z Abstract >>
(3'-5')-Cyclic diguanylate (c-di-GMP) is a bacterial second messenger with immunomodulatory activities in mice suggesting potential applications as a vaccine adjuvant and as a therapeutic agent. Clinical studies in larger animals or humans will require larger doses that are difficult and expensive to generate by currently available chemical or enzymatic synthesis and purification methods. Here we report the production of c-di-GMP at the multi-gram scale from the economical precursors guanosine monophosphate (GMP) and adenosine triphosphate by a "one-pot" three enzyme cascade consisting of GMP kinase, nucleoside diphosphate kinase, and a mutated form of diguanylate cyclase engineered to lack product inhibition. The c-di-GMP was purified to apparent homogeneity by a combination of anion exchange chromatography and solvent precipitation and was characterized by reversed phase high performance liquid chormatography and mass spectrometry, nuclear magnetic resonance spectroscopy, and further compositional analyses. The immunomodulatory activity of the c-di-GMP preparation was confirmed by its potentiating effect on the lipopolysaccharide-induced interleukin 1�], tumor necrosis factor �\, and interleukin 6 messenger RNA expression in J774A.1 mouse macrophages.
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884. |
Curiao T,
Cantón R,
Garcillán-Barcia MP,
de la Cruz F,
Baquero F,
Coque TM,
( 2011 ) Association of composite IS26-sul3 elements with highly transmissible IncI1 plasmids in extended-spectrum-beta-lactamase-producing Escherichia coli clones from humans. PMID : 21343460 : DOI : 10.1128/AAC.01448-10 PMC : PMC3088239 Abstract >>
The association of an IS440-sul3 platform with Tn21 class 1 integrons carried by IncI1 plasmids encoding extended-spectrum �]-lactamases (ESBLs; mainly SHV-12 and CTX-M-14) among worldwide Escherichia coli clones of phylogroups A (ST10, ST23, and ST46), B1 (ST155, ST351, and ST359), and D/B2 (ST131) is reported. An in silico comparative analysis of sul3 elements available in the GenBank database shows the evolution of sul3 platforms by hosting different transposable elements facilitating the potential genesis of IS26 composite transposons and further insertion element-mediated promoted arrangements.
|
885. |
Peirano G,
Ahmed-Bentley J,
Woodford N,
Pitout JD,
( 2011 ) New Delhi metallo-beta-lactamase from traveler returning to Canada. PMID : 21291595 : DOI : 10.3201/eid1702.101313 PMC : PMC3204781 Abstract >>
An Escherichia coli isolate with New Delhi metallo-beta-lactamase was isolated from a patient with pyelonephritis and prostatitis who returned to Canada after recent hospitalization in India. The patient was successfully treated with ertapenem and fosfomycin. This patient highlights the role of international travel in the spread of antimicrobial drug resistance and blaNDM-1.
|
886. |
Deng Y,
He L,
Chen S,
Zheng H,
Zeng Z,
Liu Y,
Sun Y,
Ma J,
Chen Z,
Liu JH,
( 2011 ) F33:A-:B- and F2:A-:B- plasmids mediate dissemination of rmtB-blaCTX-M-9 group genes and rmtB-qepA in Enterobacteriaceae isolates from pets in China. PMID : 21788459 : DOI : 10.1128/AAC.00133-11 PMC : PMC3186975 Abstract >>
This study investigated the prevalence of 16S rRNA methylase genes in 267 Enterobacteriaceae isolates collected from pets. The rmtB gene was detected in 69 isolates, most of which were clonally unrelated. The coexistence of the rmtB gene with the bla(CTX-M-9) group genes and/or qepA within the same IncFII replicons was commonly detected. The two dominant types of IncF plasmids, F2:A-:B-, carrying rmtB-qepA, and F33:A-:B-, carrying the rmtB-bla(CTX-M-9) group genes (and especially bla(CTX-M-65)), shared restriction patterns within each incompatibility group.
|
887. |
Salgado PS,
Taylor JD,
Cota E,
Matthews SJ,
( 2011 ) Extending the usability of the phasing power of diselenide bonds: SeCys SAD phasing of CsgC using a non-auxotrophic strain. PMID : 21206057 : DOI : 10.1107/S0907444910042022 PMC : PMC3522112 Abstract >>
The CsgC protein is a component of the curli system in Escherichia coli. Reported here is the successful incorporation of selenocysteine (SeCys) and selenomethionine (SeMet) into recombinant CsgC, yielding derivatized crystals suitable for structural determination. Unlike in previous reports, a standard autotrophic expression strain was used and only single-wavelength anomalous dispersion (SAD) data were required for successful phasing. The level of SeCys/SeMet incorporation was estimated by mass spectrometry to be about 80%. The native protein crystallized in two different crystal forms (form 1 belonging to space group C222(1) and form 2 belonging to space group C2), which diffracted to 2.4 and 2.0 ? resolution, respectively, whilst Se-derivatized protein crystallized in space group C2 and diffracted to 1.7 ? resolution. The Se-derivatized crystals are suitable for SAD structure determination using only the anomalous signal derived from the SeCys residues. These results extend the usability of SeCys labelling to more general and less favourable cases, rendering it a suitable alternative to traditional phasing approaches.
|
888. |
Djamdjian L,
Naas T,
Tandé D,
Cuzon G,
Hanrotel-Saliou C,
Nordmann P,
( 2011 ) CTX-M-93, a CTX-M variant lacking penicillin hydrolytic activity. PMID : 21343457 : DOI : 10.1128/AAC.01656-10 PMC : PMC3088237 Abstract >>
Extended-spectrum �]-lactamases (ESBLs) of the CTX-M type are increasingly being reported worldwide, with more than 90 known variants. Clinical Escherichia coli isolate Bre-1 was isolated in 2009 and displayed an unusual ESBL phenotype, made of a synergy image between expanded cephalosporins and clavulanic acid discs and susceptibility to penicillins. E. coli Bre-1 harbored a novel CTX-M-encoding gene, designated bla(CTX-M-93). CTX-M-93 differed from CTX-M-27 by only a single L169Q substitution. Compared to CTX-M-27, CTX-M-93 conferred higher MICs of ceftazidime for E. coli (MIC of 8 versus 1.5 �gg/ml) and decreased MICs of other expanded-cephalosporins (MIC of cefotaxime of 1 versus 32 �gg/ml) and penicillins (MIC of ticarcillin of 0.5 versus >256 �gg/ml). A comparison of enzymatic properties revealed that the L169Q substitution led to a decreased Km for ceftazidime (25.5 versus 330 �gM) but decreased hydrolytic activity against good substrates, such as cefotaxime (kcat of 0.6 versus 113 s(-1)), probably owing to the alteration of the omega loop positioning during the catalytic process. The blaCTX-M-93 gene was surrounded by the ISEcp1 and IS903 elements and inserted onto a 150-kb non-self-transferrable IncF-type plasmid. E. coli Bre-1 belongs to phylogroup D and is of multilocus sequence type (MLST) 624, a sequence type found only in rare Spanish CTX-M-14-producing E. coli isolates. We have characterized a novel CTX-M variant, CTX-M-93, lacking significant penicillin hydrolysis but with increased ceftazidime hydrolysis.
|
889. |
Rocha SP,
Abe CM,
Sperandio V,
Bando SY,
Elias WP,
( 2011 ) Atypical enteropathogenic Escherichia coli that contains functional locus of enterocyte effacement genes can be attaching-and-effacing negative in cultured epithelial cells. PMID : 21343354 : DOI : 10.1128/IAI.00693-10 PMC : PMC3088124 Abstract >>
Enteropathogenic Escherichia coli (EPEC) induces a characteristic histopathology on enterocytes known as the attaching-and-effacing (A/E) lesion, which is triggered by proteins encoded by the locus of enterocyte effacement (LEE). EPEC is currently classified as typical EPEC (tEPEC) and atypical EPEC (aEPEC), based on the presence or absence of the EPEC adherence factor plasmid, respectively. Here we analyzed the LEE regions of three aEPEC strains displaying the localized adherence-like (LAL), aggregative adherence (AA), and diffuse adherence (DA) patterns on HEp-2 cells as well as one nonadherent (NA) strain. The adherence characteristics and the ability to induce A/E lesions were investigated with HeLa, Caco-2, T84, and HT29 cells. The adherence patterns and fluorescent actin staining (FAS) assay results were reproducible with all cell lines. The LEE region was structurally intact and functional in all strains regardless of their inability to cause A/E lesions. An EspF(U)-expressing plasmid (pKC471) was introduced into all strains, demonstrating no influence of this protein on either the adherence patterns or the capacity to cause A/E of the adherent strains. However, the NA strain harboring pKC471 expressed the LAL pattern and was able to induce A/E lesions on HeLa cells. Our data indicate that FAS-negative aEPEC strains are potentially able to induce A/E in vivo, emphasizing the concern about this test for the determination of aEPEC virulence. Also, the presence of EspF(U) was sufficient to provide an adherent phenotype for a nonadherent aEPEC strain via the direct or indirect activation of the LEE4 and LEE5 operons.
|
890. |
Solé M,
Pitart C,
Roca I,
F?brega A,
Salvador P,
Muñoz L,
Oliveira I,
Gascón J,
Marco F,
Vila J,
( 2011 ) First description of an Escherichia coli strain producing NDM-1 carbapenemase in Spain. PMID : 21730115 : DOI : 10.1128/AAC.00642-11 PMC : PMC3165357 Abstract >>
A carbapenem-resistant Escherichia coli strain (DVR22) was recovered from a stool specimen from a patient with traveler's diarrhea who had traveled to India. Molecular screening led to the first identification of NDM-1 in Spain. The bla(NDM-1) gene was located in a conjugative plasmid of ca. 300 kb that also contained the bla(CTX-M-15), bla(TEM-1), �Gbla(DHA-1), and armA genes. In addition, bla(NDM-1) was preceded by an ISAba125 insertion element only found in Acinetobacter spp.
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891. |
Dhanji H,
Patel R,
Wall R,
Doumith M,
Patel B,
Hope R,
Livermore DM,
Woodford N,
( 2011 ) Variation in the genetic environments of bla(CTX-M-15) in Escherichia coli from the faeces of travellers returning to the United Kingdom. PMID : 21393166 : DOI : 10.1093/jac/dkr041 Abstract >>
The genetic surroundings of bla(CTX-M-15) in Escherichia coli recovered from faeces of travellers returning to the UK from overseas were compared with those among established UK strains to provide further insights into the spread of bla(CTX-M-15) in the UK. From August 2006 to January 2008, 1031 faecal specimens were collected at the North West London NHS Trust from general practice patients with a clinical history of diarrhoea following recent international travel. Cefuroxime-resistant E. coli were isolated on cystine-lactose-electrolyte deficient agar and those that produced extended-spectrum �]-lactamases (ESBLs) were identified by double disc synergy test (DDST). The molecular environments surrounding bla(CTX-M-15) were investigated by PCR, DNA sequencing, gene cloning and northern blotting. 182/1031 (18%) E. coli isolated from returning travellers gave a positive DDST, and were confirmed by PCR to produce CTX-M ESBLs; 174 (96%) had bla(CTX-M-15), including 21 belonging to clone ST131. Among these 174 isolates, the environment upstream of bla(CTX-M-15) consisted of either: (i) an intact ISEcp1 (n = 108); (ii) various lengths of truncated ISEcp1 (n = 58); or (iii) a 24 bp remnant of ISEcp1 (n = 8). Two different promoters were found to transcribe bla(CTX-M-15), resulting in different levels of cephalosporin resistance. E. coli with CTX-M-15 ESBL from returning travellers harboured previously seen UK bla(CTX-M-15) genetic environments (intact or 24 bp remnant of ISEcp1) as well as bla(CTX-M-15) genetic environments previously unseen in the UK (various lengths of truncated ISEcp1), which suggest overseas acquisition and highlight the difficulty of control in a time of population mobility and travel.
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892. |
Herrero M,
de Lorenzo V,
Timmis KN,
( 1990 ) Transposon vectors containing non-antibiotic resistance selection markers for cloning and stable chromosomal insertion of foreign genes in gram-negative bacteria. PMID : 2172216 : DOI : 10.1128/jb.172.11.6557-6567.1990 PMC : PMC526845 Abstract >>
A simple procedure for cloning and stable insertion of foreign genes into the chromosomes of gram-negative eubacteria was developed by combining in two sets of plasmids (i) the transposition features of Tn10 and Tn5; (ii) the resistances to the herbicide bialaphos, to mercuric salts and organomercurial compounds, and to arsenite, and (iii) the suicide delivery properties of the R6K-based plasmid pGP704. The resulting constructions contained unique NotI or SfiI sites internal to either the Tn10 or the Tn5 inverted repeats. These sites were readily used for cloning DNA fragments with the help of two additional specialized cloning plasmids, pUC18Not and pUC18Sfi. The newly derived constructions could be maintained only in donor host strains that produce the R6K-specified pi protein, which is an essential replication protein for R6K and plasmids derived therefrom. Donor plasmids containing hybrid transposons were transformed into a specialized lambda pir lysogenic Escherichia coli strain with a chromosomally integrated RP4 that provided broad-host-range conjugal transfer functions. Delivery of the donor plasmids into selected host bacteria was accomplished through mating with the target strain. Transposition of the hybrid transposon from the delivered suicide plasmid to a replicon in the target cell was mediated by the cognate transposase encoded on the plasmid at a site external to the transposon. Since the transposase function was not maintained in target cells, such cells were not immune to further transposition rounds. Multiple insertions in the same strain are therefore only limited by the availability of distinct selection markers. The utility of the system was demonstrated with a kanamycin resistance gene as a model foreign insert into Pseudomonas putida and a melanin gene from Streptomyces antibioticus into Klebsiella pneumoniae. Because of the modular nature of the functional parts of the cloning vectors, they can be easily modified and further selection markers can be incorporated. The cloning system described here will be particularly useful for the construction of hybrid bacteria that stably maintain inserted genes, perhaps in competitive situations (e.g., in open systems and natural environments), and that do not carry antibiotic resistance markers characteristic of most available cloning vectors (as is currently required of live bacterial vaccines).
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893. |
López-Cerero L,
Egea P,
Serrano L,
Navarro D,
Mora A,
Blanco J,
Doi Y,
Paterson DL,
Rodríguez-Baño J,
Pascual A,
( 2011 ) Characterisation of clinical and food animal Escherichia coli isolates producing CTX-M-15 extended-spectrum �]-lactamase belonging to ST410 phylogroup A. PMID : 21330111 : DOI : 10.1016/j.ijantimicag.2011.01.001 Abstract >>
Seven phylogroup A CTX-M-15-producing Escherichia coli isolates recovered from clinical and meat samples were further characterised. All of them belonged to sequence type ST410. Only 2 of the 22 virulence genes investigated were detected. All isolates carried the fimH gene encoding type 1 fimbriae, and five isolates harboured the iucD gene encoding aerobactin siderophore. A group of five isolates showed 81.2% similarity by pulsed-field gel electrophoresis (PFGE), comprising three clinical isolates belonging to ONT:H9 and two food isolates belonging to O55:H9. Different HpaI digestion patterns were observed for plasmids, but all of them belonged to IncFIB group and harboured bla(CTX-M-15) associated with bla(OXA-1), bla(TEM), tetA, catB3 and aac(6')-Ib surrounded by an identical genetic environment. These findings showed the possibility of lateral gene transfer of bla(CTX-M-15) as well as other antibiotic resistance determinants between low-virulence food and clinical isolates.
|
894. |
Zong Z,
Yu R,
( 2011 ) bla(CTX-M)-carrying Escherichia coli of the O25b ST131 clonal group have emerged in China. PMID : 21251572 : DOI : 10.1016/j.diagmicrobio.2010.10.007 Abstract >>
Six Escherichia coli O25b ST131 isolates, which were mostly hospital-acquired but from various types of samples, were found to carry bla(CTX-M-3a,)bla(CTX-M-14), or bla(CTX-M-65) genes, demonstrating that such isolates have emerged in China. The bla(CTX-M) genes were mostly carried by IncFII-related conjugative plasmids. All isolates were also resistant to ciprofloxacin and gentamicin and had substitutions in GyrA and carried an aac(3)-II gene.
|
895. |
Li G,
Wei Q,
Wang Y,
Du X,
Zhao Y,
Jiang X,
( 2011 ) Novel genetic environment of the plasmid-mediated KPC-3 gene detected in Escherichia coli and Citrobacter freundii isolates from China. PMID : 21153909 : DOI : 10.1007/s10096-010-1124-7 PMC : PMC3052496 Abstract >>
The imipenem and meropenem-resistant strains Citrobacter freundii HS70 and Escherichia coli HS510 were isolated from patients in Shanghai, China. By isoelectric focusing, PCR amplification and sequencing, these strains were each found to produce four �]-lactamases: TEM-1, KPC-3, SHV-7 and CTX-M-14. A conjugation experiment and plasmid restriction digestion revealed that the bla (KPC-3) gene was located on the same plasmid in both isolates. Bidirectional primer walking sequencing showed that the nucleotide sequence surrounding the 3.8 kb bla(KPC-3) contained a 671-bp insertion similar to that previously characterized in China. The insertion was located between the promoter and the coding region of the bla(KPC-3) gene. Susceptibility testing performed on recombinant strains carrying the bla(KPC-3) gene with or without the insertion revealed that minimum inhibitory concentrations of imipenem, meropenem, cefepime, and cefotaxime for E. coli EMU-KPC3 (without insertion) were four times higher than that of E. coli EKPC3 (with insertion). The 671 bp insertion reduced bla(KPC-3) expression significantly. Taken together, these results suggest that KPC-3-producing C. freundii and E. coli have begun to emerge in our hospital.
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896. |
Tiedeman AA,
DeMarini DJ,
Parker J,
Smith JM,
( 1990 ) DNA sequence of the purC gene encoding 5'-phosphoribosyl-5-aminoimidazole-4-N-succinocarboxamide synthetase and organization of the dapA-purC region of Escherichia coli K-12. PMID : 2120198 : DOI : 10.1128/jb.172.10.6035-6041.1990 PMC : PMC526926 Abstract >>
5'-Phosphoribosyl-5-aminoimidazole-4-N-succinocarboxamide synthetase (EC 6.3.2.6), encoded by the purC gene of Escherichia coli K-12, catalyzes the synthesis of 5'-phosphoribosyl-5-aminoimidazole-4-N-succinocarboxamide from 5'-phosphoribosyl-5-aminoimidazole-4-carboxylic acid. The mature protein, as deduced from the purC structural gene sequence, contains 237 amino acids and has a calculated Mr of 26,998. The control region of the purC gene was identified by primer extension mapping of the 5' end of the purC mRNA. The purC control region contains a binding site for and is regulated by the purine repressor, the product of the purR gene. An unusual feature of the 5' untranslated region of the purC mRNA is the presence of a repetitive extragenic palindrome sequence normally found in intercistronic or 3' untranslated regions. The DNA sequence was extended 1.281 kilobases upstream of the purC structural gene and overlapped with the previously determined dapA sequence. Termination of transcription from the dapA-purC intercistronic region may occur within the -35 region of the purC control region. The purC gene has been positioned on the E. coli restriction map and is transcribed in a counterclockwise direction.
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897. |
Li Y,
Perepelov AV,
Guo D,
Shevelev SD,
Senchenkova SN,
Shahskov AS,
Liu B,
Wang L,
Knirel YA,
( 2011 ) Structural and genetic relationships of two pairs of closely related O-antigens of Escherichia coli and Salmonella enterica: E. coli O11/S. enterica O16 and E. coli O21/S. enterica O38. PMID : 21205000 : DOI : 10.1111/j.1574-695X.2010.00771.x Abstract >>
The O-antigen is a part of the lipopolysaccharide molecule present in the outer membrane of Gram-negative bacteria, and is essential for the full function of the microorganisms. Salmonella enterica and Escherichia coli are taxonomically closely related species. In this study, the O-antigen structures of S. enterica O16 and O38 and E. coli O11 were determined. Salmonella enterica O38 and E. coli O21 were found to have identical O-antigen structures, whereas S. enterica O16 and E. coli O11 had closely related structures, differing only in the presence of a lateral glucose residue and O-acetylation of a mannose residue in the former. The O-antigen gene clusters of S. enterica O16 and O38 and E. coli O11 were sequenced and analyzed together with that of E. coli O21 retrieved from the GenBank. Each S. enterica/E. coli pair was found to contain the same set of genes organized in the same manner and to share 56-78% overall DNA identity. These data suggest that the O-antigen gene clusters of each pair studied originated from a common ancestor. Thus, it has become evident that in the past, the degree of relatedness between the O-antigens of S. enterica and E. coli was underestimated.
|
898. |
Fratamico PM,
Yan X,
Caprioli A,
Esposito G,
Needleman DS,
Pepe T,
Tozzoli R,
Cortesi ML,
Morabito S,
( 2011 ) The complete DNA sequence and analysis of the virulence plasmid and of five additional plasmids carried by Shiga toxin-producing Escherichia coli O26:H11 strain H30. PMID : 21212019 : DOI : 10.1016/j.ijmm.2010.09.002 Abstract >>
Shiga toxin-producing Escherichia coli (STEC) strains belonging to serogroup O26 have been associated with sporadic cases and outbreaks of hemorrhagic colitis and hemolytic uremic syndrome. In addition to chromosomal virulence genes, STEC strains usually harbor a large plasmid that carries genes associated with pathogenicity. The complete nucleotide sequence and genetic organization of 6 plasmids carried by STEC O26:H11 strain H30 were determined. The large virulence plasmid (pO26-Vir) was approximately 168 kb in size and contained 196 open reading frames (ORFs). pO26-Vir possesses a mosaic structure and shows similarity to the virulence plasmids in locus of enterocyte effacement (LEE)-negative STEC O113:H21 EH41 (pO113), in E. coli clinical strain C1096 (pSERB1), and in E. coli O157:H7 RIMD 0509952 (pO157). Plasmid pO26-Vir shares several highly conserved regions with pO157 and carries important virulence genes, including toxB, katP, espP, and the hly gene cluster. In addition, pO26-Vir possesses genes encoding for type IV pili (pilL-V). The second largest plasmid, pO26-L (73 kb) contains 101 ORFs. pO26-L carries the tetracycline resistance gene and has regions that show similarity to the E. coli conjugative resistance plasmid NR1. The third largest plasmid, pO26-S4 (5.8 kb), is homologous to the ColE2 colicinogenic plasmid that encodes for colicin E2. The remaining 3 plasmids, pO26-S1 (1.5 kb), pO26-S2 (3.1 kb), and pO26-S3 (4.2 kb), carry very little genetic information except for putative proteins involved in plasmid replication and DNA maintenance. The data presented underscore the diversity among the STEC virulence plasmids and provide insights into the evolution of these plasmids in STEC strains that cause serious human illness.
|
899. |
Fuller CA,
Pellino CA,
Flagler MJ,
Strasser JE,
Weiss AA,
( 2011 ) Shiga toxin subtypes display dramatic differences in potency. PMID : 21199911 : DOI : 10.1128/IAI.01182-10 PMC : PMC3067513 Abstract >>
Purified Shiga toxin (Stx) alone is capable of producing systemic complications, including hemolytic-uremic syndrome (HUS), in animal models of disease. Stx includes two major antigenic forms (Stx1 and Stx2), with minor variants of Stx2 (Stx2a to -h). Stx2a is more potent than Stx1. Epidemiologic studies suggest that Stx2 subtypes also differ in potency, but these differences have not been well documented for purified toxin. The relative potencies of five purified Stx2 subtypes, Stx2a, Stx2b, Stx2c, Stx2d, and activated (elastase-cleaved) Stx2d, were studied in vitro by examining protein synthesis inhibition using Vero monkey kidney cells and inhibition of metabolic activity (reduction of resazurin to fluorescent resorufin) using primary human renal proximal tubule epithelial cells (RPTECs). In both RPTECs and Vero cells, Stx2a, Stx2d, and elastase-cleaved Stx2d were at least 25 times more potent than Stx2b and Stx2c. In vivo potency in mice was also assessed. Stx2b and Stx2c had potencies similar to that of Stx1, while Stx2a, Stx2d, and elastase-cleaved Stx2d were 40 to 400 times more potent than Stx1.
|
900. |
Wandersman C,
Delepelaire P,
( 1990 ) TolC, an Escherichia coli outer membrane protein required for hemolysin secretion. PMID : 2112747 : DOI : 10.1073/pnas.87.12.4776 PMC : PMC54200 Abstract >>
Secretion of Escherichia coli alpha-hemolysin into the medium does not require the cleavage of an N-terminal signal peptide. The specific secretion apparatus was shown to consist of two proteins, HlyB and HlyD, both located in the inner membrane and encoded by genes contiguous to the hemolysin structural gene (hlyA). It was proposed that these two proteins constitute a membrane-bound translocator for hemolysin [Mackman, N., Nicaud, J. M., Gray, L. & Holland, I. B. (1986) Curr. Top. Microbiol. Immunol. 125, 159-181]. We show here that an E. coli outer membrane protein, the TolC protein, encoded by a gene not located in the hly cluster, is specifically required for hemolysin secretion. This result suggests that an outer membrane protein might be a component of the secretion apparatus allowing a specific interaction between the inner and the outer membrane.
|
901. |
Zong Z,
Yu R,
Wang X,
Lü X,
( 2011 ) blaCTX-M-65 is carried by a Tn1722-like element on an IncN conjugative plasmid of ST131 Escherichia coli. PMID : 21163826 : DOI : 10.1099/jmm.0.026997-0 Abstract >>
Escherichia coli clinical isolate WCE307 was found to belong to phylogroup B2, O25b and sequence type (ST) 131 and had blaCTX-M-65, which was carried by an IncN conjugative plasmid, pWCE307. On pWCE307, the ISEcp1�G-blaCTX-M-65-IS903D-iron�G structure was located in a Tn1722-like element flanked by 5 bp direct target repeats. This context was highly similar to that on pKC396, an IncN plasmid from E. coli isolate KC396 of ST131 from Germany. The Tn1722-like elements carrying blaCTX-M-65 on pWCE307 and pKC396 are likely to be hybrids of Tn1722 and Tn5051, resulting from double crossover at the res sites and the tnpA genes. The left end of the Tn1722-like elements was partially missing. As ISEcp1 and Tn1722 were both incomplete, blaCTX-M-65 might have been trapped in this context. A plasmid multilocus sequence typing (pMLST) scheme with five targets, repN, stbB, traI, traB and the korB-orfI spacer region, was developed for IncN plasmid typing. pMLST revealed that the five alleles of pWCE307 and pKC396 were identical, indicating that WCE307 and KC396 are likely to have originated very recently from a common strain, suggesting the international spread of blaCTX-M-65 mediated by ST131 E. coli.
|
902. |
Réjiba S,
Mercuri PS,
Power P,
Kechrid A,
( 2011 ) Emergence and dominance of CTX-M-15 extended spectrum beta-lactamase among Escherichia coli isolates from children. PMID : 21288137 : DOI : 10.1089/mdr.2010.0098 Abstract >>
Of forty-seven extended-spectrum cephalosporin-resistant Escherichia coli isolates, collected from children at the Children's Hospital in 2006 (Tunis, Tunisia), we analyzed 32 isolates that were genotypically different by enterobacterial repetitive intergenic consensus -polymerase chain reaction. For all isolates, the double-disk diffusion test revealed synergy between clavulanate and cefotaxime and/or ceftazidime, suggesting the production of extended-spectrum beta-lactamases. Polymerase chain reaction experiments, performed on plasmid DNA, and sequencing revealed the presence of bla(TEM-1B) (26 isolates, 81%), bla(TEM-34(IRT-6)) (3 isolates, 9%), bla(SHV-12) (2 isolates, 6%), and bla(CTX-M-15) (31 isolates, 97%). Further, the insertion sequence ISEcp1 was found upstream from the bla(CTX-M-15) gene in 11 isolates. The bla genes were found alone or in various combinations in a single isolate. bla(TEM-1B) and bla(CTX-M-15) genes were detected in 26 out of the 32 isolates. Three isolates harbored both bla(TEM-34(IRT-6)) and bla(CTX-M-15). bla(SHV-12) was identified either alone or with bla(CTX-M-15) in a single isolate. Our investigation showed the dominance of CTX-M-type extended-spectrum beta-lactamases, with CTX-M-15 particularly common, and to our best knowledge, this is the first report of the coexistence of CTX-M-15 and IRT-6 in E. coli isolates from children in Tunisia.
|
903. |
Nuk MR,
Reisner A,
Neuwirth M,
Schilcher K,
Arnold R,
Jehl A,
Rattei T,
Zechner EL,
( 2011 ) Functional analysis of the finO distal region of plasmid R1. PMID : 21145347 : DOI : 10.1016/j.plasmid.2010.12.002 Abstract >>
The intergenic region linking conjugative transfer and replication copy control modules of IncF plasmids shows conservation of gene homology and organization. Genes distal to finO are coordinately expressed with the upstream transfer operon encoding the majority of conjugation genes in related plasmids. Here we investigate potential functions for these genes in copy number control and in processes related to conjugation: gene transfer, pilus specific phage infection and plasmid-promoted biofilm formation by an Escherichia coli host. We find that insertional inactivation of genes in the finO distal region reduced transcriptional read through into the downstream copB gene of plasmid R1. The mutant plasmid derivatives exhibited a reduced copy number compared to the wild type. Moreover all insertion mutant derivatives of plasmid R1-16 with aberrantly low copy numbers conferred poor biofilm forming ability to their hosts. The general mutagenesis thus identified plasmid stability genes as the only plasmid functions besides conjugation genes linked to plasmid-promoted biofilm production under these laboratory conditions. Our findings imply that a novel component of cis- or trans-regulation on the transcriptional level is important to normal R1 plasmid copy number regulation.
|
904. |
Fornili A,
Giabbai B,
Garau G,
Degano M,
( 2010 ) Energy landscapes associated with macromolecular conformational changes from endpoint structures. PMID : 21082835 : DOI : 10.1021/ja107640u Abstract >>
Conformational changes modulate macromolecular function by promoting the specific binding of ligands (such as in antigen recognition) or the stabilization of transition states in enzymatic reactions. However, quantitative characterization of the energetics underlying dynamic structural interconversions is still challenging and lacks a unified method. Here, we introduce a novel in silico approach based on the combined use of essential dynamics sampling and nonequilibrium free-energy calculations to obtain quantitative data on conformational energy landscapes. This technique allows the unbiased investigation of highly complex rearrangements, and does not require the crucial definition of user-defined collective variables. We show that free-energy values derived from profiles connecting the unliganded and ligand-bound X-ray structures of a bacterial nucleoside hydrolase match the experimental binding constant. This approach also provides first evidence for a rate-limiting character of the conformational transition in this enzyme, and an unexpected role of the protonation state of a single residue in regulating substrate binding and product release.
|
905. |
Zhao J,
Dang H,
( 2011 ) Identification of a globally distributed clinical streptomycin-resistance plasmid and other resistance determinants in a coastal bay of China. PMID : 21054449 : DOI : 10.1111/j.1472-765X.2010.02958.x Abstract >>
To study streptomycin-resistant bacteria isolated from Jiaozhou Bay and their molecular determinants of resistance. Twenty-seven tetracycline-resistant and 49 chloramphenicol-resistant bacterial isolates from surface seawater of Jiaozhou Bay were selected for investigation. More than 88% of these isolates were resistant to streptomycin. Half of the streptomycin-resistant bacteria harboured the strA-strB gene pair, and six isolates carried Tn5393-like transposons by PCR detection. The p9123-related plasmids containing the sul2-strA-strB gene cluster were characterized in two environmental Escherichia coli isolates. Transposon Tn5393 was first identified on a Klebsiella pneumoniae plasmid, which also carried Tn1721, estP and umu genes responsible for antimicrobial and insecticide resistance. Coresistance to streptomycin and tetracycline or chloramphenicol was found with high frequency. p9123-related plasmid and Tn5393 transposon may contribute to the wide distribution and spread of the strA-strB gene pair in Jiaozhou Bay. The detection of streptomycin-resistance plasmid pQ1-1 from Jiaozhou Bay seawater bacteria and human bacterial pathogens from USA indicates its global dissemination and transmission, across different components of the microbiota on earth. Streptomycin resistance can be recognized as an important bioindicator of environmental quality, owing to its association with anthropogenic pollution and the multidrug-resistant microbiota.
|
906. |
Perepelov AV,
Ni Z,
Wang Q,
Shevelev SD,
Senchenkova SN,
Shahskov AS,
Wang L,
Knirel YA,
( 2011 ) Structure and gene cluster of the O-antigen of Escherichia coli O109; chemical and genetic evidences of the presence of L-RhaN3N derivatives in the O-antigens of E. coli O109 and O119. PMID : 20964722 : DOI : 10.1111/j.1574-695X.2010.00745.x Abstract >>
O-antigen representing the O-polysaccharide chain of the lipopolysaccharide is the most variable constituent on the cell surface of Gram-negative bacteria and a player in their pathogenicity. The O-polysaccharide of Escherichia coli O109 was studied by sugar analysis and nuclear magnetic resonance spectroscopy and found to contain a rarely occurring monosaccharide, 2,3-diacetamido-2,3,6-trideoxy-l-mannose (l-RhaNAc3NAc). The following structure of the tetrasaccharide repeating unit of the O-polysaccharide was established, which is closely related to that of Proteus penneri O66: Ac--4-�]-L-RhapNAc3NAc -->4)-�\-D-Glcp-(1-->3)-�\-L-6dTalp-(1-->3)-�]-D-GlcpNAc-(1-->. The O-antigen gene cluster of E. coli O109 was sequenced and all 14 genes found were assigned functions based on their similarity to genes from the available databases. Putative genes for synthesis of l-RhaN3N were found in E. coli O109 and their homologues in E. coli O119, whose O-antigen has been reported earlier to contain 2-acetamido-2,3,6-trideoxy-3-formamido-d-mannose (d-RhaNAc3NFo). Analysis by GLC of the (S)-2-octyl glycosides confirmed that the absolute configuration of RhaN3N in E. coli O119 should be revised from D TO L.
|
907. |
Rajkhowa S,
Scaria J,
Garcia DL,
Musser KA,
Akey BL,
Chang YF,
( 2010 ) Analysis of Escherichia coli O157 clinical isolates by multilocus sequence typing. PMID : 21176142 : DOI : 10.1186/1756-0500-3-343 PMC : PMC3016269 Abstract >>
Although many strain typing methods exist for pathogenic Escherichia coli, most have drawbacks in terms of resolving power, interpretability, or scalability. For this reason, multilocus sequence typing (MLST) is an appealing alternative especially when applied to the typing of temporal and spatially separated isolates. This method relies on an unambiguous DNA sequence analysis of nucleotide polymorphisms in housekeeping genes and has shown a high degree of intraspecies discriminatory power for bacterial and fungal pathogens. Here we used the MLST method to study the genetic diversity among E. coli O157 isolates collected from humans from two different locations of USA over a period of several years (2000-2008). MLST analysis of 33 E. coli O157 patient isolates using the eBurst algorithm distinguished 26 different sequence types (STs), which were clustered into two clonal groups and 11 singletons. The predominant ST was ST2, which consisted of 5 isolates (14.28%) followed by ST1 (11.42%). All the isolates under clonal group I exhibited a virtually similar virulence profile except for two strains, which tested negative for the presence of stx genes. The isolates that were assigned to clonal group II in addition to the 11 singletons were found to be phylogenetically distant from clonal group I. Furthermore, we observed a positive correlation between the virulence profile of the isolates and their clonal origin. Our data suggests the presence of genetic diversity among E. coli O157 isolates from humans shows no measurable correlation to the geographic origin of the isolates.
|
908. |
Aoki SK,
Diner EJ,
de Roodenbeke CT,
Burgess BR,
Poole SJ,
Braaten BA,
Jones AM,
Webb JS,
Hayes CS,
Cotter PA,
Low DA,
( 2010 ) A widespread family of polymorphic contact-dependent toxin delivery systems in bacteria. PMID : 21085179 : DOI : 10.1038/nature09490 PMC : PMC3058911 Abstract >>
Bacteria have developed mechanisms to communicate and compete with one another in diverse environments. A new form of intercellular communication, contact-dependent growth inhibition (CDI), was discovered recently in Escherichia coli. CDI is mediated by the CdiB/CdiA two-partner secretion (TPS) system. CdiB facilitates secretion of the CdiA 'exoprotein' onto the cell surface. An additional small immunity protein (CdiI) protects CDI(+) cells from autoinhibition. The mechanisms by which CDI blocks cell growth and by which CdiI counteracts this growth arrest are unknown. Moreover, the existence of CDI activity in other bacteria has not been explored. Here we show that the CDI growth inhibitory activity resides within the carboxy-terminal region of CdiA (CdiA-CT), and that CdiI binds and inactivates cognate CdiA-CT, but not heterologous CdiA-CT. Bioinformatic and experimental analyses show that multiple bacterial species encode functional CDI systems with high sequence variability in the CdiA-CT and CdiI coding regions. CdiA-CT heterogeneity implies that a range of toxic activities are used during CDI. Indeed, CdiA-CTs from uropathogenic E. coli and the plant pathogen Dickeya dadantii have different nuclease activities, each providing a distinct mechanism of growth inhibition. Finally, we show that bacteria lacking the CdiA-CT and CdiI coding regions are unable to compete with isogenic wild-type CDI(+) cells both in laboratory media and on a eukaryotic host. Taken together, these results suggest that CDI systems constitute an intricate immunity network with an important function in bacterial competition.
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909. |
Haiko J,
Laakkonen L,
Juuti K,
Kalkkinen N,
Korhonen TK,
( 2010 ) The omptins of Yersinia pestis and Salmonella enterica cleave the reactive center loop of plasminogen activator inhibitor 1. PMID : 20639337 : DOI : 10.1128/JB.00458-10 PMC : PMC2937412 Abstract >>
Plasminogen activator inhibitor 1 (PAI-1) is a serine protease inhibitor (serpin) and a key molecule that regulates fibrinolysis by inactivating human plasminogen activators. Here we show that two important human pathogens, the plague bacterium Yersinia pestis and the enteropathogen Salmonella enterica serovar Typhimurium, inactivate PAI-1 by cleaving the R346-M347 bait peptide bond in the reactive center loop. No cleavage of PAI-1 was detected with Yersinia pseudotuberculosis, an oral/fecal pathogen from which Y. pestis has evolved, or with Escherichia coli. The cleavage and inactivation of PAI-1 were mediated by the outer membrane proteases plasminogen activator Pla of Y. pestis and PgtE protease of S. enterica, which belong to the omptin family of transmembrane endopeptidases identified in Gram-negative bacteria. Cleavage of PAI-1 was also detected with the omptins Epo of Erwinia pyrifoliae and Kop of Klebsiella pneumoniae, which both belong to the same omptin subfamily as Pla and PgtE, whereas no cleavage of PAI-1 was detected with omptins of Shigella flexneri or E. coli or the Yersinia chromosomal omptins, which belong to other omptin subfamilies. The results reveal a novel serpinolytic mechanism by which enterobacterial species expressing omptins of the Pla subfamily bypass normal control of host proteolysis.
|
910. |
Burgos Y,
Beutin L,
( 2010 ) Common origin of plasmid encoded alpha-hemolysin genes in Escherichia coli. PMID : 20637130 : DOI : 10.1186/1471-2180-10-193 PMC : PMC2918590 Abstract >>
Alpha (alpha)-hemolysin is a pore forming cytolysin and serves as a virulence factor in intestinal and extraintestinal pathogenic strains of E. coli. It was suggested that the genes encoding alpha-hemolysin (hlyCABD) which can be found on the chromosome and plasmid, were acquired through horizontal gene transfer. Plasmid-encoded alpha-hly is associated with certain enterotoxigenic (ETEC), shigatoxigenic (STEC) and enteropathogenic E. coli (EPEC) strains. In uropathogenic E. coli (UPEC), the alpha-hly genes are located on chromosomal pathogenicity islands. Previous work suggested that plasmid and chromosomally encoded alpha-hly may have evolved independently. This was explored in our study. We have investigated 11 alpha-hly plasmids from animal and human ETEC, STEC and EPEC strains. The size of alpha-hly plasmids ranges from 48-157 kb and eight plasmids are conjugative. The regulatory gene (hlyR) located upstream of the hlyCABD gene operon and an IS911 element located downstream of hlyD are conserved. Chromosomally-encoded alpha-hly operons lack the hlyR and IS911 elements. The DNA sequence of hlyC and hlyA divided the plasmid- and chromosomally-encoded alpha-hemolysins into two clusters. The plasmid-encoded alpha-hly genes could be further divided into three groups based on the insertion of IS1 and IS2 in the regulatory region upstream of the alpha-hly operon. Transcription of the hlyA gene was higher than the housekeeping icdA gene in all strains (rq 4.8 to 143.2). Nucleotide sequence analysis of a chromosomally located alpha-hly determinant in Enterobacter cloacae strain indicates that it originates from an E. coli alpha-hly plasmid. Our data indicate that plasmids encoding alpha-hly in E. coli descended from a common ancestor independent of the plasmid size and the origin of the strains. Conjugative plasmids could contribute to the spread of the alpha-hly determinant to Enterobacter cloacae. The presence of IS-elements flanking the plasmid-encoded alpha-hly indicate that they might be mobile genetic elements.
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911. |
Du XD,
Liu HB,
Wu CM,
Li XS,
Cao XY,
Cui BA,
Zhang SM,
Hu GZ,
Shen JZ,
( 2010 ) The genetic environment of armA on pHNE, an IncN plasmid, in one Escherichia coli isolate from a chicken. PMID : 20947619 : DOI : 10.1093/jac/dkq382 Abstract >>
N/A
|
912. |
Graus-Göldner A,
Graus H,
Schlacher T,
Högenauer G,
( 1990 ) The sequences of genes bordering oriT in the enterotoxin plasmid P307: comparison with the sequences of plasmids F and R1. PMID : 2096398 : Abstract >>
The nucleotide sequences of the enterotoxin plasmid P307 transfer genes traM, finP, traJ, traY, and gene 19 were determined. Gene 19 is highly conserved; its product is very similar to that coded by the F and R1 plasmids. The TraM protein is similar in P307 and in F; the R1 sequence shows differences in the 40 N-terminal amino acids. The traJ product is very different in P307, F, and R1. The traY gene from P307, which in F is almost twice as long, is similar in size to that from R1. The finP RNA shows a high degree of homology with that from R1 and F, except for the two loop regions where base changes were observed. The genes coding for proteins, except traY, could be expressed in minicell- and T7 promoter-driven expression systems, whereas traJ and gene 19 could be expressed only in the latter system.
|
913. |
Bergholz PW,
Noar JD,
Buckley DH,
( 2011 ) Environmental patterns are imposed on the population structure of Escherichia coli after fecal deposition. PMID : 21075897 : DOI : 10.1128/AEM.01880-10 PMC : PMC3019742 Abstract >>
The intestinal microbe Escherichia coli is subject to fecal deposition in secondary habitats, where it persists transiently, allowing for the opportunity to colonize new hosts. Selection in the secondary habitat can be postulated, but its impact on the genomic diversity of E. coli is unknown. Environmental selective pressure on extrahost E. coli can be revealed by landscape genetic analysis, which examines the influences of dispersal processes, landscape features, and the environment on the spatiotemporal distribution of genes in natural populations. We conducted multilocus sequence analysis of 353 E. coli isolates from soil and fecal samples obtained in a recreational meadow to examine the ecological processes controlling their distributions. Soil isolates, as a group, were not genetically distinct from fecal isolates, with only 0.8% of genetic variation and no fixed mutations attributed to the isolate source. Analysis of the landscape genetic structure of E. coli populations showed a patchy spatial structure consistent with patterns of fecal deposition. Controlling for the spatial pattern made it possible to detect environmental gradients of pH, moisture, and organic matter corresponding to the genetic structure of E. coli in soil. Ecological distinctions among E. coli subpopulations (i.e., E. coli reference collection [ECOR] groups) contributed to variation in subpopulation distributions. Therefore, while fecal deposition is the major predictor of E. coli distributions on the field scale, selection imposed by the soil environment has a significant impact on E. coli population structure and potentially amplifies the occasional introduction of stress-tolerant strains to new host individuals by transmission through water or food.
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914. |
Seputiene V,
Linkevicius M,
Bogdaite A,
Povilonis J,
Planciūniene R,
Giedraitiene A,
Pavilonis A,
Suziedeliene E,
( 2010 ) Molecular characterization of extended-spectrum �]-lactamase-producing Escherichia coli and Klebsiella pneumoniae isolates from hospitals in Lithuania. PMID : 20576750 : DOI : 10.1099/jmm.0.021972-0 Abstract >>
N/A
|
915. |
Ghosh PK,
Ali A,
( 2010 ) Isolation of atypical enteropathogenic Escherichia coli from children with and without diarrhoea in Delhi and the National Capital Region, India. PMID : 20634334 : DOI : 10.1099/jmm.0.014530-0 Abstract >>
A total of 17 typical and atypical enteropathogenic Escherichia coli (EPEC) were isolated from 396 children with and without diarrhoea. Out of 12 EPEC isolates from patients with diarrhoea, 3 (25 %) were atypical EPEC while 9 (75 %) were typical EPEC. It was observed that atypical EPEC strains had colonized the intestines of healthy children and its isolation rates were higher in healthy children than in children with diarrhoea. Interestingly all of the atypical EPEC isolates carried a megaplasmid, mostly comparable with the size of EPEC adherence factor (EAF) encoding gene but no virulence gene was detected in this megaplasmid. Studies also indicated that multidrug resistance EPEC are emerging and all the atypical EPEC strains showed significantly less resistance to all antimicrobial agents used in this study than typical EPEC. This study also supports the opinion that Shiga toxin-producing E. coli does not pose a major threat to human health in India. Subtyping analysis reveals that eae-�\1, eae-�]2 and eae-�f could be common EPEC subtypes prevalent in children with diarrhoea in Delhi. The present study is believed to be the first report of the detection of atypical EPEC from children without diarrhoea and records of isolation of eae-�^1, eae-�^2 and the rare eae-�f subtype in India. The data also indicated that typical EPEC are a common cause of diarrhoea and atypical EPEC are emerging as colonizers of the intestine of children with and without diarrhoea in Delhi and the National Capital Region, India.
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916. |
Husain N,
Obranic S,
Koscinski L,
Seetharaman J,
Babic F,
Bujnicki JM,
Maravic-Vlahovicek G,
Sivaraman J,
( 2011 ) Structural basis for the methylation of A1408 in 16S rRNA by a panaminoglycoside resistance methyltransferase NpmA from a clinical isolate and analysis of the NpmA interactions with the 30S ribosomal subunit. PMID : 21062819 : DOI : 10.1093/nar/gkq1033 PMC : PMC3061052 Abstract >>
NpmA, a methyltransferase that confers resistance to aminoglycosides was identified in an Escherichia coli clinical isolate. It belongs to the kanamycin-apramycin methyltransferase (Kam) family and specifically methylates the 16S rRNA at the N1 position of A1408. We determined the structures of apo-NpmA and its complexes with S-adenosylmethionine (AdoMet) and S-adenosylhomocysteine (AdoHcy) at 2.4, 2.7 and 1.68 ?, respectively. We generated a number of NpmA variants with alanine substitutions and studied their ability to bind the cofactor, to methylate A1408 in the 30S subunit, and to confer resistance to kanamycin in vivo. Residues D30, W107 and W197 were found to be essential. We have also analyzed the interactions between NpmA and the 30S subunit by footprinting experiments and computational docking. Helices 24, 42 and 44 were found to be the main NpmA-binding site. Both experimental and theoretical analyses suggest that NpmA flips out the target nucleotide A1408 to carry out the methylation. NpmA is plasmid-encoded and can be transferred between pathogenic bacteria; therefore it poses a threat to the successful use of aminoglycosides in clinical practice. The results presented here will assist in the development of specific NpmA inhibitors that could restore the potential of aminoglycoside antibiotics.
|
917. |
Corsini G,
Karahanian E,
Tello M,
Fernandez K,
Rivero D,
Saavedra JM,
Ferrer A,
( 2010 ) Purification and characterization of the antimicrobial peptide microcin N. PMID : 20979348 : DOI : 10.1111/j.1574-6968.2010.02106.x Abstract >>
Microcins are low-molecular-weight proteins secreted by certain bacteria that act by limiting the growth of other bacteria that share the same ecological niche. In the present work, the previous microcin 24 system was resequenced.We detected three nucleotide differences in the microcin-coding gene that partially change the amino acid sequence. According to the present microcin nomenclature, we renamed the five genes constituting this microcin system (mcnRINAB), which are arranged in an operon-like structure: mcnR codes for a putative histone-like nucleoid protein regulator; mcnI codes for the immunity protein; mcnN encodes microcin N; and mcnA and mcnB correspond to an ATP-binding cassette transporter system. Purified microcin N has a molecular weight of 7274.23 Da, as determined by MS. This peptide was stable up to 100�XC, resistant to treatment with lipase, lysozyme, trypsin, and chymotrypsin, and susceptible to degradation by proteinase K.
|
918. |
Wang S,
Niu C,
Shi Z,
Xia Y,
Yaqoob M,
Dai J,
Lu C,
( 2011 ) Effects of ibeA deletion on virulence and biofilm formation of avian pathogenic Escherichia coli. PMID : 20974831 : DOI : 10.1128/IAI.00821-10 PMC : PMC3019902 Abstract >>
The ibeA gene is located on a genomic island, GimA, which is involved in the pathogenesis of neonatal meningitis Escherichia coli (NMEC) and avian pathogenic E. coli (APEC). The prevalence of ibeA in the APEC collection in China was investigated, and 20 of 467 strains (4.3%) were positive. In addition, analysis of the association of the E. coli reference (ECOR) groups with positive strains revealed that ibeA was linked to group B2. The ibeA gene in DE205B was analyzed and compared to those of APEC and NMEC, which indicated that the specificity of ibeA was not consistent along pathotypes. The invasion of chicken embryo fibroblast DF-1 cells by APEC DE205B and RS218 was observed, which suggested that DF-1 cells could be a model to study the mechanism of APEC invasion. The inactivation of ibeA in APEC DE205B led to the reduced capacity to invade DF-1 cells, defective virulence in vivo, and decreased biofilm formation compared to the wild-type strain. In addition, strain AAEC189 expressing ibeA exhibited enhanced invasion capacity and biofilm formation. The results of the quantitative real-time reverse transcription-PCR (qRT-PCR) analysis and animal system infection experiments indicated that the loss of ibeA decreased the colonization and proliferation capacities of APEC in the brain during system infection.
|
919. |
Ayala Cde O,
Ramos Moreno AC,
Martinez MB,
Burgos YK,
Pestana de Castro AF,
Bando SY,
( 2012 ) Determination of flagellar types by PCR-RFLP analysis of enteropathogenic Escherichia coli (EPEC) and Shiga toxin-producing E. coli (STEC) strains isolated from animals in S?o Paulo, Brazil. PMID : 21094508 : DOI : 10.1016/j.rvsc.2010.10.025 Abstract >>
This study evaluated the polymerase chain reaction- restriction fragment length polymorphism (PCR-RFLP) analysis of fliC for typing flagella antigen (H) of Shiga toxin-producing Escherichia coli (STEC) and enteropathogenic E. coli (EPEC) strains isolated from different animals. The molecular typing of the H type was efficient in the determination of 93 (85%) strains. Two nonmotile (H-) E. coli strains showed a PCR-RFLP electrophoretic profile that did not match known H type patterns. The fliC nucleotide sequence of strains B2N and 4a revealed a nucleotide substitution at the restriction site and a nucleotide insertion that generated a stop codon, respectively. The results of this study showed that PCR-RFLP analysis of fliC is faster, less laborious and as efficient for the determination of H type E. coli isolated from animals, compared to serotyping and that it is useful in determining H type in nonmotile strains and strains expressing non-reactive H antigens. Moreover, the fliC sequence of strain B2N suggests that we could have found a new flagellin antigen type.
|
920. |
Merkel V,
Ohder B,
Bielaszewska M,
Zhang W,
Fruth A,
Menge C,
Borrmann E,
Middendorf B,
Müthing J,
Karch H,
Mellmann A,
( 2010 ) Distribution and phylogeny of immunoglobulin-binding protein G in Shiga toxin-producing Escherichia coli and its association with adherence phenotypes. PMID : 20547747 : DOI : 10.1128/IAI.00006-10 PMC : PMC2916290 Abstract >>
eibG in Shiga toxin-producing Escherichia coli (STEC) O91 encodes a protein (EibG) which binds human immunoglobulins G and A and contributes to bacterial chain-like adherence to human epithelial cells. We investigated the prevalence of eibG among STEC, the phylogeny of eibG, and eibG allelic variations and their impact on the adherence phenotype. eibG was found in 15.0% of 240 eae-negative STEC strains but in none of 157 eae-positive STEC strains. The 36 eibG-positive STEC strains belonged to 14 serotypes and to eight multilocus sequence types (STs), with serotype O91:H14/H(-) and ST33 being the most common. Sequences of the complete eibG gene (1,527 bp in size) from eibG-positive STEC resulted in 21 different alleles with 88.11% to 100% identity to the previously reported eibG sequence; they clustered into three eibG subtypes (eibG-alpha, eibG-beta, and eibG-gamma). Strains expressing EibG-alpha and EibG-beta displayed a mostly typical chain-like adherence pattern (CLAP), with formation of long chains on both human and bovine intestinal epithelial cells, whereas strains with EibG-gamma adhered in short chains, a pattern we termed atypical CLAP. The same adherence phenotypes were displayed by E. coli BL21(DE3) clones containing the respective eibG-alpha, eibG-beta, and eibG-gamma subtypes. We propose two possible evolutionary scenarios for eibG in STEC: a clonal development of eibG in strains with the same phylogenetic background or horizontal transfer of eibG between phylogenetically unrelated STEC strains.
|
921. |
Kamenšek S,
Podlesek Z,
Gillor O,
Zgur-Bertok D,
( 2010 ) Genes regulated by the Escherichia coli SOS repressor LexA exhibit heterogeneous expression. PMID : 21070632 : DOI : 10.1186/1471-2180-10-283 PMC : PMC2994835 Abstract >>
Phenotypic heterogeneity may ensure that a small fraction of a population survives environmental perturbations or may result in lysis in a subpopulation, to increase the survival of siblings. Genes involved in DNA repair and population dynamics play key roles in rapid responses to environmental conditions. In Escherichia coli the transcriptional repressor LexA controls a coordinated cellular response to DNA damage designated the SOS response. Expression of LexA regulated genes, e.g. colicin encoding genes, recA, lexA and umuDC, was examined utilizing transcription fusions with the promoterless gfp at the single cell level. The investigated LexA regulated genes exhibited heterogeneity, as only in a small fraction of the population more intense fluorescence was observed. Unlike recA and lexA, the pore forming and nuclease colicin activity genes as well as umuDC, exhibited no basal level activity. However, in a lexA defective strain high level expression of the gene fusions was observed in the large majority of the cells. All of the investigated genes were expressed in a recA defective strain, albeit at lower levels, revealing expression in the absence of a spontaneous SOS response. In addition, the simultaneous expression of cka, encoding the pore forming colicin K, and lexA, investigated at the single cell level revealed high level expression of only cka in rare individual cells. LexA regulated genes exhibit phenotypic heterogeneity as high level expression is observed in only a small subpopulation of cells. Heterogeneous expression is established primarily by stochastic factors and the binding affinity of LexA to SOS boxes.
|
922. |
Lu SY,
Zhang YL,
Geng SN,
Li TY,
Ye ZM,
Zhang DS,
Zou F,
Zhou HW,
( 2010 ) High diversity of extended-spectrum beta-lactamase-producing bacteria in an urban river sediment habitat. PMID : 20639374 : DOI : 10.1128/AEM.00711-10 PMC : PMC2935080 Abstract >>
Antibiotic-resistant bacteria (ARB) have been surveyed widely in water bodies, but few studies have determined the diversity of ARB in sediment, which is the most taxon-abundant habitat in aquatic environments. We isolated 56 extended-spectrum beta-lactamase (ESBL)-producing bacteria from a single sediment sample taken from an urban river in China. All strains were confirmed for ESBL-producing capability by both the clavulanic acid combination disc method and MIC determination. Of the isolated strains, 39 were classified as Enterobacteriaceae (consisting of the genera Escherichia, Klebsiella, Serratia, and Aeromonas) by 16S rRNA gene sequencing and biochemical analysis. The present study identifies, for the first time, ESBL-producing strains from the families Brucellaceae and Moraxellaceae. The bla(CTX-M) gene was the most dominant of the ESBL genes (45 strains), while the bla(TEM) gene was the second-most dominant (22 strains). A total of five types of bla(CTX-M) fragments were identified, with both known and novel sequences. A library of bla(CTX-M) cloned from the sediment DNA showed an even higher diversity of bla(CTX-M) sequences. The discovery of highly diverse ESBL-producing bacteria and ESBL genes, particularly bla(CTX), in urban river sediment raises alarms for potential dissemination of ARB in communities through river environments.
|
923. |
Liu B,
Perepelov AV,
Guo D,
Shevelev SD,
Senchenkova SN,
Feng L,
Shashkov AS,
Wang L,
Knirel YA,
( 2010 ) Structural and genetic relationships between the O-antigens of Escherichia coli O118 and O151. PMID : 21039922 : DOI : 10.1111/j.1574-695X.2010.00738.x Abstract >>
O-antigen (O-polysaccharide) is a highly variable part of the lipopolysaccharide present in the outer membrane of Gram-negative bacteria, which is used as the basis for bacterial serotyping and is essential for the full function and virulence of bacteria. In this work, the structure and genetics of the O-antigens of Escherichia coli O118 and O151 were investigated. Both O-polysaccharides were found to contain ribitol phosphate and have similar structures, the only difference between their backbones being one linkage mode (�]1��3 in E. coli O118 vs. �]1��2 in E. coli O151), which, most probably, is the linkage between the oligosaccharide repeats (O-units). The O-antigen gene clusters of the two bacteria are organized in the same manner and share high-level identity (>99%). Analysis of the wzy genes from E. coli O118 and O151 strains, which are responsible for the linkage between O-units, revealed only one nucleotide substitution, resulting in one amino acid residue substitution. The possible genetic events that may lead to the structural difference between two O-antigen structures are discussed. Salmonella O47 has the same O-unit backbone and a similar O-antigen gene cluster (OGC) (the DNA identity ranges from 74% to 83%) as E. coli O118 and O151. It was suggested that the OGCs of the three bacteria studied originated from a common ancestor.
|
924. |
Bhagwat AS,
Johnson B,
Weule K,
Roberts RJ,
( 1990 ) Primary sequence of the EcoRII endonuclease and properties of its fusions with beta-galactosidase. PMID : 2104830 : Abstract >>
The EcoRII endonuclease cleaves DNA containing the sequence CC(A/T)GG before the first cytosine. The methylation of the second cytosine in the sequence by either the EcoRII methylase or Dcm, a chromosomally coded protein in Escherichia coli, inhibits the cleavage. The gene for the EcoRII endonuclease was mapped by analysis of derivatives containing linker insertions, transposon insertions, and restriction fragment deletions. Surprisingly, plasmids carrying the wild-type endonuclease gene and the EcoRII methylase gene interrupted by transposon insertions appeared to be lethal to dcm+ strains of E. coli. We conclude that not all the EcoRII/Dcm recognition sites in the cellular DNA are methylated in dcm+ strains. The DNA sequence of a 1650-base pair fragment containing the endonuclease gene was determined. It revealed an open reading frame that could code for a 45.6-kDa protein. This predicted size is consistent with the known size of the endonuclease monomer (44 kDa). The endonuclease and methylase genes appear to be transcribed convergently from separate promoters. The reading frame of the endonuclease gene was confirmed at three points by generating random protein fusions between the endonuclease and beta-galactosidase, followed by an analysis of the sequence at the junctions. One of these fusions is missing 18 COOH-terminal amino acids of the endonuclease but still displays significant ability to restrict incoming phage in addition to beta-galactosidase activity. No striking similarity between the sequence of the endonuclease and any other protein in the PIR data base was found. The knowledge of the primary sequence of the endonuclease and the availability of the various constructs involving its gene should be helpful in the study of the interaction of the enzyme with its substrate DNA.
|
925. |
López J,
Salazar L,
Andrés I,
Ortiz JM,
Rodríguez JC,
( 1991 ) Nucleotide sequence of the oriT-traM-finP region of the haemolytic plasmid pSU316: comparison to F. PMID : 2062659 : DOI : 10.1093/nar/19.12.3451 PMC : PMC328347 Abstract >>
N/A
|
926. |
Li R,
Harada T,
Honjoh K,
Miyamoto T,
( 2010 ) Phylogenetic analysis and Shiga toxin production profiling of Shiga toxin-producing/enterohemorrhagic Escherichia coli clinical isolates. PMID : 20558273 : DOI : 10.1016/j.micpath.2010.06.005 Abstract >>
Shiga toxin-producing Escherichia coli (STEC) can cause severe illnesses in humans such as hemorrhagic colitis and hemolytic-uremic syndrome. In this study, we carried out genotypic analysis of the Shiga toxin (stx) gene in 120 clinical isolates of STEC and enterohemorrhagic E. coli (EHEC) from patients in a southern district of Japan. We identified 88 stx(1)(+) and 103 stx(2)(+) strains. We further identified 12 stx(1)(+) and stx(2)(+) isolates expressing little or no Shiga toxin 1 (Stx(1)) and/or 2 (Stx(2)) by reversed passive latex agglutination (RPLA) and Vero cell toxicity assays. Among them, 1 strain could not produce Stx(1), 8 could not produce Stx(2), and 3 strains could produce neither. Two of the latter three strains were of the non-O157 serotype. Most of the Stx RPLA-negative strains belonged to the stx(1)/stx(2) subtype (11/12, [91.7%]). Our quantitative reverse transcription PCR analysis indicated that the stx genes were not effectively transcribed in the RPLA-negative strains. This is the first report of the isolation of stx-positive strains showing Stx-negative phenotype from stx(1)-bearing strains and non-O157 strains.
|
927. |
Hultdin UW,
Lindberg S,
Grundström C,
Huang S,
Uhlin BE,
Sauer-Eriksson AE,
( 2010 ) Structure of FocB--a member of a family of transcription factors regulating fimbrial adhesin expression in uropathogenic Escherichia coli. PMID : 20646069 : DOI : 10.1111/j.1742-4658.2010.07742.x Abstract >>
In uropathogenic Escherichia coli, UPEC, different types of fimbriae are expressed to mediate interactions with host tissue. FocB belongs to the PapB family of transcription factors involved in the regulation of fimbriae gene clusters. Recent findings suggest that members from this family of proteins may form homomeric or heteromeric complexes and exert both positive and negative effects on the transcription of fimbriae genes. To elucidate the detailed function of FocB, we have determined its crystal structure at 1.4 A resolution. FocB is an all alpha-helical protein with a helix-turn-helix motif. Interestingly, conserved residues important for DNA-binding are located not in the postulated recognition helix of the motif, but in the preceding helix. Results from protein-DNA-binding studies suggest that FocB interacts with the minor groove of its cognate DNA target, which is indicative of a DNA interaction that is unusual for this motif. FocB crystallizes in the form of dimers. Packing interactions in the crystals give two plausible dimerization interfaces. Conserved residues, known to be important for protein oligomerization, are present at both interfaces, suggesting that both sites could play a role in a functional FocB protein.
|
928. |
Berçot B,
Poirel L,
Silva-Sanchez J,
Nordmann P,
( 2010 ) Association of the extended-spectrum beta-lactamase gene blaTLA-1 with a novel ISCR element, ISCR20. PMID : 20585120 : DOI : 10.1128/AAC.00075-10 PMC : PMC2934968 Abstract >>
The bla(TLA-1) gene encoding an extended-spectrum beta-lactamase was identified in 11 enterobacterial isolates from Mexico City, Mexico. This gene was located on different plasmids and plasmid types with different sizes and incompatibility groups. It was associated with a novel insertion sequence, ISCR20, encoding a putative transposase that shared only 20% amino acid identity with the most closely related transposase of ISCR1. The ISCR20 element provided specific promoter sequences for expression of the bla(TLA-1) gene.
|
929. |
Villa L,
García-Fernández A,
Fortini D,
Carattoli A,
( 2010 ) Replicon sequence typing of IncF plasmids carrying virulence and resistance determinants. PMID : 20935300 : DOI : 10.1093/jac/dkq347 Abstract >>
IncF plasmids are frequently encountered in clinical enterobacterial strains associated with the dissemination of relevant antimicrobial resistance and virulence genes. These plasmids are usually heterogeneous in size and carry multiple replicons, and technical difficulties can impair the comparison and detection of related plasmids by restriction fragment length polymorphism analysis. We devised a rapid sequence-based typing scheme to categorize the members of this plasmid family into homogeneous groups. We compared the available IncF replicon sequences, identifying the combination of the different IncF replicon alleles as the discriminating characteristic of these plasmid scaffolds. An IncF typing method based on PCR amplification and sequence typing of the IncF replicons was devised. A collection of IncF plasmids carrying resistance and/or virulence genes, identified in strains from different sources and geographical origins, was tested with this typing system. We devised a replicon sequence typing (RST) scheme discriminating IncF plasmid variants. This system was tested on the collection of IncF plasmids, demonstrating that it was useful for the discrimination of plasmids carrying the same resistance gene (i.e. the bla(CTX-M-15) gene), but also recognized strictly related virulence plasmids (i.e. IncFIme plasmids). The PCR-based replicon typing (PBRT) system was also updated by including new primer pairs to allow the identification of the Salmonella, Klebsiella and Yersinia IncF plasmids. The ability to recognize and sub-categorize IncF plasmids by RST in homogeneous groups on the basis of their phylogenetic relatedness can be helpful in analysing their distribution in nature and discovering their evolutionary origin.
|
930. |
Smet A,
Van Nieuwerburgh F,
Vandekerckhove TT,
Martel A,
Deforce D,
Butaye P,
Haesebrouck F,
( 2010 ) Complete nucleotide sequence of CTX-M-15-plasmids from clinical Escherichia coli isolates: insertional events of transposons and insertion sequences. PMID : 20585456 : DOI : 10.1371/journal.pone.0011202 PMC : PMC2887853 Abstract >>
CTX-M-producing Escherichia coli strains are regarded as major global pathogens. The nucleotide sequence of three plasmids (pEC_B24: 73801-bp; pEC_L8: 118525-bp and pEC_L46: 144871-bp) from Escherichia coli isolates obtained from patients with urinary tract infections and one plasmid (pEC_Bactec: 92970-bp) from an Escherichia coli strain isolated from the joint of a horse with arthritis were determined. Plasmid pEC_Bactec belongs to the IncI1 group and carries two resistance genes: bla(TEM-1) and bla(CTX-M-15). It shares more than 90% homology with a previously published bla(CTX-M)-plasmid from E. coli of human origin. Plasmid pEC_B24 belongs to the IncFII group whereas plasmids pEC_L8 and pEC_L46 represent a fusion of two replicons of type FII and FIA. On the pEC_B24 backbone, two resistance genes, bla(TEM-1) and bla(CTX-M-15), were found. Six resistance genes, bla(TEM-1), bla(CTX-M-15), bla(OXA-1), aac6'-lb-cr, tetA and catB4, were detected on the pEC_L8 backbone. The same antimicrobial drug resistance genes, with the exception of tetA, were also identified on the pEC_L46 backbone. Genome analysis of all 4 plasmids studied provides evidence of a seemingly frequent transposition event of the bla(CTX-M-15)-ISEcp1 element. This element seems to have a preferred insertion site at the tnpA gene of a bla(TEM)-carrying Tn3-like transposon, the latter itself being inserted by a transposition event. The IS26-composite transposon, which contains the bla(OXA-1), aac6'-lb-cr and catB4 genes, was inserted into plasmids pEC_L8 and pEC_L46 by homologous recombination rather than a transposition event. Results obtained for pEC_L46 indicated that IS26 also plays an important role in structural rearrangements of the plasmid backbone and seems to facilitate the mobilisation of fragments from other plasmids. Collectively, these data suggests that IS26 together with ISEcp1 could play a critical role in the evolution of diverse multiresistant plasmids found in clinical Enterobacteriaceae.
|
931. |
Hordijk J,
Bosman AB,
van Essen-Zandbergen A,
Veldman K,
Dierikx C,
Wagenaar JA,
Mevius D,
( 2011 ) qnrB19 gene bracketed by IS26 on a 40-kilobase IncR plasmid from an Escherichia coli isolate from a veal calf. PMID : 20956595 : DOI : 10.1128/AAC.00866-10 PMC : PMC3019637 Abstract >>
N/A
|
932. |
Papagiannitsis CC,
Tzouvelekis LS,
Kotsakis SD,
Tzelepi E,
Miriagou V,
( 2011 ) Sequence of pR3521, an IncB plasmid from Escherichia coli encoding ACC-4, SCO-1, and TEM-1 beta-lactamases. PMID : 20956594 : DOI : 10.1128/AAC.00875-10 PMC : PMC3019680 Abstract >>
The sequence of pR3521, a self-transmissible plasmid from Escherichia coli, was determined. pR3521 (110,416 bp) comprised a contiguous IncB sequence (84,034 bp) sharing extensive similarities with IncI replicons and an acquired region (26,382 bp) carrying sequences of diverse origin, containing bla(ACC-4), bla(SCO-1), bla(TEM-1b) (two copies), strA, strB, sul2, and aacC2.
|
933. |
Biserci? M,
Feutrier JY,
Reeves PR,
( 1991 ) Nucleotide sequences of the gnd genes from nine natural isolates of Escherichia coli: evidence of intragenic recombination as a contributing factor in the evolution of the polymorphic gnd locus. PMID : 2050640 : DOI : 10.1128/jb.173.12.3894-3900.1991 PMC : PMC208022 Abstract >>
Nine natural isolates of Escherichia coli were examined, and the sequence of the entire 1,404 bases of the gnd gene (6-phosphogluconate dehydrogenase, EC 1.1.1.44) was determined. These isolates, along with E. coli K-12, constitute 10 strains for analysis. (The sequence of the E. coli K-12 gnd gene is known.) A total of 184 sites were polymorphic, and up to 6% sequence divergence was observed between pairs of strains. The deduced amino acid sequences showed much more variation than had been shown by multilocus enzyme electrophoresis, and in addition the net charge calculated did not correlate strongly with electrophoretic mobility. A phylogenetic tree for the sequences that was based on maximum parsimony differed significantly from a tree for the same strains that was based on multilocus enzyme electrophoresis for 35 enzymes (R. K. Selander, D. A. Caugant, and T. S. Whittam, p. 1625-1648, in F. C. Neidhardt, J. L. Ingraham, K. B. Low, B. Magasanik, M. Schaechter, and H. E. Umbarger, ed., Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology, 1987). These data, together with analysis of sequence variation between the strains, indicated that intragenic recombination and transfer of the whole of gnd have occurred in the evolution of these strains. There is evidence of one recombination event between E. coli and Salmonella typhimurium.
|
934. |
Perepelov AV,
Li D,
Liu B,
Senchenkova SN,
Guo D,
Shashkov AS,
Feng L,
Knirel YA,
Wang L,
( 2011 ) Structural and genetic characterization of the closely related O-antigens of Escherichia coli O85 and Salmonella enterica O17. PMID : 20501518 : DOI : 10.1177/1753425910369270 Abstract >>
O-Antigen is a part of the lipopolysaccharide present in the outer membrane of Gram-negative bacteria, which confers major antigenic variability to the cell surface. In this study, we report on a previously undefined pair of Escherichia coli and Salmonella enterica with closely related O-antigens. The O-polysaccharides were isolated from the lipopolysaccharides of E. coli O85 and S. enterica O17 by mild acid degradation and studied by sugar analysis and NMR spectroscopy. The following structure was established for the O-unit of the E. coli O85-polysaccharide: The S. enterica O17-polysaccharide has the same carbohydrate backbone and, in addition, contains an O-acetyl group at position 2 of ~80% �]-Galf residues. The O-antigen gene cluster of E. coli O85 was found to be closely related to that of S. enterica O17. Screening of type strains of all E. coli and S. enterica O-serogroups revealed two genes specific to the E. coli O85 O-antigen gene cluster, which can be used for development of PCR-based assays for identification and detection of E. coli O85 strains.
|
935. |
Geue L,
Schares S,
Mintel B,
Conraths FJ,
Müller E,
Ehricht R,
( 2010 ) Rapid microarray-based genotyping of enterohemorrhagic Escherichia coli serotype O156:H25/H-/Hnt isolates from cattle and clonal relationship analysis. PMID : 20581183 : DOI : 10.1128/AEM.00743-10 PMC : PMC2918981 Abstract >>
Since enterohemorrhagic Escherichia coli (EHEC) isolates of serogroup O156 have been obtained from human diarrhea patients and asymptomatic carriers, we studied cattle as a potential reservoir for these bacteria. E. coli isolates serotyped by agglutination as O156:H25/H-/Hnt strains (n = 32) were isolated from three cattle farms during a period of 21 months and characterized by rapid microarray-based genotyping. The serotyping by agglutination of the O156 isolates was not confirmed in some cases by the results of DNA-based serotyping as only 25 of the 32 isolates were conclusively identified as O156:H25. In the multilocus sequence typing (MLST) analysis, all EHEC O156:H25 isolates were characterized as sequence type 300 (ST300) and ST688, which differ by a single-nucleotide exchange in the purA gene. Oligonucleotide microarrays allow simultaneous detection of a wider range of EHEC-associated and other E. coli virulence markers than other methods. All O156:H25 isolates showed a wide spectrum of virulence factors typical for EHEC. The stx(1) genes combined with the EHEC hlyA (hlyA(EHEC)) gene, the eae gene of the zeta subtype, as well as numerous other virulence markers were present in all EHEC O156:H25 strains. The behavior of eight different cluster groups, including four that were EHEC O156:H25, was monitored in space and time. Variations in the O156 cluster groups were detected. The results of the cluster analysis suggest that some O156:H25 strains had the genetic potential for a long persistence in the host and on the farm, while other strains did not. As judged by their pattern of virulence markers, E. coli O156:H25 isolates of bovine origin may represent a considerable risk for human infection. Our results showed that the miniaturized E. coli oligonucleotide arrays are an excellent tool for the rapid detection of a large number of virulence markers.
|
936. |
Macmaster R,
Zelinskaya N,
Savic M,
Rankin CR,
Conn GL,
( 2010 ) Structural insights into the function of aminoglycoside-resistance A1408 16S rRNA methyltransferases from antibiotic-producing and human pathogenic bacteria. PMID : 20639535 : DOI : 10.1093/nar/gkq627 PMC : PMC2995053 Abstract >>
X-ray crystal structures were determined of the broad-spectrum aminoglycoside-resistance A1408 16S rRNA methyltransferases KamB and NpmA, from the aminoglycoside-producer Streptoalloteichus tenebrarius and human pathogenic Escherichia coli, respectively. Consistent with their common function, both are Class I methyltransferases with additional highly conserved structural motifs that embellish the core SAM-binding fold. In overall structure, the A1408 rRNA methyltransferase were found to be most similar to a second family of Class I methyltransferases of distinct substrate specificity (m(7)G46 tRNA). Critical residues for A1408 rRNA methyltransferase activity were experimentally defined using protein mutagenesis and bacterial growth assays with kanamycin. Essential residues for SAM coenzyme binding and an extended protein surface that likely interacts with the 30S ribosomal subunit were thus revealed. The structures also suggest potential mechanisms of A1408 target nucleotide selection and positioning. We propose that a dynamic extended loop structure that is positioned adjacent to both the bound SAM and a functionally critical structural motif may mediate concerted conformational changes in rRNA and protein that underpin the specificity of target selection and activation of methyltransferase activity. These new structures provide important new insights that may provide a starting point for strategies to inhibit these emerging causes of pathogenic bacterial resistance to aminoglycosides.
|
937. |
Miquel S,
Peyretaillade E,
Claret L,
de Vallée A,
Dossat C,
Vacherie B,
Zineb el H,
Segurens B,
Barbe V,
Sauvanet P,
Neut C,
Colombel JF,
Medigue C,
Mojica FJ,
Peyret P,
Bonnet R,
Darfeuille-Michaud A,
( 2010 ) Complete genome sequence of Crohn's disease-associated adherent-invasive E. coli strain LF82. PMID : 20862302 : DOI : 10.1371/journal.pone.0012714 PMC : PMC2941450 Abstract >>
Ileal lesions of Crohn's disease (CD) patients are abnormally colonized by pathogenic adherent-invasive Escherichia coli (AIEC) able to invade and to replicate within intestinal epithelial cells and macrophages. We report here the complete genome sequence of E. coli LF82, the reference strain of adherent-invasive E. coli associated with ileal Crohn's disease. The LF82 genome of 4,881,487 bp total size contains a circular chromosome with a size of 4,773,108 bp and a plasmid of 108,379 bp. The analysis of predicted coding sequences (CDSs) within the LF82 flexible genome indicated that this genome is close to the avian pathogenic strain APEC_01, meningitis-associated strain S88 and urinary-isolated strain UTI89 with regards to flexible genome and single nucleotide polymorphisms in various virulence factors. Interestingly, we observed that strains LF82 and UTI89 adhered at a similar level to Intestine-407 cells and that like LF82, APEC_01 and UTI89 were highly invasive. However, A1EC strain LF82 had an intermediate killer phenotype compared to APEC-01 and UTI89 and the LF82 genome does not harbour most of specific virulence genes from ExPEC. LF82 genome has evolved from those of ExPEC B2 strains by the acquisition of Salmonella and Yersinia isolated or clustered genes or CDSs located on pLF82 plasmid and at various loci on the chromosome. LF82 genome analysis indicated that a number of genes, gene clusters and pathoadaptative mutations which have been acquired may play a role in virulence of AIEC strain LF82.
|
938. |
Ong CL,
Beatson SA,
Totsika M,
Forestier C,
McEwan AG,
Schembri MA,
( 2010 ) Molecular analysis of type 3 fimbrial genes from Escherichia coli, Klebsiella and Citrobacter species. PMID : 20576143 : DOI : 10.1186/1471-2180-10-183 PMC : PMC2900259 Abstract >>
Catheter-associated urinary tract infection (CAUTI) is the most common nosocomial infection in the United States and is caused by a range of uropathogens. Biofilm formation by uropathogens that cause CAUTI is often mediated by cell surface structures such as fimbriae. In this study, we characterised the genes encoding type 3 fimbriae from CAUTI strains of Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Citrobacter koseri and Citrobacter freundii. Phylogenetic analysis of the type 3 fimbrial genes (mrkABCD) from 39 strains revealed they clustered into five distinct clades (A-E) ranging from one to twenty-three members. The majority of sequences grouped in clade A, which was represented by the mrk gene cluster from the genome sequenced K. pneumoniae MGH78578. The E. coli and K. pneumoniae mrkABCD gene sequences clustered together in two distinct clades, supporting previous evidence for the occurrence of inter-genera lateral gene transfer. All of the strains examined caused type 3 fimbriae mediated agglutination of tannic acid treated human erythrocytes despite sequence variation in the mrkD-encoding adhesin gene. Type 3 fimbriae deletion mutants were constructed in 13 representative strains and were used to demonstrate a direct role for type 3 fimbriae in biofilm formation. The expression of functional type 3 fimbriae is common to many Gram-negative pathogens that cause CAUTI and is strongly associated with biofilm growth. Our data provides additional evidence for the spread of type 3 fimbrial genes by lateral gene transfer. Further work is now required to substantiate the clade structure reported here by examining more strains as well as other bacterial genera that make type 3 fimbriae and cause CAUTI.
|
939. |
Cimini D,
De Rosa M,
Viggiani A,
Restaino OF,
Carlino E,
Schiraldi C,
( 2010 ) Improved fructosylated chondroitin production by kfoC overexpression in E. coli K4. PMID : 20888875 : DOI : 10.1016/j.jbiotec.2010.09.954 Abstract >>
Escherichia coli K4 is one of the bacteria expressing a surface polysaccharide, indicated as capsular polysaccharide (K-antigen), showing a chemical structure that resembles that of metabolites commonly used in pharmaceutical applications. In this study we provide evidence that homologous overexpression of the chondroitin polymerase, encoded by the kfoC gene, acts on a potential bottleneck for production of capsular polysaccharide, and increases productivity by 100%. However, we also demonstrate that genetic engineering and scale-up of the production process with E. coli K4 is not straight forward due to genetic instability of recombinant strains, partly overcome by multiple additions of antibiotic throughout fermentation that prove to increase plasmid maintenance inside the cells. A lower resistance to the antibiotic was nevertheless highlighted in the stationary phase suggesting other concomitant causes for plasmid instability. The latter might partly be related to a newly discovered endogenous mobile element that we indicate as pK4EC05. Sequencing and analysis of a 1900 bp fragment of pK4EC05 shows a high percentage of sequence similarity to large conjugative plasmids isolated from Shigella, Salmonella and E. coli strains.
|
940. |
Dai J,
Wang S,
Guerlebeck D,
Laturnus C,
Guenther S,
Shi Z,
Lu C,
Ewers C,
( 2010 ) Suppression subtractive hybridization identifies an autotransporter adhesin gene of E. coli IMT5155 specifically associated with avian pathogenic Escherichia coli (APEC). PMID : 20828376 : DOI : 10.1186/1471-2180-10-236 PMC : PMC2944236 Abstract >>
Extraintestinal pathogenic E. coli (ExPEC) represent a phylogenetically diverse group of bacteria which are implicated in a large range of infections in humans and animals. Although subgroups of different ExPEC pathotypes, including uropathogenic, newborn meningitis causing, and avian pathogenic E. coli (APEC) share a number of virulence features, there still might be factors specifically contributing to the pathogenesis of a certain subset of strains or a distinct pathotype. Thus, we made use of suppression subtractive hybridization and compared APEC strain IMT5155 (O2:K1:H5; sequence type complex 95) with human uropathogenic E. coli strain CFT073 (O6:K2:H5; sequence type complex 73) to identify factors which may complete the currently existing model of APEC pathogenicity and further elucidate the position of this avian pathotype within the whole ExPEC group. Twenty-eight different genomic loci were identified, which are present in IMT5155 but not in CFT073. One of these loci contained a gene encoding a putative autotransporter adhesin. The open reading frame of the gene spans a 3,498 bp region leading to a putative 124-kDa adhesive protein. A specific antibody was raised against this protein and expression of the adhesin was shown under laboratory conditions. Adherence and adherence inhibition assays demonstrated a role for the corresponding protein in adhesion to DF-1 chicken fibroblasts. Sequence analyses revealed that the flanking regions of the chromosomally located gene contained sequences of mobile genetic elements, indicating a probable spread among different strains by horizontal gene transfer. In accordance with this hypothesis, the adhesin was found to be present not only in different phylogenetic groups of extraintestinal pathogenic but also of commensal E. coli strains, yielding a significant association with strains of avian origin. We identified a chromosomally located autotransporter gene in a highly virulent APEC strain which confers increased adherence of a non-fimbriated E. coli K-12 strain to a chicken fibroblast cell line. Even though flanked by mobile genetic elements and three different genetic regions upstream of the gene, most probably indicating horizontal gene transfer events, the adhesin gene was significantly linked with strains of avian origin. Due to the nucleotide sequence similarity of 98% to a recently published adhesin-related gene, located on plasmid pAPEC-O1-ColBM, the name aatA (APEC autotransporter adhesin A) was adopted from that study.Our data substantiate that AatA might not only be of relevance in APEC pathogenicity but also in facilitating their reservoir life style in the chicken intestine, which might pave the way for future intestinal preventive strategies.
|
941. |
Wang Q,
Ruan X,
Wei D,
Hu Z,
Wu L,
Yu T,
Feng L,
Wang L,
( 2010 ) Development of a serogroup-specific multiplex PCR assay to detect a set of Escherichia coli serogroups based on the identification of their O-antigen gene clusters. PMID : 20561581 : DOI : 10.1016/j.mcp.2010.06.002 Abstract >>
The Escherichia coli serogroups O115, O126, O137, O158, O165, and O173 are pathogenic strains associated with diarrhea. Molecular approaches such as PCR have been proven to be rapid, inexpensive, and accurate. The sequences of the O-antigen-processing genes wzx and wzy are specific for different O antigens and are generally used as the target genes for the detection and identification of E. coli strains belonging to different O serogroups. In this report, the O-antigen gene clusters of these 6 O serogroups were sequenced, and genes were identified on the basis of homology. By screening these sequences against all 186 E. coli and Shigella strains, we found that the sequences of the wzx and wzy genes were serogroup-specific, and 2 specific primer pairs for each serogroup were screened out. A multiplex PCR assay targeting all 6 serogroups was developed. Twenty-nine strains were used to validate the specificity of the assay. The detection sensitivity was 1ng genomic DNA. As the assay was shown to be accurate and sensitive, it can be used for the identification and detection of strains belonging to these serogroups in stool and other environmental samples after being isolated by culture.
|
942. |
Li D,
Liu B,
Chen M,
Guo D,
Guo X,
Liu F,
Feng L,
Wang L,
( 2010 ) A multiplex PCR method to detect 14 Escherichia coli serogroups associated with urinary tract infections. PMID : 20434495 : DOI : 10.1016/j.mimet.2010.04.008 Abstract >>
Urinary tract infections (UTIs) are one of the most common bacterial infections and are predominantly caused by uropathogenic Escherichia coli (UPEC). E. coli strains belonging to 14 serogroups, including O1, O2, O4, O6, O7, O8, O15, O16, O18, O21, O22, O25, O75 and O83, are the most frequently detected UPEC strains in a diverse range of clinical urine specimens. In the current study, the O-antigen gene clusters of E. coli serogroups O1, O2, O18 and O75 were characterized. A multiplex PCR method based on O-antigen-specific genes was developed for the simultaneous detection of all 14 E. coli serogroups. The multiplex PCR method was shown to be highly specific and reproducible when tested against 186 E. coli and Shigella O-serogroup reference strains, 47 E. coli clinical isolates and 10 strains of other bacterial species. The sensitivity of the multiplex PCR method was analyzed and shown to detect O-antigen-specific genes in samples containing 25 ng of genomic DNA or in mock urine specimens containing 40 colony-forming units (CFUs) per ml. Five urine specimens from hospital were examined using this multiplex PCR method, and the result for one sample was verified by the conventional serotyping methods. The multiplex PCR method developed herein can be used for the detection of relevant E. coli strains from clinical and/or environmental samples, and it is particularly useful for epidemiologic analysis of urine specimens from patients with UTIs.
|
943. |
Woodward R,
Yi W,
Li L,
Zhao G,
Eguchi H,
Sridhar PR,
Guo H,
Song JK,
Motari E,
Cai L,
Kelleher P,
Liu X,
Han W,
Zhang W,
Ding Y,
Li M,
Wang PG,
( 2010 ) In vitro bacterial polysaccharide biosynthesis: defining the functions of Wzy and Wzz. PMID : 20418877 : DOI : 10.1038/nchembio.351 PMC : PMC2921718 Abstract >>
Polysaccharides constitute a major component of bacterial cell surfaces and play critical roles in bacteria-host interactions. The biosynthesis of such molecules, however, has mainly been characterized through in vivo genetic studies, thus precluding discernment of the details of this pathway. Accordingly, we present a chemical approach that enabled reconstitution of the E. coli O-polysaccharide biosynthetic pathway in vitro. Starting with chemically prepared undecaprenyl-diphospho-N-acetyl-D-galactosamine, the E. coli O86 oligosaccharide repeating unit was assembled by means of sequential enzymatic glycosylation. Successful expression of the putative polymerase Wzy using a chaperone coexpression system then allowed demonstration of polymerization in vitro using this substrate. Analysis of more substrates revealed a defined mode of recognition for Wzy toward the lipid moiety. Specific polysaccharide chain length modality was furthermore demonstrated to result from the action of Wzz. Collectively, polysaccharide biosynthesis was chemically reconstituted in vitro, providing a well defined system for further underpinning molecular details of this biosynthetic pathway.
|
944. |
Li X,
Perepelov AV,
Wang Q,
Senchenkova SN,
Liu B,
Shevelev SD,
Guo X,
Shashkov AS,
Chen W,
Wang L,
Knirel YA,
( 2010 ) Structural and genetic characterization of the O-antigen of Escherichia coli O161 containing a derivative of a higher acidic diamino sugar, legionaminic acid. PMID : 20510395 : DOI : 10.1016/j.carres.2010.04.008 Abstract >>
The O-antigen is an essential component of lipopolysaccharide on the surface of Gram-negative bacteria and plays an important role in its pathogenicity. Composition and structure of the O-antigens of Escherichia coli are highly diverse mainly due to genetic variations in the O-antigen gene cluster. In this work, the chemical structure and the gene cluster of the O-antigen of E. coli O161 were studied. Chemical degradations, sugar analyses, and NMR spectroscopy showed that the O161 antigen possesses a trisaccharide O-repeating unit containing a 5-N-acetyl-7-N-(d-alanyl) derivative of 5,7-diamino-3,5,7,9-tetradeoxy-d-glycero-d-galacto-non-2-ulosonic (legionaminic) acid (Leg5Ac7Ala) and having the following structure: The O-antigen gene cluster of E. coli O161 was sequenced. In addition to the genes encoding sugar transferases, O-repeating unit flippase (Wzx) and O-antigen polymerase (Wzy), the genes involved in the biosynthesis of a legionaminic acid derivative were identified based on database similarities.
|
945. |
Prager R,
Fruth A,
Busch U,
Tietze E,
( 2011 ) Comparative analysis of virulence genes, genetic diversity, and phylogeny of Shiga toxin 2g and heat-stable enterotoxin STIa encoding Escherichia coli isolates from humans, animals, and environmental sources. PMID : 20728406 : DOI : 10.1016/j.ijmm.2010.06.003 Abstract >>
An analysis for stx(2) variants among the 2010 human stx(2)-positive Shiga toxin-producing Escherichia coli (STEC) strains from Germany collected at the National Reference Centre 1999-2008 revealed 0.6% to possess the recently described stx(2g) gene. Sequencing of the whole stx(2g) operons showed new alleles and pseudogenes. The further molecular, phenotypic, and phylogenetic comparison of 12 human stx(2g)-harbouring isolates with 12 stx(2g)-harbouring isolates from animals or environmental sources demonstrated that both groups are closely related, indicating the human infections as a potential zoonotic disease. Although originating from various different sources, the stx(2g)-containing strains belong to only 3 phylogenetic lineages, represented by 4 serovars belonging to 4 sequence types. In view of the huge diversity among other STEC, this suggests the emergence of the stx(2g) variant as a rather recent microevolutionary event. Interestingly, in the strains under investigation, Stx2g was not expressed. However, all of them contained the estIa gene which typically is associated with enterotoxin-producing E. coli and did express STIa. By this combination of virulence genes of different pathotypes of intestinal pathogenic E. coli, these strains represent a new, intermediate pathotype and emerging pathogens. Given a rising number of intermediate pathotypes becoming described among E. coli, a wider range of virulence markers should be included in the regular pathotype diagnostics.
|
946. |
Dawes FE,
Kuzevski A,
Bettelheim KA,
Hornitzky MA,
Djordjevic SP,
Walker MJ,
( 2010 ) Distribution of class 1 integrons with IS26-mediated deletions in their 3'-conserved segments in Escherichia coli of human and animal origin. PMID : 20856797 : DOI : 10.1371/journal.pone.0012754 PMC : PMC2939871 Abstract >>
Class 1 integrons play a role in the emergence of multi-resistant bacteria by facilitating the recruitment of gene cassettes encoding antibiotic resistance genes. 512 E. coli strains sourced from humans (n = 202), animals (n = 304) and the environment (n = 6) were screened for the presence of the intI1 gene. In 31/79 integron positive E. coli strains, the gene cassette regions could not be PCR amplified using standard primers. DNA sequence analysis of 6 serologically diverse strains revealed atypical integrons harboured the dfrA5 cassette gene and only 24 bp of the integron 3'-conserved segment (CS) remained, due to the insertion of IS26. PCR targeting intI1 and IS26 followed by restriction fragment length polymorphism (RFLP) analysis identified the integron-dfrA5-IS26 element in 27 E. coli strains of bovine origin and 4 strains of human origin. Southern hybridization and transformation studies revealed the integron-dfrA5-IS26 gene arrangement was either chromosomally located or plasmid borne. Plasmid location in 4/9 E. coli strains and PCR linkage of Tn21 transposition genes with the intI1 gene in 20/31 strains, suggests this element is readily disseminated by horizontal transfer.
|
947. |
Chak KF,
Kuo WS,
Lu FM,
James R,
( 1991 ) Cloning and characterization of the ColE7 plasmid. PMID : 2045785 : DOI : 10.1099/00221287-137-1-91 Abstract >>
The 6.2 kb ColE7-K317 plasmid was mapped and the DNA fragments of the colicin E7 operon subcloned into pUC18 and pUC19. The size of the functional colicin E7 operon deduced by subcloning was 2.3 kb. The colicin E7 gene product was purified by carboxymethylcellulose chromatography. Both colicin E7 and E9 were demonstrated to exhibit a non-specific DNAase-type activity by in vitro biological assay. The molecular mass of colicin E7 was 61 kDa, as determined by SDS-PAGE. From DNA sequence data, the estimated sizes of the E7 immunity protein and the E7 lysis protein were 9926 Da and 4847 Da, respectively. Comparison of restriction maps and DNA sequence data suggests that ColE7 and ColE2 are more closely related than other E colicin plasmids.
|
948. |
Mak AN,
Lambert AR,
Stoddard BL,
( 2010 ) Folding, DNA recognition, and function of GIY-YIG endonucleases: crystal structures of R.Eco29kI. PMID : 20800503 : DOI : 10.1016/j.str.2010.07.006 PMC : PMC2955809 Abstract >>
The GIY-YIG endonuclease family comprises hundreds of diverse proteins and a multitude of functions; none have been visualized bound to DNA. The structure of the GIY-YIG restriction endonuclease R.Eco29kI has been solved both alone and bound to its target site. The protein displays a domain-swapped homodimeric structure with several extended surface loops encircling the DNA. Only three side chains from each protein subunit contact DNA bases, two directly and one via a bridging solvent molecule. Both tyrosine residues within the GIY-YIG motif are positioned in the catalytic center near a putative nucleophilic water; the remainder of the active site resembles the HNH endonuclease family. The structure illustrates how the GIY-YIG scaffold has been adapted for the highly specific recognition of a DNA restriction site, in contrast to nonspecific DNA cleavage by GIY-YIG domains in homing endonucleases or structure-specific cleavage by DNA repair enzymes such as UvrC.
|
949. |
Li D,
Yu T,
Zhang Y,
Yang M,
Li Z,
Liu M,
Qi R,
( 2010 ) Antibiotic resistance characteristics of environmental bacteria from an oxytetracycline production wastewater treatment plant and the receiving river. PMID : 20400569 : DOI : 10.1128/AEM.02964-09 PMC : PMC2876458 Abstract >>
We characterized the bacterial populations in surface water receiving effluent from an oxytetracycline (OTC) production plant. Additional sampling sites included the receiving river water 5 km upstream and 20 km downstream from the discharge point. High levels of OTC were found in the wastewater (WW), and the antibiotic was still detectable in river water downstream (RWD), with undetectable levels in river water upstream (RWU). A total of 341 bacterial strains were isolated using nonselective media, with the majority being identified as Gammaproteobacteria. The MICs were determined for 10 antibiotics representing seven different classes of antibiotics, and the corresponding values were significantly higher for the WW and RWD isolates than for the RWU isolates. Almost all bacteria (97%) from the WW and RWD samples demonstrated multidrug-resistant (MDR) phenotypes, while in RWU samples, these were less frequent (28%). The WW and RWD isolates were analyzed for the presence of 23 tetracycline (tet) resistance genes. The majority of isolates (94.2% and 95.4% in WW and RWD, respectively) harbored the corresponding genes, with tet(A) being the most common (67.0%), followed by tet(W), tet(C), tet(J), tet(L), tet(D), tet(Y), and tet(K) (in the range between 21.0% and 40.6%). Class I integrons were detected in the majority of WW and RWD isolates (97.4% and 86.2%, respectively) but were not associated with the tet genes. We hypothesize that the strong selective pressure imposed by a high concentration of OTC contributes to the wide dissemination of tetracycline resistance genes and other antibiotic resistance genes, possibly through mobile genetic elements.
|
950. |
Nakata K,
Koh MM,
Tsuchido T,
Matsumura Y,
( 2010 ) All genomic mutations in the antimicrobial surfactant-resistant mutant, Escherichia coli OW66, are involved in cell resistance to surfactant. PMID : 20480162 : DOI : 10.1007/s00253-010-2638-8 Abstract >>
The spontaneous antimicrobial surfactant-resistant mutant, Escherichia coli OW66, has been isolated, and its physiological properties have been characterized in our previous paper (Ishikawa et al., J Appl Microbiol 92:261-268, 2002b). This report revealed that strain OW66 had seven mutations in their chromosomal DNA by comparative genomic hybridization microarray, and that their alternative functions were involved in cell resistance to antimicrobial surfactants. These mutations were located in oppB, ydcR, IVR(vacJ-yfdC), rpoN, rpoB, rpoC, and soxR. Furthermore, seven of the single-mutated isogenic strains and seven of the six-mutated isogenic strains were constructed from strains OW6 (NBRC106482) and OW66, respectively, through homologous recombination, and their resistances to an antimicrobial surfactant were measured using the minimum inhibitory concentration method. These results revealed that all six-mutated strains were more sensitive than strain OW66, and that the soxR66 mutation was independently involved in antimicrobial surfactant resistance of E. coli cells. Expression of soxR66 and soxS was increased in both strains OW66 and OW6-soxR66 without the surfactant treatment by the quantitative real time-polymerase chain reaction analysis, compared with strain OW6. Two-dimensional polyacrylamide gel electrophoresis analysis also revealed that some proteins in the soxRS regulon, including Mn-SOD, were overexpressed in both strains OW66 and OW6-soxR66. These results indicate that the soxR66 mutation leads to the constitutive expression of the soxRS regulon, resulting in the acquired resistance of E. coli cells to an antimicrobial surfactant.
|
951. |
Wachino J,
Yamane K,
Suzuki S,
Kimura K,
Arakawa Y,
( 2010 ) Prevalence of fosfomycin resistance among CTX-M-producing Escherichia coli clinical isolates in Japan and identification of novel plasmid-mediated fosfomycin-modifying enzymes. PMID : 20404116 : DOI : 10.1128/AAC.01834-09 PMC : PMC2897269 Abstract >>
We evaluated the in vitro activity of fosfomycin against a total of 192 CTX-M beta-lactamase-producing Escherichia coli strains isolated in 70 Japanese clinical settings. Most of the isolates (96.4%) were found to be susceptible to fosfomycin. On the other hand, some of the resistant isolates were confirmed to harbor the novel transferable fosfomycin resistance determinants named FosA3 and FosC2, which efficaciously inactivate fosfomycin through glutathione S-transferase activity.
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952. |
Povilonis J,
?eputien? V,
Ružauskas M,
?iugždinien? R,
Virgailis M,
Pavilonis A,
Sužied?lien? E,
( 2010 ) Transferable class 1 and 2 integrons in Escherichia coli and Salmonella enterica isolates of human and animal origin in Lithuania. PMID : 20578916 : DOI : 10.1089/fpd.2010.0536 Abstract >>
Antibiotic-resistant Escherichia coli (n = 191) and Salmonella enterica (n = 87) isolates of human and animal origin obtained in Lithuania during 2005-2008 were characterized for the presence and diversity of class 1 and 2 integrons. E. coli isolates were obtained from patients with urinary tract infections (UTIs) (n = 59) and both healthy and diseased farm animals, including poultry (n = 54), swine (n = 35), and cattle (n = 43). Isolates of non-typhoidal S. enterica were recovered from salmonellosis patients (n = 37) and healthy animals, including poultry (n = 31) and swine (n = 19). The presence of integrons, their gene cassette structure, and genome location were investigated by polymerase chain reaction, restriction fragment-length polymorphism, DNA sequencing, Southern blot hybridization, and conjugation experiments. Forty percent of the E. coli and 11% of the S. enterica isolates carried class 1 integrons, whereas class 2 integrons were found in E. coli isolates (9%) only. The incidence of integrons in human UTIs and cattle isolates was most frequent (p < 0.01). A total of 23 different gene cassettes within 15 different variable regions were observed. Seven different integron types, all of them transferable by conjugation, were common for isolates from human infections and for one or more groups of animal isolates. The most prevalent integron types contained arrays dfrA1-aadA1 (36%), dfrA17-aadA5 (23%), and dfrA1-sat1-aadA1 (78%). Two E. coli isolates from humans with UTIs harbored class 1 integron on conjugative plasmid with the novel array type of 4800 bp/dfrA17-aadA5�G-IS26-�GintI1-aadB-aadA1-cmlA residing on the Tn21-like transposon. Three S. enterica isolates from swine contained class 1 integron with the newly observed array type of 1800 bp/aadA7-aadA7. Integrons of 10 different types of both classes were located on transferable plasmids in E. coli and S. enterica. Our study demonstrated the existence of a considerable and common pool of transferable integrons in E. coli and S. enterica present in clinical and livestock environment in Lithuania.
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953. |
Bardiau M,
Grégoire F,
Muylaert A,
Nahayo A,
Duprez JN,
Mainil J,
Linden A,
( 2010 ) Enteropathogenic (EPEC), enterohaemorragic (EHEC) and verotoxigenic (VTEC) Escherichia coli in wild cervids. PMID : 20880133 : DOI : 10.1111/j.1365-2672.2010.04855.x Abstract >>
The aim of this study was to investigate the presence of enteropathogenic (EPEC), enterohaemorragic (EHEC) and verotoxigenic (VTEC) Escherichia coli strains in free-ranging wild ruminants in Belgium and to characterize the positive isolates (serogroups and virulence-associated factor-encoding genes). Escherichia coli strains isolated from faeces of wild cervids were characterized by PCR targeting genes coding for the main virulence properties of EPEC, EHEC and VTEC strains. The prevalence rate of these pathogenic strains in faecal samples obtained from the wild ruminants was found to be 15%. No pathogenic isolate was found to belong to the O157, O26, O111, O103 or O145 serogroups. Moreover, a new gene, eibH, showing 88% identity with eibG was detected in VTEC strains. The results reveal that wild ruminants could be considered as a potential source of VTEC and EPEC infection for humans and possibly also for domestic ruminants. Our study suggests the potential risk of transmission of VTEC, EHEC and EPEC strains from wild ruminants to humans via the consumption of venison and to domestic ruminants because of sharing of the same pasture. Indeed, many serogroups other than O157 EHEC have also been shown to be responsible for outbreaks in humans in several countries, and studies focusing solely on O157:H7 EHEC tend to underestimate this risk of transmission.
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954. |
Klaasen P,
de Graaf FK,
( 1990 ) Characterization of FapR, a positive regulator of expression of the 987P operon in enterotoxigenic Escherichia coli. PMID : 2077360 : DOI : 10.1111/j.1365-2958.1990.tb00556.x Abstract >>
Expression of the 987P gene cluster is activated by the adjacent IS1 element of an STpa transposon. Nucleotide sequence analysis of the 987P-DNA region contiguous with this IS1 element revealed the presence of an open reading frame designated fapR, encoding a basic protein of 260 amino acid residues with a molecular mass of 30,349 Daltons. The gene product, FapR, possesses similarity to a number of positive regulators of gene expression: VirF, Rns, AppY and EnvY. Moreover, a 43-amino-acid residue sequence in the C-terminal part of FapR is similar to the C-terminal domain of AraC, RhaR, and RhaS. Expression of fapR is dependent on the adjacent IS1 element. The FapR protein appears to be required for activation of the silent promoter of the fimbrial subunit gene, fapC.
|
955. |
Oke M,
Carter LG,
Johnson KA,
Liu H,
McMahon SA,
Yan X,
Kerou M,
Weikart ND,
Kadi N,
Sheikh MA,
Schmelz S,
Dorward M,
Zawadzki M,
Cozens C,
Falconer H,
Powers H,
Overton IM,
van Niekerk CA,
Peng X,
Patel P,
Garrett RA,
Prangishvili D,
Botting CH,
Coote PJ,
Dryden DT,
Barton GJ,
Schwarz-Linek U,
Challis GL,
Taylor GL,
White MF,
Naismith JH,
( 2010 ) The Scottish Structural Proteomics Facility: targets, methods and outputs. PMID : 20419351 : DOI : 10.1007/s10969-010-9090-y PMC : PMC2883930 Abstract >>
The Scottish Structural Proteomics Facility was funded to develop a laboratory scale approach to high throughput structure determination. The effort was successful in that over 40 structures were determined. These structures and the methods harnessed to obtain them are reported here. This report reflects on the value of automation but also on the continued requirement for a high degree of scientific and technical expertise. The efficiency of the process poses challenges to the current paradigm of structural analysis and publication. In the 5 year period we published ten peer-reviewed papers reporting structural data arising from the pipeline. Nevertheless, the number of structures solved exceeded our ability to analyse and publish each new finding. By reporting the experimental details and depositing the structures we hope to maximize the impact of the project by allowing others to follow up the relevant biology.
|
956. |
Lehti TA,
Bauchart P,
Heikkinen J,
Hacker J,
Korhonen TK,
Dobrindt U,
Westerlund-Wikström B,
( 2010 ) Mat fimbriae promote biofilm formation by meningitis-associated Escherichia coli. PMID : 20522494 : DOI : 10.1099/mic.0.039610-0 Abstract >>
The mat (or ecp) fimbrial operon is ubiquitous and conserved in Escherichia coli, but its functions remain poorly described. In routine growth media newborn meningitis isolates of E. coli express the meningitis-associated and temperature-regulated (Mat) fimbria, also termed E. coli common pilus (ECP), at 20 degrees C, and here we show that the six-gene (matABCDEF)-encoded Mat fimbria is needed for temperature-dependent biofilm formation on abiotic surfaces. The matBCDEF deletion mutant of meningitis E. coli IHE 3034 was defective in an early stage of biofilm development and consequently unable to establish a detectable biofilm, contrasting with IHE 3034 derivatives deleted for flagella, type 1 fimbriae or S-fimbriae, which retained the wild-type biofilm phenotype. Furthermore, induced production of Mat fimbriae from expression plasmids enabled biofilm-deficient E. coli K-12 cells to form biofilm at 20 degrees C. No biofilm was detected with IHE 3034 or MG1655 strains grown at 37 degrees C. The surface expression of Mat fimbriae and the frequency of Mat-positive cells in the IHE 3034 population from 20 degrees C were high and remained unaltered during the transition from planktonic to biofilm growth and within the matured biofilm community. Considering the prevalence of the highly conserved mat locus in E. coli genomes, we hypothesize that Mat fimbria-mediated biofilm formation is an ancestral characteristic of E. coli.
|
957. |
Goncharoff P,
Saadi S,
Chang CH,
Saltman LH,
Figurski DH,
( 1991 ) Structural, molecular, and genetic analysis of the kilA operon of broad-host-range plasmid RK2. PMID : 2045366 : DOI : 10.1128/jb.173.11.3463-3477.1991 PMC : PMC207960 Abstract >>
The kil loci (kilA, kilB, kilC, and kilE) of incompatibility group P (IncP), broad-host-range plasmid RK2 were originally detected by their potential lethality to Escherichia coli host cells. Expression of the kil determinants is controlled by different combinations of kor functions (korA, korB, korC, and korE). This system of regulated genes, known as the kil-kor regulon, includes trfA, which encodes the RK2 replication initiator. The functions of the kil loci are unknown, but their coregulation with an essential replication function suggests that they have a role in the maintenance or host range of RK2. In this study, we have determined the nucleotide sequence of a 3-kb segment of RK2 that encodes the entire kilA locus. The region encodes three genes, designated klaA, klaB, and klaC. The phage T7 RNA polymerase-dependent expression system was use to identify three polypeptide products. The estimated masses of klaA and klaB products were in reasonable agreement with the calculated molecular masses of 28,407 and 42,156 Da, respectively. The klaC product is calculated to be 32,380 Da, but the observed polypeptide exhibited an apparent mass of 28 kDa on sodium dodecyl sulfate-polyacrylamide gels. Mutants of klaC were used to confirm that initiation of translation of the observed product occurs at the first ATG in the klaC open reading frame. Hydrophobicity analysis indicated that the KlaA and KlaB polypeptides are likely to be soluble, whereas the KlaC polypeptide was predicted to have four potential membrane-spanning domains. The only recognizable promoter sequences in the kilA region were those of the kilA promoter located upstream of klaA and the promoter for the korA-korB operon located just downstream of a rho-independent terminatorlike sequence following klaC. The transcriptional start sites for these promoters were determined by primer extension. Using isogenic sets of plasmids with nonpolar mutations, we found that klaA, klaB, and klaC are each able to express a host-lethal (Kil+) phenotype in the absence of kor functions. Inactivation of the kilA promoter causes loss of the lethal phenotype, demonstrating that all three genes are expressed from the kilA promoter as a multicistronic operon. We investigated two other phenotypes that have been mapped to the kilA region of RK2 or the closely related IncP plasmids RP1 and RP4: inhibition of conjugal transfer of IncW plasmids (fwB) and resistance to potassium tellurite. The cloned kilA operon was found to express both phenotypes, even in the presence of korA and korB, whose functions are known to regulate the kilA promoter. In addition, mutant and complementation analyses showed that the kilA promoter and the products of all three kla genes are necessary for expression of both phenotypes. Therefore, host lethality, fertility inhibition, and tellurite resistance are all properties of the kilA operon. We discuss the possible role of the kilA operon for RK2.
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958. |
Hu B,
Perepelov AV,
Liu B,
Shevelev SD,
Guo D,
Senchenkova SN,
Shashkov AS,
Feng L,
Knirel YA,
Wang L,
( 2010 ) Structural and genetic evidence for the close relationship between Escherichia coli O71 and Salmonella enterica O28 O-antigens. PMID : 20482625 : DOI : 10.1111/j.1574-695X.2010.00676.x Abstract >>
O-antigen is the most variable cell wall constituent of Gram-negative bacteria. Escherichia coli and Salmonella enterica are closely related species. In this work, we present structural and genetic evidence for the close relationship between O-antigens of E. coli O71 and S. enterica O28. The E. coli O71 O-antigen was found to consist of tetrasaccharide-repeating units containing d-GalpNAc, d-Galp, l-Rhap, and d-Quip3NAc, with multiple O-acetyl lateral groups. It is very similar to the known structure of the S. enterica O28 O-antigen, which has the same backbone units, but with a lateral Glc residue instead of O-acetyl groups. The O-antigen gene clusters of E. coli O71 and S. enterica O28 were sequenced and found to contain the same genes with high-level similarity. All of the genes expected for the synthesis of the common backbone structure of the two O-antigens were identified based on homology. It is proposed that the two gene clusters had originated from the same ancestor, and diverged by acquiring prophage genes to carry out side-chain modifications. This is a new pair of the closely related E. coli and S. enterica O-serogroups. The serogroup-specific genes of E. coli O71 and S. enterica O28 were also identified.
|
959. |
Berthiaume F,
Leblond MF,
Harel J,
Mourez M,
( 2010 ) Growth-phase-dependent expression of the operon coding for the glycosylated autotransporter adhesin AIDA-I of pathogenic Escherichia coli. PMID : 20831592 : DOI : 10.1111/j.1574-6968.2010.02088.x Abstract >>
The adhesin involved in diffuse adherence (AIDA-I) is an autotransporter found in pathogenic strains of Escherichia coli causing diarrhea in humans and pigs. The AIDA-I protein is glycosylated by a specific enzyme, the AIDA-associated heptosyltransferase (Aah). The aah gene is immediately upstream of the aidA gene, suggesting that they form an operon. However, the mechanisms of regulation of the aah and aidA genes are unknown. Using a clinical E. coli isolate expressing AIDA-I, we identified two putative promoters 149 and 128 nucleotides upstream of aah. Using qRT-PCR, we observed that aah and aidA are transcribed in a growth-dependent fashion, mainly at the start of the stationary phase. Western blotting confirmed that protein expression follows the same pattern. Using a fusion to a reporter gene, we observed that the regulation of the isolated aah promoter matched this transcription and expression pattern. Lastly, we found glucose to be a repressor and nutrient starvation to be an inducer. Taken together, our results suggest that, in the strain and the conditions we studied, aah-aidA is transcribed as a bicistronic message from a promoter upstream of aah, with maximal expression under conditions of nutrient limitation such as high cell density.
|
960. |
Fratamico PM,
Yan X,
Liu Y,
DebRoy C,
Byrne B,
Monaghan A,
Fanning S,
Bolton D,
( 2010 ) Escherichia coli serogroup O2 and O28ac O-antigen gene cluster sequences and detection of pathogenic E. coli O2 and O28ac by PCR. PMID : 20453897 : DOI : 10.1139/w10-010 Abstract >>
The O-antigen gene clusters of Escherichia coli serogroups O2 and O28ac were sequenced, and PCR assays were developed to identify strains belonging to these 2 serogroups. Sixteen and 8 open reading frames were mapped to these loci in E. coli O2:H4 U 9-41 and E. coli O28ac:H25 96-3286, respectively. The wzx (O-antigen flippase) and wzy (O-antigen polymerase) genes in the E. coli O2 and O28ac O-antigen gene clusters were selected as targets for PCR assays for their identification. PCR assays targeting the wzx and wzy genes were specific for these serogroups, with one exception. Escherichia coli serogroup O42 strains gave positive results with wzx and wzy PCR assays targeting E. coli O28ac, and antiserum raised against O42 cross-reacted with serogroup O28ac strains. The O-antigen gene cluster of a strain of E. coli serogroup O42 was sequenced, and there were only 3 nt differences between the O-antigen gene clusters of the O28ac and O42 strains. Multiplex PCR assays targeting the O2 wzx gene, the stx1, stx2, hly, eae, and saa genes, and the O28ac wzx, ial, ipaC, and ipaH genes were developed for detecting Shiga toxin-producing E. coli O2 strains and enteroinvasive E. coli O28ac strains, respectively. The O2 and O28ac wzx and wzy genes can be used as diagnostic markers in PCR assays for rapid identification of these serogroups as an alternative to serotyping, and the multiplex PCR assays targeting serogroup-specific genes in combination with virulence genes can be used to identify and to detect pathogenic serogroup O2 and O28ac strains.
|
961. |
Bailey JK,
Pinyon JL,
Anantham S,
Hall RM,
( 2010 ) Commensal Escherichia coli of healthy humans: a reservoir for antibiotic-resistance determinants. PMID : 20671087 : DOI : 10.1099/jmm.0.022475-0 Abstract >>
This study examined in detail the population structure of Escherichia coli from healthy adults with respect to the prevalence of antibiotic resistance and specific resistance determinants. E. coli isolated from the faeces of 20 healthy adults not recently exposed to antibiotics was tested for resistance to ten antibiotics and for carriage of integrons and resistance determinants using PCR. Strain diversity was assessed using biochemical and molecular criteria. E. coli was present in 19 subjects at levels ranging from 2.0��10(4) to 1.7��10(8) c.f.u. (g faeces)(-1). Strains resistant to one to six antibiotics were found at high levels (>30 %) in only ten individuals, but at significant levels (>0.5 %) in 14. Resistant isolates with the same phenotype from the same individual were indistinguishable, but more than one susceptible strain was sometimes found. Overall, individuals harboured one to four E. coli strains, although in 17 samples one strain was dominant (>70 % of isolates). Eighteen strains resistant to ampicillin, sulfamethoxazole, tetracycline and trimethoprim in 15 different combinations were observed. One resistant strain was carried by two unrelated individuals and a susceptible strain was shared by two cohabiting subjects. Two minority strains were derivatives of a more abundant resistant strain in the same sample, showing that continuous evolution is occurring in vivo. The trimethoprim-resistance genes dfrA1, dfrA5, dfrA7, dfrA12 or dfrA17 were in cassettes in a class 1 or class 2 integron. Ampicillin resistance was conferred by the bla(TEM) gene, sulfamethoxazole resistance by sul1, sul2 or sul3 and tetracycline resistance by tetA(A) or tetA(B). Chloramphenicol resistance (cmlA1 gene) was detected only once. Phylogenetic groups A and B2 were more common than B1 and D. Commensal E. coli of healthy humans represent an important reservoir for numerous antibiotic-resistance genes in many combinations. However, measuring the true extent of resistance carriage in commensal E. coli requires in-depth analysis.
|
962. |
Llosa M,
Bolland S,
de la Cruz F,
( 1991 ) Structural and functional analysis of the origin of conjugal transfer of the broad-host-range IncW plasmid R388 and comparison with the related IncN plasmid R46. PMID : 2038309 : DOI : 10.1007/bf00260661 Abstract >>
We cloned and sequenced a 402 bp DNA segment containing the origin of conjugal transfer (oriT) of the IncW plasmid R388. Progressive deletions from each end of the sequence were assayed for oriT activity. Stepwise reductions in mobilization frequencies, representing the loss of functional elements, correlated with deletion of structural motifs in the sequence. A sequence of 330 bp of oriT was sufficient for efficient mobilization. The first 86 bp of the sequence contains five tandemly repeated DNA sequences of 11 bp, followed by a 10 bp perfect inverted repeat. Deletion of the first 95 bp reduced the frequency of transfer by a hundred-fold. The sequence between bp 183 and 218 was necessary and sufficient for low frequency mobilization and, thus, it was assumed to contain the nick site. This basis core was cloned as a 60 bp segment (from bp 176-236) that could be mobilized at low frequency. It includes two inverted repeats and a perfect integration host factor (IHF) consensus binding site. A third functionally important segment in oriT was located between bp 260 and 330. The DNA sequence of the oriT of R388 could be aligned with that of the broad-host-range IncN plasmid R46. Moreover, the relative positions of the three inverted repeats are also conserved. Overall sequence similarity was 52%, but was significantly higher in particular regions, which coincided with the functionally important segments mapped by deletion analysis. Conservation of these segments provided independent support for their essential role in oriT function.
|
963. |
Wang Y,
Tang C,
Yu X,
Xia M,
Yue H,
( 2010 ) Distribution of serotypes and virulence-associated genes in pathogenic Escherichia coli isolated from ducks. PMID : 20706886 : DOI : 10.1080/03079457.2010.495742 Abstract >>
The objective of the present study was to investigate the serotypes and virulence-associated genes of avian pathogenic Escherichia coli (APEC) isolated from duck colibacillosis cases. Two hundred and fifty-four APEC isolates from duck colibacillosis cases were serotyped and amplified for 12 known virulence-associated genes and the betA gene (encoding choline dehydrogenase) by polymerase chain reaction assays. One hundred and forty-three E. coli isolates from cloacal swabs of healthy ducks were also amplified for the same genes. A total of 53 O-serogroups were found in 254 APEC isolates, among which O93, O78 and O92 were predominant serogroups. Polymerase chain reaction results showed that Shiga-toxin-producing E. coli distributed in only 2.4% of ducks compared with 49.2% of the APEC isolates harbouring the irp2 gene, and 44.9% the fyuA gene, respectively. The ibeA gene was only present in 27 APEC isolates and was not found in healthy ducks. The rfaH gene was detected in 20.5% of APEC isolates, whereas 5.6% was found in healthy ducks. A total 79.5% of APEC isolates harboured the betA gene, which was significantly higher than in healthy ducks (16.1%), suggesting that betA may be associated with virulence.
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964. |
Landman D,
Babu E,
Shah N,
Kelly P,
Bäcker M,
Bratu S,
Quale J,
( 2010 ) Activity of a novel aminoglycoside, ACHN-490, against clinical isolates of Escherichia coli and Klebsiella pneumoniae from New York City. PMID : 20667885 : DOI : 10.1093/jac/dkq278 Abstract >>
Reports of Enterobacteriaceae resistant to all commonly used antimicrobial agents, including �]-lactams, fluoroquinolones and aminoglycosides, are increasing in hospitals worldwide. The activity of ACHN-490, a next-generation aminoglycoside, was examined against clinical isolates of Escherichia coli and Klebsiella pneumoniae from hospitals in New York City, an area where multidrug-resistant organisms are endemic. Unique patient isolates of E. coli and K. pneumoniae were gathered from 16 hospitals located in New York City in 2009 and underwent susceptibility testing to aminoglycosides and ACHN-490. Subsets of isolates were characterized by PCR for the presence of genes encoding aminoglycoside-modifying enzymes, ribosomal methylases and KPC-type carbapenemases. Although most isolates of E. coli were susceptible to the aminoglycosides, the MIC(90) values of gentamicin, tobramycin and amikacin were 32, 8 and 4 mg/L, respectively. The MIC(90) of ACHN-490 was 1 mg/L. Multidrug resistance, including resistance to aminoglycosides and the presence of bla(KPC), was much more common in isolates of K. pneumoniae. However, the MIC(90) of ACHN-490 for K. pneumoniae was also 1 mg/L. The MICs of ACHN-490 did not correlate with the presence of commonly recovered aminoglycoside-modifying enzymes. Bactericidal activity was evident in most isolates at concentrations 4�� the MIC. The novel aminoglycoside ACHN-490 retains activity against most isolates of E. coli and K. pneumoniae, including multidrug-resistant strains. Additional studies examining the roles of efflux systems and outer membrane permeability alterations are recommended in isolates with reduced susceptibility to this agent.
|
965. |
Yan JJ,
Wu JJ,
Lee CC,
Ko WC,
Yang FC,
( 2010 ) Prevalence and characteristics of ertapenem-nonsusceptible Escherichia coli in a Taiwanese university hospital, 1999 to 2007. PMID : 20700614 : DOI : 10.1007/s10096-010-1020-1 Abstract >>
The present study was conducted to investigate the prevalence and characteristics of ertapenem-nonsusceptible (ETP-NS) Escherichia coli in a Taiwanese university. A total of 9,722 isolates collected in 1999, 2003, 2005, and 2007 were examined. Overall, 1.0% of all isolates from 66 patients were interpreted as ETP-NS on the basis of the disk diffusion test result. Most of these isolates were clonally unrelated and showed low-level ertapenem resistance, the production of CMY-2 cephalosporinase (86.4%), and decreased expression of the OmpF (97.0%) and/or OmpC (56.1%) porins. No carbapenemase was detected. The decreased porin expression was associated with disruptions of ompF or ompC in only about one-third of the ETP-NS isolates. During the study period, the prevalence of ETP-NS strains increased from 0.1 to 1.7%, accompanying an increase (0.8 to 17.6%) in the prevalence of CMY-2 producers. Coexistent or pre-existing clonally related ertapenem-susceptible (ETP-S) E. coli isolates were identified in 47.0% of all case patients, and almost all of the ETP-S isolates had the same �]-lactamases as the ETP-NS isolates. Our study results suggest the restricted use of extended-spectrum cephalosporins to hinder the emergence and prevalence of carbapenem resistance in E. coli, which may arise by the accumulation of multiple resistance determinants.
|
966. |
Zhao J,
Chen Z,
Chen S,
Deng Y,
Liu Y,
Tian W,
Huang X,
Wu C,
Sun Y,
Sun Y,
Zeng Z,
Liu JH,
( 2010 ) Prevalence and dissemination of oqxAB in Escherichia coli isolates from animals, farmworkers, and the environment. PMID : 20696876 : DOI : 10.1128/AAC.00139-10 PMC : PMC2944610 Abstract >>
OqxAB has recently been identified as one of the mechanisms of plasmid-mediated quinolone resistance (PMQR). Compared to what is observed for other PMQR determinants, there is a paucity of data with regard to the prevalence and epidemiology of OqxAB and its contribution to resistance to different antimicrobials. In this study, the prevalence and dissemination of oqxAB and other PMQR genes in Escherichia coli isolates from animals, farmworkers, and the environment in 2002 in China were investigated. Of the 172 E. coli isolates, 39.0% carried oqxA, while only 4.1%, 2.9%, and 0.6% carried qnr (1 qnrB6 isolate, 5 qnrS1 isolates, and 1 qnrD isolate), qepA, and aac(6')-Ib-cr, respectively. Among the 33 isolates from farmworkers, 10 (30.3%) were positive for oqxA. oqxAB was associated with IS26 and was carried on the 43- to 115-kb IncF transferable plasmid. Transconjugants carrying oqxAB showed 4- to 16-fold increases in the MICs of quinolones, 16- to 64-fold increases in the MICs of quinoxalines, 8- to 32-fold increases in the MICs of chloramphenicol and trimethoprim-sulfamethoxazole, and 4- to 8-fold increases in the MICs of florfenicol compared to the levels for the recipient. The pulsed-field gel electrophoresis (PFGE) analysis showed that the high levels of prevalence and dissemination of oqxAB in E. coli in animal farms were primarily due to the transmission of plasmids carrying oqxAB, although clonal transmission between human and swine E. coli isolates was observed. It is concluded that oqxAB was widespread in animal farms in China, which may be due to the overuse of quinoxalines in animals. This study warrants the prudent use of quinoxalines in food animals.
|
967. |
Corvec S,
Crémet L,
Leprince C,
Dauvergne S,
Reynaud A,
Lepelletier D,
Caroff N,
( 2010 ) Epidemiology of Escherichia coli clinical isolates producing AmpC plasmidic beta-lactamase during a 5-year period in a French teaching Hospital. PMID : 20462724 : DOI : 10.1016/j.diagmicrobio.2010.02.007 Abstract >>
We investigated the prevalence and epidemiology of AmpC plasmidic cephalosporinases in Escherichia coli clinical strains resistant to third-generation cephalosporins during a 5-year period at Nantes University Hospital, France (3100 beds). The prevalence and diversity of plasmidic cephalosporinase did not increase during the study period (0.09% of 25 861 E. coli isolates); only CMY-2 producers were detected (and 1 new variant, with a Y-to-C substitution at position 219). CMY-2-producing strains belonged to the 4 main phylogenetic groups and to 11 different sequence types. Three sequence types included more than 1 isolate (ST156, ST46, and ST354).
|
968. |
Poirel L,
Lagrutta E,
Taylor P,
Pham J,
Nordmann P,
( 2010 ) Emergence of metallo-�]-lactamase NDM-1-producing multidrug-resistant Escherichia coli in Australia. PMID : 20823289 : DOI : 10.1128/AAC.00878-10 PMC : PMC2976126 Abstract >>
A multidrug-resistant Escherichia coli isolate recovered in Australia produced a carbapenem-hydrolyzing �]-lactamase. Molecular investigations revealed the first identification of the bla(NDM-1) metallo-�]-lactamase gene in that country. In addition, this E. coli isolate expressed the extended-spectrum �]-lactamase CTX-M-15, together with two 16S rRNA methylases, namely, ArmA and RmtB, conferring a high level of resistance to aminoglycosides.
|
969. |
Davis MA,
Baker KN,
Orfe LH,
Shah DH,
Besser TE,
Call DR,
( 2010 ) Discovery of a gene conferring multiple-aminoglycoside resistance in Escherichia coli. PMID : 20368404 : DOI : 10.1128/AAC.01743-09 PMC : PMC2876372 Abstract >>
Bovine-origin Escherichia coli isolates were tested for resistance phenotypes using a disk diffusion assay and for resistance genotypes using a DNA microarray. An isolate with gentamicin and amikacin resistance but with no corresponding genes detected yielded a 1,056-bp DNA sequence with the closest homologues for its inferred protein sequence among a family of 16S rRNA methyltransferase enzymes. These enzymes confer high-level aminoglycoside resistance and have only recently been described in Gram-negative bacteria.
|
970. |
Zalewska-Piatek B,
Kur M,
Wilkanowicz S,
Piatek R,
Kur J,
( 2010 ) The DraC usher in Dr fimbriae biogenesis of uropathogenic E. coli Dr(+) strains. PMID : 20349311 : DOI : 10.1007/s00203-010-0564-x Abstract >>
Biogenesis of Dr fimbriae encoded by the dra gene cluster of uropathogenic Escherichia coli strains requires the chaperone-usher pathway. This secretion system is based on two non-structural assembly components, the DraB periplasmic chaperone and DraC outer-membrane usher. The DraB controls the folding of DraE subunits, and DraC forms the assembly and secretion platform for polymerization of subunits in linear fibers. In this study, mutagenesis of the DraC N-terminus was undertaken to select residues critical for Dr fimbriae bioassembly. The DraC-F4A, DraC-C64, DraC-C100A and DraC-W142A significantly reduced the adhesive ability of E. coli strains. The biological activity of the DraC mutants as a assembly platform for Dr fimbriae polymerization was verified by agglutination of human erythrocytes and adhesion to DAF localized at the surface of CHO-DAF(+) and HeLa cells. The residue F4 of the DraC usher conserved among FGL and FGS chaperone-assembled adhesive organelles can be used to design pillicides blocking the biogenesis of Dr fimbriae. Because the draC and afaC-III genes share 100% identity the range of the virulence determinant inhibitors could also be extended to E. coli strains encoding afa-3 gene cluster. The investigations performed showed that the usher N-terminus plays an important role in biogenesis of complete fiber.
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971. |
Schwab M,
Gruber H,
Högenauer G,
( 1991 ) The TraM protein of plasmid R1 is a DNA-binding protein. PMID : 2041477 : DOI : 10.1111/j.1365-2958.1991.tb02127.x Abstract >>
The TraM protein of the resistance plasmid R1 was purified to homogeneity and used for DNA-binding studies. Both gel retardation- and footprint experiments showed that TraM specifically binds to DNA of plasmid R1 comprising the region between the origin of transfer and the traM gene. Several TraM molecules bind and, according to the footprint experiments, two distinct sites of specific binding exist. The two sites are separated from each other by 12 nucleotides and each contains an inverted repeat. DNase I protection assays showed that the initial TraM binding occurs at these palindromic sequences. At higher protein concentrations the lengths of the DNA segments protected by TraM were increased towards the traM gene. In one region this extension leads to binding of TraM protein at its own promoters.
|
972. |
Smith AN,
Boulnois GJ,
Roberts IS,
( 1990 ) Molecular analysis of the Escherichia coli K5 kps locus: identification and characterization of an inner-membrane capsular polysaccharide transport system. PMID : 2082146 : DOI : 10.1111/j.1365-2958.1990.tb02035.x Abstract >>
The complete nucleotide sequence has been determined of a region of the Escherichia coli K5 antigen gene cluster postulated to encode functions for the translocation of capsular polysaccharide across the inner membrane. This revealed two genes, designated kpsM and kpsT, organized in a single transcriptional unit. Analysis of the predicted amino acid sequence of the KpsM and KpsT proteins indicates that they may function as dual components in a polysaccharide export system analogous to the periplasmic binding protein-dependent transport systems of Gram-negative bacteria. We propose that the KpsT protein acts as an energizer, coupling ATP hydrolysis to the transport process mediated by the KpsM protein. Extensive sequence homology between the KpsM and KpsT proteins and the products of the bexB and bexA genes present in the capsulation (cap) locus of Haemophilus influenzae, indicates that a common mechanism for the export of polysaccharide across the inner membrane may exist in these two micro-organisms.
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973. |
Nash RP,
Habibi S,
Cheng Y,
Lujan SA,
Redinbo MR,
( 2010 ) The mechanism and control of DNA transfer by the conjugative relaxase of resistance plasmid pCU1. PMID : 20448025 : DOI : 10.1093/nar/gkq303 PMC : PMC2943615 Abstract >>
Bacteria expand their genetic diversity, spread antibiotic resistance genes, and obtain virulence factors through the highly coordinated process of conjugative plasmid transfer (CPT). A plasmid-encoded relaxase enzyme initiates and terminates CPT by nicking and religating the transferred plasmid in a sequence-specific manner. We solved the 2.3 A crystal structure of the relaxase responsible for the spread of the resistance plasmid pCU1 and determined its DNA binding and nicking capabilities. The overall fold of the pCU1 relaxase is similar to that of the F plasmid and plasmid R388 relaxases. However, in the pCU1 structure, the conserved tyrosine residues (Y18,19,26,27) that are required for DNA nicking and religation were displaced up to 14 A out of the relaxase active site, revealing a high degree of mobility in this region of the enzyme. In spite of this flexibility, the tyrosines still cleaved the nic site of the plasmid's origin of transfer, and did so in a sequence-specific, metal-dependent manner. Unexpectedly, the pCU1 relaxase lacked the sequence-specific DNA binding previously reported for the homologous F and R388 relaxase enzymes, despite its high sequence and structural similarity with both proteins. In summary, our work outlines novel structural and functional aspects of the relaxase-mediated conjugative transfer of plasmid pCU1.
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974. |
Poirel L,
Carattoli A,
Bernabeu S,
Bruderer T,
Frei R,
Nordmann P,
( 2010 ) A novel IncQ plasmid type harbouring a class 3 integron from Escherichia coli. PMID : 20525990 : DOI : 10.1093/jac/dkq166 Abstract >>
To determine the genetic structures at the origin of the mobilization of the extended-spectrum beta-lactamase (ESBL) bla(GES-1) gene in an Escherichia coli clinical isolate. ESBL-encoding genes and class 1 or class 3 integron-specific motifs were screened. Conjugation experiments were performed to determine whether the plasmid-carrying bla(GES-1) gene was self-transferable. Plasmid sequencing was achieved by a primer-walking approach. The bla(GES-1) gene was located in a class 3 integron. That unusual genetic structure was itself located on an approximately 9 kb plasmid, pQ7, which was not self-transferable. Sequence analysis revealed that plasmid pQ7 belonged to a novel subtype of the IncQ group. This study identified for the first time the bla(GES-1) gene in E. coli and in Switzerland. It describes a novel IncQ-type plasmid subgroup that possesses original features, in particular iteron sequences that constitute a hot spot for integration of foreign DNA.
|
975. |
Singer JT,
Phennicie RT,
Sullivan MJ,
Porter LA,
Shaffer VJ,
Kim CH,
( 2010 ) Broad-host-range plasmids for red fluorescent protein labeling of gram-negative bacteria for use in the zebrafish model system. PMID : 20363780 : DOI : 10.1128/AEM.01679-09 PMC : PMC2876450 Abstract >>
To observe real-time interactions between green fluorescent protein-labeled immune cells and invading bacteria in the zebrafish (Danio rerio), a series of plasmids was constructed for the red fluorescent protein (RFP) labeling of a variety of fish and human pathogens. The aim of this study was to create a collection of plasmids that would express RFP pigments both constitutively and under tac promoter regulation and that would be nontoxic and broadly transmissible to a variety of Gram-negative bacteria. DNA fragments encoding the RFP dimeric (d), monomeric (m), and tandem dimeric (td) derivatives d-Tomato, td-Tomato, m-Orange, and m-Cherry were cloned into the IncQ-based vector pMMB66EH in Escherichia coli. Plasmids were mobilized into recipient strains by conjugal mating. Pigment production was inducible in Escherichia coli, Pseudomonas aeruginosa, Edwardsiella tarda, and Vibrio (Listonella) anguillarum strains by isopropyl-beta-d-thiogalactopyranoside (IPTG) treatment. A spontaneous mutant exconjugant of P. aeruginosa PA14 was isolated that expressed td-Tomato constitutively. Complementation analysis revealed that the constitutive phenotype likely was due to a mutation in lacI(q) carried on pMMB66EH. DNA sequence analysis confirmed the presence of five transitions, four transversions, and a 2-bp addition within a 14-bp region of lacI. Vector DNA was purified from this constitutive mutant, and structural DNA sequences for RFP pigments were cloned into the constitutive vector. Exconjugants of P. aeruginosa, E. tarda, and V. anguillarum expressed all pigments in an IPTG-independent fashion. Results from zebrafish infectivity studies indicate that RFP-labeled pathogens will be useful for the study of real-time interactions between host cells of the innate immune system and the infecting pathogen.
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976. |
Turner PC,
Miller EN,
Jarboe LR,
Baggett CL,
Shanmugam KT,
Ingram LO,
( 2011 ) YqhC regulates transcription of the adjacent Escherichia coli genes yqhD and dkgA that are involved in furfural tolerance. PMID : 20676725 : DOI : 10.1007/s10295-010-0787-5 Abstract >>
Previous results have demonstrated that the silencing of adjacent genes encoding NADPH-dependent furfural oxidoreductases (yqhD dkgA) is responsible for increased furfural tolerance in an E. coli strain EMFR9 [Miller et al., Appl Environ Microbiol 75:4315-4323, 2009]. This gene silencing is now reported to result from the spontaneous insertion of an IS10 into the coding region of yqhC, an upstream gene. YqhC shares homology with transcriptional regulators belonging to the AraC/XylS family and was shown to act as a positive regulator of the adjacent operon encoding YqhD and DkgA. Regulation was demonstrated by constructing a chromosomal deletion of yqhC, a firefly luciferase reporter plasmid for yqhC, and by a direct comparison of furfural resistance and NADPH-dependent furfural reductase activity. Closely related bacteria contain yqhC, yqhD, and dkgA orthologs in the same arrangement as in E. coli LY180. Orthologs of yqhC are also present in more distantly related Gram-negative bacteria. Disruption of yqhC offers a useful approach to increase furfural tolerance in bacteria.
|
977. |
Han Z,
Pinkner JS,
Ford B,
Obermann R,
Nolan W,
Wildman SA,
Hobbs D,
Ellenberger T,
Cusumano CK,
Hultgren SJ,
Janetka JW,
( 2010 ) Structure-based drug design and optimization of mannoside bacterial FimH antagonists. PMID : 20507142 : DOI : 10.1021/jm100438s PMC : PMC2894565 Abstract >>
FimH-mediated cellular adhesion to mannosylated proteins is critical in the ability of uropathogenic E. coli (UPEC) to colonize and invade the bladder epithelium during urinary tract infection. We describe the discovery and optimization of potent small-molecule FimH bacterial adhesion antagonists based on alpha-d-mannose 1-position anomeric glycosides using X-ray structure-guided drug design. Optimized biarylmannosides display low nanomolar binding affinity for FimH in a fluorescence polarization assay and submicromolar cellular activity in a hemagglutination (HA) functional cell assay of bacterial adhesion. X-ray crystallography demonstrates that the biphenyl moiety makes several key interactions with the outer surface of FimH including pi-pi interactions with Tyr-48 and an H-bonding electrostatic interaction with the Arg-98/Glu-50 salt bridge. Dimeric analogues linked through the biaryl ring show an impressive 8-fold increase in potency relative to monomeric matched pairs and represent the most potent FimH antagonists identified to date. The FimH antagonists described herein hold great potential for development as novel therapeutics for the effective treatment of urinary tract infections.
|
978. |
Delmas J,
Leyssene D,
Dubois D,
Birck C,
Vazeille E,
Robin F,
Bonnet R,
( 2010 ) Structural insights into substrate recognition and product expulsion in CTX-M enzymes. PMID : 20452359 : DOI : 10.1016/j.jmb.2010.04.062 Abstract >>
beta-Lactamase-mediated resistance to beta-lactam antibiotics poses a major threat to our antibiotic armamentarium. Among beta-lactamases, a significant threat comes from enzymes that hydrolyze extended-spectrum cephalosporins such as cefotaxime. Among the enzymes that exhibit this phenotype, the CTX-M family is found worldwide. These enzymes have a small active site, which makes it difficult to explain how they hydrolyze the bulky extended-spectrum cephalosporins into the binding site. We investigated noncovalent substrate recognition and product release in CTX-M enzymes using steered molecular dynamics simulation and X-ray diffraction. An arginine residue located far from the binding site favors the capture and tracking of substrates during entrance into the catalytic pocket. We show that the accommodation of extended-spectrum cephalosporins by CTX-M enzymes induced subtle changes in the active site and established a high density of electrostatic interactions. Interestingly, the product of the catalytic reaction initiates its own release because of steric hindrances and electrostatic repulsions. This suggests that there exists a general mechanism for product release for all members of the beta-lactamase family and probably for most carboxypeptidases.
|
979. |
Roche D,
Fléchard M,
Lallier N,
Répérant M,
Brée A,
Pascal G,
Schouler C,
Germon P,
( 2010 ) ICEEc2, a new integrative and conjugative element belonging to the pKLC102/PAGI-2 family, identified in Escherichia coli strain BEN374. PMID : 20675467 : DOI : 10.1128/JB.00609-10 PMC : PMC2944505 Abstract >>
The diversity of the Escherichia coli species is in part due to the large number of mobile genetic elements that are exchanged between strains. We report here the identification of a new integrative and conjugative element (ICE) of the pKLC102/PAGI-2 family located downstream of the tRNA gene pheU in the E. coli strain BEN374. Indeed, this new region, which we called ICEEc2, can be transferred by conjugation from strain BEN374 to the E. coli strain C600. We were also able to transfer this region into a Salmonella enterica serovar Typhimurium strain and into a Yersinia pseudotuberculosis strain. This transfer was then followed by the integration of ICEEc2 into the host chromosome downstream of a phe tRNA gene. Our data indicated that this transfer involved a set of three genes encoding DNA mobility enzymes and a type IV pilus encoded by genes present on ICEEc2. Given the wide distribution of members of this family, these mobile genetic elements are likely to play an important role in the diversification of bacteria.
|
980. |
Wang W,
Baker P,
Seah SY,
( 2010 ) Comparison of two metal-dependent pyruvate aldolases related by convergent evolution: substrate specificity, kinetic mechanism, and substrate channeling. PMID : 20364820 : DOI : 10.1021/bi100251u Abstract >>
HpaI and BphI are two pyruvate class II aldolases found in aromatic meta-cleavage degradation pathways that catalyze similar reactions but are not related in sequence. Steady-state kinetic analysis of the aldol addition reactions and product inhibition assays showed that HpaI exhibits a rapid equilibrium random order mechanism while BphI exhibits a compulsory order mechanism, with pyruvate binding first. Both aldolases are able to utilize aldehyde acceptors two to five carbons in length; however, HpaI showed broader specificity and had a preference for aldehydes containing longer linear alkyl chains or C2-OH substitutions. Both enzymes were able to bind 2-keto acids larger than pyruvate, but only HpaI was able to utilize both pyruvate and 2-ketobutanoate as carbonyl donors in the aldol addition reaction. HpaI lacks stereospecific control producing racemic mixtures of 4-hydroxy-2-oxopentanoate (HOPA) from pyruvate and acetaldehyde while BphI synthesizes only (4S)-HOPA. BphI is also able to utilize acetaldehyde produced by the reduction of acetyl-CoA catalyzed by the associated aldehyde dehydrogenase, BphJ. This aldehyde was directly channeled from the dehydrogenase to the aldolase active sites, with an efficiency of 84%. Furthermore, the BphJ reductive deacylation reaction increased 4-fold when BphI was catalyzing the aldol addition reaction. Therefore, the BphI-BphJ enzyme complex exhibits unique bidirectionality in substrate channeling and allosteric activation.
|
981. |
Ratiner YA,
Sihvonen LM,
Liu Y,
Wang L,
Siitonen A,
( 2010 ) Alteration of flagellar phenotype of Escherichia coli strain P12b, the standard type strain for flagellar antigen H17, possessing a new non-fliC flagellin gene flnA, and possible loss of original flagellar phenotype and genotype in the course of subculturing through semisolid media. PMID : 20174918 : DOI : 10.1007/s00203-010-0556-x Abstract >>
A practically important phenomenon, resulting in the loss of the original flagellar phenotype (genotype) of bacteria, is described in the Escherichia coli H17 type strain P12b possessing two distinct genes for H17 and H4 flagellins, respectively. By PCR, sequencing, and phylogenetic investigation, the H17 gene (originally expressed) was considered a new non-fliC flagellin gene and assigned flnA, while the H4 gene (originally cryptic) was reaffirmed as fliC. H17 and H4 flagella differed morphologically. The phenomenon consisted in the replacement of H17 cells by H4 cells during subculturing through certain semisolid media and resulted from the excision of flnA (H17) entirely or in part. The substitution rate depended on the density and nutrient composition of media and reached 100% even after a single passage through 0.3% LB agar. Such phenomenon can lead to an unexpected loss of original H17 phenotype. Our review of the literature showed that the loss of the original flagellar genotype (phenotype) of P12b has occurred in some laboratories while the authors continued to consider their cultures H17. We showed how to distinguish these alternative flagellin genotypes using popular fliC primers. Attention was also paid to possible discrepancies between serological and molecular results in flagellar typing of E. coli.
|
982. |
Sáenz Y,
Vinué L,
Ruiz E,
Somalo S,
Martínez S,
Rojo-Bezares B,
Zarazaga M,
Torres C,
( 2010 ) Class 1 integrons lacking qacEDelta1 and sul1 genes in Escherichia coli isolates of food, animal and human origins. PMID : 20176451 : DOI : 10.1016/j.vetmic.2010.01.026 Abstract >>
To study the frequency and diversity of class 1 integrons lacking the 3'-conserved segment (CS) in intI1-positive Escherichia coli isolates of different origins. The presence of intI1 was previously detected in 84 E. coli isolates of food (21 isolates), animal (32) and healthy-human volunteer (31) origins. The qacEDelta1-sul1 genes were analyzed by PCR and those isolates that lacked these genes were included in this work. The genetic structure of class 1 integrons was determined, using the PCR and sequencing primer-walking strategy. Isolates and plasmids were typed. Class 1 integrons lacking the 3'-CS were found in 13 of the 84 intI1-positive E. coli isolates (15.5%) of food, animal, and human origins. All 13 isolates showed unrelated patterns by REP-PCR. The following gene cassette arrangements were identified inside the class 1 integrons of these 13 strains: dfrA1; dfrA5; dfrA12-orfF-aadA2-cmlA1-aadA1-qacH-IS440-sul3; dfrA12-orfF-aadA2-cmlA1-aadA1-IS440-sul3; estX-psp-aadA2-cmlA1-aadA1-qacH-IS440-sul3; and a new arrangement estX-psp-aadA2-cmlA1Delta-IS1294-DeltacmlA1-aadA1-qacH-IS440-sul3 that contain the IS1294 into the cmlA1 gene (included in GenBank, number EU704128). Complete or truncated mef(B) gene was detected upstream of sul3 gene in this type of integrons. Plasmids were identified in four of the studied strains by PCR-replicon-typing, detecting different combinations of IncY, I1, FIC, FII, FIB plasmids. Non-classic integrons were located into plasmids of 100-150 kb in four studied strains. Occurrence and diversity of class 1 integrons lacking 3'-CS among the studied intI1-positive E. coli isolates of different origins were relatively high. The sul3 gene was detected in most of class 1 integrons lacking 3'-CS.
|
983. |
Ho PL,
Wong RC,
Lo SW,
Chow KH,
Wong SS,
Que TL,
( 2010 ) Genetic identity of aminoglycoside-resistance genes in Escherichia coli isolates from human and animal sources. PMID : 20185552 : DOI : 10.1099/jmm.0.015032-0 Abstract >>
A bacterial collection (n=249) obtained in Hong Kong from 2002 to 2004 was used to investigate the molecular epidemiology of aminoglycoside resistance among Escherichia coli isolates from humans and food-producing animals. Of these, 89 isolates were gentamicin-sensitive (human n=60, animal n=29) and 160 isolates were gentamicin-resistant (human n=107, animal n=53). Overall, 84.1% (90/107) and 75.5% (40/53) of the gentamicin-resistant isolates from human and animal sources, respectively, were found to possess the aacC2 gene. The aacC2 gene for 20 isolates (10 each for human and animal isolates) was sequenced. Two alleles were found that were equally distributed in human and animal isolates. PFGE showed that the gentamicin-resistant isolates exhibited diverse patterns with little clonality. In some isolates, the aacC2 gene was encoded on large transferable plasmids of multiple incompatibility groups (IncF, IncI1 and IncN). An IncFII plasmid of 140 kb in size was shared by one human and three animal isolates. In summary, this study showed that human and animal isolates share the same pool of resistance genes.
|
984. |
Sabui S,
Ghosal A,
Dutta S,
Ghosh A,
Ramamurthy T,
Nataro JP,
Hamabata T,
Chatterjee NS,
( 2010 ) Allelic variation in colonization factor CS6 of enterotoxigenic Escherichia coli isolated from patients with acute diarrhoea and controls. PMID : 20299505 : DOI : 10.1099/jmm.0.017582-0 Abstract >>
Colonization factor antigens (CFAs) are important virulence factors in enterotoxigenic Escherichia coli (ETEC). Using a multiplex PCR and RT-PCR, this study tested the presence of common colonization factor-encoding genes and their expression in 50 ETEC strains isolated from stool specimens. The samples were from patients (children) with acute diarrhoea (cases) admitted to the Infectious Disease Hospital (Kolkata, India) and from normal children (controls) under 5 years of age from the community. The results indicated that coli surface antigen 6 (CS6) was the most prevalent CFA (78 %) expressed by these ETEC strains. Sequence analysis of both of the CS6 structural genes, i.e. cssA and cssB, in different ETEC isolates revealed the presence of point mutations in a systematic fashion. Based on the analysis of these variations, it was found that CssA had three alleles and CssB had two. Based on the allelic variations, subtyping of CS6 into AIBI, AIIBII, AIIIBI, AIBII and AIIIBII is proposed. The point mutations in the different alleles were reflected in a partial alteration in the secondary structure of both subunits, as determined by computational analysis. The functional significance of these changes was confirmed with cellular binding studies in Caco-2 cells with representative ETEC isolates. CS6 with AI or AIII allelic subtypes showed a higher binding capacity than AII, whereas BI showed stronger binding than BII. The AII and BII alleles were mostly detected in controls rather than in cases. The antibody specificity of BI and BII also varied due to alteration of the amino acids. Thus, CS6 variants are formed as a result of different allelic combinations of CssA and CssB, and these changes at the functional level might be important in the development of an effective ETEC vaccine.
|
985. |
Crémet L,
Caroff N,
Giraudeau C,
Dauvergne S,
Lepelletier D,
Reynaud A,
Corvec S,
( 2010 ) Occurrence of ST23 complex phylogroup A Escherichia coli isolates producing extended-spectrum AmpC beta-lactamase in a French hospital. PMID : 20145079 : DOI : 10.1128/AAC.01580-09 PMC : PMC2863611 Abstract >>
Extended-spectrum AmpC beta-lactamase (ESAC) Escherichia coli producers were investigated over a 5-year period. Eleven isolates presenting a strong ampC promoter and different strategic AmpC mutations, including two newly described modifications (A292V and an L-A-A insertion at 295), were characterized. All the isolates belonged to phylogenetic group A and to the ST23 complex.
|
986. |
Iida M,
Okamura N,
Yamazaki M,
Yatsuyanagi J,
Kurazono T,
Suzuki R,
Hiruta N,
Isobe J,
Seto K,
Kawano K,
Narimatsu H,
Ratchtrachenchai OA,
Okabe N,
Ito K,
( 2010 ) Classification of perA sequences and their correlation with autoaggregation in typical enteropathogenic Escherichia coli isolates collected in Japan and Thailand. PMID : 20377747 : DOI : 10.1111/j.1348-0421.2010.00212.x Abstract >>
Enteropathogenic Escherichia coli (EPEC) strains produce a bundle-forming pilus (BFP) that mediates localized adherence (LA) to intestinal epithelial cells. The major structural subunit of the BFP is bundlin, which is encoded by the bfpA gene located on a large EAF plasmid. The perA gene has been shown to activate genes within the bfp operon. We analyzed perA gene polymorphism among typical (eae- and bfpA-positive) EPEC strains isolated from healthy and diarrheal persons in Japan (n=27) and Thailand (n=26) during the period 1995 to 2007 and compared this with virulence and phenotypic characteristics. Eight genotypes of perA were identified by heteroduplex mobility assay (HMA). The strains isolated in Thailand showed strong autoaggregation and had an intact perA, while most of those isolated in Japan showed weak or no autoaggregation, and had a truncated perA due to frameshift mutation. The degree of autoaggregation was well correlated with adherence to HEp-2 cells, contact hemolysis and BFP expression. Our results showed that functional deficiency due to frameshift mutation and subsequent nonsense mutation in perA reduced BFP expression in typical EPEC strains isolated in Japan.
|
987. |
Joseph TC,
Rajan LA,
Thampuran N,
James R,
( 2010 ) Functional characterization of trehalose biosynthesis genes from E. coli: an osmolyte involved in stress tolerance. PMID : 20217281 : DOI : 10.1007/s12033-010-9259-4 Abstract >>
Trehalose (1-alpha-D-glucopyranosyl-1-alpha-D-glucopyranoside), a non-reducing disaccharide is a major compatible solute, which maintains fluidity of membranes and protects the biological structure of organisms under stress. In this study, trehalose-6-phosphate synthase (otsA) and trehalose-6-phosphate phosphatase (otsB) genes encoding for trehalose biosynthesis from Escherichia coli was cloned as an operon and expressed in E. coli M15(pREP4). The recombinant E. coli strain showed a threefold increase in the activity of otsBA pathway enzymes, compared to the control strain. The transgenic E. coli accumulated up to 0.86 mg/l of trehalose. The sequence of otsA and otsB genes reported in this study contains several base substitutions with that of reported sequences in GenBank, resulting in the altered amino acid sequences of the translated proteins.
|
988. |
Fagerquist CK,
Garbus BR,
Miller WG,
Williams KE,
Yee E,
Bates AH,
Boyle S,
Harden LA,
Cooley MB,
Mandrell RE,
( 2010 ) Rapid identification of protein biomarkers of Escherichia coli O157:H7 by matrix-assisted laser desorption ionization-time-of-flight-time-of-flight mass spectrometry and top-down proteomics. PMID : 20232878 : DOI : 10.1021/ac902455d Abstract >>
Six protein biomarkers from two strains of Escherichia coli O157:H7 and one non-O157:H7, nonpathogenic strain of E. coli have been identified by matrix-assisted laser desorption ionization time-of-flight-time-of-flight tandem mass spectrometry (MALDI-TOF-TOF-MS/MS) and top-down proteomics. Proteins were extracted from bacterial cell lysates, ionized by MALDI, and analyzed by MS/MS. Protein biomarker ions were identified from their sequence-specific fragment ions by comparison to a database of in silico fragment ions derived from bacterial protein sequences. Web-based software, developed in-house, was used to rapidly compare the mass-to-charge (m/z) of MS/MS fragment ions to the m/z of in silico fragment ions derived from hundreds of bacterial protein sequences. A peak matching algorithm and a p-value algorithm were used to independently score and rank identifications on the basis of the number of MS/MS-in silico matches. The six proteins identified were the acid stress chaperone-like proteins, HdeA and HdeB; the cold shock protein, CspC; the YbgS (or homeobox protein); the putative stress-response protein YjbJ (or CsbD family protein); and a protein of unknown function, YahO. HdeA, HdeB, YbgS, and YahO proteins were found to be modified post-translationally with removal of an N-terminal signal peptide. Gene sequencing of hdeA, hdeB, cspC, ybgS, yahO, and yjbJ for 11 strains of E. coli O157:H7 and 7 strains of the "near-neighbor" serotype O55:H7 revealed a high degree sequence homology between these two serotypes. Although it was not possible to distinguish O157:H7 from O55:H7 from these six biomarkers, it was possible to distinguish E. coli O157:H7 from a nonpathogenic E. coli by top-down proteomics of the YahO and YbgS. In the case of the YahO protein, a single amino acid residue substitution in its sequence (resulting in a molecular weight difference of only 1 Da) was sufficient to distinguish E. coli O157:H7 from a non-O157:H7, nonpathogenic E. coli by MALDI-TOF-TOF-MS/MS, whereas this would be difficult to distinguish by MALDI-TOF-MS. Finally, a protein biomarker ion at m/z approximately 9060 observed in the MS spectra of non-O157:H7 E. coli strains but absent from MS spectra of E. coli O157:H7 strains was identified by top-down analysis to be the HdeB acid stress chaperone-like protein consistent with previous identifications by gene sequencing and bottom-up proteomics.
|
989. |
Sukumaran SK,
Fu NY,
Tin CB,
Wan KF,
Lee SS,
Yu VC,
( 2010 ) A soluble form of the pilus protein FimA targets the VDAC-hexokinase complex at mitochondria to suppress host cell apoptosis. PMID : 20347420 : DOI : 10.1016/j.molcel.2010.02.015 Abstract >>
Inhibition of apoptotic response of host cells during an early phase of infection is a strategy used by many enteroinvasive bacterial pathogens to enhance their survival. Here, we report the identification of a soluble form of the pilus protein FimA from the culture supernatants of E. coli K1, Salmonella, and Shigella that can potently inhibit Bax-mediated release of cytochrome c from isolated mitochondria. Similar to the infected cells, HCT116 cells stably expressing FimA display a delay in the integration of Bax into outer mitochondrial membrane induced by apoptotic stimuli. FimA targets to mitochondria through binding to VDAC1, which is a prerequisite step for E. coli K1 to render the short-term blockade of apoptotic death in the host cells. Interestingly, FimA strengthens the VDAC1-hexokinase interaction and prevents dissociation of hexokinase from VDAC1 triggered by apoptotic stimuli. Together, these data thus reveal a paradigm of antiapoptosis mechanism undertaken by the enteroinvasive bacteria.
|
990. |
Wang Q,
Wang S,
Beutin L,
Cao B,
Feng L,
Wang L,
( 2010 ) Development of a DNA microarray for detection and serotyping of enterotoxigenic Escherichia coli. PMID : 20351209 : DOI : 10.1128/JCM.02014-09 PMC : PMC2884529 Abstract >>
Enterotoxigenic Escherichia coli (ETEC) is a common pathogen worldwide causing infectious diarrhea, especially traveler's diarrhea. Traditional physiological assays, immunoassays, and PCR-based methods for the detection of ETEC target the heat-labile enterotoxin and/or the heat-stable enterotoxin. Separate serotyping methods using antisera are required to determine the ETEC serogroup. In this study, we developed a DNA microarray that can simultaneously detect enterotoxin genes and the 19 most common O serogroup genes in ETEC strains. The specificity and reproducibility of this approach were verified by hybridization to 223 strains: 50 target reference or clinical strains and 173 other strains, including those belonging to other E. coli O serogroups and closely related species. The sensitivity of detection was determined to be 50 ng of genomic DNA or 10(8) CFU per ml of organisms in pure culture. The random PCR strategy used in this study with minimal bias provides an effective alternative to multiplex PCR for the detection of pathogens using DNA microarrays. The assay holds promise for applications in the clinical diagnosis and epidemiological surveillance of pathogenic microorganisms.
|
991. |
Liu B,
Perepelov AV,
Li D,
Senchenkova SN,
Han Y,
Shashkov AS,
Feng L,
Knirel YA,
Wang L,
( 2010 ) Structure of the O-antigen of Salmonella O66 and the genetic basis for similarity and differences between the closely related O-antigens of Escherichia coli O166 and Salmonella O66. PMID : 20185508 : DOI : 10.1099/mic.0.037325-0 Abstract >>
O-antigen is a component of the outer membrane of Gram-negative bacteria and is one of the most variable cell surface constituents, leading to major antigenic variability. The O-antigen forms the basis for bacterial serotyping. In this study, the O-antigen structure of Salmonella O66 was established, which differs from the known O-antigen structure of Escherichia coli O166 only in one linkage (most likely the linkage between the O-units) and O-acetylation. The O-antigen gene clusters of Salmonella O66 and E. coli O166 were found to have similar organizations, the only exception being that in Salmonella O66, the wzy gene is replaced by a non-coding region. The function of the wzy gene in E. coli O166 was confirmed by the construction and analysis of deletion and trans-complementation mutants. It is proposed that a functional wzy gene located outside the O-antigen gene cluster is involved in Salmonella O66 O-antigen biosynthesis, as has been reported previously in Salmonella serogroups A, B and D1. The sequence identity for the corresponding genes between the O-antigen gene clusters of Salmonella O66 and E. coli O166 ranges from 64 to 70 %, indicating that they may originate from a common ancestor. It is likely that after the species divergence, Salmonella O66 got its specific O-antigen form by inactivation of the wzy gene located in the O-antigen gene cluster and acquisition of two new genes (a wzy gene and a prophage gene for O-acetyl modification) both residing outside the O-antigen gene cluster.
|
992. |
Mellata M,
Ameiss K,
Mo H,
Curtiss R,
( 2010 ) Characterization of the contribution to virulence of three large plasmids of avian pathogenic Escherichia coli chi7122 (O78:K80:H9). PMID : 20086082 : DOI : 10.1128/IAI.00981-09 PMC : PMC2849417 Abstract >>
Despite the fact that the presence of multiple large plasmids is a defining feature of extraintestinal pathogenic Escherichia coli (ExPEC), such as avian pathogenic E. coli (APEC), and despite the fact that these bacteria pose a considerable threat to both human and animal health, characterization of these plasmids is still limited. In this study, after successfully curing APEC of its plasmids, we were able to investigate, for the first time, the contribution to virulence of three plasmids, pAPEC-1 (103 kb), pAPEC-2 (90 kb), and pAPEC-3 (60 kb), from APEC strain chi7122 individually as well as in all combinations in the wild-type background. Characterization of the different strains revealed unique features of APEC virulence. In vivo assays showed that curing the three plasmids resulted in severe attenuation of virulence. The presence of different plasmids and combinations of plasmids resulted in strains with different pathotypes and levels of virulence, reflecting the diversity of APEC strains associated with colibacillosis in chickens. Unexpectedly, our results associated the decrease in growth of some strains in some media with the virulence of APEC, and the mechanism was associated with some combinations of plasmids that included pAPEC-1. This study provided new insights into the roles of large plasmids in the virulence, growth, and evolution of APEC by showing for the first time that both the nature of plasmids and combinations of plasmids have an effect on these phenomena. It also provided a plausible explanation for some of the conflicting results related to the virulence of ExPEC strains. This study should help us understand the virulence of other ExPEC strains and design more efficient infection control strategies.
|
993. |
Ruvolo PP,
Keating KM,
Williams KR,
Chase JW,
( 1991 ) Single-stranded DNA binding proteins (SSBs) from prokaryotic transmissible plasmids. PMID : 2008432 : DOI : 10.1002/prot.340090206 Abstract >>
The DNA and protein sequences of single-stranded DNA binding proteins (SSBs) encoded by the plP71a, plP231a, and R64 conjugative plasmids have been determined and compared to Escherichia coli SSB and the SSB encoded by F-plasmid. Although the amino acid sequences of all of these proteins are highly conserved within the NH2-terminal two-thirds of the protein, they diverge in the COOH-terminal third region. A number of amino acid residues which have previously been implicated as being either directly or indirectly involved in DNA binding are conserved in all of these SSBs. These residues include Trp-40, Trp-54, Trp-88, His-55, and Phe-60. On the basis of these sequence comparisons and DNA binding studies, a role for Tyr-70 in DNA binding is suggested for the first time. Although the COOH-terminal third of these proteins diverges more than their NH2-terminal regions, the COOH-terminal five amino acid residues of all five of these proteins are identical. In addition, all of these proteins share the characteristic property of having a protease resistant, NH2-terminal core and an acidic COOH-terminal region. Despite the high degree of sequence homology among the plasmid SSB proteins, the F-plasmid SSB appears unique in that it was the only SSB tested that neither bound well to poly(dA) nor was able to stimulate DNA polymerase III holoenzyme elongation rates. Poly [d(A-T)] melting studies suggest that at least three of the plasmid encoded SSBs are better helix-destabilizing proteins than is the E. coli SSB protein.
|
994. |
Johnson TJ,
Jordan D,
Kariyawasam S,
Stell AL,
Bell NP,
Wannemuehler YM,
Alarcón CF,
Li G,
Tivendale KA,
Logue CM,
Nolan LK,
( 2010 ) Sequence analysis and characterization of a transferable hybrid plasmid encoding multidrug resistance and enabling zoonotic potential for extraintestinal Escherichia coli. PMID : 20160015 : DOI : 10.1128/IAI.01174-09 PMC : PMC2863545 Abstract >>
ColV plasmids of extraintestinal pathogenic Escherichia coli (ExPEC) encode a variety of fitness and virulence factors and have long been associated with septicemia and avian colibacillosis. These plasmids are found significantly more often in ExPEC, including ExPEC associated with human neonatal meningitis and avian colibacillosis, than in commensal E. coli. Here we describe pAPEC-O103-ColBM, a hybrid RepFIIA/FIB plasmid harboring components of the ColV pathogenicity island and a multidrug resistance (MDR)-encoding island. This plasmid is mobilizable and confers the ability to cause septicemia in chickens, the ability to cause bacteremia resulting in meningitis in the rat model of human disease, and the ability to resist the killing effects of multiple antimicrobial agents and human serum. The results of a sequence analysis of this and other ColV plasmids supported previous findings which indicated that these plasmid types arose from a RepFIIA/FIB plasmid backbone on multiple occasions. Comparisons of pAPEC-O103-ColBM with other sequenced ColV and ColBM plasmids indicated that there is a core repertoire of virulence genes that might contribute to the ability of some ExPEC strains to cause high-level bacteremia and meningitis in a rat model. Examination of a neonatal meningitis E. coli (NMEC) population revealed that approximately 58% of the isolates examined harbored ColV-type plasmids and that 26% of these plasmids had genetic contents similar to that of pAPEC-O103-ColBM. The linkage of the ability to confer MDR and the ability contribute to multiple forms of human and animal disease on a single plasmid presents further challenges for preventing and treating ExPEC infections.
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995. |
Allsopp LP,
Totsika M,
Tree JJ,
Ulett GC,
Mabbett AN,
Wells TJ,
Kobe B,
Beatson SA,
Schembri MA,
( 2010 ) UpaH is a newly identified autotransporter protein that contributes to biofilm formation and bladder colonization by uropathogenic Escherichia coli CFT073. PMID : 20145097 : DOI : 10.1128/IAI.01010-09 PMC : PMC2849410 Abstract >>
Escherichia coli is the primary cause of urinary tract infection (UTI) in the developed world. The major factors associated with virulence of uropathogenic E. coli (UPEC) are fimbrial adhesins, which mediate specific attachment to host receptors and trigger innate host responses. Another group of adhesins is represented by the autotransporter (AT) subgroup of proteins. In this study, we identified a new AT-encoding gene, termed upaH, present in a 6.5-kb unannotated intergenic region in the genome of the prototypic UPEC strain CFT073. Cloning and sequencing of the upaH gene from CFT073 revealed an intact 8.535-kb coding region, contrary to the published genome sequence. The upaH gene was widely distributed among a large collection of UPEC isolates as well as the E. coli Reference (ECOR) strain collection. Bioinformatic analyses suggest beta-helix as the predominant structure in the large N-terminal passenger (alpha) domain and a 12-strand beta-barrel for the C-terminal beta-domain of UpaH. We demonstrated that UpaH is expressed at the cell surface of CFT073 and promotes biofilm formation. In the mouse UTI model, deletion of the upaH gene in CFT073 and in two other UPEC strains did not significantly affect colonization of the bladder in single-challenge experiments. However, in competitive colonization experiments, CFT073 significantly outcompeted its upaH isogenic mutant strain in urine and the bladder.
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996. |
Serventi F,
Ramazzina I,
Lamberto I,
Puggioni V,
Gatti R,
Percudani R,
( 2010 ) Chemical basis of nitrogen recovery through the ureide pathway: formation and hydrolysis of S-ureidoglycine in plants and bacteria. PMID : 20038185 : DOI : 10.1021/cb900248n Abstract >>
While some organisms, including humans, eliminate oxidized purines to get rid of excess nitrogen, for many others the recovery of the purine ring nitrogen is vital. In the so-called ureide pathway, nitrogen is released as ammonia from allantoate through a series of reactions starting with allantoate amidohydrolase (AAH), a manganese-dependent enzyme found in plants and bacteria. We report NMR evidence that the true product of the AAH reaction is S-ureidoglycine, a nonstandard alpha-amino acid that spontaneously releases ammonia in vitro. Using gene proximity and logical genome analysis, we identified a candidate gene (ylbA) for S-ureidoglycine metabolism. The proteins encoded by Escherichia coli and Arabidopsis thaliana genes catalyze the manganese-dependent release of ammonia through hydrolysis of S-ureidoglycine. Hydrolysis then inverts the configuration and yields S-ureidoglycolate. S-Ureidoglycine aminohydrolase (UGHY) is cytosolic in bacteria, whereas in plants it is localized, like allantoate amidohydrolase, in the endoplasmic reticulum. These findings strengthen the basis for the known sensitivity of the ureide pathway to Mn availability and suggest a further rationale for the active transport of Mn in the endoplasmic reticulum of plant cells.
|
997. |
Zhou H,
Liao X,
Wang T,
Du G,
Chen J,
( 2010 ) Enhanced l-phenylalanine biosynthesis by co-expression of pheA(fbr) and aroF(wt). PMID : 20137911 : DOI : 10.1016/j.biortech.2010.01.043 Abstract >>
A beta-2-thienylalanine-resistant E. coli K12 mutant carrying a Thr326Pro mutation in the regulation (R) domain of pheA (pheA(fbr)) was obtained by N-methyl-N'-nitro-N-nitrosoguanidine (NTG) mutagenesis. In the presence of 200mM l-phenylalanine (l-Phe), a recombinant E. coli WSH-Z06 (pAP-B03) carrying pheA(fbr) as well as wild-type aroF (aroF(wt)) exhibited more than 70% of the chorismate mutase-prephenate dehydratase (CM-PDT) activity as observed in the absence of this amino acid. The l-Phe titer of WSH-Z06 (pAP-B03) reached 35.38g/L in a 3-L fermentor, which was 2.81-fold higher than that of the original strain E. coli WSH-Z06. Furthermore, the l-Phe yield on glucose of WSH-Z06 (pAP-B03) (0.26mol/mol) was twice that of E. coli WSH-Z06. This recombinant E. coli WSH-Z06 (pAP-B03) is a potential strain for over-production of l-Phe.
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998. |
Vinué L,
Sáenz Y,
Rojo-Bezares B,
Olarte I,
Undabeitia E,
Somalo S,
Zarazaga M,
Torres C,
( 2010 ) Genetic environment of sul genes and characterisation of integrons in Escherichia coli isolates of blood origin in a Spanish hospital. PMID : 20188519 : DOI : 10.1016/j.ijantimicag.2010.01.012 Abstract >>
The prevalence and characterisation of integrons and the genetic environment of sulphonamide resistance genes were studied in 135 Escherichia coli isolates recovered from blood cultures in a Spanish hospital during 2007. Class 1 and 2 integrons were identified in 54 isolates (intI1, 52 isolates; intI2, 1 isolate; and intI1+intI2, 1 isolate). Of the 53 intI1-positive isolates, 36 (67.9%) contained the classic class 1 integron including the qacEDelta1-sul1 region, and 11 different gene cassette arrangements were demonstrated in 33 of these isolates. Seventeen intI1-positive isolates lacked the qacEDelta1-sul1 region, and 8 gene cassette arrangements were demonstrated in 12 of these isolates. Seventy-one isolates showed a sulphonamide-resistant phenotype, 63 of which contained sul genes. The sul1 gene was associated with intI1 in 36 of 42 sul1-positive isolates, and the sul3 gene was associated with non-classic class 1 integrons in 5 of 7 sul3-positive isolates. Finally, sul2 was found associated with strA-strB genes in 32 of 35 sul2-positive isolates, identifying 11 genetic structures, 1 of them presenting the IS150 element disrupting the strB gene; this structure was included in GenBank with accession no. FJ705354. Almost one-half of the E. coli isolates from blood cultures contained integrons and sul genes. Moreover, sul genes were detected in different structures, one of them new, and could be important determinants in antibiotic resistance dissemination.
|
999. |
Mochida S,
Tsuchiya H,
Mori K,
Kaji A,
( 1991 ) Three short fragments of Rts1 DNA are responsible for the temperature-sensitive growth phenotype (Tsg) of host bacteria. PMID : 2013575 : DOI : 10.1128/jb.173.8.2600-2607.1991 PMC : PMC207826 Abstract >>
Rts1 is a multiphenotype drug resistance factor, and one of its phenotypes is temperature-sensitive growth (Tsg) of host bacteria. A 3.65-kb fragment from Rts1 DNA was shown to cause the Tsg phenotype in host cells. This tsg fragment was split by a restriction enzyme, HincII, into four fragments. Two of these fragments were called HincII-S (short) and HincII-L (long), respectively. Each of these two fragments conferred the Tsg phenotype, indicating that, in fact, these two independent regions were responsible for the Tsg phenotype. The HincII-S 783-bp and HincII-L 1,479-bp fragments were sequenced. The region in the HincII-S fragment to which the Tsg phenotype was attributed was narrowed to a 146-bp (nucleotides 1 to 146) fragment by various restriction enzyme digestions. Further digestion of the 146-bp fragment with Bal 31 suggested that the 116-bp (nucleotides 9 to 124) fragment is the minimum sequence required for Tsg. On the other hand, in the HincII-L fragment, a fragment of 249 bp (nucleotides 1210 to 1458) and a fragment of 321 bp (nucleotides 1942 to 2262) contained separate temperature-sensitive growth activity. None of three tsg fragments contained open reading frames. The 249-bp fragment had very weak Tsg activity, while the 321-bp fragment had no Tsg activity. On the other hand, when these two fragments were together in the pUC19 vector, they exhibited very strong Tsg activity equivalent to that of the original 1,479-bp fragment. In addition, two of the 249-bp fragments gave similar, strong Tsg activity. The HincII-L 1,479-bp fragment contained an open reading frame for kanamycin resistance which was found between nucleotides 1423 and 2238. This kanamycin resistance gene sequence was different from that of the reported kanamycin resistance gene of Tn903 at 12 positions which were deduced to change seven amino acids.
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1000. |
Cullik A,
Pfeifer Y,
Prager R,
von Baum H,
Witte W,
( 2010 ) A novel IS26 structure surrounds blaCTX-M genes in different plasmids from German clinical Escherichia coli isolates. PMID : 20093380 : DOI : 10.1099/jmm.0.016188-0 Abstract >>
This report focuses on the molecular characterization of 22 extended-spectrum beta-lactamase-producing Escherichia coli isolates collected in a German university hospital during a period of 9 months in 2006. Relationship analysis of clinical isolates was done via PFGE, multilocus sequence typing, plasmid profiling and additionally PCR for bla(ESBL) detection and determination of phylogroups. After conjugal transfer, plasmid isolation and subsequent PCR for bla(ESBL) detection and determination of incompatibility groups were performed. Using one-primer walking, up to 3600 bp upstream and downstream of different bla(CTX-M) genes could be sequenced. beta-Lactamases found were TEM-1 (n=14), SHV-5 (n=1) and a wide variety of CTX-M types (n=21), i.e. CTX-M-15 (n=12), CTX-M-1 (n=4), CTX-M-14 (n=2), CTX-M-9 (n=1), CTX-M-3 (n=1) and one new type, CTX-M-65 (n=1). In 18 isolates, bla(ESBL) genes were located on conjugative plasmids of sizes between 40 and 180 kbp belonging to incompatibility groups FII (n=9), N (n=5) and I1 (n=4). bla(CTX-M) was found to be associated with the common elements ISEcp1, IS26 and IS903-D, but with unusual spacer sequences for ISEcp1 in two isolates. These insertion sequences, connected to bla(CTX-M) as well as other genes, were located between two IS26 elements in a configuration that has not yet been described. The results reveal the emergence of bla(ESBL), predominantly bla(CTX-M), located on different plasmids harboured by genotypically different E. coli strains. The identical gene arrangement in the bla(CTX-M) neighbourhood in plasmids of different incompatibility groups indicates a main role of IS26 in distribution of mobile resistance elements between different plasmids.
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1001. |
Kokotek W,
Lotz W,
( 1991 ) Construction of a mobilizable cloning vector for site-directed mutagenesis of gram-negative bacteria: application to Rhizobium leguminosarum. PMID : 2013412 : DOI : 10.1016/0378-1119(91)90097-u Abstract >>
A mobilizable cloning vector was constructed from defined fragments to serve as a suicide plasmid for site-directed mutagenesis. The new vector, pKOK4, closely resembles plasmid pBR325. However, the inverted duplication existing in the latter was not introduced. The useful cloning sites of pBR325 (EcoRI, HindIII, EcoRV, BamHI, SalI, PstI and PvuI) were retained and are located in one of the three resistance markers, ApR, CmR or TcR, respectively. Also, in pKOK4 the CmR gene retains its own promoter. The mob site of plasmid RP4 was introduced as a 760-bp fragment at a defined location. The mobilization frequency of pKOK4 within Escherichia coli strains is approx. 4 x 10(-2) per recipient cell. The size of pKOK4, deduced from the construction, is 6368 bp. We used pKOK4 for site-directed mutagenesis of hup-specific DNA from Rhizobium leguminosarum B10. Integration of the vector could be distinguished reliably from marker exchange by screening for the antibiotic resistance(s) of the plasmid. This reduced the number of clones to be retested by colony and Southern hybridization to approx. 1% of the original number. Of these, almost 70% contained the desired marker exchange.
|
1002. |
Snelling AM,
Macfarlane-Smith LR,
Fletcher JN,
Okeke IN,
( 2009 ) The commonly-used DNA probe for diffusely-adherent Escherichia coli cross-reacts with a subset of enteroaggregative E. coli. PMID : 20025771 : DOI : 10.1186/1471-2180-9-269 PMC : PMC2803494 Abstract >>
The roles of diffusely-adherent Escherichia coli (DAEC) and enteroaggregative E. coli (EAEC) in disease are not well understood, in part because of the limitations of diagnostic tests for each of these categories of diarrhoea-causing E. coli. A HEp-2 adherence assay is the Gold Standard for detecting both EAEC and DAEC but DNA probes with limited sensitivity are also employed. We demonstrate that the daaC probe, conventionally used to detect DAEC, cross-reacts with a subset of strains belonging to the EAEC category. The cross hybridization is due to 84% identity, at the nucleotide level, between the daaC locus and the aggregative adherence fimbriae II cluster gene, aafC, present in some EAEC strains. Because aaf-positive EAEC show a better association with diarrhoea than other EAEC, this specific cross-hybridization may have contributed to an over-estimation of the association of daaC with disease in some studies. We have developed a discriminatory PCR-RFLP protocol to delineate EAEC strains detected by the daaC probe in molecular epidemiological studies. A PCR-RFLP protocol described herein can be used to identify aaf-positive EAEC and daaC-positive DAEC and to delineate these two types of diarrhoeagenic E. coli, which both react with the daaC probe. This should help to improve current understanding and future investigations of DAEC and EAEC epidemiology.
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1003. |
Lescat M,
Hoede C,
Clermont O,
Garry L,
Darlu P,
Tuffery P,
Denamur E,
Picard B,
( 2009 ) aes, the gene encoding the esterase B in Escherichia coli, is a powerful phylogenetic marker of the species. PMID : 20040078 : DOI : 10.1186/1471-2180-9-273 PMC : PMC2805673 Abstract >>
Previous studies have established a correlation between electrophoretic polymorphism of esterase B, and virulence and phylogeny of Escherichia coli. Strains belonging to the phylogenetic group B2 are more frequently implicated in extraintestinal infections and include esterase B2 variants, whereas phylogenetic groups A, B1 and D contain less virulent strains and include esterase B1 variants. We investigated esterase B as a marker of phylogeny and/or virulence, in a thorough analysis of the esterase B-encoding gene. We identified the gene encoding esterase B as the acetyl-esterase gene (aes) using gene disruption. The analysis of aes nucleotide sequences in a panel of 78 reference strains, including the E. coli reference (ECOR) strains, demonstrated that the gene is under purifying selection. The phylogenetic tree reconstructed from aes sequences showed a strong correlation with the species phylogenetic history, based on multi-locus sequence typing using six housekeeping genes. The unambiguous distinction between variants B1 and B2 by electrophoresis was consistent with Aes amino-acid sequence analysis and protein modelling, which showed that substituted amino acids in the two esterase B variants occurred mostly at different sites on the protein surface. Studies in an experimental mouse model of septicaemia using mutant strains did not reveal a direct link between aes and extraintestinal virulence. Moreover, we did not find any genes in the chromosomal region of aes to be associated with virulence. Our findings suggest that aes does not play a direct role in the virulence of E. coli extraintestinal infection. However, this gene acts as a powerful marker of phylogeny, illustrating the extensive divergence of B2 phylogenetic group strains from the rest of the species.
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1004. |
Call DR,
Singer RS,
Meng D,
Broschat SL,
Orfe LH,
Anderson JM,
Herndon DR,
Kappmeyer LS,
Daniels JB,
Besser TE,
( 2010 ) blaCMY-2-positive IncA/C plasmids from Escherichia coli and Salmonella enterica are a distinct component of a larger lineage of plasmids. PMID : 19949054 : DOI : 10.1128/AAC.00055-09 PMC : PMC2812137 Abstract >>
Large multidrug resistance plasmids of the A/C incompatibility complex (IncA/C) have been found in a diverse group of Gram-negative commensal and pathogenic bacteria. We present three completed sequences from IncA/C plasmids that originated from Escherichia coli (cattle) and Salmonella enterica serovar Newport (human) and that carry the cephamycinase gene blaCMY-2. These large plasmids (148 to 166 kbp) share extensive sequence identity and synteny. The most divergent plasmid, peH4H, has lost several conjugation-related genes and has gained a kanamycin resistance region. Two of the plasmids (pAM04528 and peH4H) harbor two copies of blaCMY-2, while the third plasmid (pAR060302) harbors a single copy of the gene. The majority of single-nucleotide polymorphisms comprise nonsynonymous mutations in floR. A comparative analysis of these plasmids with five other published IncA/C plasmids showed that the blaCMY-2 plasmids from E. coli and S. enterica are genetically distinct from those originating from Yersinia pestis and Photobacterium damselae and distal to one originating from Yersinia ruckeri. While the overall similarity of these plasmids supports the likelihood of recent movements among E. coli and S. enterica hosts, their greater divergence from Y. pestis or Y. ruckeri suggests less recent plasmid transfer among these pathogen groups.
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1005. |
Yamamoto D,
Hernandes RT,
Blanco M,
Greune L,
Schmidt MA,
Carneiro SM,
Dahbi G,
Blanco JE,
Mora A,
Blanco J,
Gomes TA,
( 2009 ) Invasiveness as a putative additional virulence mechanism of some atypical Enteropathogenic Escherichia coli strains with different uncommon intimin types. PMID : 19622141 : DOI : 10.1186/1471-2180-9-146 PMC : PMC2724384 Abstract >>
Enteropathogenic Escherichia coli (EPEC) produce attaching/effacing (A/E) lesions on eukaryotic cells mediated by the outer membrane adhesin intimin. EPEC are sub-grouped into typical (tEPEC) and atypical (aEPEC). We have recently demonstrated that aEPEC strain 1551-2 (serotype O non-typable, non-motile) invades HeLa cells by a process dependent on the expression of intimin sub-type omicron. In this study, we evaluated whether aEPEC strains expressing other intimin sub-types are also invasive using the quantitative gentamicin protection assay. We also evaluated whether aEPEC invade differentiated intestinal T84 cells. Five of six strains invaded HeLa and T84 cells in a range of 13.3%-20.9% and 5.8%-17.8%, respectively, of the total cell-associated bacteria. The strains studied were significantly more invasive than prototype tEPEC strain E2348/69 (1.4% and 0.5% in HeLa and T84 cells, respectively). Invasiveness was confirmed by transmission electron microscopy. We also showed that invasion of HeLa cells by aEPEC 1551-2 depended on actin filaments, but not on microtubules. In addition, disruption of tight junctions enhanced its invasion efficiency in T84 cells, suggesting preferential invasion via a non-differentiated surface. Some aEPEC strains may invade intestinal cells in vitro with varying efficiencies and independently of the intimin sub-type.
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1006. |
Mairhofer J,
Cserjan-Puschmann M,
Striedner G,
Nöbauer K,
Razzazi-Fazeli E,
Grabherr R,
( 2010 ) Marker-free plasmids for gene therapeutic applications--lack of antibiotic resistance gene substantially improves the manufacturing process. PMID : 20138928 : DOI : 10.1016/j.jbiotec.2010.01.025 Abstract >>
Plasmid DNA is being considered as a promising alternative to traditional protein vaccines or viral delivery methods for gene therapeutic applications. DNA-based products are highly flexible, stable, are easily stored and can be manufactured on a large scale. Although, much safer than viral approaches, issues have been raised with regard to safety due to possible integration of plasmid DNA into cellular DNA or spread of antibiotic resistance genes to intestinal bacteria by horizontal gene transfer. Accordingly, there is interest in methods for the production of plasmid DNA that lacks the antibiotic resistance gene to further improve their safety profile. Here, we report for the first time the gram-scale manufacturing of a minimized plasmid that is devoid of any additional sequence elements on the plasmid backbone, and merely consists of the target expression cassette and the bacterial origin of replication. Three different host/vector combinations were cultivated in a fed-batch fermentation process, comparing the progenitor strain JM108 to modified strains JM108murselect, hosting a plasmid either containing the aminoglycoside phosphotransferase which provides kanamycin resistance, or a marker-free variant of the same plasmid. The metabolic load exerted by expression of the aminoglycoside phosphotransferase was monitored by measuring ppGpp- and cAMP-levels. Moreover, we revealed that JM108 is deficient of the Lon protease and thereby refined the genotype of JM108. The main consequences of Lon-deficiency with regard to plasmid DNA production are discussed herein. Additionally, we found that the expression of the aminoglycoside phosphotransferase, conferring resistance to kanamycin, was very high in plasmid DNA producing processes that actually inclusion bodies were formed. Thereby, a severe metabolic load on the host cell was imposed, detrimental for overall plasmid yield. Hence, deleting the antibiotic resistance gene from the vector backbone is not only beneficial with regards to safety and potency of the end-product but also regarding the overall process performance.
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1007. |
Liu K,
Knabel SJ,
Dudley EG,
( 2009 ) rhs genes are potential markers for multilocus sequence typing of Escherichia coli O157:H7 strains. PMID : 19633111 : DOI : 10.1128/AEM.00859-09 PMC : PMC2747862 Abstract >>
DNA sequence-based molecular subtyping methods such as multilocus sequence typing (MLST) are commonly used to generate phylogenetic inferences for monomorphic pathogens. The development of an effective MLST scheme for subtyping Escherichia coli O157:H7 has been hindered in the past due to the lack of sequence variation found within analyzed housekeeping and virulence genes. A recent study suggested that rhs genes are under strong positive selection pressure, and therefore in this study we analyzed these genes within a diverse collection of E. coli O157:H7 strains for sequence variability. Eighteen O157:H7 strains from lineages I and II and 15 O157:H7 strains from eight clades were included. Examination of these rhs genes revealed 44 polymorphic loci (PL) and 10 sequence types (STs) among the 18 lineage strains and 280 PL and 12 STs among the 15 clade strains. Phylogenetic analysis using rhs genes generally grouped strains according to their known lineage and clade classifications. These findings also suggested that O157:H7 strains from clades 6 and 8 fall into lineage I/II and that strains of clades 1, 2, 3, and 4 fall into lineage I. Additionally, unique markers were found in rhsA and rhsJ that might be used to define clade 8 and clade 6. Therefore, rhs genes may be useful markers for phylogenetic analysis of E. coli O157:H7.
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1008. |
Javier Terán F,
Alvarez M,
Suárez JE,
Mendoza MC,
( 1991 ) Characterization of two aminoglycoside-(3)-N-acetyltransferase genes and assay as epidemiological probes. PMID : 1960117 : DOI : 10.1093/jac/28.3.333 Abstract >>
Two genes encoding for aminoglycoside-(3)-N-acetyltransferases (AAC(3)s) of different substrate patterns, present in multiresistance plasmids of hospital strains of Serratia marcescens and Escherichia coli isolated from urine, have been cloned and characterized. The first, aacC1 with AAC(3)I activity, contained a 531 base pair open reading frame which encodes a polypeptide of 177 aminoacids and 19,392 daltons, confirmed by minicell analysis. Its sequence differed from previously published work in four positions. Three of the changes did not alter the aminoacid sequence while the fourth was a substitution of an alanine by a proline. The second gene, an AAC(3)II encoded by aacC2, resulted from the translation of an 858 base pair open reading frame, which encoded a 286 aminoacid polypeptide of 31,574 daltons and was identical to those from plasmids isolated in Germany and the United States. However, the homology was broken in a position between the -10 and -35 promoter sequences, which resulted in different -35 hexanucleotides and levels of resistance conferred. The assay of both genes as molecular probes has revealed their specificity with respect to other aac genes, although their usefulness was limited in the case of aacC1 derived sequences to isolated plasmid DNA, since it hybridized under stringent conditions with chromosomal DNA of some strains of E. coli.
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1009. |
Novais A,
Baquero F,
Machado E,
Cantón R,
Peixe L,
Coque TM,
( 2010 ) International spread and persistence of TEM-24 is caused by the confluence of highly penetrating enterobacteriaceae clones and an IncA/C2 plasmid containing Tn1696::Tn1 and IS5075-Tn21. PMID : 19995930 : DOI : 10.1128/AAC.00959-09 PMC : PMC2812134 Abstract >>
TEM-24 remains one of the most widespread TEM-type extended-spectrum beta-lactamases (ESBLs) among Enterobacteriaceae. To analyze the reasons influencing its spread and persistence, a multilevel population genetics study was carried out on 28 representative TEM-24 producers from Belgium, France, Portugal, and Spain (13 Enterobacter aerogenes isolates, 6 Escherichia coli isolates, 6 Klebsiella pneumoniae isolates, 2 Proteus mirabilis isolates, and 1 Klebsiella oxytoca isolate, from 1998 to 2004). Clonal relatedness (XbaI pulsed-field gel electrophoresis [PFGE] and E. coli phylogroups) and antibiotic susceptibility were determined by standard procedures. Plasmid analysis included determination of the incompatibility group (by PCR, hybridization, and/or sequencing) and comparison of restriction fragment length polymorphism (RFLP) patterns. Characterization of genetic elements conferring antibiotic resistance included integrons (classes 1, 2, and 3) and transposons (Tn3, Tn21, and Tn402). Similar PFGE patterns were identified among E. aerogenes, K. pneumoniae, and P. mirabilis isolates, while E. coli strains were diverse (phylogenetic groups A, B2, and D). Highly related 180-kb IncA/C2 plasmids conferring resistance to kanamycin, tobramycin, chloramphenicol, trimethoprim, and sulfonamides were identified. Each plasmid contained defective In0-Tn402 (dfrA1-aadA1, aacA4, or aacA4-aacC1-orfE-aadA2-cmlA1) and In4-Tn402 (aacA4 or dfrA1-aadA1) variants. These integrons were located within Tn21, Tn1696, or hybrids of these transposons, with IS5075 interrupting their IRtnp and IRmer. In all cases, blaTEM-24 was part of an IS5075-DeltaTn1 transposon within tnp1696, mimicking other genetic elements containing blaTEM-2 and blaTEM-3 variants. The international dissemination of TEM-24 is fuelled by an IncA/C2 plasmid acquired by different enterobacterial clones which seem to evolve by gaining diverse genetic elements. This work highlights the risks of a confluence between highly penetrating clones and highly promiscuous plasmids in the spread of antibiotic resistance, and it contributes to the elucidation of the origin and evolution of TEM-2 ESBL derivatives.
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1010. |
Hagelueken G,
Huang H,
Mainprize IL,
Whitfield C,
Naismith JH,
( 2009 ) Crystal structures of Wzb of Escherichia coli and CpsB of Streptococcus pneumoniae, representatives of two families of tyrosine phosphatases that regulate capsule assembly. PMID : 19616007 : DOI : 10.1016/j.jmb.2009.07.026 PMC : PMC2777267 Abstract >>
Many Gram-positive and Gram-negative bacteria utilize polysaccharide surface layers called capsules to evade the immune system; consequently, the synthesis and export of the capsule are a potential therapeutic target. In Escherichia coli K-30, the integral membrane tyrosine autokinase Wzc and the cognate phosphatase Wzb have been shown to be key for both synthesis and assembly of capsular polysaccharides. In the Gram-positive bacterium Streptococcus pneumoniae, the CpsCD complex is analogous to Wzc and the phosphatase CpsB is the corresponding cognate phosphatase. The phosphatases are known to dephosphorylate their corresponding autokinases, yet despite their functional equivalence, they share no sequence homology. We present the structure of Wzb in complex with phosphate and high-resolution structures of apo-CpsB and a phosphate-complexed CpsB. We show that both proteins are active toward Wzc and thereby demonstrate that CpsB is not specific for CpsCD. CpsB is a novel enzyme and represents the first solved structure of a tyrosine phosphatase from a Gram-positive bacterium. Wzb and CpsB have completely different structures, suggesting that they must operate by very different mechanisms. Although the mechanism of Wzb can be inferred from previous studies, CpsB appears to have a tyrosine phosphatase mechanism not observed before. We propose a chemical mechanism for CpsB based on site-directed mutagenesis and structural data.
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1011. |
Chandran V,
Fronzes R,
Duquerroy S,
Cronin N,
Navaza J,
Waksman G,
( 2009 ) Structure of the outer membrane complex of a type IV secretion system. PMID : 19946264 : DOI : 10.1038/nature08588 PMC : PMC2797999 Abstract >>
Type IV secretion systems are secretion nanomachines spanning the two membranes of Gram-negative bacteria. Three proteins, VirB7, VirB9 and VirB10, assemble into a 1.05 megadalton (MDa) core spanning the inner and outer membranes. This core consists of 14 copies of each of the proteins and forms two layers, the I and O layers, inserting in the inner and outer membrane, respectively. Here we present the crystal structure of a approximately 0.6 MDa outer-membrane complex containing the entire O layer. This structure is the largest determined for an outer-membrane channel and is unprecedented in being composed of three proteins. Unexpectedly, this structure identifies VirB10 as the outer-membrane channel with a unique hydrophobic double-helical transmembrane region. This structure establishes VirB10 as the only known protein crossing both membranes of Gram-negative bacteria. Comparison of the cryo-electron microscopy (cryo-EM) and crystallographic structures points to conformational changes regulating channel opening and closing.
|
1012. |
Endimiani A,
Doi Y,
Bethel CR,
Taracila M,
Adams-Haduch JM,
O'Keefe A,
Hujer AM,
Paterson DL,
Skalweit MJ,
Page MG,
Drawz SM,
Bonomo RA,
( 2010 ) Enhancing resistance to cephalosporins in class C beta-lactamases: impact of Gly214Glu in CMY-2. PMID : 19938877 : DOI : 10.1021/bi9015549 PMC : PMC4018810 Abstract >>
The biochemical properties of CMY-32, a class C enzyme possessing a single-amino acid substitution in the Omega loop (Gly214Glu), were compared to those of the parent enzyme, CMY-2, a widespread class C beta-lactamase. In parallel with our microbiological characterization, the Gly214Glu substitution in CMY-32 reduced catalytic efficiency (k(cat)/K(m)) by 50-70% against "good" substrates (i.e., cephalothin) while increasing k(cat)/K(m) against "poor" substrates (i.e., cefotaxime). Additionally, CMY-32 was more susceptible to inactivation by sulfone beta-lactamase inhibitors (i.e., sulbactam and tazobactam) than CMY-2. Timed electrospray ionization mass spectrometry (ESI-MS) analysis of the reaction of CMY-2 and CMY-32 with different substrates and inhibitors suggested that both beta-lactamases formed similar intermediates during catalysis and inactivation. We next showed that the carbapenems (imipenem, meropenem, and doripenem) form long-lived acyl-enzyme intermediates and present evidence that there is beta-lactamase-catalyzed elimination of the C(6) hydroxyethyl substituent. Furthermore, we discovered that the monobactam aztreonam and BAL29880, a new beta-lactamase inhibitor of the monobactam class, inactivate CMY-2 and CMY-32 by forming an acyl-enzyme intermediate that undergoes elimination of SO(3)(2-). Molecular modeling and dynamics simulations suggest that the Omega loop is more constrained in CMY-32 than CMY-2. Our model also proposes that Gln120 adopts a novel conformation in the active site while new interactions form between Glu214 and Tyr221, thus explaining the increased level of cefotaxime hydrolysis. When it is docked in the active site, we observe that BAL29880 exploits contacts with highly conserved residues Lys67 and Asn152 in CMY-2 and CMY-32. These findings highlight (i) the impact of single-amino acid substitutions on protein evolution in clinically important AmpC enzymes and (ii) the novel insights into the mechanisms by which carbapenems and monobactams interact with CMY-2 and CMY-32 beta-lactamases.
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1013. |
Zhang Y,
Laing C,
Zhang Z,
Hallewell J,
You C,
Ziebell K,
Johnson RP,
Kropinski AM,
Thomas JE,
Karmali M,
Gannon VP,
( 2010 ) Lineage and host source are both correlated with levels of Shiga toxin 2 production by Escherichia coli O157:H7 strains. PMID : 19948861 : DOI : 10.1128/AEM.01288-09 PMC : PMC2805208 Abstract >>
Escherichia coli O157:H7 strains fall into three major genetic lineages that differ in their distribution among humans and cattle. Several recent studies have reported differences in the expression of virulence factors between E. coli O157:H7 strains from these two host species. In this study, we wished to determine if important virulence-associated "mobile genetic elements" such as Shiga toxin 2 (Stx2)-encoding prophage are lineage restricted or are host source related and acquired independently of the pathogen genotype. DNA sequencing of the stx(2) flanking region from a lineage II (LII) strain, EC970520, revealed that the transcriptional activator gene Q in LI strain EDL933 (upstream of stx(2)) is replaced by a pphA (serine/threonine phosphatase) homologue and an altered Q gene in this and all other LII strains tested. In addition, nearly all LI strains carried stx(2), whereas all LII strains carried variant stx(2c) and 4 of 14 LI/II strains had copies of both stx(2) and variant stx(2c). Real-time PCR (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) demonstrated that LI and LI/II strains produce significantly more stx(2) mRNA and Stx2 than LII strains. However, among LI strains significantly more Stx2 is also produced by strains from humans than from cattle. Therefore, lineage-associated differences among E. coli O157:H7 strains such as prophage content, toxin type, and toxin expression may contribute to host isolation bias. However, the level of Stx2 production alone may also play an important role in the within-lineage association of E. coli O157:H7 strains with human clinical disease.
|
1014. |
Handford CL,
Stang CT,
Raivio TL,
Dennis JJ,
( 2009 ) The contribution of small cryptic plasmids to the antibiotic resistance of enteropathogenic Escherichia coli E2348/69. PMID : 19940931 : DOI : 10.1139/w09-079 Abstract >>
Two uncharacterized small cryptic plasmids (SCPs) were isolated from enteropathogenic Escherichia coli strain E2348/69. Genomic DNA sequence analysis of both SCPs indicated that the smaller plasmid, p5217, encoded several mobilization genes, whereas the larger plasmid, p6148, encoded several putative antibiotic resistance determinants. Complementation analysis showed that p6148 encodes functional streptomycin resistance genes but, owing to the presence of several frameshift mutations, a nonfunctional sulfonamide resistance determinant. A plasmid similar to p6148 has previously been shown to confer a slight growth advantage on E. coli. However, we were unable to observe any significant growth advantage in different E. coli strains transformed with p6148. The p6148 DNA sequence is homologous in sequence and arrangement to DNA from other plasmid families, including large conjugative plasmids and SXT integrative and conjugative elements. This study suggests that gene clusters of the sul2-strAB antibiotic resistance genes are widespread and highly transferable, owing to their presence in a wide variety of mobile genetic elements.
|
1015. |
Tozzoli R,
Caprioli A,
Cappannella S,
Michelacci V,
Marziano ML,
Morabito S,
( 2010 ) Production of the subtilase AB5 cytotoxin by Shiga toxin-negative Escherichia coli. PMID : 19940059 : DOI : 10.1128/JCM.01648-09 PMC : PMC2812301 Abstract >>
The subtilase cytotoxin (SubAB) is an AB(5) toxin described in certain Shiga toxin (Stx)-producing Escherichia coli (STEC) strains that usually lack the locus for enterocyte effacement (LEE). We report for the first time the production of SubAB by two Stx-negative E. coli strains, isolated from unrelated cases of childhood diarrhea. The characterization of the SubAB-coding genes showed a 90% nucleotide sequence similarity with that of the prototype subAB, located on the virulence plasmid of the STEC O113 strain 98NK2 (pO113). In both strains, subAB was physically associated with tia, an invasion genetic determinant of enterotoxigenic E. coli. The strains were negative for the saa gene, encoding an adhesin located on pO113 and present in many of the SubAB-positive strains described so far. PCR screening of 61 STEC and 100 Stx-negative E. coli strains in our collection revealed the presence of subAB in five LEE-negative STEC strains but not in the Stx-negative strains. subAB was contiguous to tia in three of the positive strains, which were all negative for saa. These results indicate that SubAB production is not restricted to STEC and suggest that a subAB-tia putative pathogenicity island is involved in the dissemination of subAB genes, as an alternative to plasmid pO113.
|
1016. |
Verdet C,
Gautier V,
Chachaty E,
Ronco E,
Hidri N,
Decré D,
Arlet G,
( 2009 ) Genetic context of plasmid-carried blaCMY-2-like genes in Enterobacteriaceae. PMID : 19596889 : DOI : 10.1128/AAC.00753-08 PMC : PMC2737857 Abstract >>
Analysis of 15 European clinical Enterobacteriaceae isolates showed that differences in the genetic context of blaCMY-2-like genes reflected the replicon type, usually IncA/C or IncI1. These blaCMY-2 loci may originate from the same ISEcp1-mediated mobilization from the Citrobacter freundii chromosome as structures described in earlier studies.
|
1017. |
Wang RC,
Seror SJ,
Blight M,
Pratt JM,
Broome-Smith JK,
Holland IB,
( 1991 ) Analysis of the membrane organization of an Escherichia coli protein translocator, HlyB, a member of a large family of prokaryote and eukaryote surface transport proteins. PMID : 1994034 : DOI : 10.1016/0022-2836(91)90748-u Abstract >>
Haemolysin B (HlyB) is essential for secretion of the 107 x 10(3) Mr haemolysin A protein from Escherichia coli and is a member of a family of highly conserved, apparently ATP-dependent surface proteins in many organisms. We have shown in this study that both HlyB and HlyD fractionate primarily with the cytoplasmic membrane of E. coli and are accessible to proteases after removal of the outer membrane. We have measured experimentally the topological organization of HlyB within the membrane by construction of fusions to beta-lactamase as a reporter. The predicted folding of HlyB, with a minimum of six transmembrane segments, does not always coincide with regions of highest average hydrophobicity. This suggests that HlyB may have a novel organization within the bilayer. From our data and comparative sequence analysis, we have been able to predict very similar topological models for the other members of the HlyB family.
|
1018. |
Liu B,
Wu F,
Li D,
Beutin L,
Chen M,
Cao B,
Wang L,
( 2010 ) Development of a serogroup-specific DNA microarray for identification of Escherichia coli strains associated with bovine septicemia and diarrhea. PMID : 19932572 : DOI : 10.1016/j.vetmic.2009.10.019 Abstract >>
Escherichia coli strains belonging to serogroups O8, O9, O15, O26, O35, O78, O86, O101, O115 and O119 are commonly associated with septicemia or diarrhea in calves and pose a significant threat to the cattle industry worldwide. In this study, a microarray detection system targeting O-antigen-specific genes was developed for the identification of those serogroups. By testing against 186 E. coli and Shigella O-serogroup reference strains, 36 E. coli clinical isolates, and 9 representative strains of other closely related bacterial species, the microarray was shown to be specific and reproducible. The detection sensitivity was determined to be 50 ng genomic DNA. The microarray assay developed here is suitable for the detection and identification of relevant strains from environmental and/or clinical samples, and is especially useful for epidemiologic studies.
|
1019. |
Yuan L,
Liu JH,
Hu GZ,
Pan YS,
Liu ZM,
Mo J,
Wei YJ,
( 2009 ) Molecular characterization of extended-spectrum beta-lactamase-producing Escherichia coli isolates from chickens in Henan Province, China. PMID : 19574412 : DOI : 10.1099/jmm.0.012229-0 Abstract >>
Extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli has spread rapidly worldwide and poses a serious threat to human and animal health. This study collected 51 non-replicate E. coli isolates from 14 different chicken farms in Henan Province in China from December 2007 to August 2008. The prevalence of ESBL-producing E. coli, molecular characterization of the ESBL-related bla genes, including bla(TEM), bla(SHV) and bla(CTX-M), and the susceptibilities of these bacteria to various antimicrobial agents were determined. Thirty-one of the 51 isolates were positive for an ESBL phenotype and 29 of these isolates carried one or more bla genes. Twenty-two isolates harboured bla(TEM) genes and 15 isolates carried bla(CTX-M) genes (one CTX-M-14, three CTX-M-24 and 11 CTX-M-65). One isolate carried bla(TEM)(-57); the remaining bla(TEM) isolates carried bla(TEM-1) with one silent nucleotide base variation (T18C). We believe that this is the first study to report TEM-57 in E. coli isolates. All isolates harbouring bla(CTX-M-24) and bla(CTX-M-14) and five of the bla(CTX-M-65) isolates also harboured the bla(TEM-1) gene. To our knowledge, this study is the first to describe detection of CTX-M-65-producing E. coli isolated from chickens. None of the isolates contained the bla(SHV) gene. Conjugation experiments demonstrated that bla(CTX-M) and bla(TEM) genes could be transferred to E. coli DH5 alpha. The results indicate that ESBL frequency has reached an alarming level in chicken isolates in China, with TEM-1 and CTX-M-65 enzymes being the two predominant beta-lactamases detected.
|
1020. |
Leinberger DM,
Grimm V,
Rubtsova M,
Weile J,
Schröppel K,
Wichelhaus TA,
Knabbe C,
Schmid RD,
Bachmann TT,
( 2010 ) Integrated detection of extended-spectrum-beta-lactam resistance by DNA microarray-based genotyping of TEM, SHV, and CTX-M genes. PMID : 20007393 : DOI : 10.1128/JCM.00765-09 PMC : PMC2815585 Abstract >>
Extended-spectrum beta-lactamases (ESBL) of the TEM, SHV, or CTX-M type confer resistance to beta-lactam antibiotics in gram-negative bacteria. The activity of these enzymes against beta-lactam antibiotics and their resistance against inhibitors can be influenced by genetic variation at the single-nucleotide level. Here, we describe the development and validation of an oligonucleotide microarray for the rapid identification of ESBLs in gram-negative bacteria by simultaneously genotyping bla(TEM), bla(SHV), and bla(CTX-M). The array consists of 618 probes that cover mutations responsible for 156 amino acid substitutions. As this comprises unprecedented genotyping coverage, the ESBL array has a high potential for epidemiological studies and infection control. With an assay time of 5 h, the ESBL microarray also could be an attractive option for the development of rapid antimicrobial resistance tests in the future. The validity of the DNA microarray was demonstrated with 60 blinded clinical isolates, which were collected during clinical routines. Fifty-eight of them were characterized phenotypically as ESBL producers. The chip was characterized with regard to its resolution, phenotype-genotype correlation, and ability to resolve mixed genotypes. ESBL phenotypes could be correctly ascribed to ESBL variants of bla(CTX-M) (76%), bla(SHV) (22%), or both (2%), whereas no ESBL variant of bla(TEM) was found. The most prevalent ESBLs identified were CTX-M-15 (57%) and SHV-12 (18%).
|
1021. |
Xia X,
Meng J,
McDermott PF,
Ayers S,
Blickenstaff K,
Tran TT,
Abbott J,
Zheng J,
Zhao S,
( 2010 ) Presence and characterization of shiga toxin-producing Escherichia coli and other potentially diarrheagenic E. coli strains in retail meats. PMID : 20080990 : DOI : 10.1128/AEM.01968-09 PMC : PMC2837998 Abstract >>
To determine the presence of Shiga toxin-producing Escherichia coli (STEC) and other potentially diarrheagenic E. coli strains in retail meats, 7,258 E. coli isolates collected by the U.S. National Antimicrobial Resistance Monitoring System (NARMS) retail meat program from 2002 to 2007 were screened for Shiga toxin genes. In addition, 1,275 of the E. coli isolates recovered in 2006 were examined for virulence genes specific for other diarrheagenic E. coli strains. Seventeen isolates (16 from ground beef and 1 from a pork chop) were positive for stx genes, including 5 positive for both stx(1) and stx(2), 2 positive for stx(1), and 10 positive for stx(2). The 17 STEC strains belonged to 10 serotypes: O83:H8, O8:H16, O15:H16, O15:H17, O88:H38, ONT:H51, ONT:H2, ONT:H10, ONT:H7, and ONT:H46. None of the STEC isolates contained eae, whereas seven carried enterohemorrhagic E. coli (EHEC) hlyA. All except one STEC isolate exhibited toxic effects on Vero cells. DNA sequence analysis showed that the stx(2) genes from five STEC isolates encoded mucus-activatable Stx2d. Subtyping of the 17 STEC isolates by pulsed-field gel electrophoresis (PFGE) yielded 14 distinct restriction patterns. Among the 1,275 isolates from 2006, 11 atypical enteropathogenic E. coli (EPEC) isolates were identified in addition to 3 STEC isolates. This study demonstrated that retail meats, mainly ground beef, were contaminated with diverse STEC strains. The presence of atypical EPEC strains in retail meat is also of concern due to their potential to cause human infections.
|
1022. |
Sampaio SC,
Gomes TA,
Pichon C,
du Merle L,
Guadagnini S,
Abe CM,
Sampaio JL,
Le Bouguénec C,
( 2009 ) The flagella of an atypical enteropathogenic Escherichia coli strain are required for efficient interaction with and stimulation of interleukin-8 production by enterocytes in vitro. PMID : 19620340 : DOI : 10.1128/IAI.00177-09 PMC : PMC2747955 Abstract >>
The ability of some typical enteropathogenic Escherichia coli (EPEC) strains to adhere to, invade, and increase interleukin-8 (IL-8) production in intestinal epithelial cells in vitro has been demonstrated. However, few studies regarding these aspects have been performed with atypical EPEC (aEPEC) strains, which are emerging enteropathogens in Brazil. In this study, we evaluated a selected aEPEC strain (1711-4) of serotype O51:H40, the most prevalent aEPEC serotype in Brazil, in regard to its ability to adhere to and invade Caco-2 and T84 cells and to elicit IL-8 production in Caco-2 cells. The role of flagella in aEPEC 1711-4 adhesion, invasion, and IL-8 production was investigated by performing the same experiments with an isogenic aEPEC mutant unable to produce flagellin (FliC), the flagellum protein subunit. We demonstrated that this mutant (fliC mutant) had a marked decrease in the ability to adhere to T84 cells and invade both T84 and Caco-2 cells in gentamicin protection assays and by transmission electron microscopy. In addition, the aEPEC 1711-4 fliC mutant had a reduced ability to stimulate IL-8 production by Caco-2 cells in early (3-h) but not in late (24-h) infections. Our findings demonstrate that flagella of aEPEC 1711-4 are required for efficient adhesion, invasion, and early but not late IL-8 production in intestinal epithelial cells in vitro.
|
1023. |
Schmitt CK,
McKee ML,
O'Brien AD,
( 1991 ) Two copies of Shiga-like toxin II-related genes common in enterohemorrhagic Escherichia coli strains are responsible for the antigenic heterogeneity of the O157:H- strain E32511. PMID : 1997410 : PMC : PMC258368 Abstract >>
Thirty-two clinical isolates of Shiga-like toxin (SLT)-producing Escherichia coli associated with single cases or outbreaks of bloody diarrhea, hemorrhagic colitis, the hemolytic uremic syndrome, or edema disease of swine were examined for multiple copies of genes belonging to the slt-I or slt-II toxin families. Five of 19 strains that were known to produce SLT-II or to hybridize to slt-II-specific probes by colony blot were found by Southern hybridization to contain two copies of toxin genes related to slt-II. The genes for two toxins closely related to slt-II were cloned from one of the isolates, Escherichia coli O157:H- strain E32511. One copy of the operon was found to be essentially identical to slt-II; it differed from slt-II by only one nucleotide base. This single nucleotide difference did not affect the predicted amino acid sequence. The predicted amino acid sequence of the A subunit of the second operon was identical to that of SLT-II, but the predicted amino acid sequence of the B subunit was identical to that of the B2F1 toxin VT2ha. We designated this second operon slt-IIc. Neutralization assays using several monoclonal antibodies and polyclonal antiserum prepared against SLT-II showed that SLT-IIc was antigenically related to but distinct from SLT-II.
|
1024. |
Robin F,
Aggoune-Khinache N,
Delmas J,
Naim M,
Bonnet R,
( 2010 ) Novel VIM metallo-beta-lactamase variant from clinical isolates of Enterobacteriaceae from Algeria. PMID : 19901092 : DOI : 10.1128/AAC.00017-09 PMC : PMC2798476 Abstract >>
Five different strains of bacteria belonging to the family Enterobacteriaceae were isolated from two patients hospitalized in the intensive care unit of the Central Military Hospital of Algiers, Algeria. All five strains, one Providencia stuartii strain, two Escherichia coli strains, and two Klebsiella pneumoniae strains, were intermediate or resistant to all beta-lactams, including carbapenems. Synergy between imipenem and EDTA was observed for all five strains. The results of the PCR experiment confirmed the presence of a bla(VIM) gene in all five strains. The bla(VIM) genes were located as part of a class 1 integron on a 180-kb conjugative plasmid. They encoded a novel metallo-beta-lactamase designated VIM-19, which differed from the parental enzyme VIM-1 by only two substitutions: Ser228Arg, previously observed in the closely related enzyme VIM-4, and Asn215Lys, not previously observed in other VIM-type carbapenemases. VIM-19 was further characterized after purification through determination of its kinetic constants. This enzyme was inhibited by EDTA and hydrolyzed penicillins, cephalosporins, and carbapenems, as observed for other VIM-type carbapenemases but with greater catalytic efficiency against penicillins than VIM-1. VIM-19 is the first carbapenemase enzyme identified from an isolate from Algeria. These results confirm the emergence of VIM-4-like enzymes in members of the family Enterobacteriaceae from Mediterranean countries.
|
1025. |
Kawasaki Y,
Wada C,
Yura T,
( 1991 ) Mini-F plasmid mutants able to replicate in the absence of sigma 32: mutations in the repE coding region producing hyperactive initiator protein. PMID : 1991708 : DOI : 10.1128/jb.173.3.1064-1072.1991 PMC : PMC207225 Abstract >>
Mini-F plasmids cannot replicate in Escherichia coli strains (delta rpoH) lacking sigma 32, presumably because transcription of the repE gene encoding the replication initiator protein (RepE protein) depends mostly on RNA polymerase containing sigma 32. We have isolated and characterized mini-F mutants able to replicate in delta rpoH cells. Contrary to the initial expectation, five mutants with mutations in the repE coding region that produce altered RepE proteins were obtained. The mutations caused replacement of a single amino acid: the 92nd glutamic acid was replaced by lysine (repE10, repE16, and repE25) or glycine (repE22) or the 109th glutamic acid was replaced by lysine (repE26). These plasmids overproduced RepE protein and exhibited very high copy numbers. Two major activities of mutated RepE proteins have been determined in vivo; the autogenous repressor activity was significantly reduced, whereas the initiator activity was much enhanced in all mutants. These results indicate the importance of a small central region of RepE protein for both initiator and repressor activities. Thus the decreased repE transcription in delta rpoH cells can be compensated for by an increased initiator activity and a decreased repressor activity of RepE, resulting in the increased synthesis of hyperactive RepE protein.
|
1026. |
Pallecchi L,
Riccobono E,
Sennati S,
Mantella A,
Bartalesi F,
Trigoso C,
Gotuzzo E,
Bartoloni A,
Rossolini GM,
( 2010 ) Characterization of small ColE-like plasmids mediating widespread dissemination of the qnrB19 gene in commensal enterobacteria. PMID : 20008783 : DOI : 10.1128/AAC.01160-09 PMC : PMC2812132 Abstract >>
In this work, we have characterized two small ColE-like plasmids (pECY6-7, 2.7 kb in size, and pECC14-9, of 3.0 kb), encoding the QnrB19 quinolone resistance determinant, that were carried by several clonally unrelated quinolone-resistant commensal Escherichia coli strains isolated from healthy children living in different urban areas of Peru and Bolivia. The two plasmids are closely related to each other and carry the qnrB19 gene as the sole resistance determinant, located in a conserved genetic context between the plasmid RNAII sequence (which controls plasmid replication) and the plasmid Xer site (involved in plasmid dimer resolution). ISEcp1-like or other putative insertion sequences are not present in the qnrB19-flanking regions or elsewhere on the plasmids. Since we previously observed a high prevalence (54%) of qnrB genes in the metagenomes of commensal enterobacteria from the same population of healthy children, the presence of pECY6-7- and pECC14-9-like plasmids in those qnrB-positive metagenomes was investigated by PCR mapping. Both plasmids were found to be highly prevalent (67% and 16%, respectively) in the qnrB-positive metagenomes, suggesting that dissemination of these small plasmids played a major role in the widespread dissemination of qnrB genes observed in commensal enterobacteria from healthy children living in those areas.
|
1027. |
Dias RC,
Moreira BM,
Riley LW,
( 2010 ) Use of fimH single-nucleotide polymorphisms for strain typing of clinical isolates of Escherichia coli for epidemiologic investigation. PMID : 20018817 : DOI : 10.1128/JCM.01858-09 PMC : PMC2815601 Abstract >>
Strain typing methods that compare electrophoresis banding patterns are commonly used but are difficult to standardize and poorly portable. Multilocus sequence typing (MLST) is a sequence-based alternative, but it is not practical for large-scale epidemiological studies. In the present study, the usefulness of fimH single-nucleotide polymorphisms (SNPs) for Escherichia coli typing was explored. fimH SNPs were determined for 345 E. coli clinical isolates (including 3 reference strains) and compared to PCR-based ECOR (E. coli reference collection) phylogrouping. The fimH gene could be amplified for 316 (92%) of the 345 isolates. fimH SNP analysis found 46 distinct terminal groups in the nucleotide sequence-based phylogenetic tree (fimH types). A subset of the E. coli isolates (162 clinical isolates and the 3 reference strains) were compared by fimH type, PCR phylogroup, and MLST. These isolates fell into 27 fimH types and 18 MLST clonal complexes (CCs) that contained 2 to 28 isolates per complex. The combination of PCR phylogroup and fimH type corresponded to a single CC for 113 (68%) isolates and 2 or 3 CCs for the other 52 (32%) isolates. We propose that the combination of PCR phylogrouping and fimH SNP analysis may be a useful method to type a large collection of clinical E. coli isolates for epidemiologic studies.
|
1028. |
Chen SL,
Hung CS,
Pinkner JS,
Walker JN,
Cusumano CK,
Li Z,
Bouckaert J,
Gordon JI,
Hultgren SJ,
( 2009 ) Positive selection identifies an in vivo role for FimH during urinary tract infection in addition to mannose binding. PMID : 20018753 : DOI : 10.1073/pnas.0902179106 PMC : PMC2794649 Abstract >>
FimH, the type 1 pilus adhesin of uropathogenic Escherichia coli (UPEC), contains a receptor-binding domain with an acidic binding pocket specific for mannose. The fim operon, and thus type 1 pilus production, is under transcriptional control via phase variation of an invertible promoter element. FimH is critical during urinary tract infection for mediating colonization and invasion of the bladder epithelium and establishment of intracellular bacterial communities (IBCs). In silico analysis of FimH gene sequences from 279 E. coli strains identified specific amino acids evolving under positive selection outside of its mannose-binding pocket. Mutating two of these residues (A27V/V163A) had no effect on phase variation, pilus assembly, or mannose binding in vitro. However, compared to wild-type, this double mutant strain exhibited a 10,000-fold reduction in mouse bladder colonization 24 h after inoculation and was unable to form IBCs even though it bound normally to mannosylated receptors in the urothelium. In contrast, the single A62S mutation altered phase variation, reducing the proportion of piliated cells, reduced mannose binding 8-fold, and decreased bladder colonization 30-fold in vivo compared to wild-type. A phase-locked ON A62S mutant restored virulence to wild-type levels even though in vitro mannose binding remained impaired. Thus, positive selection analysis of FimH has separated mannose binding from in vivo fitness, suggesting that IBC formation is critical for successful infection of the mammalian bladder, providing support for more general use of in silico positive selection analysis to define the molecular underpinnings of bacterial pathogenesis.
|
1029. |
Derakhshandeh A,
Zahraei Salehi T,
Tadjbakhsh H,
Karimi V,
( 2009 ) Identification, cloning and sequencing of Escherichia coli strain chi1378 (O78:K80) iss gene isolated from poultry colibacillosis in Iran. PMID : 19622075 : DOI : 10.1111/j.1472-765X.2009.02681.x Abstract >>
To identify, clone and sequence the iss (increased serum survival) gene from E. coli strain chi1378 isolated from Iranian poultry and to predict its protein product, Iss. The iss gene from E. coli strain chi1378 was amplified and cloned into the pTZ57R/T vector and sequenced. From the DNA sequence, the Iss predictive protein was evaluated using bioinformatics. Iss from strain chi1378 had 100% identity with other E. coli serotypes and isolates from different origins and also 98% identity with E. coli O157:H7 Iss protein. Phylogenetic analysis showed no significant different phylogenic groups among E. coli strains. The strong association of predicted Iss protein among different E. coli strains suggests that it could be a good antigen to control and detect avian pathogenic E. coli (APEC). Because the exact pathogenesis and the role of virulence factors are unknown, the Iss protein could be used as a target for vaccination in the future, but further research is required.
|
1030. |
Huang Z,
Sutton SE,
Wallenfang AJ,
Orchard RC,
Wu X,
Feng Y,
Chai J,
Alto NM,
( 2009 ) Structural insights into host GTPase isoform selection by a family of bacterial GEF mimics. PMID : 19620963 : DOI : 10.1038/nsmb.1647 PMC : PMC5130228 Abstract >>
The Escherichia coli type III effector Map belongs to a large family of bacterial virulence factors that activate host Rho GTPase signaling pathways through an unknown molecular mechanism. Here we report direct evidence that Map functions as a potent and selective guanine-nucleotide exchange factor (GEF) for Cdc42. The 2.3-A structure of the Map-Cdc42 complex revealed that Map mimics the GEF strategy of the mammalian Dbl family but has a three-dimensional architecture that is nearly identical to the bacterial GEF Salmonella spp. SopE. A comparative analysis between human and bacterial GEFs revealed a previously uncharacterized pairing mechanism between Map and the variable beta2-3 interswitch region of Cdc42. We propose a GTPase selection model that is experimentally validated by the preferential activation Rac1 and RhoA by the Shigella spp. effectors IpgB1 and IpgB2, respectively. These results significantly expand the repertoire of bacterial GEF mimics and unify a GEF selection mechanism for host GTPase substrates.
|
1031. |
Tao T,
Bourne JC,
Blumenthal RM,
( 1991 ) A family of regulatory genes associated with type II restriction-modification systems. PMID : 1995588 : DOI : 10.1128/jb.173.4.1367-1375.1991 PMC : PMC207272 Abstract >>
Restriction-modification systems must be regulated to avoid autorestriction and death of the host cell. An open reading frame (ORF) in the PvuII restriction-modification system appears to code for a regulatory protein from a previously unrecognized family. First, interruptions of this ORF result in a nonrestricting phenotype. Second, this ORF can restore restriction competence to such interrupted mutants in trans. Third, the predicted amino acid sequence of this ORF resembles those of known DNA-binding proteins and includes a probable helix-turn-helix motif. A survey of unattributed ORFs in 15 other type II restriction-modification systems revealed three that closely resemble the PvuII ORF. All four members of this putative regulatory gene family have a common position relative to the endonuclease genes, suggesting a common regulatory mechanism.
|
1032. |
Regni CA,
Roush RF,
Miller DJ,
Nourse A,
Walsh CT,
Schulman BA,
( 2009 ) How the MccB bacterial ancestor of ubiquitin E1 initiates biosynthesis of the microcin C7 antibiotic. PMID : 19494832 : DOI : 10.1038/emboj.2009.146 PMC : PMC2711183 DOI : 10.1038/emboj.2009.146 PMC : PMC2711183 Abstract >>
The 39-kDa Escherichia coli enzyme MccB catalyses a remarkable posttranslational modification of the MccA heptapeptide during the biosynthesis of microcin C7 (MccC7), a 'Trojan horse' antibiotic. The approximately 260-residue C-terminal region of MccB is homologous to ubiquitin-like protein (UBL) activating enzyme (E1) adenylation domains. Accordingly, MccB-catalysed C-terminal MccA-acyl-adenylation is reminiscent of the E1-catalysed activation reaction. However, unlike E1 substrates, which are UBLs with a C-terminal di-glycine sequence, MccB's substrate, MccA, is a short peptide with an essential C-terminal Asn. Furthermore, after an intramolecular rearrangement of MccA-acyl-adenylate, MccB catalyses a second, unique reaction, producing a stable phosphoramidate-linked analogue of acyl-adenylated aspartic acid. We report six-crystal structures of MccB in apo, substrate-, intermediate-, and inhibitor-bound forms. Structural and kinetic analyses reveal a novel-peptide clamping mechanism for MccB binding to heptapeptide substrates and a dynamic-active site for catalysing dual adenosine triphosphate-consuming reactions. The results provide insight into how a distinctive member of the E1 superfamily carries out two-step activation for generating the peptidyl-antibiotic MccC7.
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1033. |
Rodriguez-Martinez JM,
Nordmann P,
Fortineau N,
Poirel L,
( 2010 ) VIM-19, a metallo-beta-lactamase with increased carbapenemase activity from Escherichia coli and Klebsiella pneumoniae. PMID : 19917750 : DOI : 10.1128/AAC.00458-09 PMC : PMC2798500 Abstract >>
Two carbapenem-resistant isolates, one Escherichia coli isolate and one Klebsiella pneumoniae isolate, recovered from an Algerian patient expressed a novel VIM-type metallo-beta-lactamase (MBL). The identified bla(VIM-19) gene was located on a ca. 160-kb plasmid and located inside a class 1 integron in both isolates. VIM-19 differed from VIM-1 by the Asn215Lys and Ser228Arg substitutions, increasing its hydrolytic activity toward carbapenems. Site-directed mutagenesis experiments showed that both substitutions were necessary for the increased carbapenemase activity of VIM-19. This study indicates that MBLs with enhanced activity toward carbapenems may be obtained as a result of very few amino acid substitutions.
|
1034. |
Miñana-Galbis D,
Urbizu-Serrano A,
Farfán M,
Fusté MC,
Lorén JG,
( 2009 ) Phylogenetic analysis and identification of Aeromonas species based on sequencing of the cpn60 universal target. PMID : 19567585 : DOI : 10.1099/ijs.0.005413-0 Abstract >>
An analysis of the universal target (UT) sequence from the cpn60 gene was performed in order to evaluate its usefulness in phylogenetic and taxonomic studies and as an identification marker for the genus Aeromonas. Sequences of 555 bp, corresponding to the UT region, were obtained from a collection of 35 strains representing all of the species and subspecies of Aeromonas. From the analysis of these sequences, a range of divergence of 0-23.3% was obtained, with a mean of 11.2+/-0.9%. Comparative analyses between cpn60 and gyrB, rpoD and 16S rRNA gene sequences were carried out from the same Aeromonas strain collection. Sequences of the cpn60 UT region showed similar discriminatory power to gyrB and rpoD sequences. The phylogenetic relationships inferred from cpn60 sequence distances indicated an excellent correlation with the present affiliation of Aeromonas species with the exception of Aeromonas hydrophila subsp. dhakensis, which appeared in a separate phylogenetic line, and Aeromonas sharmana, which exhibited a very loose phylogenetic relationship to the genus Aeromonas. Sequencing of cpn60 from 33 additional Aeromonas strains also allowed us to establish intra- and interspecific threshold values. Intraspecific divergence rates were
|
1035. |
Ajiboye RM,
Solberg OD,
Lee BM,
Raphael E,
Debroy C,
Riley LW,
( 2009 ) Global spread of mobile antimicrobial drug resistance determinants in human and animal Escherichia coli and Salmonella strains causing community-acquired infections. PMID : 19538087 : DOI : 10.1086/600301 Abstract >>
Antimicrobial drugs used in human infection treatment and animal husbandry may select for drug-resistant bacterial pathogens, which are increasingly observed worldwide. We sought to examine the extent to which identical mobile drug resistance elements are shared across common pathogens isolated from human and animal sources. We compared the distribution of one class of mobile elements--integrons and gene cassettes--among uropathogenic Escherichia coli isolates, Salmonella enterica serovar Typhimurium isolates from human diarrhea cases, and E. coli and Salmonella isolates from nonhuman sources in the United States. The sequences of the gene cassettes were also compared with those deposited in GenBank. Class 1 integrons were detected in 68 (49%) of 139 uropathogenic E. coli isolates, 16 (72%) of 22 human and animal Salmonella isolates, and 120 (28%) of 436 nonhuman E. coli isolates. The most prevalent cassettes were those encoding aminoglycoside adenyltransferase A (aadA) and dihydrofolate reductase A (dfrA). Sequences of aadA1, dfrA12-orfF-aadA2, and dfrA17-aadA5 gene cassettes from 35 urinary tract infection E. coli isolates and of aadA2 and aadA12 gene cassettes from 7 Salmonella isolates were 100% identical to the corresponding cassette sequences from food animal E. coli isolates and those deposited in GenBank from a wide variety of bacteria isolated from animal hosts from distinct regions of the world. Common community-acquired human drug-resistant infections are caused by bacterial strains that harbor mobile drug resistance determinants of identical sequences that are found in diverse bacterial species from varied animal sources worldwide.
|
1036. |
Tennant SM,
Tauschek M,
Azzopardi K,
Bigham A,
Bennett-Wood V,
Hartland EL,
Qi W,
Whittam TS,
Robins-Browne RM,
( 2009 ) Characterisation of atypical enteropathogenic E. coli strains of clinical origin. PMID : 19490652 : DOI : 10.1186/1471-2180-9-117 PMC : PMC2700815 Abstract >>
Enteropathogenic E. coli (EPEC) is a prominent cause of diarrhoea, and is characterised in part by its carriage of a pathogenicity island: the locus for enterocyte effacement (LEE). EPEC is divided into two subtypes according to the presence of bundle-forming pili (BFP), a fimbrial adhesin that is a virulence determinant of typical EPEC (tEPEC), but is absent from atypical EPEC (aEPEC). Because aEPEC lack BFP, their virulence has been questioned, as they may represent LEE-positive Shiga toxin-producing E. coli (STEC) that have lost the toxin-encoding prophage, or tEPEC that have lost the genes for BFP. To determine if aEPEC isolated from humans in Australia or New Zealand fall into either of these categories, we undertook phylogenetic analysis of 75 aEPEC strains, and compared them with reference strains of EPEC and STEC. We also used PCR and DNA hybridisation to determine if aEPEC carry virulence determinants that could compensate for their lack of BFP. The results showed that aEPEC are highly heterogeneous. Multilocus sequence typing revealed that 61 of 75 aEPEC strains did not belong to known tEPEC or STEC clades, and of those that did, none expressed an O:H serotype that is frequent in tEPEC or STEC strains associated with disease. PCR for each of 18 known virulence-associated determinants of E. coli was positive in less than 15% of strains, apart from NleB which was detected in 30%. Type I fimbriae were expressed by all aEPEC strains, and 12 strains hybridised with DNA probes prepared from either bfpA or bfpB despite being negative in the PCR for bfpA. Our findings indicate that clinical isolates of aEPEC obtained from patients in Australia or New Zealand are not derived from tEPEC or STEC, and suggest that functional equivalents of BFP and possibly type I fimbriae may contribute to the virulence of some aEPEC strains.
|
1037. |
Niki H,
Jaffé A,
Imamura R,
Ogura T,
Hiraga S,
( 1991 ) The new gene mukB codes for a 177 kd protein with coiled-coil domains involved in chromosome partitioning of E. coli. PMID : 1989883 : PMC : PMC452628 Abstract >>
An Escherichia coli temperature sensitive mutant which produces spontaneously normal size anucleate cells at low temperature was isolated. The mutant is defective in a previously undescribed gene, named mukB, located at 21 min on the chromosome. The mukB gene codes for a large protein (approximately 180 kd). A 1534 amino acid protein (176,826 daltons) was deduced from the nucleotide sequence of the mukB gene. Computer analysis revealed that the predicted MukB protein has distinct domains: an amino-terminal globular domain containing a nucleotide binding sequence, a central region containing two alpha-helical coiled-coil domains and one globular domain, and a carboxyl-terminal globular domain which is rich in Cys, Arg and Lys. A 180 kd protein detected in wild-type cell extracts by electrophoresis is absent in mukB null mutants. Although the null mutants are not lethal at low temperature, the absence of MukB leads to aberrant chromosome partitioning. At high temperature the mukB null mutants cannot form colonies and many nucleoids are distributed irregularly along elongated cells. We conclude that the MukB protein is required for chromosome partitioning in E. coli.
|
1038. |
DebRoy C,
Sidhu MS,
Sarker U,
Jayarao BM,
Stell AL,
Bell NP,
Johnson TJ,
( 2010 ) Complete sequence of pEC14_114, a highly conserved IncFIB/FIIA plasmid associated with uropathogenic Escherichia coli cystitis strains. PMID : 19887083 : DOI : 10.1016/j.plasmid.2009.10.003 Abstract >>
Extraintestinal pathogenic Escherichia coli (ExPEC) are known to cause important diseases of humans and animals, and they have been shown to carry a variety of plasmids associated with increased virulence and decreased antimicrobial susceptibility. Here, the completed DNA sequence of a human uropathogenic E. coli (UPEC; O6:H31 isolate) plasmid, pEC14_114, was determined. The plasmid was 114,222bp in length and was highly similar to plasmid sequences or draft contiguous sequences from three other human cystitis-associated UPEC isolates. pEC14_114 contained 141 coding regions, including a number of genes associated with mobile genetic elements, F-type transfer, plasmid maintenance and stability, colicin immunity, and plasmid replication. This plasmid also possessed a "genetic load" region containing genes with predicted similarity to iron acquisition systems and virulence factors. The prevalence of pEC14-associated genes was determined for a collection of 1456 E. coli isolates, including those from food products, humans, dogs, cats, pigs, chickens, and turkeys. pEC14_114-associated genes were found significantly more often (16-35%) among human UPEC and neonatal meningitis-associated isolates than among food- and animal-source isolates (0-8%). Overall, this plasmid represents a novel IncFIB/FIIA plasmid type associated with human ExPEC belonging to the B2 phylogenetic group. The overall role of this plasmid, if any, in human ExPEC infections remains to be determined.
|
1039. |
Mata C,
Miró E,
Rivera A,
Mirelis B,
Coll P,
Navarro F,
( 2010 ) Prevalence of acquired AmpC beta-lactamases in Enterobacteriaceae lacking inducible chromosomal ampC genes at a Spanish hospital from 1999 to 2007. PMID : 19523051 : DOI : 10.1111/j.1469-0691.2009.02864.x Abstract >>
In 2007, a significant increase in acquired ampC genes in Enterobacteriaceae from 0.06% in 1999 to 1.3% was observed. Proteus mirabilis showed the highest prevalence (0.95%) and CMY-2 was the most prevalent AmpC enzyme (66.7%). Other enzymes such as CMY-4, DHA-1, ACC-1, and three new enzymes called CMY-25, CMY-27 and CMY-40 were detected. Seven out of the 117 isolates (6%) also produced an extended-spectrum beta-lactamase. As acquired AmpC enzymes are likely to become a serious public health issue worldwide, close surveillance is necessary to curb their spread.
|
1040. |
Perez-Casal J,
Swartley JS,
Scott JR,
( 1990 ) Gene encoding the major subunit of CS1 pili of human enterotoxigenic Escherichia coli. PMID : 1977705 : PMC : PMC313703 Abstract >>
Some enterotoxigenic strains of Escherichia coli (ETEC) utilize the CS1 pilus for colonization of human intestinal epithelium. We have cloned the gene which encodes the major CS1 subunit and called it cooA (for coli surface antigen one). Hybridization showed that the ETEC strain from which it was cloned carried cooA on a plasmid different from the one encoding its positive regulator, rns. Based on the cooA DNA sequence, cleavage with signal peptidase would be expected to produce a mature protein of 15.2 kDa; a 16-kDa polypeptide that reacted with CS1-specific antiserum was observed on electrophoresis. At the protein level, there was 92% similarity and 55% identity between cooA and cfaB, the major colonization factor antigen I (CFA/I) antigen. However, CS1-specific antisera did not react with CfaB. No hybridization was seen between either of two different cooA probes and total DNA from ETEC strains expressing AFA-1, CFA/I, CS2, CS3, CS4, CS5, or CS6.
|
1041. |
Simons BL,
Willemsen PT,
Bakker D,
Roosendaal B,
De Graaf FK,
Oudega B,
( 1990 ) Structure, localization and function of FanF, a minor component of K99 fibrillae of enterotoxigenic Escherichia coli. PMID : 1982454 : DOI : 10.1111/j.1365-2958.1990.tb00564.x Abstract >>
The DNA sequence of the K99 fanF gene, encoding FanF, was determined. An open reading frame of 999 bp was found. The primary structure of FanF was deduced and analysis revealed the presence of a signal sequence of 22 amino acid residues. The mature protein contains 311 amino acid residues (Mr 33,905 D). The amino acid sequence of FanF showed similarity with the K88ab major subunit FaeG. A specific mouse antiserum against FanF was prepared by constructing and purifying a hybrid Cro-LacZ-FanF protein. Minicell analysis, immunoblotting and immunoelectronmicroscopy revealed a pool of FanF in the periplasm of K99-producing cells and showed, furthermore, that FanF is a minor component of K99 fibrillae, present at the top and in or along the shaft of the K99 fibrillar structures. A fanF mutant plasmid was constructed. Cells harbouring this plasmid produced all K99-specific proteins, except FanF, but produced 0.1% of the K99 fibrillae relative to 'normal' K99-producing cells. Electron microscopic observations showed that cells defective in fanF produce only a few (apparently short) K99 fibrillae. FanF, therefore, was supposed to play a role in initiation and elongation of K99 fibrillae formation. Thin-layer chromatography experiments involving purified receptor material showed that FanF is not required for binding of K99 fibrillae to the ganglioside receptor. Fibrillae produced by an adhesion-negative strain carrying a mutation in the K99 major fibrillar subunit were shown to contain a normal amount of FanF.
|
1042. |
Di Laurenzio L,
Frost LS,
Finlay BB,
Paranchych W,
( 1991 ) Characterization of the oriT region of the IncFV plasmid pED208. PMID : 1943709 : DOI : 10.1111/j.1365-2958.1991.tb01927.x Abstract >>
DNA sequence analysis of a 2.2kb EcoRI-HindIII fragment from pED208, the derepressed form of the IncFV plasmid Folac, revealed sequences highly homologous to the oriT region, traM, and traJ genes of other IncF plasmids. The TraM protein was purified and immunoblots of fractionated cells containing pED208 or Folac showed that TraM was predominantly in the cytoplasm. Using DNA retardation assays and the DNase I footprinting technique, the TraM protein was found to bind to three large motifs in the oriT region: (I) an inverted repeat, (II) two direct repeats, and (III) the traM promoter region. These three footprint regions contained a Hinfl-like sequence (GANTC) that appeared 16 times, spaced 11-12 bp (or multiples thereof) apart, suggesting that TraM protein binds in a complex manner over this entire region.
|
1043. |
Lu L,
Dai L,
Wang Y,
Wu C,
Chen X,
Li L,
Qi Y,
Xia L,
Shen J,
( 2010 ) Characterization of antimicrobial resistance and integrons among Escherichia coli isolated from animal farms in Eastern China. PMID : 19744463 : DOI : 10.1016/j.actatropica.2009.08.028 Abstract >>
A total of 182 Escherichia coli isolates from animals, environment and workers of dairy cattle, swine and chicken farms in Shandong which locates in Eastern China, were investigated for antimicrobial resistance as well as prevalence and the transfer mechanisms of integrons. The results revealed isolates from swine and chicken farm exhibited high levels of resistance to antimicrobial agents. The positive rate of gene cassette of class 1 integron in dairy cattle, swine and chicken farms was 5%, 20% and 41.94%, respectively. Only four isolates possessed class 2 integron, all of which were from chicken farm. Nine distinct cassette arrays were detected and two novel gene cassette arrays yheSDelta-yheR-kefBDelta and chrADelta-sul1-qacEDelta1-orf5-aadA5-dfrA17 were identified in class 1 integron for the first time. Class 1 integrons were found to be located mostly in both chromosomal and conjugative plasmid through southern hybridization and conjugation. PFGE revealed clonal relatedness among the isolates from different sources, especially within the same farm. The results confirmed the antimicrobial resistance and prevalence of integrons were strongly associated with the selection pressure of antimicrobial agents, and resistance genes in animal farms were probably spread by both vertical and horizontal transfer.
|
1044. |
Müller D,
Benz I,
Liebchen A,
Gallitz I,
Karch H,
Schmidt MA,
( 2009 ) Comparative analysis of the locus of enterocyte effacement and its flanking regions. PMID : 19506015 : DOI : 10.1128/IAI.00090-09 PMC : PMC2715695 Abstract >>
The attaching-and-effacing (A/E) phenotype mediated by factors derived from the locus of enterocyte effacement (LEE) is a hallmark of clinically important intestinal pathotypes of Escherichia coli, including enteropathogenic (EPEC), atypical EPEC (ATEC), and enterohemorrhagic E. coli strains. Epidemiological studies indicate that the frequency of diarrhea outbreaks caused by ATEC is increasing. Hence, it is of major importance to further characterize putative factors contributing to the pathogenicity of these strains and to gain additional insight into the plasticity and evolutionary aspects of this emerging pathotype. Here, we analyzed the two clinical ATEC isolates B6 (O26:K60) and 9812 (O128:H2) and compared the genetic organizations, flanking regions, and chromosomal insertion loci of their LEE with those of the LEE of other A/E pathogens. Our analysis shows that the core LEE is largely conserved-particularly among genes coding for the type 3 secretion system-whereas genes encoding effector proteins display a higher variability. Chromosomal insertion loci appear to be restricted to selC, pheU, and pheV. In contrast, striking differences were found between the 5'- and 3'-associated flanking regions reflecting the different histories of the various strains and also possibly indicating different lines in evolution.
|
1045. |
Fricke WF,
Welch TJ,
McDermott PF,
Mammel MK,
LeClerc JE,
White DG,
Cebula TA,
Ravel J,
( 2009 ) Comparative genomics of the IncA/C multidrug resistance plasmid family. PMID : 19482926 : DOI : 10.1128/JB.00189-09 PMC : PMC2715731 Abstract >>
Multidrug resistance (MDR) plasmids belonging to the IncA/C plasmid family are widely distributed among Salmonella and other enterobacterial isolates from agricultural sources and have, at least once, also been identified in a drug-resistant Yersinia pestis isolate (IP275) from Madagascar. Here, we present the complete plasmid sequences of the IncA/C reference plasmid pRA1 (143,963 bp), isolated in 1971 from the fish pathogen Aeromonas hydrophila, and of the cryptic IncA/C plasmid pRAx (49,763 bp), isolated from Escherichia coli transconjugant D7-3, which was obtained through pRA1 transfer in 1980. Using comparative sequence analysis of pRA1 and pRAx with recent members of the IncA/C plasmid family, we show that both plasmids provide novel insights into the evolution of the IncA/C MDR plasmid family and the minimal machinery necessary for stable IncA/C plasmid maintenance. Our results indicate that recent members of the IncA/C plasmid family evolved from a common ancestor, similar in composition to pRA1, through stepwise integration of horizontally acquired resistance gene arrays into a conserved plasmid backbone. Phylogenetic comparisons predict type IV secretion-like conjugative transfer operons encoded on the shared plasmid backbones to be closely related to a group of integrating conjugative elements, which use conjugative transfer for horizontal propagation but stably integrate into the host chromosome during vegetative growth. A hipAB toxin-antitoxin gene cluster found on pRA1, which in Escherichia coli is involved in the formation of persister cell subpopulations, suggests persistence as an early broad-spectrum antimicrobial resistance mechanism in the evolution of IncA/C resistance plasmids.
|
1046. |
Moura RA,
Sircili MP,
Leomil L,
Matté MH,
Trabulsi LR,
Elias WP,
Irino K,
Pestana de Castro AF,
( 2009 ) Clonal relationship among atypical enteropathogenic Escherichia coli strains isolated from different animal species and humans. PMID : 19801470 : DOI : 10.1128/AEM.00636-09 PMC : PMC2786407 Abstract >>
Forty-nine typical and atypical enteropathogenic Escherichia coli (EPEC) strains belonging to different serotypes and isolated from humans, pets (cats and dogs), farm animals (bovines, sheep, and rabbits), and wild animals (monkeys) were investigated for virulence markers and clonal similarity by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). The virulence markers analyzed revealed that atypical EPEC strains isolated from animals have the potential to cause diarrhea in humans. A close clonal relationship between human and animal isolates was found by MLST and PFGE. These results indicate that these animals act as atypical EPEC reservoirs and may represent sources of infection for humans. Since humans also act as a reservoir of atypical EPEC strains, the cycle of mutual infection of atypical EPEC between animals and humans, mainly pets and their owners, cannot be ruled out since the transmission dynamics between the reservoirs are not yet clearly understood.
|
1047. |
Yao Y,
Xie Y,
Perace D,
Zhong Y,
Lu J,
Tao J,
Guo X,
Kim KS,
( 2009 ) The type III secretion system is involved in the invasion and intracellular survival of Escherichia coli K1 in human brain microvascular endothelial cells. PMID : 19758329 : DOI : 10.1111/j.1574-6968.2009.01763.x PMC : PMC2829324 Abstract >>
Type III secretion systems (T3SSs) have been documented in many Gram-negative bacteria, including enterohemorrhagic Escherichia coli. We have previously shown the existence of a putative T3SS in meningitis-causing E. coli K1 strains, referred to as E. coli type III secretion 2 (ETT2). The sequence of ETT2 in meningitis-causing E. coli K1 strain EC10 (O7:K1) revealed that ETT2 comprises the epr, epa and eiv genes, but bears mutations, deletions and insertions. We constructed the EC10 mutants deleted of ETT2 or eivA gene, and their contributions to bacterial pathogenesis were evaluated in human brain microvascular endothelial cells (HBMECs). The deletion mutant of ETT2 exhibited defects in invasion and intracellular survival compared with the parental E. coli K1 strain EC10. The mutant deleted of eivA within ETT2 was also significantly defective in invasion and intracellular survival in HBMECs, and the defects of the eiv mutant were restored to the levels of the parent strain EC10 by transcomplementation. These findings suggest that ETT2 plays a role in the pathogenesis of E. coli K1 infection, including meningitis.
|
1048. |
Kaur S,
Kamli MR,
Ali A,
( 2009 ) Diversity of arsenate reductase genes (arsC Genes) from arsenic-resistant environmental isolates of E. coli. PMID : 19484295 : DOI : 10.1007/s00284-009-9432-9 Abstract >>
A polymerase chain reaction (PCR) approach was used to assess the occurrence and diversity of arsenate reductase gene (arsC gene) in arsenic-resistant environmental E. coli strains. For this purpose, two different sets of primers were designed for the specific amplification of approximately 370-bp fragments from the arsC gene. These primers were used to screen a collection of 25 environmental arsenic-resistant strains isolated from different geographical regions of India, as well as Bangladesh. The PCR results showed that 17 out of the 25 environmental isolates (68%) contained a gene related to the arsC family. Phylogenetic analysis of the protein sequences deduced from the amplicons indicated a prevalence of arsC genes in the isolated strains. A significant divergence in the DNA sequence was found in the arsC genes among As-resistant environmental E. coli strains from this study, and arsenic resistance, a genetic character, arose from a common ancestral background.
|
1049. |
Yamaguchi Y,
Sato G,
Yamagata Y,
Doi Y,
Wachino J,
Arakawa Y,
Matsuda K,
Kurosaki H,
( 2009 ) Structure of AmpC beta-lactamase (AmpCD) from an Escherichia coli clinical isolate with a tripeptide deletion (Gly286-Ser287-Asp288) in the H10 helix. PMID : 19478427 : DOI : 10.1107/S1744309109014249 PMC : PMC2688406 Abstract >>
The X-ray crystal structure of AmpC beta-lactamase (AmpC(D)) with a tripeptide deletion (Gly286-Ser287-Asp288) produced by Escherichia coli HKY28, a ceftazidime-resistant strain, was determined at a resolution of 1.7 A. The structure of AmpC(D) suggests that the tripeptide deletion at positions 286-288 located in the H10 helix causes a structural change of the Asn289-Asn294 region from the alpha-helix present in the native AmpC beta-lactamase of E. coli to a loop structure, which results in a widening of the substrate-binding site.
|
1050. |
Mshana SE,
Imirzalioglu C,
Hossain H,
Hain T,
Domann E,
Chakraborty T,
( 2009 ) Conjugative IncFI plasmids carrying CTX-M-15 among Escherichia coli ESBL producing isolates at a University hospital in Germany. PMID : 19534775 : DOI : 10.1186/1471-2334-9-97 PMC : PMC2708165 Abstract >>
Multi-drug-resistant, extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae, constitute an emerging public-health concern. Little data on the molecular epidemiology of ESBL producing Escherichia coli is available in Germany. Here we describe the prevalence and molecular epidemiology of ESBL producing-Escherichia coli isolates at a German University hospital. We analysed 63 non-duplicate clinical ESBL isolates obtained over an 8-month period using PCR and sequence-based ESBL allele typing, plasmid replicon typing, phylogenetic group typing. Pulsed-field gel electrophoresis (PFGE) based genotyping and plasmid profiling was performed, as well as confirmatory DNA-based hybridization assays. Examination of the 63 Escherichia coli isolates revealed an almost equal distribution among the E. coli phylogenetic groups A, B1, B2 and D. High prevalence (36/63) of the CTX-M-15 gene was observed and an analysis of PFGE-based patterns revealed the presence of this CTX-M allele in multiple clones. Resistance to cefotaxime was a transferable trait and a commonly occurring 145.5 kb conjugative IncFI plasmid was detected in 65% of E. coli carrying the CTX-M-15 allele. The rate of transferable antibiotic resistances for GM, SXT, TET, GM-SXT-TET, SXT-TET and GM-TET was 33%, 61%, 61%, 27%, 44% and 11%, respectively. The remaining strains did not have a common IncFI plasmid but harboured transferable IncFI plasmids with sizes that ranged from 97 to 242.5 kb. Our data demonstrate the presence of IncFI plasmids within the prevailing E. coli population in a hospital setting and suggest that the dissemination of CTX-M-15 allele is associated to lateral transfer of these well-adapted, conjugative IncFI plasmids among various E. coli genotypes.
|
1051. |
Leopold SR,
Magrini V,
Holt NJ,
Shaikh N,
Mardis ER,
Cagno J,
Ogura Y,
Iguchi A,
Hayashi T,
Mellmann A,
Karch H,
Besser TE,
Sawyer SA,
Whittam TS,
Tarr PI,
( 2009 ) A precise reconstruction of the emergence and constrained radiations of Escherichia coli O157 portrayed by backbone concatenomic analysis. PMID : 19439656 : DOI : 10.1073/pnas.0812949106 PMC : PMC2689004 Abstract >>
Single nucleotide polymorphisms (SNPs) in stable genome regions provide durable measurements of species evolution. We systematically identified each SNP in concatenations of all backbone ORFs in 7 newly or previously sequenced evolutionarily instructive pathogenic Escherichia coli O157:H7, O157:H(-), and O55:H7. The 1,113 synonymous SNPs demonstrate emergence of the largest cluster of this pathogen only in the last millennium. Unexpectedly, shared SNPs within circumscribed clusters of organisms suggest severely restricted survival and limited effective population sizes of pathogenic O157:H7, tenuous survival of these organisms in nature, source-sink evolutionary dynamics, or, possibly, a limited number of mutations that confer selective advantage. A single large segment spanning the rfb-gnd gene cluster is the only backbone region convincingly acquired by recombination as O157 emerged from O55. This concatenomic analysis also supports using SNPs to differentiate closely related pathogens for infection control and forensic purposes. However, constrained radiations raise the possibility of making false associations between isolates.
|
1052. |
Kim HB,
Wang M,
Park CH,
Kim EC,
Jacoby GA,
Hooper DC,
( 2009 ) oqxAB encoding a multidrug efflux pump in human clinical isolates of Enterobacteriaceae. PMID : 19528276 : DOI : 10.1128/AAC.01574-08 PMC : PMC2715617 Abstract >>
The genes for multidrug efflux pump OqxAB, which is active on fluoroquinolones, were found in human clinical isolates on a plasmid in Escherichia coli and on the chromosome of Klebsiella pneumoniae. IS26-like sequences flanked the plasmid-mediated oqxAB genes, suggesting that they had been mobilized as part of a composite transposon.
|
1053. |
Du XD,
Wu CM,
Liu HB,
Li XS,
Beier RC,
Xiao F,
Qin SS,
Huang SY,
Shen JZ,
( 2009 ) Plasmid-mediated ArmA and RmtB 16S rRNA methylases in Escherichia coli isolated from chickens. PMID : 19808234 : DOI : 10.1093/jac/dkp354 Abstract >>
N/A
|
1054. |
Jagura-Burdzy G,
Ibbotson JP,
Thomas CM,
( 1991 ) The korF region of broad-host-range plasmid RK2 encodes two polypeptides with transcriptional repressor activity. PMID : 1987165 : DOI : 10.1128/jb.173.2.826-833.1991 PMC : PMC207077 Abstract >>
Broad-host-range IncP plasmid RK2 possesses a series of operons involved in plasmid maintenance, whose expression is coordinated by a number of regulators, most of which are encoded in the central regulatory korA-korB operon. The nucleotide sequence of two new cistrons in this operon, comprising what we have previously designated the korF locus located between coordinates 57.0 and 56.0 kb on the genome of the IncP alpha plasmid RK2, is presented. The cistrons encode polypeptides of 173 and 175 amino acids. Each can repress transcription from the promoters for the kfrA (a monocistronic operon which follows the korA-korB operon) and trfA (a polycistronic operon encoding a putative single-stranded-DNA-binding protein as well as the essential plasmid replication protein TrfA) operons. In addition, the korF loci allow korB to repress kfrA transcription. Both polypeptides contain hydrophobic segments, suggesting that they may be membrane associated. KorFI is highly basic protein whose predicted properties are similar to those of histone like proteins.
|
1055. |
Arbeloa A,
Blanco M,
Moreira FC,
Bulgin R,
López C,
Dahbi G,
Blanco JE,
Mora A,
Alonso MP,
Mamani RC,
Gomes TA,
Blanco J,
Frankel G,
( 2009 ) Distribution of espM and espT among enteropathogenic and enterohaemorrhagic Escherichia coli. PMID : 19528152 : DOI : 10.1099/jmm.0.010231-0 PMC : PMC2884945 Abstract >>
Enterohaemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC) translocate dozens of type III secretion system effectors, including the WxxxE effectors Map, EspM and EspT that activate Rho GTPases. While map, which is carried on the LEE pathogenicity island, is absolutely conserved among EPEC and EHEC strains, the prevalence of espM and espT is not known. Here we report the results of a large screen aimed at determining the prevalence of espM and espT among clinical EPEC and EHEC isolates. The results suggest that espM, detected in 51 % of the tested strains, is more commonly found in EPEC and EHEC serogroups that are linked to severe human infections. In contrast, espT was absent from all the EHEC isolates and was found in only 1.8 % of the tested EPEC strains. Further characterization of the virulence gene repertoire of the espT-positive strains led to the identification of a new zeta2 intimin variant. All the espT-positive strains but two contained the tccP gene. espT was first found in Citrobacter rodentium and later in silico in EPEC E110019, which is of particular interest as this strain was responsible for a particularly severe diarrhoeal outbreak in Finland in 1987 that affected 650 individuals in a school complex and an additional 137 associated household members. Comparing the protein sequences of EspT to that of E110019 showed a high level of conservation, with only three strains encoding EspT that differed in 6 amino acids. At present, it is not clear why espT is so rare, and what impact EspM and EspT have on EPEC and EHEC infection.
|
1056. |
Miller EN,
Jarboe LR,
Yomano LP,
York SW,
Shanmugam KT,
Ingram LO,
( 2009 ) Silencing of NADPH-dependent oxidoreductase genes (yqhD and dkgA) in furfural-resistant ethanologenic Escherichia coli. PMID : 19429550 : DOI : 10.1128/AEM.00567-09 PMC : PMC2704836 Abstract >>
Low concentrations of furfural are formed as a side product during the dilute acid hydrolysis of hemicellulose. Growth is inhibited by exposure to furfural but resumes after the complete reduction of furfural to the less toxic furfuryl alcohol. Growth-based selection was used to isolate a furfural-resistant mutant of ethanologenic Escherichia coli LY180, designated strain EMFR9. Based on mRNA expression levels in the parent and mutant in response to furfural challenge, genes encoding 12 oxidoreductases were found to vary by more than twofold (eight were higher in EMFR9; four were higher in the parent). All 12 genes were cloned. When expressed from plasmids, none of the eight genes in the first group increased furfural tolerance in the parent (LY180). Expression of three of the silenced genes (yqhD, dkgA, and yqfA) in EMFR9 was found to decrease furfural tolerance compared to that in the parent. Purified enzymes encoded by yqhD and dkgA were shown to have NADPH-dependent furfural reductase activity. Both exhibited low K(m) values for NADPH (8 microM and 23 microM, respectively), similar to those of biosynthetic reactions. Furfural reductase activity was not associated with yqfA. Deleting yqhD and dkgA in the parent (LY180) increased furfural tolerance, but not to the same extent observed in the mutant EMFR9. Together, these results suggest that the process of reducing furfural by using an enzyme with a low K(m) for NADPH rather than a direct inhibitory action is the primary cause for growth inhibition by low concentrations of furfural.
|
1057. |
Valverde A,
Cantón R,
Garcillán-Barcia MP,
Novais A,
Galán JC,
Alvarado A,
de la Cruz F,
Baquero F,
Coque TM,
( 2009 ) Spread of bla(CTX-M-14) is driven mainly by IncK plasmids disseminated among Escherichia coli phylogroups A, B1, and D in Spain. PMID : 19786598 : DOI : 10.1128/AAC.01706-08 PMC : PMC2786348 Abstract >>
Since its first description in 2000, CTX-M-14 has become one of the most widespread extended-spectrum beta-lactamases in Spain. In the present Escherichia coli multilevel population genetic study involving the characterization of phylogroups, clones, plasmids, and genetic platforms, 61 isolates from 16 hospitalized patients and 40 outpatients and healthy volunteers recovered from 2000 to 2005 were analyzed. Clonal relatedness (XbaI pulsed-field gel electrophoresis [PFGE] type, phylogenetic group, multilocus sequence type [MLST]) was established by standard methods. Analysis of transferred plasmids (I-CeuI; S1 nuclease; restriction fragment length polymorphism analysis; and analysis of RNA interference, replicase, and relaxase) was performed by PCR, sequencing, and hybridization. The genetic environment of bla(CTX-M-14) was characterized by PCR on the basis of known associated structures (ISEcp1, IS903, ISCR1). The isolates were mainly recovered from patients in the community (73.8%; 45/61) with urinary tract infections (62.2%; 28/45). They were clonally unrelated by PFGE and corresponded to phylogenetic groups A (36.1%), D (34.4%), and B1 (29.5%). MLST revealed a high degree of sequence type (ST) diversity among phylogroup D isolates and the overrepresentation of the ST10 complex among phylogroup A isolates and ST359/ST155 among phylogroup B1 isolates. Two variants of bla(CTX-M-14) previously designated bla(CTX-M-14a) (n = 59/61) and bla(CTX-M-14b) (n = 2/61) were detected. bla(CTX-M-14a) was associated with either ISEcp1 within IncK plasmids (n = 27), ISCR1 linked to an IncHI2 plasmid (n = 1), or ISCR1 linked to IncI-like plasmids (n = 3). The bla(CTX-M-14b) identified was associated with an ISCR1 element located in an IncHI2 plasmid (n = 1) or with ISEcp1 located in IncK (n = 1). The CTX-M-14-producing E. coli isolates in our geographic area are frequent causes of community-acquired urinary tract infections. The increase in the incidence of such isolates is mostly due to the dissemination of IncK plasmids among E. coli isolates of phylogroups A, B1, and D.
|
1058. |
Tennent JM,
Lindberg F,
Normark S,
( 1990 ) Integrity of Escherichia coli P pili during biogenesis: properties and role of PapJ. PMID : 1975085 : DOI : 10.1111/j.1365-2958.1990.tb00645.x Abstract >>
The papJ gene of uropathogenic Escherichia coli is required to maintain the integrity of Gal alpha (1-4)Gal-binding P pili. Electron microscopy and ELISA have established that strains carrying the papJ1 mutant allele have a large amount of pilus antigen free of the cells. In contrast to the whole pili released by strains unable to produce the PapH pilus anchor, the free papJ1 pili consist of variably sized segments that appear to result from internal breakages to the pilus. The DNA sequence of papJ is presented and its gene product identified as an 18kD periplasmic protein that possesses homology with nucleotide-binding proteins. PapJ may function as a 'molecular chaperone' directly or indirectly establishing the correct assembly of PapA subunits in the P pilus.
|
1059. |
Wolf MK,
Boedeker EC,
( 1990 ) Cloning of the genes for AF/R1 pili from rabbit enteroadherent Escherichia coli RDEC-1 and DNA sequence of the major structural subunit. PMID : 1969392 : PMC : PMC258593 Abstract >>
AF/R1 pili on the surface of Escherichia coli RDEC-1 promote attachment of the bacteria to rabbit intestinal brush borders. In order to characterize AF/R1 pili and manipulate their expression, we cloned the genes necessary for AF/R1 expression; determined the size of proteins produced in minicells; located the gene encoding the major structural subunit, named AfrA; and determined the DNA sequence of afrA as well as the sequence of 700 additional nucleotides upstream of afrA. Two contiguous EcoRI fragments spanning 7.9 kilobases were cloned from the 86-megadalton plasmid of RDEC-1 into vector pUC19 to make plasmid pW1. Bacteria carrying pW1 produced AF/R1 pili that were recognized by AF/R1-specific antiserum and promoted adherence of bacteria to brush borders prepared from rabbit intestine. Proteins with a molecular weight of 17,000 (17K proteins), which was the size of AfrA, as well as 15K, 15.5K, 26K, 28K, and 80K proteins were detected in minicells carrying pW1. The gene afrA was located by using an oligonucleotide probe, and its DNA sequence was determined. The DNA sequence of 700 additional nucleotides upstream was determined because this sequence may be important in the regulation of AF/R1 expression.
|
1060. |
Fratamico PM,
DebRoy C,
Miyamoto T,
Liu Y,
( 2009 ) PCR detection of enterohemorrhagic Escherichia coli O145 in food by targeting genes in the E. coli O145 O-antigen gene cluster and the shiga toxin 1 and shiga toxin 2 genes. PMID : 19435408 : DOI : 10.1089/fpd.2008.0254 Abstract >>
Shiga toxin-producing Escherichia coli (STEC) strains belonging to serogroup O145 are an important cause of hemorrhagic colitis and hemolytic uremic syndrome worldwide. Cattle and other animals are potential reservoirs for this pathogen. To develop PCR assays for detection and identification of E. coli O145, the wzx (O-antigen flippase) and wzy (O-antigen polymerase) genes in the O145 O-antigen gene cluster that are specific for this serogroup were selected as targets. Oligonucleotide primers complementary to regions in the E. coli O145 wzx and wzy genes were designed to perform PCR assays with DNA from strains of E. coli O145, non-O145 E. coli serogroups, and other bacterial genera. The assays were highly specific for E. coli O145. A multiplex PCR assay targeting the E. coli O145 wzx and wzy genes and the Shiga toxin 1 (stx(1)) and Shiga toxin 2 (stx(2)) genes and a real-time multiplex PCR assay targeting the O145 wzy, stx(1), and stx(2) genes were developed for detection of STEC O145. The assays were used for detecting STEC O145 in seeded ground beef, lettuce, and raw milk initially inoculated with ca. 2, 20, or 200 CFU/25 g or 25 mL after 8 or 20 h of enrichment at 42 degrees C in modified EC broth containing 20 mg/L of novobiocin. STEC O145 was detected in all samples inoculated with 2 CFU/25 g or 25 mL. The detection limit of the multiplex PCR assays was
|
1061. |
Waye MM,
Mui F,
Hodge K,
Li VK,
( 1991 ) A phagemid vector library for cloning DNA with four-nucleotide 5' or 3' overhangs. PMID : 1946750 : Abstract >>
A phagemid vector library for cloning DNA with four nucleotide 5' or 3' overhangs has been constructed. This library is based on the pT7T3 vector (Pharmacia) which is a modification of the phagemid pTZ18U vector. We have chosen pT7T3 as the parent vector because it can be used for Sanger's dideoxy sequencing and for the generation of RNA probes with either the T7 or T3 promoter. Each member of the cloning vector series pBM has recognition sites for both of the restriction enzymes BspM1 and BstX1 in addition to the basic multiple cloning sites. BspM1 recognizes the sequence 5'...ACCTGC NNNN/NNNN...3' whereas BstX1 recognizes the sequence 5'...CCAN NNNN/NTGG...3'. Thus these two sites can be overlapped, so that only 256 vectors (instead of 512 vectors) need be constructed to cover all the theoretical possible combinations of sites which give complementary cohesive ends for cloning DNA with four nucleotide 5' or 3' overhangs. This vector library can be used for amplification cloning of DNA in a tandem array by choosing appropriate vectors which have nonpalindromic sequences. We have obtained approximately 200 members of the 256 possible clones and have organized the vectors using a MacIntosh HyperCard program for easy retrieval.
|
1062. |
Clarke BR,
Greenfield LK,
Bouwman C,
Whitfield C,
( 2009 ) Coordination of polymerization, chain termination, and export in assembly of the Escherichia coli lipopolysaccharide O9a antigen in an ATP-binding cassette transporter-dependent pathway. PMID : 19734145 : DOI : 10.1074/jbc.M109.052878 PMC : PMC2781620 Abstract >>
The Escherichia coli O9a O-polysaccharide (O-PS) is a prototype for O-PS synthesis and export by the ATP-binding cassette transporter-dependent pathway. Comparable systems are widespread in Gram-negative bacteria. The polymannose O9a O-PS is assembled on a polyisoprenoid lipid intermediate by mannosyltransferases located at the cytoplasmic membrane, and the final polysaccharide chain length is determined by the chain terminating dual kinase/methyltransferase, WbdD. The WbdD protein is tethered to the membrane via a C-terminal region containing amphipathic helices located between residues 601 and 669. Here, we establish that the C-terminal domain of WbdD plays an additional pivotal role in assembly of the O-PS by forming a complex with the chain-extending mannosyltransferase, WbdA. Membrane preparations from a DeltawbdD mutant had severely diminished mannosyltransferase activity in vitro, and no significant amounts of the WbdA protein are targeted to the membrane fraction. Expression of a polypeptide comprising the WbdD C-terminal region was sufficient to restore both proper localization of WbdA and mannosyltransferase activity. In contrast to WbdA, the other required mannosyltransferases (WbdBC) are targeted to the membrane independent of WbdD. A bacterial two-hybrid system confirmed the interaction of WbdD and WbdA and identified two regions in the C terminus of WbdD that contributed to the interaction. Therefore, in the O9a assembly export system, the WbdD protein orchestrates the critical localization and coordination of activities involved in O-PS chain extension and termination at the cytoplasmic membrane.
|
1063. |
Song JS,
Jang SJ,
Bae IK,
Lee HJ,
Jeong BC,
Lee SH,
( 2010 ) New complex class 1 integron carrying an ISCR1 element in Escherichia coli clinical isolates harbouring the blaCMY-11 gene. PMID : 19729458 : DOI : 10.1099/jmm.0.012559-0 Abstract >>
N/A
|
1064. |
Van Molle I,
Moonens K,
Garcia-Pino A,
Buts L,
De Kerpel M,
Wyns L,
Bouckaert J,
De Greve H,
( 2009 ) Structural and thermodynamic characterization of pre- and postpolymerization states in the F4 fimbrial subunit FaeG. PMID : 19799915 : DOI : 10.1016/j.jmb.2009.09.059 DOI : 10.1016/j.jmb.2009.09.059 Abstract >>
Enterotoxigenic Escherichia coli expressing F4 fimbriae are the major cause of porcine colibacillosis and are responsible for significant death and morbidity in neonatal and postweaned piglets. Via the chaperone-usher pathway, F4 fimbriae are assembled into thin, flexible polymers mainly composed of the single-domain adhesin FaeG. The F4 fimbrial system has been labeled eccentric because the F4 pilins show some features distinct from the features of pilins of other chaperone-usher-assembled structures. In particular, FaeG is much larger than other pilins (27 versus approximately 17 kDa), grafting an additional carbohydrate binding domain on the common immunoglobulin-like core. Structural data of FaeG during different stages of the F4 fimbrial biogenesis process, combined with differential scanning calorimetry measurements, confirm the general principles of the donor strand complementation/exchange mechanisms taking place during pilus biogenesis via the chaperone-usher pathway.
|
1065. |
Sandmeier H,
Iida S,
Hübner P,
Hiestand-Nauer R,
Arber W,
( 1991 ) Gene organization in the multiple DNA inversion region min of plasmid p15B of E.coli 15T-: assemblage of a variable gene. PMID : 1945872 : DOI : 10.1093/nar/19.21.5831 PMC : PMC329034 Abstract >>
The bacteriophage P1-related plasmid p15B of E. coli 15T- contains a 3.5 kb long region which frequently undergoes complex rearrangements by DNA inversion. Site-specific recombination mediated by the Min DNA invertase occurs at six crossover sites and it eventually results in a population of 240 isomeric configurations of this region. We have determined 8.3-kb sequences of the invertible DNA and its flanking regions. The result explains how DNA inversion fuses variable 3' parts to a constant 5' part, thereby alternatively assembling one out of six different open reading frames (ORF). The resulting variable gene has a coding capacity of between 739 and 762 amino acids. A large portion of its constant part is composed of repeated sequences. The p15B sequences in front of the variable fusion gene encode a small ORF and a phage-specific late promoter and are highly homologous to P1 DNA. Adjacent to the DNA invertase gene min, we have found a truncated 5' region of a DNA invertase gene termed psi cin which is highly homologous to the phage P1 cin gene. Its recombinational enhancer segment is inactive, but it can be activated by the substitution of two nucleotides.
|
1066. |
Bielaszewska M,
Stoewe F,
Fruth A,
Zhang W,
Prager R,
Brockmeyer J,
Mellmann A,
Karch H,
Friedrich AW,
( 2009 ) Shiga toxin, cytolethal distending toxin, and hemolysin repertoires in clinical Escherichia coli O91 isolates. PMID : 19403777 : DOI : 10.1128/JCM.00201-09 PMC : PMC2708519 Abstract >>
Shiga toxin (Stx)-producing Escherichia coli (STEC) strains of serogroup O91 are the most common human pathogenic eae-negative STEC strains. To facilitate diagnosis and subtyping of these pathogens, we genotypically and phenotypically characterized 100 clinical STEC O91 isolates. Motile strains expressed flagellar antigens H8 (1 strain), H10 (2 strains), H14 (52 strains), and H21 (20 strains) or were H nontypeable (Hnt) (10 strains); 15 strains were nonmotile. All nonmotile and Hnt strains possessed the fliC gene encoding the flagellin subunit of the H14 antigen (fliC(H14)). Most STEC O91 strains possessed enterohemorrhagic E. coli hlyA and expressed an enterohemolytic phenotype. Among seven stx alleles identified, stx(2dact), encoding mucus- and elastase-activatable Stx2d, was present solely in STEC O91:H21, whereas most strains of the other serotypes possessed stx(1). Moreover, only STEC O91:H21 possessed the cdt-V cluster, encoding cytolethal distending toxin V; the toxin was regularly expressed and was lethal to human microvascular endothelial cells. Infection with STEC O91:H21 was associated with hemolytic-uremic syndrome (P = 0.0015), whereas strains of the other serotypes originated mostly in patients with nonbloody diarrhea. We conclude that STEC O91 clinical isolates belong to at least four lineages that differ by H antigens/fliC types, stx genotypes, and non-stx putative virulence factors, with accumulation of virulence determinants in the O91:H21 lineage. Isolation of STEC O91 from patients' stools on enterohemolysin agar and the rapid initial subtyping of these isolates using fliC genotyping facilitate the identification of these emerging pathogens in clinical and epidemiological studies and enable prediction of the risk of a severe clinical outcome.
|
1067. |
Kist ML,
Salit IE,
Hofmann T,
( 1990 ) Purification and characterization of the Dr hemagglutinins expressed by two uropathogenic Escherichia coli strains. PMID : 1968432 : PMC : PMC258521 Abstract >>
The fibrillar Dr hemagglutinins expressed by two uropathogenic Escherichia coli isolates were mechanically sheared from whole cells and subsequently purified by using anion-exchange high-pressure liquid chromatography. The isolated hemagglutinins were proteins with apparent subunit molecular masses of 14,500 daltons by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric points of 5.4 in denaturing isoelectric focusing gels. The two proteins were serologically related to each other but distinct from P fimbriae, as assessed by bacterial agglutination and immunoblotting. The amino acid compositions of the two hemagglutinins were highly similar both to each other and to other Dr hemagglutinins. N-terminal amino acid sequencing of the major hemagglutinin subunit proteins demonstrated homology with afimbrial E. coli adhesins.
|
1068. |
Pope CE,
Carter PE,
Heffernan HM,
( 2009 ) CMY-29 and CMY-30, two novel plasmid-mediated AmpC beta-lactamases. PMID : 19414570 : DOI : 10.1128/AAC.01586-08 PMC : PMC2704635 Abstract >>
N/A
|
1069. |
Le Gac M,
Doebeli M,
( 2010 ) Environmental viscosity does not affect the evolution of cooperation during experimental evolution of colicigenic bacteria. PMID : 19674096 : DOI : 10.1111/j.1558-5646.2009.00814.x Abstract >>
Cooperation should be favored under environmental conditions allowing the preferential interaction of cooperators among themselves and limiting interactions with defectors. Bacteria cooperating to kill competitors by secreting a toxin evolved during several hundred generations in two environments: a viscous environment that should promote cooperator assortment, and a nonviscous environment that should not allow such preferential interaction. A quantitative decrease in cooperation was observed in all populations, but as expected, cooperation was maintained at a higher level in the viscous environment. Mutants that are resistant against but not producing the toxin were identified at a low frequency in a few populations from the viscous environment and at a high frequency in all the populations from the nonviscous environment. The underlying mutations were identified. Relative fitness of the cooperator and mutant genotypes were obtained with bacteria that were isogenic, except for the identified mutations. Competition experiments indicated that cooperation is not favored by environmental viscosity as imposed in our system and suggested that when it comes to cooperation, environmental viscosity should be considered not only in terms of individual movement, but also in terms of the distribution of the public good.
|
1070. |
Savelkoul PH,
Willshaw GA,
McConnell MM,
Smith HR,
Hamers AM,
van der Zeijst BA,
Gaastra W,
( 1990 ) Expression of CFA/I fimbriae is positively regulated. PMID : 1971911 : Abstract >>
Production of the plasmid-coded fimbrial antigen CFA/I of Escherichia coli requires both CFA/I region 1 and CFA/I region 2, which are separated by about 40 kb on the wildtype plasmid. The nucleotide sequence of region 2 was determined and contains an open reading frame (cfa d), encoding a protein of 265 amino acids. The protein has no signal sequence and upon sequence analysis appeared to be a DNA-binding protein. A plasmid was constituted, with a promoterless beta-galactosidase gene preceded by the promoter of region 1. Introduction of a plasmid, carrying the cfa d gene, into a strain containing this construct enhanced expression of beta-galactosidase by at least five-fold indicating that the cfa d protein was enhancing expression from the promoter of region 1. The cfa d gene sequence differed at 28 positions from the Rns gene, which encodes a protein that is a positive regulator of the expression of CS1 or CS2 fimbriae. It was shown that the cfa d gene and the Rns gene can functionally substitute each other in regulating fimbrial synthesis.
|
1071. |
Chen Y,
Shoichet BK,
( 2009 ) Molecular docking and ligand specificity in fragment-based inhibitor discovery. PMID : 19305397 : DOI : 10.1038/nchembio.155 PMC : PMC4006998 Abstract >>
Fragment screens have successfully identified new scaffolds in drug discovery, often with relatively high hit rates (5%) using small screening libraries (1,000-10,000 compounds). This raises two questions: would other noteworthy chemotypes be found were one to screen all commercially available fragments (>300,000), and does the success rate imply low specificity of fragments? We used molecular docking to screen large libraries of fragments against CTX-M beta-lactamase. We identified ten millimolar-range inhibitors from the 69 compounds tested. The docking poses corresponded closely to the crystallographic structures subsequently determined. Notably, these initial low-affinity hits showed little specificity between CTX-M and an unrelated beta-lactamase, AmpC, which is unusual among beta-lactamase inhibitors. This is consistent with the idea that the high hit rates among fragments correlate to a low initial specificity. As the inhibitors were progressed, both specificity and affinity rose together, yielding to our knowledge the first micromolar-range noncovalent inhibitors against a class A beta-lactamase.
|
1072. |
Perepelov AV,
Li D,
Liu B,
Senchenkova SN,
Guo D,
Shevelev SD,
Shashkov AS,
Guo X,
Feng L,
Knirel YA,
Wang L,
( 2009 ) Structural and genetic characterization of Escherichia coli O99 antigen. PMID : 19682076 : DOI : 10.1111/j.1574-695X.2009.00584.x Abstract >>
O-antigen is part of the lipopolysaccharide present in the outer membrane of Gram-negative bacteria, and contributes the major antigenic variability to the cell surface. Screening for the Escherichia coli O-serogroup is the conventional method for identifying E. coli clones. In this study, we investigated the structural characteristics of the E. coli O99 O-antigen and the organization of the genes involved in its synthesis. On the basis of sugar and methylation analysis and nuclear magnetic resonance spectroscopy data, we established the structure of the branched hexasaccharide repeat unit of the O-polysaccharide. This unit consists of four d-rhamnose (d-Rha) moieties in the backbone and two d-glucose (d-Glc) moieties in the side chain, as shown below: [carbohydrate structure: see text]. The O-antigen gene cluster of E. coli O99, which was located between galF and gnd, was found to contain putative genes for the synthesis of d-Rha, genes encoding sugar transferases, and ATP-binding cassette (ABC) transporter genes (wzm and wzt). Our findings indicate that in E. coli O99, the synthesis and translocation of the O-antigen occurs by an ABC transporter-dependent process.
|
1073. |
Nawaz M,
Khan AA,
Khan S,
Sung K,
Kerdahi K,
Steele R,
( 2009 ) Molecular characterization of tetracycline-resistant genes and integrons from avirulent strains of Escherichia coli isolated from catfish. PMID : 19388830 : DOI : 10.1089/fpd.2008.0204 Abstract >>
A study was undertaken to investigate the occurrence of tetracycline-resistant genes and to characterize the integrons present in Escherichia coli isolated from catfish. Sixty-three tetracycline-resistant E. coli strains were isolated from the intestinal contents of 407 farm-raised catfish. All strains were resistant to multiple antibiotics. A polymerase chain reaction (PCR) assay detected tetA in the DNA of 15 of 63 (25.0%) isolates by amplifying a PCR amplicon measuring 957 bp. Oligonucleotide primers targeting a 436-bp region of tetB successfully amplified a PCR amplicon from 47 of 63 (77.0%) isolates, indicating that tetB was predominant. Oligonucleotide primers specific for tetC amplified a 589-bp PCR amplicon from 3 of 63 (5%) isolates. Eleven (17.0%) of the isolates contained both tetA and tetB genes. Class I integrons amplified from the genomic DNA of 14 of 63 (22.0%) isolates measured 1.6 and 1.8 kb. Sequence analysis of the 1.6 kb integrons indicated the presence of three different gene cassettes: a dfrA12, conferring resistance to trimethoprim; an open reading frame, orfF, a hypothetical protein of unknown function; and aadA2, conferring resistance to aminoglycosides. Sequence analysis of the 1.8-kb integron indicated the presence of dfrA17 and aadA5. PCR assays for the detection of the six predominant virulence genes failed to amplify any genes from the genomic DNA. Pulsed-field gel electrophoresis using XbaI identified 16 distinct macro restriction patterns among the 63 isolates. The dendrogram analysis indicated that the DNA from 4 of 16 isolates had a similarity index of 90.0%. Our results indicate that the use of oxytetracycline and Romet 30 (sulfadimethoxine and ormetoprim) in farm-raised catfish may select for multiple antibiotic-resistant E. coli that could serve as a reservoir of tetracycline, trimethoprim, and aminoglycoside resistance genes.
|
1074. |
Doi Y,
Paterson DL,
Adams-Haduch JM,
Sidjabat HE,
O'Keefe A,
Endimiani A,
Bonomo RA,
( 2009 ) Reduced susceptibility to cefepime among Escherichia coli clinical isolates producing novel variants of CMY-2 beta-lactamase. PMID : 19414578 : DOI : 10.1128/AAC.00133-09 PMC : PMC2704647 Abstract >>
Here we describe three Escherichia coli clinical isolates with reduced susceptibility to cefepime. Sequencing of the bla(CMY) genes revealed two novel variants (CMY-33 and -44) with two- to four-amino-acid deletions in the H-10 helix. The deletions were responsible for 12- to 24-fold increases in the MICs of cefepime.
|
1075. |
Christenson JK,
Gordon DM,
( 2009 ) Evolution of colicin BM plasmids: the loss of the colicin B activity gene. PMID : 19372169 : DOI : 10.1099/mic.0.026666-0 Abstract >>
Colicins, a class of antimicrobial compounds produced by bacteria, are thought to be important mediators of intra- and interspecific interactions, and are a significant factor in maintaining microbial diversity. Colicins B and M are among the most common colicins produced by Escherichia coli, and are usually encoded adjacently on the same plasmid. In this study, the characterization of a collection of E. coli isolated from Australian vertebrates revealed that a significant fraction of colicin BM strains lack an intact colicin B activity gene. The colicin B and M gene region was sequenced in 60 strains and it was found (with one exception) that all plasmids lacking an intact colicin B activity gene have an identical colicin gene structure, possessing a complete colicin B immunity gene and a 130 bp remnant of the B activity gene. A phylogenetic analysis of the colicin M and B operons and characterization of the plasmids suggested that ColBM plasmids with a truncated B activity gene have evolved on at least three separate occasions. Colicin B immunity was found to be non-functional in strains that have lost colicin B activity, and colicin M was still produced despite the absence of the SOS box believed to regulate its production in colicin BM strains. The presence of a remnant of the microcin V operon next to the truncated colicin B activity gene indicated that these plasmids evolved as a consequence of gene transfer between colicin BM and microcin V plasmids. We suggest that these transfer events most likely involved the transfer of some microcin V genes and associated virulence factors onto ColBM plasmids.
|
1076. |
Liu W,
Chen L,
Li H,
Duan H,
Zhang Y,
Liang X,
Li X,
Zou M,
Xu L,
Hawkey PM,
( 2009 ) Novel CTX-M {beta}-lactamase genotype distribution and spread into multiple species of Enterobacteriaceae in Changsha, Southern China. PMID : 19297379 : DOI : 10.1093/jac/dkp068 Abstract >>
The aim of this study was to undertake a survey of the occurrence of CTX-M and SHV extended-spectrum beta-lactamase (ESBL) genotypes in Enterobacteriaceae from Hunan Province, China. Clinical isolates (425) from three major hospitals in Changsha, Hunan Province, were collected between October 2004 and July 2005, and their antimicrobial susceptibilities of the genotype of bla(CTX-M) and bla(SHV) were determined. Random amplified polymorphic DNA was used to characterize the clonality of all of the isolates. The overall rate of ESBL-positive isolates was 33.4% (142/425). The dominant ESBLs were CTX-M types, and were found in 109/142 (76.8%) isolates comprising seven different genera/species, namely Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae, Enterobacter aerogenes, Citrobacter freundii, Proteus vulgaris and Providencia stuartii. The most common bla(CTX-M) genotypes were bla(CTX-M-14) (47.7%), bla(CTX-M-3) (29.4%) and bla(CTX-M-15) (17.4%). A novel gene derived from bla(CTX-M-15), bla(CTX-M-82) (Ala-40-->Pro), was identified. The dominant ESBL genotype in Hunan Province was bla(CTX-M). The high prevalence (17.4%) of bla(CTX-M-15) has not previously been reported from China. Our results identify that an epidemic of bla(CTX-M) in Changsha, Hunan Province, has evolved with the appearance and spread of bla(CTX-M-15) against the dominant genotypes bla(CTX-M-14) and bla(CTX-M-3.) The worldwide dominance of bla(CTX-M-15) could be poised to spread to China, displacing the current prevailing genotypes.
|
1077. |
Blackburn D,
Husband A,
Saldaña Z,
Nada RA,
Klena J,
Qadri F,
Girón JA,
( 2009 ) Distribution of the Escherichia coli common pilus among diverse strains of human enterotoxigenic E. coli. PMID : 19357209 : DOI : 10.1128/JCM.00260-09 PMC : PMC2691072 Abstract >>
The Escherichia coli common pilus (ECP) is produced by commensal and pathogenic E. coli strains. This pilus is unrelated to any of the known colonization factors (CFs) of enterotoxigenic E. coli (ETEC). In this study, we investigated the distribution and production of ECP among a collection of 136 human CF-positive and CF-negative ETEC strains of different geographic origins. The major pilus subunit gene, ecpA, was found in 109 (80%) of these strains, suggesting that it is widely distributed among ETEC strains. Phenotypic analysis of a subset of 43 strains chosen randomly showed that 58% of them produced ECP independently of the presence or absence of CFs, a percentage even higher than that of the most prevalent CFs. These data suggest an important role for ECP in the biology of ETEC, particularly in CF-negative strains, and in human infection.
|
1078. |
Bogaerts P,
Rodriguez-Villalobos H,
Laurent C,
Deplano A,
Struelens MJ,
Glupczynski Y,
( 2009 ) Emergence of extended-spectrum-AmpC-expressing Escherichia coli isolates in Belgian hospitals. PMID : 19240070 : DOI : 10.1093/jac/dkp046 Abstract >>
N/A
|
1079. |
Soufi L,
Abbassi MS,
Sáenz Y,
Vinué L,
Somalo S,
Zarazaga M,
Abbas A,
Dbaya R,
Khanfir L,
Ben Hassen A,
Hammami S,
Torres C,
( 2009 ) Prevalence and diversity of integrons and associated resistance genes in Escherichia coli isolates from poultry meat in Tunisia. PMID : 19642918 : DOI : 10.1089/fpd.2009.0284 Abstract >>
Fifty-five Escherichia coli isolates were acquired from chicken and turkey meat obtained from two slaughterhouses in Tunis. Eighty-nine percent, 80%, 78%, 67%, 45%, 27%, 7%, 4%, and 2% of these isolates showed resistance to tetracycline, trimethoprim/sulfamethoxazole, streptomycin, nalidixic acid, ampicillin, chloramphenicol, ciprofloxacin, colistine, and gentamicin, respectively. No resistance was detected to cefotaxime, ceftazidime, or amikacin. bla(TEM) gene was found in 22 of 25 ampicillin-resistant isolates, and 1 isolate harbored bla(OXA-1) gene. Tetracycline resistance was predominately mediated by the tetA gene. The sul1, sul2, and sul3 genes, alone or combined, were detected in 46 of 48 sulfonamide-resistant isolates, and sul1 and sul3 were included in class 1 integrons in some cases. Sixty percent of isolates harbored integrons (class 1, 30 isolates; class 2, 5 isolates). Class 2 integrons contained in all cases the dfrA1-sat1-aadA1-orfX gene cassette arrangement. Nine gene cassette arrangements have been detected among class 1 integrons, containing different alleles of dfrA (five alleles) and aadA (2 alleles) genes, which encode trimethoprim and streptomycin resistance, respectively. An uncommon gene cassette array (sat-psp-aadA2-cmlA1-aadA1-qacH-IS440-sul3) has been identified in three class 1 integron-positive isolates, and one additional isolate had this same structure with the insertion of IS26 inside the aadA1 gene (included in GenBank with accession no. FJ160769). The 55 studied isolates belong to the four phylogenic groups of E. coli, and phylogroups A and D were the most prevalent ones. At least one virulence-associated gene (fimA, papC, or aer) was detected in 44 of the 55 (80%) studied isolates. E. coli isolates of poultry origin could be a reservoir of antimicrobial-resistance genes and of integrons, and its evolution should be tracked in the future.
|
1080. |
Tivendale KA,
Noormohammadi AH,
Allen JL,
Browning GF,
( 2009 ) The conserved portion of the putative virulence region contributes to virulence of avian pathogenic Escherichia coli. PMID : 19202093 : DOI : 10.1099/mic.0.023143-0 Abstract >>
Colibacillosis is a common systemic disease of worldwide economic importance in poultry, caused by Escherichia coli. E. coli are normally found in the intestines of poultry, but some strains are able to cause extraintestinal disease. Plasmid pVM01 is essential for virulence in avian pathogenic Escherichia coli (APEC) strain E3 in chickens after aerosol exposure and contains the virulence-associated genes iucA, iss and tsh in distinct regions. The determination of the complete sequence of this plasmid identified many ORFs that were highly similar to genes found in the APEC O1 plasmid, as well as many hypothetical ORFs. Truncated versions of pVM01 were constructed and introduced into avirulent APEC strain E3/2.4 and the pathogenicity of these strains was assessed by aerosol exposure. The function of the region of pVM01 that contains the genes for conjugation was confirmed. Strains carrying the truncated plasmids appeared to be of intermediate virulence compared to the wild-type APEC strain E3. The conserved portion of the putative virulence region was found to contribute to the colonization of and generation of lesions in the air sacs. Both the conserved and variable portions of the putative virulence region were shown to contribute to the colonization of the trachea, but the variable portion of the putative virulence region was not required for the strain to confer a virulent phenotype. These results reveal that deletion of the conserved portion of the putative virulence region, but not the variable portion of the putative virulence region, is associated with a decrease in virulence of APEC.
|
1081. |
De Toni F,
de Souza EM,
Pedrosa FO,
Klassen G,
Irino K,
Un Rigo L,
Steffens MB,
Fialho OB,
Farah SM,
Fadel-Picheth CM,
( 2009 ) A prospective study on Shiga toxin-producing Escherichia coli in children with diarrhea in Paran? State, Brazil. PMID : 19228288 : DOI : 10.1111/j.1472-765X.2009.02569.x Abstract >>
To examine stool specimens from children with diarrhea from Paran? State, southern Brazil, for presence of STEC. A PCR screening assay for stx genes was used to examine a loopful of confluent colonies of 306 stool samples cultures. In six (1.96%) of them, DNA fragments of the expected size were observed, and the presence of stx was confirmed by DNA sequencing. Then up to 100 single colonies from each of the six stool cultures were analyzed using the same PCR protocol. However, stx-positive colonies were found only in two of the cultures. The E. coli strains belonged to serotypes O69:H11 and O178:H19, and presented genotypes stx(1)eae ehxA and stx(1) respectively. Shiga toxin production was confirmed using the VTEC Screen Seiken. Except ampicillin, they were susceptible to all the antimicrobials tested. These results show that STEC may be an important cause of diarrhea in children of Paran? State, and that they are present in low numbers in stools. The strains belonged to serotypes not commonly found associated with STEC and probably present low virulence. These results indicate that molecular methods are required to diagnosis of STEC infections.
|
1082. |
Ozgumus OB,
Sandalli C,
Sevim A,
Celik-Sevim E,
Sivri N,
( 2009 ) Class 1 and class 2 integrons and plasmid-mediated antibiotic resistance in coliforms isolated from ten rivers in northern Turkey. PMID : 19229487 : DOI : 10.1007/s12275-008-0206-z Abstract >>
We aimed to determine the molecular mechanisms of antibiotic resistance in coliforms isolated from ten rivers in northern region of Turkey. A total of 183 isolates were tested for antimicrobial susceptibility by disk diffusion and agar dilution methods. Resistance to ampicillin, streptomycin, trimethoprim, tetracycline, and chloramphenicol was detected in 58%, 51.9%, 24%, 28.4%, and 12.5%, respectively. Twelve (6.5%) phylogenetically distant organisms were detected to harbor self-transmissible plasmids ranging 52 to >147 kb in sizes. Resistances to ampicillin, tetracycline, trimethoprim, streptomycin, and nalidixic acid were commonly transferable traits. Transferable nalidixic acid-resistant strains harbored qnrS gene, which was the first report of plasmid-mediated quinolone resistance in bacteria of environmental origin in Turkey. Fourteen and five coliforms harbored class 1 and class 2 integrons, respectively, and some of them were located on transferable plasmids. Sequence analyses of variable regions of the class 1 and 2 integrons harbored various gene cassettes, dfrA1, dfr2d, dfrA7, dfrA16, dfrA17, aadA1, aadA5, bla(oxA-30), and sat1. A gene cassette array, dfrA16 has been demonstrated for the first time in a Citrobacter koseri isolate. Class 1 and class 2-bearing strains were clustered in different groups by BOX-PCR fingerprinting. Rivers in the northern Turkey may act as receptacle for the multi-drug resistant enterobacteria and can serve as reservoirs of the antimicrobial resistance determinants in the environment. The actual risk to public health is the transfer of resistance genes from the environmental bacteria to human pathogens.
|
1083. |
Korotkov KV,
Pardon E,
Steyaert J,
Hol WG,
( 2009 ) Crystal structure of the N-terminal domain of the secretin GspD from ETEC determined with the assistance of a nanobody. PMID : 19217396 : DOI : 10.1016/j.str.2008.11.011 PMC : PMC2662362 Abstract >>
Secretins are among the largest bacterial outer membrane proteins known. Here we report the crystal structure of the periplasmic N-terminal domain of GspD (peri-GspD) from the type 2 secretion system (T2SS) secretin in complex with a nanobody, the VHH domain of a heavy-chain camelid antibody. Two different crystal forms contained the same compact peri-GspD:nanobody heterotetramer. The nanobody contacts peri-GspD mainly via CDR3 and framework residues. The peri-GspD structure reveals three subdomains, with the second and third subdomains exhibiting the KH fold which also occurs in ring-forming proteins of the type 3 secretion system. The first subdomain of GspD is related to domains in phage tail proteins and outer membrane TonB-dependent receptors. A dodecameric peri-GspD model is proposed in which a solvent-accessible beta strand of the first subdomain interacts with secreted proteins and/or T2SS partner proteins by beta strand complementation.
|
1084. |
Hammad AM,
Ishida Y,
Shimamoto T,
( 2009 ) Prevalence and molecular characterization of ampicillin-resistant Enterobacteriaceae isolated from traditional Egyptian Domiati cheese. PMID : 19343954 : DOI : 10.4315/0362-028x-72.3.624 Abstract >>
The aim of this study was to address the prevalence and the molecular characteristics of antibiotic-resistant enteric bacteria isolated from one of the most popular types of Egyptian cheese. A total of 215 ampicillin-resistant enterobacterial isolates were obtained from 80 samples of Domiati cheese, and they were screened by PCR for a large pool of antibiotic resistance markers, including extended-spectrum beta-lactamases (ESBLs), class 1 and class 2 integrons, and plasmid-mediated quinolone resistance genes. It was determined that the most frequent mechanism of ampicillin resistance was from a TEM-1-type beta-lactamase. As well, SHV beta-lactamases, including SHV-1, SHV-25, and SHV-26, showed a high prevalence, and two novel SHV beta-lactamases, SHV-110 and SHV-111, were identified. Type CTX-M-14, OXY-1, OXA-1, and CMY-4 beta-lactamases were also detected in a few isolates. In addition, a novel AmpC beta-lactamase was detected that was designated CMY-41. Sequencing results of class 1 integrons revealed that the uncommon aminoglycoside resistance gene cassette aadA22 was found for the first time in an Escherichia coli strain. The other class 1 integrons harbored various common gene cassettes, including aadA1, aadA1a, aadA2, aadA12, dfr5, dfr7, dfr12, and dfr15. The only isolate that carried a class 2 integron contained dfrA1, sat2, and aadA1. Plasmid-mediated quinolone resistance determinants qnrS and qnrB showed a low prevalence. This study provides meaningful data on high antimicrobial resistance contained in Domiati cheese samples and reports for the first time the presence of beta-lactamases, plasmid-mediated quinolone resistance, and integrons in isolates from food of Egyptian animal origin.
|
1085. |
Zhang XX,
Zhang T,
Zhang M,
Fang HH,
Cheng SP,
( 2009 ) Characterization and quantification of class 1 integrons and associated gene cassettes in sewage treatment plants. PMID : 19224208 : DOI : 10.1007/s00253-009-1886-y Abstract >>
Class 1 integrons and gene cassettes containing antibiotic resistance genes (ARGs) in five different sewage treatment plants (STPs) were characterized and quantified using polymerase chain reaction (PCR), sequencing, and quantitative real-time PCR (qRT-PCR) in this study. Class 1 integronase gene (intI1) was found commonly occurring in all of activated sludge samples from the five STPs, as well as in influent and effluent of two STPs at Hong Kong. One hundred and nine lactose-fermenting Enterobacteriaceae (LFE) strains were isolated from activated sludge of Shatin STP. Among them, 36 strains (33.0%) were found to carry class 1 integrons. PCR assays showed that 11 of the 36 intI1-carrying isolates harbored a common type of gene cassette array of about 1,600 bps, as well as the static genes (sulI and qacEDelta1) on class 1 integrons. This gene cassette array was found phylogenetically close to antibiotic resistance genes dfr17 and aadA5, encoding dihydrofolate reductase conferring resistance to trimethoprim and adenylyltransferase conferring resistance to spectinomycin/streptomycin, respectively. Antimicrobial susceptibility analysis demonstrated that all the 11 LFEs carrying gene cassette were multi-resistant, especially having common resistance to trimethoprim and streptomycin. qRT-PCR assay showed that genes copies of both class 1 integron and the gene cassette varied significantly among the activated sludge sampled from different STPs, at different time points or different treatment steps. More than 90% of class 1 integrons and the gene cassette were removed by activated sludge processes in two STPs, while the disinfection process removed 94% integron and 77% gene cassette in one STP.
|
1086. |
Literacka E,
Bedenic B,
Baraniak A,
Fiett J,
Tonkic M,
Jajic-Bencic I,
Gniadkowski M,
( 2009 ) blaCTX-M genes in escherichia coli strains from Croatian Hospitals are located in new (blaCTX-M-3a) and widely spread (blaCTX-M-3a and blaCTX-M-15) genetic structures. PMID : 19188377 : DOI : 10.1128/AAC.01431-08 PMC : PMC2663074 Abstract >>
CTX-M-producing Escherichia coli isolates from three Croatian hospitals were analyzed. All bla(CTX-M-15) genes and one bla(CTX-M-3a) gene resided in widely spread ISEcp1 transposition modules, but other bla(CTX-M-3a) genes were in a new configuration with two IS26 copies, indicating a new event of gene mobilization from a Kluyvera ascorbata genome. The study confirmed the role of the E. coli ST131 clonal group with IncFII-type plasmids in the spread of bla(CTX-M-15) and of IncL/M pCTX-M3-type plasmids in the dissemination of bla(CTX-M-3a).
|
1087. |
Tian GB,
Wang HN,
Zou LK,
Tang JN,
Zhao YW,
Ye MY,
Tang JY,
Zhang Y,
Zhang AY,
Yang X,
Xu CW,
Fu YJ,
( 2009 ) Detection of CTX-M-15, CTX-M-22, and SHV-2 extended-spectrum beta-lactamases (ESBLs) in Escherichia coli fecal-sample isolates from pig farms in China. PMID : 19272004 : DOI : 10.1089/fpd.2008.0164 Abstract >>
The aim of the present study was to investigate the antibiotic resistance profiles and the molecular epidemiology of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli isolates from two production swine operations in Sichuan Province, China, between August 2002 and February 2007. The prevalence of ESBL-producing E. coli increased dramatically from 2.2% to 10.7% during this period. This increase appeared mostly related to dissemination of CTX-M-type ESBLs among E. coli isolates. Of 212 E. coli isolates studied, 14 harbored ESBL genes. Among them, 13 harbored bla(CTX-M-15/22) and one harbored bla(SHV-2). To our knowledge, this is the first study to identify bla(CTX-M-22) from production animals. One isolate in 2002 harbored bla(SHV-2), indicating that ESBL genes have been present in farm animals in China since at least 2002. Molecular characterization and pulsed-field gel electrophoresis of the ESBL-producing isolates suggested that different mechanisms may be involved in the dissemination of the CTX-M genes and revealed that additional resistance determinants for non-beta-lactam antibiotics were carried by plasmids encoding certain ESBL genes. Results of this study provide an example of how ESBL genes, particularly those of CTX-M lineages, are rapidly spreading among E. coli isolates from commercial pig farms in Sichuan province of China.
|
1088. |
Ghosal A,
Bhowmick R,
Banerjee R,
Ganguly S,
Yamasaki S,
Ramamurthy T,
Hamabata T,
Chatterjee NS,
( 2009 ) Characterization and studies of the cellular interaction of native colonization factor CS6 purified from a clinical isolate of enterotoxigenic Escherichia coli. PMID : 19237522 : DOI : 10.1128/IAI.01397-08 PMC : PMC2681727 Abstract >>
CS6 is a widely expressed colonization factor of enterotoxigenic Escherichia coli (ETEC). To date, CS6 has not been well characterized in its native state. Here, we purified CS6 for the first time from an ETEC clinical isolate. Purified CS6 was composed of two structural subunits, CssA and CssB, which were present in equal amounts and tightly linked through noncovalent, detergent-stable association. The CssA subunit was poorly immunogenic, whereas CssB was highly immunogenic. Although the predicted molecular mass of CssA is 15 kDa, the purified CssA has an effective molecular mass of 18.5 kDa due to fatty acid modification. When purified CS6 was screened for its ability to bind with different extracellular matrix proteins, fibronectin (Fn) was found to interact with CS6 as well as CssA in a dose-dependent and saturable manner. This interaction was inhibited both by a synthetic peptide corresponding to the C-terminal hydrophilic, surface-exposed region of CssA (positions 112 to 126) and by the antibody derived against this region. Enzyme-linked immunosorbent assay results showed that CssA interacted with the 70-kDa N-terminal domain of Fn. The modifications on CssA probably do not play a role in Fn binding. Preincubation of INT 407 cells with CssA, but not CssB, inhibited ETEC binding to these cells. The results suggested that CS6-expressing ETEC binds to Fn of INT 407 cells through the C-terminal region of CssA. Purified CS6 was found to colocalize with Fn along the junctions of INT 407 cells. Based on the results obtained, we propose that CS6-expressing ETEC binds to the intestinal cells through Fn for colonization.
|
1089. |
Schmitt E,
Galimand M,
Panvert M,
Courvalin P,
Mechulam Y,
( 2009 ) Structural bases for 16 S rRNA methylation catalyzed by ArmA and RmtB methyltransferases. PMID : 19303884 : DOI : 10.1016/j.jmb.2009.03.034 Abstract >>
Aminoglycosides are used extensively for the treatment of severe infections due to Gram-negative bacteria. However, certain species have become highly resistant after acquisition of genes for methyltransferases which catalyze post-transcriptional methylation of N7-G1405 in 16 S rRNA of 30 S ribosomal subunits. Inactivation of this enzymatic activity is therefore an important challenge for development of an effective therapy. The present work describes the crystallographic structures of methyltransferases RmtB and ArmA from clinical isolates. Together with biochemical experiments, the 3D structures indicate that the N-terminal domain specific for this family of methyltransferases is required for enzymatic activity. Site-directed mutagenesis has enabled important residues for catalysis and RNA binding to be identified. These high-resolution structures should underpin the design of potential inhibitors of these enzymes, which could be used to restore the activity of aminoglycosides against resistant pathogens.
|
1090. |
Aslani MM,
Bouzari S,
( 2009 ) Characterization of virulence genes of non-O157 Shiga toxin-producing Escherichia coli isolates from two provinces of Iran. PMID : 19168953 : Abstract >>
Shiga toxin-producing Escherichia coli (STEC) include O157:H7 and non-O157 serotypes. The public health impact of STEC infections is high because of their ability to cause severe infections. We characterized our STEC strains isolated from diarrheal and asymptomatic persons in northern and southwest Iran. The 29 STEC strains were examined for the presence of virulence genes by polymerase chain reaction (PCR) and their H type was analyzed by PCR-restriction fragment length polymorphism (RFLP) of the fliC gene. Moreover, the adherence properties of these strains were checked by HeLa cell adherence assay. The presence of non-O157 isolates under these conditions was again verified. The stx1 gene was present in 93% of the isolates, and the gene encoding intimin (eae) was not found to be present among the isolates. Almost all of the STEC isolates, with the exception of three, were non-adherent upon tissue culture assay. The serogrouping revealed the presence of seven different O types among non-O157 isolates. PCR-RFLP results for the fliC gene and classical serology examination with H antisera indicated the presence of nine different H types. In this study, three new serotypes of non-O157:H7 (i.e., O25:H3, O85:H32, and O162:H21) were found to be Shiga toxin producers. These findings reconfirm the results of our previously reported studies showing that non-O157:H7 serotypes are more prevalent under the present conditions. More detailed characterization of these isolates will require additional genetic studies.
|
1091. |
Cheng C,
Tennant SM,
Azzopardi KI,
Bennett-Wood V,
Hartland EL,
Robins-Browne RM,
Tauschek M,
( 2009 ) Contribution of the pst-phoU operon to cell adherence by atypical enteropathogenic Escherichia coli and virulence of Citrobacter rodentium. PMID : 19255191 : DOI : 10.1128/IAI.01246-08 PMC : PMC2681749 Abstract >>
Strains of enteropathogenic Escherichia coli (EPEC) generally employ the adhesins bundle-forming pili (Bfp) and intimin to colonize the intestine. Atypical EPEC strains possess intimin but are negative for Bfp and, yet, are able to cause disease. To identify alternative adhesins to Bfp in atypical EPEC, we constructed a transposon mutant library of atypical EPEC strain E128012 (serotype O114:H2) using TnphoA. Six mutants that had lost the ability to adhere to HEp-2 cells were identified, and in all six mutants TnphoA had inserted into the pstSCAB-phoU (Pst) operon. To determine if the Pst operon is required for adherence, we used site-directed mutagenesis to construct a pstCA mutant of E128012. The resultant mutant showed a reduced ability to adhere to HEp-2 cells and T84 intestinal epithelial cells, which was restored by trans-complementation with intact pstCA. To determine if pst contributes to bacterial colonization in vivo, a pstCA mutation was made in the EPEC-like murine pathogen, Citrobacter rodentium. C57BL/6 mice infected perorally with the pstCA mutant of C. rodentium excreted significantly lower numbers of C. rodentium than those given the wild-type strain. Moreover, colonic hyperplasia and diarrhea, which are features of infections with C. rodentium, were not observed in mice infected with the pstCA mutant but did occur in mice given the trans-complemented mutant. As mutations in pst genes generally lead to constitutive expression of the Pho regulon, our findings suggested that the Pho regulon may contribute to the reduced virulence of the pstCA mutants. To investigate this, we inactivated phoB in the pstCA mutants of EPEC E128012 and C. rodentium and found that the phoB mutation restored the adherent phenotype of both mutant strains. These results demonstrate that Pst contributes to the virulence of atypical EPEC and C. rodentium, probably by causing increased expression of an unidentified, Pho-regulated adhesin.
|
1092. |
Hagelueken G,
Ingledew WJ,
Huang H,
Petrovic-Stojanovska B,
Whitfield C,
ElMkami H,
Schiemann O,
Naismith JH,
( 2009 ) PELDOR spectroscopy distance fingerprinting of the octameric outer-membrane protein Wza from Escherichia coli. PMID : 19294709 : DOI : 10.1002/anie.200805758 PMC : PMC3312575 Abstract >>
Distance fingerprinting: Pulsed electron-electron double resonance spectroscopy (PELDOR) is applied to the octameric membrane protein complex Wza of E. coli. The data yielded a detailed distance fingerprint of its periplasmic region that compares favorably to the crystal structure. These results provide the foundation to study conformation changes from interaction with partner proteins.
|
1093. |
Peng Y,
Kumar S,
Hernandez RL,
Jones SE,
Cadle KM,
Smith KP,
Varela MF,
( 2009 ) Evidence for the transport of maltose by the sucrose permease, CscB, of Escherichia coli. PMID : 19294451 : DOI : 10.1007/s00232-009-9161-9 PMC : PMC2661012 Abstract >>
The purpose of this study was to examine the sugar recognition and transport properties of the sucrose permease (CscB), a secondary active transporter from Escherichia coli. We tested the hypothesis that maltose transport is conferred by the wild-type CscB transporter. Cells of E. coli HS4006 harboring pSP72/cscB were red on maltose MacConkey agar indicator plates. We were able to measure "downhill" maltose transport and establish definitive kinetic behavior for maltose entry in such cells. Maltose was an effective competitor of sucrose transport in cells with CscB, suggesting that the respective maltose and sucrose binding sites and translocation pathways through the CscB channel overlap. Accumulation ("uphill" transport) of maltose by cells with CscB was profound, demonstrating active transport of maltose by CscB. Sequencing of cscB encoded on plasmid pSP72/cscB used in cells for transport studies indicate an unaltered primary CscB structure, ruling out the possibility that mutation conferred maltose transport by CscB. We conclude that maltose is a bona fide substrate for the sucrose permease of E. coli. Thus, future studies of sugar binding, transport, and permease structure should consider maltose, as well as sucrose.
|
1094. |
Buss C,
Müller D,
Rüter C,
Heusipp G,
Schmidt MA,
( 2009 ) Identification and characterization of Ibe, a novel type III effector protein of A/E pathogens targeting human IQGAP1. PMID : 19134119 : DOI : 10.1111/j.1462-5822.2009.01284.x Abstract >>
Enteropathogenic Escherichia coli (EPEC), atypical enteropathogenic Escherichia coli (ATEC) and enterohemorrhagic Escherichia coli (EHEC) belong to the family of attaching and effacing (A/E) pathogens. Pathogenicity is mediated by subversion of host cell functions involving type III secretion system (TTSS)-dependent effector proteins. In this study, we have identified and characterized a novel TTSS-dependent effector protein encoded at the 5'-end of the locus of enterocyte effacement (LEE) pathogenicity island (PAI) of ATEC strain 3431-4/86 (O8:H(-)). Using affinity purification we identified IQGAP1, a scaffolding protein involved in the regulation of the actin cytoskeleton, as a putative host cell target. Accordingly, we termed the novel effector protein 'Ibe' for IQGAP1-binding effector. The interaction of Ibe and IQGAP1 was confirmed by co-immunoprecipitation from ATEC-infected cells and immunofluorescence analysis, which revealed colocalization of Ibe and IQGAP1 in ATEC-induced pedestals and actin-rich membrane ruffles. This suggests that the putative effector function of Ibe is mediated via IQGAP1. The Ibe-independent recruitment of IQGAP1 to ATEC-induced pedestals implies a general role for IQGAP1 in the subversion of host cell functions during infection. Homologues of the novel effector Ibe are widely distributed among EPEC, ATEC and EHEC strains but are not necessarily genetically linked to the LEE as they have occasionally also been found to be encoded within lambdoid prophages.
|
1095. |
Zheng J,
Sagar V,
Smolinsky A,
Bourke C,
LaRonde-LeBlanc N,
Cropp TA,
( 2009 ) Structure and function of the macrolide biosensor protein, MphR(A), with and without erythromycin. PMID : 19265703 : DOI : 10.1016/j.jmb.2009.02.058 Abstract >>
The regulatory protein MphR(A) has recently seen extensive use in synthetic biological applications, such as metabolite sensing and exogenous control of gene expression. This protein negatively regulates the expression of a macrolide 2'-phosphotransferase I resistance gene (mphA) via binding to a 35-bp DNA operator upstream of the start codon and is de-repressed by the presence of erythromycin. Here, we present the refined crystal structure of the MphR(A) protein free of erythromycin and that of the MphR(A) protein with bound erythromycin at 2.00- and 1.76-A resolutions, respectively. We also studied the DNA binding properties of the protein and identified mutants of MphR(A) that are defective in gene repression and ligand binding in a cell-based reporter assay. The combination of these two structures illustrates the molecular basis of erythromycin-induced gene expression and provides a framework for additional applied uses of this protein in the isolation and engineered biosynthesis of polyketide natural products.
|
1096. |
Sankar TS,
Neelakanta G,
Sangal V,
Plum G,
Achtman M,
Schnetz K,
( 2009 ) Fate of the H-NS-repressed bgl operon in evolution of Escherichia coli. PMID : 19266030 : DOI : 10.1371/journal.pgen.1000405 PMC : PMC2646131 Abstract >>
In the enterobacterial species Escherichia coli and Salmonella enterica, expression of horizontally acquired genes with a higher than average AT content is repressed by the nucleoid-associated protein H-NS. A classical example of an H-NS-repressed locus is the bgl (aryl-beta,D-glucoside) operon of E. coli. This locus is "cryptic," as no laboratory growth conditions are known to relieve repression of bgl by H-NS in E. coli K12. However, repression can be relieved by spontaneous mutations. Here, we investigated the phylogeny of the bgl operon. Typing of bgl in a representative collection of E. coli demonstrated that it evolved clonally and that it is present in strains of the phylogenetic groups A, B1, and B2, while it is presumably replaced by a cluster of ORFans in the phylogenetic group D. Interestingly, the bgl operon is mutated in 20% of the strains of phylogenetic groups A and B1, suggesting erosion of bgl in these groups. However, bgl is functional in almost all B2 isolates and, in approximately 50% of them, it is weakly expressed at laboratory growth conditions. Homologs of bgl genes exist in Klebsiella, Enterobacter, and Erwinia species and also in low GC-content Gram-positive bacteria, while absent in E. albertii and Salmonella sp. This suggests horizontal transfer of bgl genes to an ancestral Enterobacterium. Conservation and weak expression of bgl in isolates of phylogenetic group B2 may indicate a functional role of bgl in extraintestinal pathogenic E. coli.
|
1097. |
Hu CX,
Xu ZR,
Li WF,
Niu D,
Dong N,
Lu P,
Fu LL,
( 2009 ) Secretory expression of K88 (F4) fimbrial adhesin FaeG by recombinant Lactococcus lactis for oral vaccination and its protective immune response in mice. PMID : 19277476 : DOI : 10.1007/s10529-009-9966-8 Abstract >>
K88 (F4) fimbrial adhesin, FaeG, was expressed extracellularly in Lactococcus lactis using a nisin-controlled gene expression system. The antibody response and protective efficacy of the recombinant bacteria (L. lactis [spNZ8048-faeG]) against live enterotoxigenic E. coli (ETEC) C(83549) challenge were evaluated in ICR mice. Mice vaccinated with L. lactis [spNZ8048-faeG] had a significantly increased antigen-specific IgG level in the serum and decreased mortality rate (P < 0.05) compared with the control. This indicates that oral immunization of L. lactis [spNZ8048-faeG] can induce an immune-response protection upon challenge with live ETEC in ICR mice.
|
1098. |
Varsaki A,
Moncalián G,
Garcillán-Barcia Mdel P,
Drainas C,
de la Cruz F,
( 2009 ) Analysis of ColE1 MbeC unveils an extended ribbon-helix-helix family of nicking accessory proteins. PMID : 19114496 : DOI : 10.1128/JB.01342-08 PMC : PMC2648203 Abstract >>
MbeC is a 13-kDa ColE1-encoded protein required for efficient mobilization of ColE1, a plasmid widely used in cloning vector technology. MbeC protein was purified and used for in vitro DNA binding, which showed that it binds specifically double-stranded DNA (dsDNA) containing the ColE1 oriT. Amino acid sequence comparison and secondary structure prediction imply that MbeC is related to the ribbon-helix-helix (RHH) protein family. Alignment with RHH members pointed to a conserved arginine (R13 in MbeC) that was mutated to alanine. The mutant MbeC(R13A) was unable to bind either single-stranded DNA or dsDNA. Limited proteolysis fragmented MbeC in two stable folding domains: the N-terminal domain, which contains the RHH motif, and the C-terminal domain, which comprises a signature shared by nicking accessory proteins. The results indicate that MbeC plays a similar role in conjugation as TraY and TrwA of plasmids F and R388, respectively. Thus, it appears that an extended, possibly universal mechanism of DNA conjugative processing exists, in which oriT-processing is carried out by relaxases assisted by homologous nicking accessory proteins. This mechanism seems to be shared by all major conjugative systems analyzed thus far.
|
1099. |
Trobos M,
Christensen H,
Sunde M,
Nordentoft S,
Agersø Y,
Simonsen GS,
Hammerum AM,
Olsen JE,
( 2009 ) Characterization of sulphonamide-resistant Escherichia coli using comparison of sul2 gene sequences and multilocus sequence typing. PMID : 19246754 : DOI : 10.1099/mic.0.024190-0 Abstract >>
The sul2 gene encodes sulphonamide resistance (Sul(R)) and is commonly found in Escherichia coli from different hosts. We typed E. coli isolates by multilocus sequence typing (MLST) and compared the results to sequence variation of sul2, in order to investigate the relation to host origin of pathogenic and commensal E. coli strains and to investigate whether transfer of sul2 into different genomic lineages has happened multiple times. Sixty-eight E. coli isolated in Denmark and Norway from different hosts and years were MLST typed and sul2 PCR products were sequenced and compared. PFGE was performed in a subset of isolates. All isolates were divided into 45 different sequence types (STs), with clonal complexes CC10, CC23, CC168, CC350 and CC69 being the most frequent. The sul2 gene from the majority of E. coli strains had only two point mutations, at positions 159 and 197, leading to a synonymous and a non-synonymous change, respectively. Five strains had extra single mutations. All poultry, poultry meat, and Danish human blood isolates had the same sul2 ST and some of these strains clustered under the same MLST STs, indicating that they shared habitats. Most PFGE profiles clustered according to source, but some included different sources. Sul(R) E. coli from different animals, food, human faeces and infections did not cluster according to their origin, suggesting that these habitats share E. coli and sul2 gene types. However, while pig isolates on one occasion clustered with urinary tract infection isolates, poultry isolates seemed more related to isolates from bloodstream infections in humans. Presence of mainly two types of the sul2 gene in both human and animal isolates, irrespective of date and geography, and the presence of both types in the same clonal lineages, suggest horizontal transfer of sul2.
|
1100. |
Cummings HS,
Sands JF,
Foreman PC,
Fraser J,
Hershey JW,
( 1991 ) Structure and expression of the infA operon encoding translational initiation factor IF1. Transcriptional control by growth rate. PMID : 1909328 : Abstract >>
The cellular levels of the three translational initiation factors, IF1, IF2, and IF3, increase as a function of growth rate in parallel with those of ribosomes. Therefore both ribosomal and initiation factor gene expression is under metabolic control. To address how expression of the Escherichia coli gene for IF1, infA, is regulated, a 3-kilobase region of the genome surrounding infA was sequenced. The 5' and 3' termini of in vivo infA transcripts were defined by S1 nuclease mapping, and mRNA size was measured by Northern blot hybridization. The infA gene is transcribed by two promoters, P1 and P2, which generate transcripts of 525 and 330 nucleotides, apparently ending at the same rho-independent terminator. Analyses of operon and protein fusions to lacZ demonstrate that neither infA transcription nor translation is affected by high cellular levels of IF1. However, P2, but not P1, increases in activity as a function of the growth rate of the cell and is the dominant promoter in rich medium. Therefore, metabolic control of infA expression occurs exclusively at the level of transcription by the P2 promoter.
|
1101. |
Lee SG,
Jeong SH,
Lee H,
Kim CK,
Lee Y,
Koh E,
Chong Y,
Lee K,
( 2009 ) Spread of CTX-M-type extended-spectrum beta-lactamases among bloodstream isolates of Escherichia coli and Klebsiella pneumoniae from a Korean hospital. PMID : 19073302 : DOI : 10.1016/j.diagmicrobio.2008.09.002 Abstract >>
This study was to determine the prevalence and characteristics of CTX-M-type extended-spectrum beta-lactamases (ESBLs) in nonduplicate Escherichia coli (n=760) and Klebsiella pneumoniae (n=379) bloodstream isolates collected during January 2005 to October 2007 at a university hospital (2000 beds) in Seoul, Korea. Antimicrobial susceptibilities were determined by disk diffusion and agar dilution methods. The double-disk synergy test detected ESBLs in 8.7% (66/760) of E. coli and 11.3% (43/379) of K. pneumoniae isolates. Polymerase chain reaction detected bla(CTX-M) in 60/66 (90.9%) E. coli and 9/43 (20.9%) K. pneumoniae isolates with the ESBL phenotype. CTX-M-14 was the most common type of CTX-M ESBLs in both E. coli (n=32) and K. pneumoniae (n=6). CTX-M-15 was the 2nd most common type of CTX-M ESBLs in E. coli (n=22), but it was not detected in K. pneumoniae. In addition, CTX-M-24 (n=2), CTX-M-65 (n=2), CTX-M-27 (n=1), and CTX-M-32 (n=1) were detected for the 1st time in Korea.
|
1102. |
Cram DS,
Loh SM,
Cheah KC,
Skurray RA,
( 1991 ) Sequence and conservation of genes at the distal end of the transfer region on plasmids F and R6-5. PMID : 1916281 : DOI : 10.1016/0378-1119(91)90469-r Abstract >>
The nucleotide sequence of the region downstream of transfer gene traI, including fertility inhibition gene finO, on the conjugative plasmids F and R6-5, has been determined. Analysis of the F sequence revealed two open reading frames (ORF's), ORF248 and ORF186; ORF186 (finO) is interrupted by the insertion of IS3. The R6-5 sequence also contained ORF248 and an intact ORF186, although an additional ORF (ORF286) was located between the two genes. ORF248, which we have designated traX, and ORF186 (finO) are highly conserved on both plasmids. The organisation of these genes indicates that traI and traX on F, and traI, traX and ORF286 on R6-5 are co-transcribed from their respective promoters upstream of traI. Sequences homologous to traX were detected on a range of conjugative F-like plasmids, whereas sequences homologous to ORF286 were only found on plasmids R6-5, R100 and R1. The conservation of traX sequences suggests a functional importance for that gene and/or its product.
|
1103. |
Zheng J,
Cui S,
Teel LD,
Zhao S,
Singh R,
O'Brien AD,
Meng J,
( 2008 ) Identification and characterization of Shiga toxin type 2 variants in Escherichia coli isolates from animals, food, and humans. PMID : 18658282 : DOI : 10.1128/AEM.00503-08 PMC : PMC2547040 Abstract >>
There is considerable heterogeneity among the Shiga toxin type 2 (Stx2) toxins elaborated by Shiga toxin-producing Escherichia coli (STEC). One such Stx2 variant, the Stx2d mucus-activatable toxin (Stx2dact), is rendered more toxic by the action of elastase present in intestinal mucus, which cleaves the last two amino acids of the A2 portion of the toxin A subunit. We screened 153 STEC isolates from food, animals, and humans for the gene encoding Stx2dact by using a novel one-step PCR procedure. This method targeted the region of stx(2dact) that encodes the elastase recognition site. The presence of stx(2dact) was confirmed by DNA sequencing of the complete toxin genes. Seven STEC isolates from cows (four isolates), meat (two isolates), and a human (one isolate) that carried the putative stx(2dact) gene were identified; all were eae negative, and none was the O157:H7 serotype. Three of the isolates (CVM9322, CVM9557, and CVM9584) also carried stx(1), two (P1332 and P1334) carried stx(1) and stx(2c), and one (CL-15) carried stx(2c). One isolate, P1130, harbored only stx(2dact). The Vero cell cytotoxicities of supernatants from P1130 and stx(1) deletion mutants of CVM9322, CVM9557, and CVM9584 were increased 13- to 30-fold after treatment with porcine elastase. Thus, Stx2dact-producing strains, as detected by our one-step PCR method, can be isolated not only from humans, as previously documented, but also from food and animals. The latter finding has important public health implications based on a recent report from Europe of a link between disease severity and infection with STEC isolates that produce Stx2dact.
|
1104. |
López J,
Delgado D,
Andrés I,
Ortiz JM,
Rodríguez JC,
( 1991 ) Isolation and evolutionary analysis of a RepFVIB replicon of the plasmid pSU212. PMID : 1865183 : DOI : 10.1099/00221287-137-5-1093 Abstract >>
We have isolated at least two different replication regions from pSU401, a Tn802 insertion derivative of the IncFVI plasmid pSU212. One of the replication regions (RepFVIB) is highly homologous to the RepFIIA replicon of IncFII plasmids, and thus belongs to the RepFIIA family. We have also cloned the incompatibility determinant (incFVI) and the copy number control gene (cop) from RepFVIB and determined their nucleotide sequences. The analysis of the sequences supports the idea of a modular evolution of RepFIIA plasmids.
|
1105. |
Cattoir V,
Poirel L,
Nordmann P,
( 2008 ) Plasmid-mediated quinolone resistance pump QepA2 in an Escherichia coli isolate from France. PMID : 18644958 : DOI : 10.1128/AAC.00638-08 PMC : PMC2565908 Abstract >>
One hundred twenty-one extended-spectrum beta-lactamse-producing enterobacterial clinical isolates were screened for the qepA gene. A single CTX-M-15-positive Escherichia coli isolate (0.8%) that produced the putative pump QepA2 was identified. This qepA2 gene was located onto a 90-kb mobilizable plasmid that conferred reduced susceptibility to hydrophilic fluoroquinolones.
|
1106. |
Schubert S,
Darlu P,
Clermont O,
Wieser A,
Magistro G,
Hoffmann C,
Weinert K,
Tenaillon O,
Matic I,
Denamur E,
( 2009 ) Role of intraspecies recombination in the spread of pathogenicity islands within the Escherichia coli species. PMID : 19132082 : DOI : 10.1371/journal.ppat.1000257 PMC : PMC2606025 Abstract >>
Horizontal gene transfer is a key step in the evolution of bacterial pathogens. Besides phages and plasmids, pathogenicity islands (PAIs) are subjected to horizontal transfer. The transfer mechanisms of PAIs within a certain bacterial species or between different species are still not well understood. This study is focused on the High-Pathogenicity Island (HPI), which is a PAI widely spread among extraintestinal pathogenic Escherichia coli and serves as a model for horizontal transfer of PAIs in general. We applied a phylogenetic approach using multilocus sequence typing on HPI-positive and -negative natural E. coli isolates representative of the species diversity to infer the mechanism of horizontal HPI transfer within the E. coli species. In each strain, the partial nucleotide sequences of 6 HPI-encoded genes and 6 housekeeping genes of the genomic backbone, as well as DNA fragments immediately upstream and downstream of the HPI were compared. This revealed that the HPI is not solely vertically transmitted, but that recombination of large DNA fragments beyond the HPI plays a major role in the spread of the HPI within E. coli species. In support of the results of the phylogenetic analyses, we experimentally demonstrated that HPI can be transferred between different E. coli strains by F-plasmid mediated mobilization. Sequencing of the chromosomal DNA regions immediately upstream and downstream of the HPI in the recipient strain indicated that the HPI was transferred and integrated together with HPI-flanking DNA regions of the donor strain. The results of this study demonstrate for the first time that conjugative transfer and homologous DNA recombination play a major role in horizontal transfer of a pathogenicity island within the species E. coli.
|
1107. |
Sjöström AE,
Sondén B,
Müller C,
Rydström A,
Dobrindt U,
Wai SN,
Uhlin BE,
( 2009 ) Analysis of the sfaX(II) locus in the Escherichia coli meningitis isolate IHE3034 reveals two novel regulatory genes within the promoter-distal region of the main S fimbrial operon. PMID : 19103276 : DOI : 10.1016/j.micpath.2008.12.001 Abstract >>
We describe the expression and regulation of the gene sfaX(II) located near the Sfa(II) fimbrial determinant in the newborn meningitis Escherichia coli (NMEC) isolate IHE3034. sfaX(II) belongs to a gene family, the 17-kDa genes, typically located downstream (300-3000bp) of different fimbrial operons found in E. coli isolates of uropathogenic and newborn meningitis origin. Using transcriptional sfaX(II) reporter gene fusions we found that different environmental conditions commonly affecting expression of fimbrial genes also affected sfaX(II) expression. Analysis of the sfaX(II) transcripts showed that the gene is part of the main fimbrial operon as it is transcribed together with the rest of the fimbrial genes. In addition, the sfaX(II) gene can be expressed from a more proximal promoter and is found to be subject to strong down-regulation by the nucleoid protein H-NS. Studies with an sfaX(II) mutant derivative of IHE3034 did not reveal effects on Sfa(II) fimbrial biogenesis as monitored by e.g. immunofluorescence microscopy. Nevertheless, a mutation in sfaX(II) resulted in altered expression of other surface components. Moreover, we define a new gene, sfaY(II), coding for a putative phosphodiesterase that is located in between the sfaX(II) gene and the fimbrial biogenesis genes. Our studies by ectopic expression of sfaY(II) in Vibrio cholerae showed that the gene product caused reduced biofilm formation and it is proposed that sfaY(II) can influence cyclic-di-GMP turnover in the bacteria. Our findings demonstrate that the operons typical for S-fimbriae of extraintestinal pathogenic E. coli include previously unrecognized novel regulatory genes.
|
1108. |
Pavez M,
Neves P,
Dropa M,
Matté MH,
Grinbaum RS,
Elmor de Araújo MR,
Mamizuka EM,
Lincopan N,
( 2008 ) Emergence of carbapenem-resistant Escherichia coli producing CMY-2-type AmpC beta-lactamase in Brazil. PMID : 19018036 : DOI : 10.1099/jmm.0.2008/002774-0 Abstract >>
N/A
|
1109. |
Liu J,
Keelan P,
Bennett PM,
Enne VI,
( 2009 ) Characterization of a novel macrolide efflux gene, mef(B), found linked to sul3 in porcine Escherichia coli. PMID : 19131424 : DOI : 10.1093/jac/dkn523 Abstract >>
The aim of this study was to characterize a putative novel macrolide efflux gene located in the vicinity of sul3 in porcine Escherichia coli. Five sul3-encoding E. coli isolates of porcine origin were investigated by plasmid characterization and random amplification of polymorphic DNA (RAPD) PCR. Unknown DNA adjacent to the sul3 genes was amplified using a PCR approach, followed by sequencing of the fragments. The putative macrolide efflux gene was cloned into pK18. The cloned gene was characterized by susceptibility testing by Etest in the presence and absence of efflux inhibitors. Five sul3-encoding isolates, demonstrated to be unrelated by RAPD PCR, were characterized. The immediate genetic context of sul3 in five isolates was identical to that in plasmid pVP440, and in all cases, sul3 was associated with class 1 integrons. In three isolates, an open reading frame (orf2) encoding a putative protein with 38% amino acid identity to Mef(A) was found, while the two remaining isolates contained a fragment of orf2 truncated by IS26 insertion. In three of the isolates, this DNA region was demonstrated to be located on non-conjugative plasmids. When the complete orf2 was cloned, it conferred high-level resistance to erythromycin and azithromycin, and the resistance property could be partially inhibited using the efflux inhibitor Phe-Arg beta-naphthylamide dihydrochloride. The gene was named mef(B). A new macrolide efflux protein, Mef(B), with 38% amino acid identity to Mef(A), has been characterized and represents the second member of the mef family of genes.
|
1110. |
Aoki SK,
Webb JS,
Braaten BA,
Low DA,
( 2009 ) Contact-dependent growth inhibition causes reversible metabolic downregulation in Escherichia coli. PMID : 19124575 : DOI : 10.1128/JB.01437-08 PMC : PMC2648372 Abstract >>
Contact-dependent growth inhibition (CDI) is a mechanism identified in Escherichia coli by which bacteria expressing two-partner secretion proteins encoded by cdiA and cdiB bind to BamA in the outer membranes of target cells and inhibit their growth. A third gene in the cluster, cdiI, encodes a small protein that is necessary and sufficient to confer immunity to CDI, thereby preventing cells expressing the cdiBA genes from inhibiting their own growth. In this study, the cdiI gene was placed under araBAD promoter control to modulate levels of the immunity protein and thereby induce CDI by removal of arabinose. This CDI autoinhibition system was used for metabolic analyses of a single population of E. coli cells undergoing CDI. Contact-inhibited cells showed altered cell morphology, including the presence of filaments. Notably, CDI was reversible, as evidenced by resumption of cell growth and normal cellular morphology following induction of the CdiI immunity protein. Recovery of cells from CDI also required an energy source. Cells undergoing CDI showed a significant, reversible downregulation of metabolic parameters, including aerobic respiration, proton motive force (Deltap), and steady-state ATP levels. It is unclear whether the decrease in respiration and/or Deltap is directly involved in growth inhibition, but a role for ATP in the CDI mechanism was ruled out using an atp mutant. Consistent with the observed decrease in Deltap, the phage shock response was induced in cells undergoing CDI but not in recovering cells, based on analysis of levels of pspA mRNA.
|
1111. |
Lessl M,
Krishnapillai V,
Schilf W,
( 1991 ) Identification and characterization of two entry exclusion genes of the promiscuous IncP plasmid R18. PMID : 1904533 : DOI : 10.1007/bf00260716 Abstract >>
Two entry exclusion genes (designated eexA and eexB) from the promiscuous IncP alpha plasmid R18 have been isolated by molecular cloning. They are located between coordinates 26.6-27.4 kb and 27.4-27.6 kb, respectively and are transcribed clockwise on the conventional R18 map. The product of the eexA gene has an apparent molecular mass of 28 kDa and its N-terminus contains a putative signal sequence for protein export. A recombinant plasmid containing R18 eex genes exerted Eex activity towards another promiscuous IncP alpha plasmid, R702, about 50 times more strongly than plasmid R18 itself. Analysis of the DNA sequence revealed no similarity to the eex genes of the F plasmid of Escherichia coli. R18 eexA includes a potential korB binding site and is followed by a potential transcription terminator. A Tn7 insertion at coordinate 20.0 kb of R18 resulted in a host range mutant pM01185, which leads to loss of Eex activity and of conjugative transfer of the plasmid into some bacterial species.
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1112. |
Wang Q,
Perepelov AV,
Feng L,
Knirel YA,
Li Y,
Wang L,
( 2009 ) Genetic and structural analyses of Escherichia coli O107 and O117 O-antigens. PMID : 19040662 : DOI : 10.1111/j.1574-695X.2008.00494.x Abstract >>
The O-antigen, consisting of many repeats of an oligosaccharide, is an essential component of the lipopolysaccharide on the surface of Gram-negative bacteria. The O-antigen is one of the most variable cell constituents, and different O-antigen forms are almost entirely due to genetic variations in O-antigen gene clusters. In this paper, we present structural and genetic evidence for a close relationship between Escherichia coli O107 and E. coli O117 O antigens. The O-antigen of E. coli O107 has a pentasaccharide repeating unit with the following structure: -->4)-beta-D-GalpNAc-(1-->3)-alpha-L-Rhap-(1-->4)-alpha-D-GlcpNAc-(1-->4)-beta-D-Galp-(1-->3)-alpha-D-GalpNAc-(1-->, which differs from the known repeating unit of E. coli O117 only in the substitution of D-GlcNAc for D-Glc. The O-antigen gene clusters of E. coli O107 and O117 share 98.6% overall DNA identity and contain the same set of genes in the same organization. It is proposed that one cluster was evolved from another via mutations, and the substitution of a few amino acids residues in predicted glycosyltransferases resulted in the functional change of one such protein for transferring different sugars in O107 (D-GlcNAc) and O117 (D-Glc), leading to different O-antigen structures. This is an example of the O-antigen alteration caused by nucleotide mutations, which is less commonly reported for O-antigen variations.
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1113. |
Arnold T,
Zeth K,
Linke D,
( 2009 ) Structure and function of colicin S4, a colicin with a duplicated receptor-binding domain. PMID : 19056731 : DOI : 10.1074/jbc.M808504200 PMC : PMC2649078 Abstract >>
Colicins are plasmid-encoded toxic proteins produced by Escherichia coli strains to kill other E. coli strains that lack the corresponding immunity protein. Colicins intrude into the host cell by exploiting existing transport, diffusion, or efflux systems. We have traced the way colicin S4 takes to execute its function and show that it interacts specifically with OmpW, OmpF, and the Tol system before it inserts its pore-forming domain into the cytoplasmic membrane. The common structural architecture of colicins comprises a translocation, a receptor-binding, and an activity domain. We have solved the crystal structure of colicin S4 to a resolution of 2.5 A, which shows a remarkably compact domain arrangement of four independent domains, including a unique domain duplication of the receptor-binding domain. Finally, we have determined the residues responsible for binding to the receptor OmpW by mutating exposed charged residues in one or both receptor-binding domains.
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1114. |
Wang Q,
Ding P,
Perepelov AV,
Xu Y,
Wang Y,
Knirel YA,
Wang L,
Feng L,
( 2008 ) Characterization of the dTDP-D-fucofuranose biosynthetic pathway in Escherichia coli O52. PMID : 19019146 : DOI : 10.1111/j.1365-2958.2008.06449.x Abstract >>
D-fucofuranose (D-Fucf) is a component of Escherichia coli O52 O antigen. This uncommon sugar is also the sugar moiety of the anticancer drug--gilvocarcin V produced by many streptomycetes. In E. coli O52, rmlA, rmlB, fcf1 and fcf2 were proposed in a previous study by our group to encode the enzymes of the dTDP-D-Fucf (the nucleotide-activated form of D-Fucf) biosynthetic pathway. In this study, Fcf1 and Fcf2 from E. coli O52 were expressed, purified and assayed for their respective activities. Novel product peaks from enzyme-substrate reactions were detected by capillary electrophoresis and the structures of the product compounds were elucidated by electro-spray ionization mass spectrometry and nuclear magnetic resonance spectroscopy. Fcf1 was confirmed to be a dTDP-6-deoxy-D-xylo-hex-4-ulopyranose reductase for the conversion of dTDP-6-deoxy-D-xylo-hex-4-ulopyranose to dTDP-D-fucopyranose (dTDP-D-Fucp), and Fcf2 a dTDP-D-Fucp mutase for the conversion of dTDP-D-Fucp to dTDP-D-Fucf. The K(m) of Fcf1 for dTDP-6-deoxy-D-xylo-hex-4-ulopyranose was determined to be 0.38 mM, and of Fcf2 for dTDP-D-Fucp to be 3.43 mM. The functional role of fcf1 and fcf2 in the biosynthesis of E. coli O52 O antigen were confirmed by mutation and complementation tests. This is the first time that the biosynthetic pathway of dTDP-D-Fucf has been fully characterized.
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1115. |
Dzinic SH,
Shukla M,
Mandija I,
Ram TS,
Ram JL,
( 2008 ) Variable length tandem repeat polyglutamine sequences in the flexible tether region of the Tsr chemotaxis receptor of Escherichia coli. PMID : 18667570 : DOI : 10.1099/mic.0.2008/016303-0 Abstract >>
Methyl-accepting chemotaxis proteins (MCPs) are receptors that play an important role in bacterial chemotaxis. Methylation of Tsr, the MCP that mediates chemotaxis towards serine in Escherichia coli, is thought to be facilitated by binding of the methyltransferase to a flexible tether region at the C-terminal end of Tsr. This study analysed natural length variants of the tether that occur in E. coli due to genetic instability in tandem repeat DNA sequences that code for glutaminyl (Q) residues, creating polyQ sequences of variable lengths in the tether region. The tsr gene of E. coli K-12 (strain MG1655) codes for 4Q at the beginning of its 35 aa tether region. The tether varies in length from 35 to 47 residues among pathogenic and non-pathogenic strains of Escherichia, Shigella spp., Salmonella, Yersinia and Photorhabdus. Among previous sequences, Escherichia and Shigella mostly have 4Q and 7Q variants, and one strain (E. coli HS) has 10Q. In E. coli isolated from 50 humans and 75 animals (dogs, cats, horses, birds, etc.), polyQ up to 13Q (44 aa tether) were identified (6 strains); relative frequencies were 7Q (approximately 77 % of the total) >4Q (14 %) >13Q (5 %) >10Q (4 %). Phylogenetic analysis revealed that E. coli strains with 10Q or 13Q largely fell within two clusters. Serine chemotaxis was not significantly different among 7Q, 10Q and 13Q strains, and was comparable to chemotaxis in the frequently studied K-12 strain. These results are consistent with models indicating that polyQ sequences from 7Q to 13Q are flexible, and that longer tethers, within this range, would not change the precision of adaptation mediated by methylation. Studies of this naturally variable polyQ region in E. coli may also have relevance to mechanisms that mediate polyQ instability in human genetic diseases.
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1116. |
Mérens A,
Matrat S,
Aubry A,
Lascols C,
Jarlier V,
Soussy CJ,
Cavallo JD,
Cambau E,
( 2009 ) The pentapeptide repeat proteins MfpAMt and QnrB4 exhibit opposite effects on DNA gyrase catalytic reactions and on the ternary gyrase-DNA-quinolone complex. PMID : 19060136 : DOI : 10.1128/JB.01205-08 PMC : PMC2648189 Abstract >>
MfpA(Mt) and QnrB4 are two newly characterized pentapeptide repeat proteins (PRPs) that interact with DNA gyrase. The mfpA(Mt) gene is chromosome borne in Mycobacterium tuberculosis, while qnrB4 is plasmid borne in enterobacteria. We expressed and purified the two PRPs and compared their effects on DNA gyrase, taking into account host specificity, i.e., the effect of MfpA(Mt) on M. tuberculosis gyrase and the effect of QnrB4 on Escherichia coli gyrase. Whereas QnrB4 inhibited E. coli gyrase activity only at concentrations higher than 30 microM, MfpA(Mt) inhibited all catalytic reactions of the M. tuberculosis gyrase described for this enzyme (supercoiling, cleavage, relaxation, and decatenation) with a 50% inhibitory concentration of 2 microM. We showed that the D87 residue in GyrA has a major role in the MfpA(Mt)-gyrase interaction, as D87H and D87G substitutions abolished MfpA(Mt) inhibition of M. tuberculosis gyrase catalytic reactions, while A83S modification did not. Since MfpA(Mt) and QnrB4 have been involved in resistance to fluoroquinolones, we measured the inhibition of the quinolone effect in the presence of each PRP. QnrB4 reversed quinolone inhibition of E. coli gyrase at 0.1 microM as described for other Qnr proteins, but MfpA(Mt) did not modify M. tuberculosis gyrase inhibition by fluoroquinolones. Crossover experiments showed that MfpA(Mt) also inhibited E. coli gyrase function, while QnrB4 did not reverse quinolone inhibition of M. tuberculosis gyrase. In conclusion, our in vitro experiments showed that MfpA(Mt) and QnrB4 exhibit opposite effects on DNA gyrase and that these effects are protein and species specific.
|
1117. |
Liu Y,
Fratamico P,
Debroy C,
Bumbaugh AC,
Allen JW,
( 2008 ) DNA sequencing and identification of serogroup-specific genes in the Escherichia coli O118 O antigen gene cluster and demonstration of antigenic diversity but only minor variation in DNA sequence of the O antigen clusters of E. coli O118 and O151. PMID : 18673069 : DOI : 10.1089/fpd.2008.0096 Abstract >>
The DNA sequence of the O antigen gene cluster of an Escherichia coli serogroup O118 strain was determined, and 13 open reading frames (ORFs) were identified, encoding genes required for O antigen sugar biosynthesis, transfer, and processing. Polymerase chain reaction (PCR) assays targeting the wzx (O antigen flippase) and wzy (O antigen polymerase) genes in the O antigen gene cluster of E. coli O118 were designed for identification of these serogroups. Specificity testing using strains belonging to E. coli O118 isolated from various sources, representative strains of 167 other E. coli O serogroups, and 20 non-E. coli bacteria revealed that the PCR assays were specific for E. coli O118. Thus, the PCR assays can be used for rapid identification of E. coli O118 as an alterative to typing using antisera. However, the PCR assays targeting the E. coli O118 wzx and wzy genes were also positive using E. coli serogroup O151 DNA. Therefore, the sequence of the O antigen gene cluster of E. coli O151 was determined, and it was very similar to that of E. coli O118, with only three nucleotide differences. Although the lipopolysaccharide profiles of O118 and O151 showed differences, multilocus sequence typing of E. coli O118 and O151 strains only revealed minor variation at the nucleotide level. Since E. coli O118 strains are more frequently isolated from humans, animals, and the environment than E. coli O151, serogroup O151 may likely be a minor variant of E. coli O118. Further studies are needed to elucidate this possibility.
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1118. |
Su Z,
Dai X,
Chen J,
Kong F,
Wang H,
Li Y,
Peng S,
Wang S,
Shao Q,
Lv L,
Xu H,
( 2008 ) The bla(CTX-M-1) gene located in a novel complex class I integron bearing an ISCR1 element in Escherichia coli isolates from Zhenjiang, China. PMID : 18647745 : DOI : 10.1093/jac/dkn300 Abstract >>
N/A
|
1119. |
Petrella S,
Ziental-Gelus N,
Mayer C,
Renard M,
Jarlier V,
Sougakoff W,
( 2008 ) Genetic and structural insights into the dissemination potential of the extremely broad-spectrum class A beta-lactamase KPC-2 identified in an Escherichia coli strain and an Enterobacter cloacae strain isolated from the same patient in France. PMID : 18625772 : DOI : 10.1128/AAC.00163-08 PMC : PMC2565876 Abstract >>
Two clinical strains of Escherichia coli (2138) and Enterobacter cloacae (7506) isolated from the same patient in France and showing resistance to extended-spectrum cephalosporins and low susceptibility to imipenem were investigated. Both strains harbored the plasmid-contained bla(TEM-1) and bla(KPC-2) genes. bla(KLUC-2), encoding a mutant of the chromosomal beta-lactamase of Kluyvera cryocrescens, was also identified at a plasmid location in E. cloacae 7506, suggesting the ISEcp1-assisted escape of bla(KLUC) from the chromosome. Determination of the KPC-2 structure at 1.6 A revealed that the binding site was occupied by the C-terminal (C-ter) residues coming from a symmetric KPC-2 monomer, with the ultimate C-ter Glu interacting with Ser130, Lys234, Thr235, and Thr237 in the active site. This mode of binding can be paralleled to the inhibition of the TEM-1 beta-lactamase by the inhibitory protein BLIP. Determination of the 1.23-A structure of a KPC-2 mutant in which the five C-ter residues were deleted revealed that the catalytic site was filled by a citrate molecule. Structure analysis and docking simulations with cefotaxime and imipenem provided further insights into the molecular basis of the extremely broad spectrum of KPC-2, which behaves as a cefotaximase with significant activity against carbapenems. In particular, residues 104, 105, 132, and 167 draw a binding cavity capable of accommodating both the aminothiazole moiety of cefotaxime and the 6 alpha-hydroxyethyl group of imipenem, with the binding of the former drug being also favored by a significant degree of freedom at the level of the loop at positions 96 to 105 and by an enlargement of the binding site at the end of strand beta 3.
|
1120. |
Corvec S,
Lepelletier D,
Reynaud A,
Dauvergne S,
Giraudeau C,
Caroff N,
( 2008 ) In vivo selection of an Escherichia coli isolate highly resistant to ciprofloxacin and ceftazidime: role of a 4-bp duplication in acrR and ampC overexpression. PMID : 18571906 : DOI : 10.1016/j.ijantimicag.2008.04.001 Abstract >>
N/A
|
1121. |
Wei Q,
Jiang X,
Yang Z,
Chen N,
Chen X,
Li G,
Lu Y,
( 2009 ) dfrA27, a new integron-associated trimethoprim resistance gene from Escherichia coli. PMID : 19008256 : DOI : 10.1093/jac/dkn474 Abstract >>
N/A
|
1122. |
Tóth I,
Nougayrède JP,
Dobrindt U,
Ledger TN,
Boury M,
Morabito S,
Fujiwara T,
Sugai M,
Hacker J,
Oswald E,
( 2009 ) Cytolethal distending toxin type I and type IV genes are framed with lambdoid prophage genes in extraintestinal pathogenic Escherichia coli. PMID : 18981247 : DOI : 10.1128/IAI.00962-08 PMC : PMC2612248 Abstract >>
Five types of cytolethal distending toxin (CDT-I to CDT-V) have been identified in Escherichia coli. In the present study we cloned and sequenced the cdt-IV operon and flanking region from a porcine extraintestinal pathogenic E. coli (ExPEC) strain belonging to serogroup O75. We confirmed that similar to other CDTs, CDT-IV induced phosphorylation of host histone H2AX, a sensitive marker of DNA double-strand breaks, and blocked the HeLa cell cycle at the G(2)-M transition. The cdt-IV genes were framed by lambdoid prophage genes. We cloned and sequenced the cdt-I operon and flanking regions from a human ExPEC O18:K1:H7 strain and observed that cdt-I genes were also flanked by lambdoid prophage genes. PCR studies indicated that a gene coding for a putative protease was always associated with the cdtC-IV gene but was not associated with cdtC genes in strains producing CDT-I, CDT-III, and CDT-V. Our results suggest that the cdt-I and cdt-IV genes might have been acquired from a common ancestor by phage transduction and evolved in their bacterial hosts. The lysogenic bacteriophages have the potential to carry nonessential "cargo" genes or "morons" and therefore play a crucial role in the generation of genetic diversity within ExPEC.
|
1123. |
Binh CT,
Heuer H,
Kaupenjohann M,
Smalla K,
( 2008 ) Piggery manure used for soil fertilization is a reservoir for transferable antibiotic resistance plasmids. PMID : 18557938 : DOI : 10.1111/j.1574-6941.2008.00526.x Abstract >>
In this study, the prevalence and types of transferable antibiotic resistance plasmids in piggery manure were investigated. Samples from manure storage tanks of 15 farms in Germany were analysed, representing diverse sizes of herds, meat or piglet production. Antibiotic resistance plasmids from manure bacteria were captured in gfp-tagged rifampicin-resistant Escherichia coli and characterized. The occurrence of plasmid types was also detected in total community DNA by PCR and hybridization. A total of 228 transconjugants were captured from 15 manures using selective media supplemented with amoxicillin, sulfadiazine or tetracycline. The restriction patterns of 81 plasmids representing different antibiotic resistance patterns or different samples clustered into seven groups. Replicon probing revealed that 28 of the plasmids belonged to IncN, one to IncW, 13 to IncP-1 and 19 to the recently discovered pHHV216-like plasmids. The amoxicillin resistance gene bla-TEM was detected on 44 plasmids, and sulphonamide resistance genes sul1, sul2 and/or sul3 on 68 plasmids. Hybridization of replicon-specific sequences amplified from community DNA revealed that IncP-1 and pHHV216-like plasmids were detected in all manures, while IncN and IncW ones were less frequent. This study showed that 'field-scale' piggery manure is a reservoir of broad-host range plasmids conferring multiple antibiotic resistance genes.
|
1124. |
McFall E,
Nikam SS,
Palchaudhuri S,
( 1991 ) Effects of structural changes in the dsdA-dsdC intergenic region on D-serine deaminase synthesis. PMID : 1899415 : DOI : 10.1128/jb.173.3.1161-1167.1991 PMC : PMC207237 Abstract >>
Single-base-pair changes well upstream of its transcription initiation site resulted in partially to fully constitutive expression of the D-serine deaminase structural gene, dsdA, independently of the cyclic AMP-cyclic AMP-binding protein complex and of the specific D-serine deaminase activator protein. These promoter mutations appear to define a consensus sequence that is repeated several times. Basal expression of dsdA+ was also strongly enhanced by subcloning on multicopy plasmids, by the DNA gyrase inhibitor novobiocin, and in dsdC(Con) mutants by increasing growth temperature. These results suggest that activation of dsdA+ expression by the dsdC-encoded protein involves distortion of promoter DNA. A dsdA translation start at bp -731 was verified by subcloning of dsdC+. Plasmid-specified activator at a high concentration interfered with chromosomal dsdC(Con) expression, and the interference was enhanced by deletion of most of the intergenic region from the plasmid. Even at a high concentration, however, plasmid-specified activator did not activate expression of chromosomal dsdA+, and in one case it was actually repressive. These results confirm the strong cis tropism of plasmid-specified dsdC-encoded protein and suggest that it is mediated by multiple sites in the dsdA-dsdC intergenic region.
|
1125. |
Diestra K,
Juan C,
Curiao T,
Moyá B,
Miró E,
Oteo J,
Coque TM,
Pérez-Vázquez M,
Campos J,
Cantón R,
Oliver A,
Navarro F,
N/A N/A,
( 2009 ) Characterization of plasmids encoding blaESBL and surrounding genes in Spanish clinical isolates of Escherichia coli and Klebsiella pneumoniae. PMID : 18988679 : DOI : 10.1093/jac/dkn453 Abstract >>
The aim of the study was to characterize plasmids that harbour blaESBL genes and their genetic environment in Escherichia coli and Klebsiella pneumoniae clones circulating in Spain. The incompatibility group of plasmids within 58 strains harbouring blaCTX-M (n=45) and blaSHV (n=15) genes was determined by rep-typing-PCR and hybridization. The blaESBL genetic environment was determined by PCR and sequencing. The blaCTX-M-9 genes (n=14) were linked to In60 located in IncI1 (50%) or IncHI2 plasmids (28%). All blaCTX-M-14 genes (n=13) were flanked by ISEcp1 and IS903 and 12 were associated with IncK plasmids. One of two blaCTX-M-10 genes was present in an IncK plasmid, but both genes were linked to a phage-related element. Five of seven blaCTX-M-1 (71%), all three blaCTX-M-32 and one of two blaCTX-M-3 genes were linked to IncN plasmids. The other blaCTX-M-3 gene was linked to IncA/C and the remaining two blaCTX-M-1 genes to IncFII plasmids. Three blaCTX-M-15 genes were associated with IncF (repFIA) and one with IncFII plasmids. All these genes from blaCTX-M group-1 showed the ISEcp1 upstream truncated by different insertion sequences. Forty-three percent of blaSHV-12 genes (n=14) were located in IncI1 plasmids, all flanked by the IS26 and DEOR region. The only detected blaSHV-5 gene was located in an IncFII plasmid and flanked by recF and DEOR regions. A diversity of the plasmid incompatibility groups that harbour blaESBL genes was observed, except for the blaCTX-M-14 gene. Moreover, a high variability was confirmed in the genetic environment of these genes as a result of insertion and deletion events.
|
1126. |
Byres E,
Paton AW,
Paton JC,
Löfling JC,
Smith DF,
Wilce MC,
Talbot UM,
Chong DC,
Yu H,
Huang S,
Chen X,
Varki NM,
Varki A,
Rossjohn J,
Beddoe T,
( 2008 ) Incorporation of a non-human glycan mediates human susceptibility to a bacterial toxin. PMID : 18971931 : DOI : 10.1038/nature07428 PMC : PMC2723748 Abstract >>
AB(5) toxins comprise an A subunit that corrupts essential eukaryotic cell functions, and pentameric B subunits that direct target-cell uptake after binding surface glycans. Subtilase cytotoxin (SubAB) is an AB(5) toxin secreted by Shiga toxigenic Escherichia coli (STEC), which causes serious gastrointestinal disease in humans. SubAB causes haemolytic uraemic syndrome-like pathology in mice through SubA-mediated cleavage of BiP/GRP78, an essential endoplasmic reticulum chaperone. Here we show that SubB has a strong preference for glycans terminating in the sialic acid N-glycolylneuraminic acid (Neu5Gc), a monosaccharide not synthesized in humans. Structures of SubB-Neu5Gc complexes revealed the basis for this specificity, and mutagenesis of key SubB residues abrogated in vitro glycan recognition, cell binding and cytotoxicity. SubAB specificity for Neu5Gc was confirmed using mouse tissues with a human-like deficiency of Neu5Gc and human cell lines fed with Neu5Gc. Despite lack of Neu5Gc biosynthesis in humans, assimilation of dietary Neu5Gc creates high-affinity receptors on human gut epithelia and kidney vasculature. This, and the lack of Neu5Gc-containing body fluid competitors in humans, confers susceptibility to the gastrointestinal and systemic toxicities of SubAB. Ironically, foods rich in Neu5Gc are the most common source of STEC contamination. Thus a bacterial toxin's receptor is generated by metabolic incorporation of an exogenous factor derived from food.
|
1127. |
Maas R,
Oppenheim J,
Saadi S,
Fuchs T,
Maas WK,
( 1991 ) Isolation and properties of the RepA1 protein of the IncFII replicon, RepFIC. PMID : 1857211 : DOI : 10.1111/j.1365-2958.1991.tb00767.x Abstract >>
The initiator protein RepA1 of the IncFII replicon RepFIC derived from the enterotoxin plasmid EntP307 has been cloned under the control of the lambda PL promoter. This has enabled us to overproduce this protein and study its properties. Here we show that RepA1 is a soluble basic protein with an experimentally determined molecular weight of 40,000. Deletion analysis indicates that the overproduced protein originates from the open reading frame which we previously designated as coding for RepA1. We have also shown that the replication function of the replicon RepFIC depends on the intact RepA1 coding frame.
|
1128. |
Bielaszewska M,
Prager R,
Vandivinit L,
Müsken A,
Mellmann A,
Holt NJ,
Tarr PI,
Karch H,
Zhang W,
( 2009 ) Detection and characterization of the fimbrial sfp cluster in enterohemorrhagic Escherichia coli O165:H25/NM isolates from humans and cattle. PMID : 18978078 : DOI : 10.1128/AEM.01815-08 PMC : PMC2612197 Abstract >>
The sfp cluster, encoding Sfp fimbriae and located in the large plasmid of sorbitol-fermenting (SF) enterohemorrhagic Escherichia coli (EHEC) O157 (pSFO157), has been considered a unique characteristic of this organism. We discovered and then characterized the sfp cluster in EHEC O165:H25/NM (nonmotile) isolates of human and bovine origin. All seven strains investigated harbored a complete sfp cluster (carrying sfpA, sfpH, sfpC, sfpD, sfpJ, sfpF, and sfpG) of 6,838 bp with >99% nucleotide sequence homology to the sfp cluster of SF EHEC O157:NM. The sfp cluster in EHEC O165:H25/NM strains was located in an approximately 80-kb (six strains) or approximately 120-kb (one strain) plasmid which differed in structure, virulence genes, and sfp flanks from pSFO157. All O165:H25/NM strains belonged to the same multilocus sequence type (ST119) and were only distantly phylogenetically related to SF EHEC O157:NM (ST11). The highly conserved sfp cluster in different clonal backgrounds suggests that this segment was acquired independently by EHEC O165:H25 and SF EHEC O157:NM. Its presence in an additional EHEC serotype extends the diagnostic utility of PCR targeting sfpA as an easy and efficient approach to seek EHEC in patients' stools. The reasons for the convergence of pathogenic EHEC strains on a suite of virulence loci remain unknown.
|
1129. |
Karanam VR,
Reddy HP,
Subba Raju BV,
Rao JC,
Kavikishore PB,
Vijayalakshmi M,
( 2008 ) Detection of indicator pathogens from pharmaceutical finished products and raw materials using multiplex PCR and comparison with conventional microbiological methods. PMID : 18521641 : DOI : 10.1007/s10295-008-0376-z Abstract >>
A multiplex PCR assay was devised and compared with standard conventional methods for quality evaluation of pharmaceutical raw materials and finished products with low levels of microbial contamination. Samples which were artificially contaminated with <10 colony forming units of Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Salmonella species and possibly contaminated samples were incubated for 16 h with different enrichment media. Primers that deduce 559 bp fragment of the 16S rRNA gene was employed in amplifying E. coli species, similarly invasion protein gene with 275 bp fragment size was used as target for detecting Salmonella spp., in case of S. aureus a 461 bp amplicon from m-RNA nuclease gene, and an 709 bp fragment from oprL gene was used for amplifying P. aeruginosa. The detection limits for artificially contaminants by multiplex PCR was 1 CFU/g, where as in case of conventional method the detection limit was >2 CFU/g. Similarly, when tested with possibly contaminated samples, 35% were detected for E. coli, Salmonella spp., S. aureus and P. aeruginosa species with multiplex PCR, while only 21% were detected with standard conventional microbial methods. Multiplex PCR assay provides sensitive and reliable results and allows for the cost-effective detection of all four bacterial pathogens in single reaction tube.
|
1130. |
Pavelka MS,
Wright LF,
Silver RP,
( 1991 ) Identification of two genes, kpsM and kpsT, in region 3 of the polysialic acid gene cluster of Escherichia coli K1. PMID : 1856162 : DOI : 10.1128/jb.173.15.4603-4610.1991 PMC : PMC208135 Abstract >>
The polysialic acid capsule of Escherichia coli K1, a causative agent of neonatal septicemia and meningitis, is an essential virulence determinant. The 17-kb kps gene cluster, which is divided into three functionally distinct regions, encodes proteins necessary for polymer synthesis and expression at the cell surface. The central region, 2, encodes products required for synthesis, activation, and polymerization of sialic acid, while flanking regions, 1 and 3, are thought to be involved in polymer assembly and transport. In this study, we identified two genes in region 3, kpsM and kpsT, which encode proteins with predicted sizes of 29.6 and 24.9 kDa, respectively. The hydrophobicity profile of KpsM suggests that it is an integral membrane protein, while KpsT contains a consensus ATP-binding domain. KpsM and KpsT belong to a family of prokaryotic and eukaryotic proteins involved with a variety of biological processes, including membrane transport. A previously described kpsT chromosomal mutant that accumulates intracellular polysialic acid was characterized and could be complemented in trans. Results of site-directed mutagenesis of the putative ATP-binding domain of KpsT are consistent with the view that KpsT is a nucleotide-binding protein. KpsM and KpsT have significant similarity to BexB and BexA, two proteins that are essential for polysaccharide capsule expression in Haemophilus influenzae type b. We propose that KpsM and KpsT constitute a system for transport of polysialic acid across the cytoplasmic membrane.
|
1131. |
Brockhausen I,
Riley JG,
Joynt M,
Yang X,
Szarek WA,
( 2008 ) Acceptor substrate specificity of UDP-Gal: GlcNAc-R beta1,3-galactosyltransferase (WbbD) from Escherichia coli O7:K1. PMID : 18536883 : DOI : 10.1007/s10719-008-9127-7 Abstract >>
Most of the glycosyltransferases involved in O antigen biosynthesis have not yet been characterized. We recently demonstrated that the wbbD gene of the O7 lipopolysaccharide biosynthesis cluster in E. coli strain VW187 (O7:K1) encodes WbbD, a UDP-Gal: GlcNAcalpha-pyrophosphate-lipid beta1,3-Gal-transferase (EC 2.4.1., accession number AAC27537) that transfers the second sugar moiety in the assembly of the O7 repeating unit. The enzyme utilizes undecaprenol-pyrophosphate-GlcNAc as a natural acceptor substrate, but can also transfer Gal to GlcNAcalpha-PO(3)-PO(3)-(CH(2))(11)-O-phenyl (GlcNAc-PP-PhU). A number of acceptor substrate analogs have now been tested to further characterize the acceptor specificity of WbbD and to determine the roles of the pyrophosphate bond and the lipid moiety in the acceptor substrate. The enzyme was found to have a low activity with a substrate containing only one phosphate group directly alpha-linked to GlcNAc, and the enzyme was inactive when the phosphate was absent or further removed from the anomeric carbon of GlcNAc. Modifications of the lipid chain yielded substrates with variable activities. GlcNAc derivatives that were inactive as substrates did not inhibit WbbD suggesting that these compounds did not bind to the active site of the enzyme. The specificity of mammalian beta4-galactosyltransferase I has been compared to that of WbbD. The results indicate that the bacterial WbbD enzyme has a distinct specificity for GlcNAc-PP-lipid, and that WbbD recognition of its acceptor substrate is very different from that of the ubiquitous mammalian beta4-galactosyltransferase I. These studies help to understand mechanisms of O antigen synthesis, to develop methods to synthesize defined oligosaccharide structures and to develop specific O antigen inhibitors.
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1132. |
Sunde M,
Tharaldsen H,
Slettemeås JS,
Norström M,
Carattoli A,
Bjorland J,
( 2009 ) Escherichia coli of animal origin in Norway contains a blaTEM-20-carrying plasmid closely related to blaTEM-20 and blaTEM-52 plasmids from other European countries. PMID : 18971216 : DOI : 10.1093/jac/dkn445 Abstract >>
N/A
|
1133. |
Cattoir V,
Nordmann P,
Silva-Sanchez J,
Espinal P,
Poirel L,
( 2008 ) ISEcp1-mediated transposition of qnrB-like gene in Escherichia coli. PMID : 18519717 : DOI : 10.1128/AAC.00349-08 PMC : PMC2493098 Abstract >>
A novel QnrB-like plasmid-mediated resistance determinant, QnrB19, was identified from an Escherichia coli clinical isolate from Colombia. Its gene was associated with an ISEcp1-like insertion element that did not act as a promoter for its expression. Using an in vitro model of transposition, we showed that the ISEcp1-like element was able to mobilize the qnrB19 gene.
|
1134. |
Warburton P,
Roberts AP,
Allan E,
Seville L,
Lancaster H,
Mullany P,
( 2009 ) Characterization of tet(32) genes from the oral metagenome. PMID : 18955517 : DOI : 10.1128/AAC.00788-08 PMC : PMC2612163 Abstract >>
tet(32) Was identified in three bacterial isolates and in metagenomic DNA from the human oral cavity. The regions immediately flanking the gene were found to have similarities to the mobile elements TnB1230 from Butyrivibrio fibrisolvens, ATE-3 from Arcanobacterium pyogenes, and CTn5 from Clostridium difficile.
|
1135. |
Le Turnier S,
Nordmann P,
Eb F,
Mammeri H,
( 2009 ) Potential evolution of hydrolysis spectrum for AmpC beta-lactamase of Escherichia coli. PMID : 18957396 : DOI : 10.1093/jac/dkn443 Abstract >>
N/A
|
1136. |
Fratamico PM,
Bhagwat AA,
Injaian L,
Fedorka-Cray PJ,
( 2008 ) Characterization of Shiga toxin-producing Escherichia coli strains isolated from swine feces. PMID : 18991545 : DOI : 10.1089/fpd.2008.0147 Abstract >>
The virulence gene and antibiotic resistance profiles of Shiga toxin-producing Escherichia coli (STEC) strains belonging to 58 different O:H serotypes (219 strains) isolated from swine feces were determined. Of the 219 isolates, 29 (13%) carried the stx(1) gene, 14 (6%) stx(2), 176 (80%) stx(2e), 46 (21%) estIa, 14 (6.4%) estIb, 10 (4.6%) fedA, 94 (42.9%) astA, 25 (11.4%) hly(933), and one (0.46%) cdt-III. None of the strains possessed the elt, bfp, faeG, fanA, fasA, fimF(41a), cnf-1, cnf-2, eae, cdt-I, or cdt-IV genes. The strains were also tested for antibiotic susceptibility using 16 antibiotics. The STEC isolates displayed resistance most often to tetracycline (95.4%), sulfamethoxazole (53.4%), kanamycin (38.4%), streptomycin (34.7%), and chloramphenicol (22.4%). An E. coli serotype O20:H42 strain, which was positive for stx(2e) and astA, was resistant to all of the antibiotics tested except for amikacin. In addition, 52 of the swine isolates, representing 16 serogroups and 30 different serotypes, were examined for their ability to withstand acid challenge by three types of acid resistance (AR) pathways, AR1 (rpoS dependent), AR2 (glutamate dependent), and AR3 (arginine dependent). None of the strains was defective in the AR1 resistance pathway, while one strain was defective in the AR2 pathway under aerobic growth conditions but fully functional under anaerobic growth conditions. Among the three AR pathways, the AR3 pathway offered the least protection, and 8 out of 52 strains were defective in this pathway. The strain that was defective in AR2 was fully functional in the AR3 pathway. Since AR plays a vital role in the survival and virulence of these strains, differences among the isolates to induce AR pathways may play a significant role in determining their infective dose. This study demonstrates that swine STEC comprise a heterogeneous group of organisms, and the possession of different combinations of E. coli virulence genes indicate that some swine STEC can potentially cause human illness.
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1137. |
Vinué L,
Lantero M,
Sáenz Y,
Somalo S,
de Diego I,
Pérez F,
Ruiz-Larrea F,
Zarazaga M,
Torres C,
( 2008 ) Characterization of extended-spectrum beta-lactamases and integrons in Escherichia coli isolates in a Spanish hospital. PMID : 18566158 : DOI : 10.1099/jmm.0.47723-0 Abstract >>
N/A
|
1138. |
Inoue N,
Uchida H,
( 1991 ) Transcription and initiation of ColE1 DNA replication in Escherichia coli K-12. PMID : 1846858 : DOI : 10.1128/jb.173.3.1208-1214.1991 PMC : PMC207244 Abstract >>
By S1 nuclease protection mapping, we characterized RNA transcripts and nascent ColE1 DNA synthesized in wild-type Escherichia coli cells after infection with lambda-mini-ColE1 hybrid bacteriophages. Transcription of the RNA II region of ColE1 DNA in vivo starts mostly from the RNA II promoter, which was identified by in vitro experiments, and ends at or near the ori site. Synthesis of the leading strand of ColE1 DNA was found to start at the ori site. Nevertheless, the molar ratio of the nascent DNA to the synthesized transcripts ending at the ori site was less than 0.05. In bacterial rnh mutants whose RNase H activities were less than 0.06% of that of the wild type, transcription patterns, as well as nascent DNA synthesis, were still similar to those in rnh+ cells. However, in bacteria whose rnh gene was interrupted by insertion of a drug resistance gene, the number of transcripts ending at the ori site was much reduced and that of transcripts reading through the ori site was definitely increased relative to that observed in wild-type bacteria. These results suggested that cleavage of the RNA transcript at the ori site in vivo is dependent on RNase H activity, as demonstrated in the in vitro system, but most of the cleaved RNA is unable to prime initiation of ColE1 DNA synthesis efficiently.
|
1139. |
Chang HW,
Nam YD,
Jung MY,
Kim KH,
Roh SW,
Kim MS,
Jeon CO,
Yoon JH,
Bae JW,
( 2008 ) Statistical superiority of genome-probing microarrays as genomic DNA-DNA hybridization in revealing the bacterial phylogenetic relationship compared to conventional methods. PMID : 18782592 : DOI : 10.1016/j.mimet.2008.08.003 Abstract >>
The genomic DNA-DNA hybridization (DDH) method has been widely used as a practical method for the determination of phylogenetic relationships between closely related biological strains. Traditional DDH methods have serious limitations including low reproducibility, a high background and a time-consuming procedure. The DDH method using a genome-probing microarray (GPM) has been recently developed to complement conventional methods and could be used to overcome the limitations that are typically encountered. It is necessary to compare the GPM-based DDH method to the conventional methods before using the GPM for the estimation of genomic similarities since all of the previous scientific data have been entirely dependent on conventional DDH methods. In order to address this issue we compared the DDH values obtained using the GPM, microplate and nylon membrane methods to multi-locus sequence typing (MLST) data for 9 Salmonella genomes and an Escherichia coli type strain. The results showed that the genome similarity values and the degrees of standard deviation obtained using the GPM method were lower than those obtained with the microplate and nylon membrane methods. The dendrogram from the cluster analysis of GPM DDH values was consistent with the phylogenetic tree obtained from the multi-locus sequence typing (MLST) data but was not similar to those obtained using the microplate and nylon membrane methods. Although the signal intensity had to be maximal when the targets were hybridized to their own probe, the methods using membranes and microplates frequently produced higher signals in the heterologous hybridizations than those obtained in the homologous hybridizations. Only the GPM method produced the highest signal intensity in homologous hybridizations. These results show that the GPM method can be used to obtain results that are more accurate than those generated by the other methods tested.
|
1140. |
Wang Q,
Cheng J,
Chen Y,
Ye Y,
Li JB,
Zhang XJ,
( 2008 ) Characterization of a novel AmpC-type plasmid-mediated beta-lactamase from an Escherichia coli strain isolated in China. PMID : 18781358 : DOI : 10.1007/s00284-008-9242-5 Abstract >>
The aim of this work was to study the phenotypic and molecular characterization of a novel plasmid-mediated AmpC beta-lactamase from Escherichia coli E384. Conjugation experiments, isoelectric focusing, pulsed-field gel electrophoresis, plasmid profiling, and Southern blot as well as PCR, sequencing techniques, and susceptibility testing were carried out to investigate the underlying mechanism of resistance. The kinetic parameters were determined to characterize the novel enzyme. MIR-4 beta-lactamase, pI 8.2, is a novel variant with four substitutions of amino acids compared with the sequence of MIR-1. E. coli E384 displays resistance to eight beta-lactam antimicrobial agents and three fluoroquinolones. The minimal inhibitory concentrations of beta-lactam in combination with beta-lactamase inhibitors show no significant synergy. Kinetic parameters suggest that the novel enzyme effectively hydrolyzes broad-spectrum beta-lactams. The same hybridization signal was detectable only in the 54-kb plasmid band that hybridized with the bla (CTX-M)- and bla(ampC)-specific probes. This is the first description of a plasmid-mediated MIR-4 enzyme in China. This study illustrates the importance of molecular surveillance in tracking AmpC-producing strains at general hospitals and emphasizes the need for epidemiological monitoring.
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1141. |
Berçot B,
Poirel L,
Nordmann P,
( 2008 ) Plasmid-mediated 16S rRNA methylases among extended-spectrum beta-lactamase-producing Enterobacteriaceae isolates. PMID : 18838598 : DOI : 10.1128/AAC.00882-08 PMC : PMC2592896 Abstract >>
N/A
|
1142. |
Sundström L,
Roy PH,
Sköld O,
( 1991 ) Site-specific insertion of three structural gene cassettes in transposon Tn7. PMID : 1850404 : DOI : 10.1128/jb.173.9.3025-3028.1991 PMC : PMC207888 Abstract >>
Transposon Tn7 has been known to carry genes for resistance to trimethoprim and spectinomycin. A poorly expressed streptothricin resistance gene, identical to the sat gene found in transposons Tn1825 and Tn1826, was localized between the two mentioned genes in Tn7. The surroundings of all three resistance genes indicated site-specific insertion of genetic cassettes.
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1143. |
Rosser T,
Dransfield T,
Allison L,
Hanson M,
Holden N,
Evans J,
Naylor S,
La Ragione R,
Low JC,
Gally DL,
( 2008 ) Pathogenic potential of emergent sorbitol-fermenting Escherichia coli O157:NM. PMID : 18852247 : DOI : 10.1128/IAI.01180-08 PMC : PMC2583558 Abstract >>
Non-sorbitol-fermenting (NSF) Escherichia coli O157:H7 is the primary Shiga toxin-producing E. coli (STEC) serotype associated with human infection. Since 1988, sorbitol-fermenting (SF) STEC O157:NM strains have emerged and have been associated with a higher incidence of progression to hemolytic-uremic syndrome (HUS) than NSF STEC O157:H7. This study investigated bacterial factors that may account for the increased pathogenic potential of SF STEC O157:NM. While no evidence of toxin or toxin expression differences between the two O157 groups was found, the SF STEC O157:NM strains adhered at significantly higher levels to a human colonic cell line. Under the conditions tested, curli were shown to be the main factor responsible for the increased adherence to Caco-2 cells. Notably, 52 of 66 (79%) European SF STEC O157:NM strains tested bound Congo red at 37 degrees C and this correlated with curli expression. In a subset of strains, curli expression was due to increased expression from the csgBAC promoter that was not always a consequence of increased csgD expression. The capacity of SF STEC O157:NM strains to express curli at 37 degrees C may have relevance to the epidemiology of human infections as curliated strains could promote higher levels of colonization and inflammation in the human intestine. In turn, this could lead to increased toxin exposure and an increased likelihood of progression to HUS.
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1144. |
Scott L,
McGee P,
Walsh C,
Fanning S,
Sweeney T,
Blanco J,
Karczmarczyk M,
Earley B,
Leonard N,
Sheridan JJ,
( 2009 ) Detection of numerous verotoxigenic E. coli serotypes, with multiple antibiotic resistance from cattle faeces and soil. PMID : 18838234 : DOI : 10.1016/j.vetmic.2008.08.008 Abstract >>
Verotoxigenic E. coli (VTEC) belong to a diverse range of serotypes. Serotypes O157 and O26 are predominately identified in VTEC-associated disease in Europe, however due to difficulty in detection little is known about the epidemiology of non-O157 serotypes. This study reports the identification of 7 VTEC serotypes from cattle faeces and soil. Cattle faeces samples (n=128) were taken from animals in 6 different farms, with soil samples (n=20) obtained from 1 farm. After sample incubation in modified tryptone soy broth (mTSB) supplemented with streptomycin sulphate samples were plated onto sorbitol MacConkey (SMAC) also supplemented with streptomycin sulphate. Bacteria detected on the plates were subjected to biochemical testing, antibiotic resistance profiling, and PCR to detect typical virulence genes, beta-lactamase and class 1 integron associated genes. Serotyping was performed on isolates positive for virulence genes. E. coli was identified from 103 samples, with verotoxin genes present in 7 E. coli isolates. Of these 7 isolates, 5 were resistant to 5 or more antibiotics. The isolate resistant to 9 antimicrobials contained a class 1 integron structure. Serotyping identified 7 separate VTEC, O2:H27, O26:H11, O63:H(-), O148:H8, O149:H1, O174:H21 and ONT:H25. Six of these VTEC have been previously associated with human disease, however with the exception of O26:H11, these serotypes have been rarely reported worldwide. Increased surveillance is required to determine the prevalence of these and other non-O157 VTEC. The presence of multi-antibiotic resistance in these isolates is of concern, and the overall implications for public health must be ascertained.
|
1145. |
Hsu Y,
Jubelin G,
Taieb F,
Nougayrède JP,
Oswald E,
Stebbins CE,
( 2008 ) Structure of the cyclomodulin Cif from pathogenic Escherichia coli. PMID : 18845161 : DOI : 10.1016/j.jmb.2008.09.051 PMC : PMC2659761 Abstract >>
Bacterial pathogens have evolved a sophisticated arsenal of virulence factors to modulate host cell biology. Enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC) use a type III protein secretion system (T3SS) to inject microbial proteins into host cells. The T3SS effector cycle inhibiting factor (Cif) produced by EPEC and EHEC is able to block host eukaryotic cell-cycle progression. We present here a crystal structure of Cif, revealing it to be a divergent member of the superfamily of enzymes including cysteine proteases and acetyltransferases that share a common catalytic triad. Mutation of these conserved active site residues abolishes the ability of Cif to block cell-cycle progression. Finally, we demonstrate that irreversible cysteine protease inhibitors do not abolish the Cif cytopathic effect, suggesting that another enzymatic activity may underlie the biological activity of this virulence factor.
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1146. |
Beutin L,
Krüger U,
Krause G,
Miko A,
Martin A,
Strauch E,
( 2008 ) Evaluation of major types of Shiga toxin 2E-producing Escherichia coli bacteria present in food, pigs, and the environment as potential pathogens for humans. PMID : 18515483 : DOI : 10.1128/AEM.00623-08 PMC : PMC2519352 Abstract >>
Shiga toxin 2e (Stx2e)-producing strains from food (n = 36), slaughtered pigs (n = 25), the environment (n = 21), diseased pigs (n = 19), and humans (n = 9) were investigated for production of Stx2e by enzyme-linked immunosorbent assay, for virulence markers by PCR, and for their serotypes to evaluate their role as potential human pathogens. Stx2e production was low in 64% of all 110 strains. Stx2e production was inducible by mitomycin C but differed considerably between strains. Analysis by nucleotide sequencing and transcription of stx(2e) genes in high- and low-Stx2e-producing strains showed that toxin production correlated with transcription rates of stx(2e) genes. DNA sequences specific for the int, Q, dam, and S genes of the stx(2e) bacteriophage P27 were found in 109 strains, indicating cryptic P27-like prophages, although 102 of these were not complete for all genes tested. Genes encoding intimin (eae), enterohemorrhagic Escherichia coli hemolysin (ehx), or other stx(1) or stx(2) variants were not found, whereas genes for heat-stable enterotoxins STI, STII, or EAST1 were present in 54.5% of the strains. Seven major serotypes that were associated with diseased pigs (O138:H14, O139:H1, and O141:H4) or with slaughter pigs, food, and the environment (O8:H4, O8:H9, O100:H30, and O101:H9) accounted for 60% of all Stx2e strains. The human Stx2e isolates did not belong to these major serotypes of Stx2e strains, and high production of Stx2e in human strains was not related to diarrheal disease. The results from this study and other studies do not point to Stx2e as a pathogenicity factor for diarrhea and hemolytic uremic syndrome in humans.
|
1147. |
Yamaguchi A,
Adachi K,
Akasaka T,
Ono N,
Sawai T,
( 1991 ) Metal-tetracycline/H+ antiporter of Escherichia coli encoded by a transposon Tn10. Histidine 257 plays an essential role in H+ translocation. PMID : 1848846 : Abstract >>
The transposon Tn10-encoded tetA gene product is a metal-tetracycline/proton antiporter (Yamaguchi, A., Udagawa, T., and Sawai, T. (1990) J. Biol. Chem. 265, 4809-4813). Its tetracycline transport activity was inhibited by a histidine-specific reagent, diethyl pyrocarbonate. Among five histidine residues in this antiporter, only His257 is located in the putative transmembrane helices. Thus, His257 was replaced by Glu or Asp. Inverted vesicles containing the Glu257 and Asp257 mutant proteins showed only 20 and 10% of the tetracycline uptake of wild-type vesicles, respectively. In contrast to wild-type vesicles, the mutant vesicles showed no tetracycline-dependent proton translocation, indicating that the mutant proteins had lost the tetracycline/H+ antiport activity. The significant 60Co2+ uptake without proton translocation by the mutant vesicles also confirmed that the mutant carriers act as uniporters of a metal-tetracycline complex. The metal-tetracycline uniport by the mutant proteins was not inhibited by diethyl pyrocarbonate, indicating that His257 is the only histidine residue essential for proton translocation. These mutant proteins conferred about half-level resistance to tetracycline, probably due to their catalyzing downhill efflux of a metal-tetracycline complex out of the cells.
|
1148. |
Zarivach R,
Deng W,
Vuckovic M,
Felise HB,
Nguyen HV,
Miller SI,
Finlay BB,
Strynadka NC,
( 2008 ) Structural analysis of the essential self-cleaving type III secretion proteins EscU and SpaS. PMID : 18451864 : DOI : 10.1038/nature06832 Abstract >>
During infection by Gram-negative pathogenic bacteria, the type III secretion system (T3SS) is assembled to allow for the direct transmission of bacterial virulence effectors into the host cell. The T3SS system is characterized by a series of prominent multi-component rings in the inner and outer bacterial membranes, as well as a translocation pore in the host cell membrane. These are all connected by a series of polymerized tubes that act as the direct conduit for the T3SS proteins to pass through to the host cell. During assembly of the T3SS, as well as the evolutionarily related flagellar apparatus, a post-translational cleavage event within the inner membrane proteins EscU/FlhB is required to promote a secretion-competent state. These proteins have long been proposed to act as a part of a molecular switch, which would regulate the appropriate chronological secretion of the various T3SS apparatus components during assembly and subsequently the transported virulence effectors. Here we show that a surface type II beta-turn in the Escherichia coli protein EscU undergoes auto-cleavage by a mechanism involving cyclization of a strictly conserved asparagine residue. Structural and in vivo analysis of point and deletion mutations illustrates the subtle conformational effects of auto-cleavage in modulating the molecular features of a highly conserved surface region of EscU, a potential point of interaction with other T3SS components at the inner membrane. In addition, this work provides new structural insight into the distinct conformational requirements for a large class of self-cleaving reactions involving asparagine cyclization.
|
1149. |
Kadlec K,
Schwarz S,
( 2008 ) Analysis and distribution of class 1 and class 2 integrons and associated gene cassettes among Escherichia coli isolates from swine, horses, cats and dogs collected in the BfT-GermVet monitoring study. PMID : 18550679 : DOI : 10.1093/jac/dkn233 Abstract >>
In the BfT-GermVet monitoring study, 417 Escherichia coli isolates collected during 2004-06 in Germany from various disease conditions of pigs (n = 87), horses (n = 102) or cats/dogs (n = 228) were investigated for their susceptibility to 24 antimicrobial agents. This study dealt with the identification of integron-associated resistance genes among these isolates. Class 1 and class 2 integrons were detected by PCR. The variable parts of the integrons were cloned and sequenced. Transformation and conjugation experiments were conducted to confirm a plasmid location of the integrons. Class 1 and/or class 2 integrons, alone or in different combinations, were detected in 79 of the 417 E. coli isolates. Four trimethoprim resistance genes (dfrA1/12/14/17), five streptomycin/spectinomycin resistance genes (aadA1/2/4/5/6), two streptothricin resistance genes (estX, sat2), one gentamicin/tobramycin/kanamycin resistance gene (aadB) and one chloramphenicol resistance gene (catB3) were detected. Seven different cassette arrangements were identified within class 1 integrons: aadA1 (21 isolates), dfrA1 + aadA1 (18 isolates), dfrA17 + aadA5 (9 isolates), dfrA12 + orfF + aadA2 (8 isolates), aadB + aadA1 (1 isolate), dfrA14 + recombined aadA6 (1 isolate) and dfrA1 + catB3 + aadA4 (1 isolate). Three different cassette arrangements in class 2 integrons, dfrA1 + sat2 + aadA1 (24 isolates), estX + sat2 + aadA1 (6 isolates) and estX + sat2 + DeltaaadA1 (1 isolate), were identified. The plasmid location of class 1 and/or class 2 integrons was confirmed in 37 isolates. Class 1 and/or class 2 integrons carrying resistance gene cassettes were detected in 18.9% of the isolates tested. This molecular analysis complements the phenotypic susceptibility testing conducted in the BfT-GermVet monitoring study and helps to explain the persistence of resistance genes even without direct selective pressure.
|
1150. |
Takahagi M,
Iwasaki H,
Nakata A,
Shinagawa H,
( 1991 ) Molecular analysis of the Escherichia coli ruvC gene, which encodes a Holliday junction-specific endonuclease. PMID : 1885548 : DOI : 10.1128/jb.173.18.5747-5753.1991 PMC : PMC208306 Abstract >>
The Escherichia coli ruvC gene is involved in DNA repair and recombination and encodes an endonuclease that resolves Holliday structure in vitro. The 2.8-kb chromosomal DNA fragment that encompasses the ruvC gene and its flanking regions was cloned and sequenced. Four open reading frames were identified in the order orf17-orf26-ruvC-orf23 immediately upstream of the ruvAB operon, and their orientations are the same as the ruvAB operon, except for orf23. Proteins encoded by orf17, orf26, and ruvC (orf19) were identified by the maxicell method, and their sizes agreed with those predicted from the DNA sequences. Among the open reading frames in this region, only ruvC is involved in the repair of UV-damaged DNA. ruvC appeared to be regulated by at least two promoters, but, in contrast to the ruvAB operon, ruvC is not regulated by the SOS system as demonstrated by operon fusions.
|
1151. |
Zong Z,
Partridge SR,
Thomas L,
Iredell JR,
( 2008 ) Dominance of blaCTX-M within an Australian extended-spectrum beta-lactamase gene pool. PMID : 18725449 : DOI : 10.1128/AAC.00107-08 PMC : PMC2573124 Abstract >>
bla(CTX-M) genes, particularly bla(CTX-M-15), are the dominant extended-spectrum beta-lactamase (ESBL) genes among clinical isolates of Escherichia coli and Klebsiella pneumoniae in Sydney, Australia, where we also found one example of bla(CTX-M-62), encoding a novel enzyme conferring ceftazidime resistance. ESBL genes were present in diverse community isolates and in a variety of associated conjugative plasmids.
|
1152. |
Norman A,
Hansen LH,
She Q,
Sørensen SJ,
( 2008 ) Nucleotide sequence of pOLA52: a conjugative IncX1 plasmid from Escherichia coli which enables biofilm formation and multidrug efflux. PMID : 18440636 : DOI : 10.1016/j.plasmid.2008.03.003 Abstract >>
The large conjugative multidrug resistance (MDR) plasmid pOLA52 was sequenced and annotated. The plasmid encodes two phenotypes normally associated with the chromosomes of opportunistic pathogens, namely MDR via a resistance-nodulation-division (RND)-type efflux-pump (oqxAB), and the formation of type 3 fimbriae (mrkABCDF). The plasmid was found to be 51,602 bp long with 68 putative genes. About half of the plasmid constituted a conserved IncX1-type backbone with predicted regions for conjugation, replication and partitioning, as well as a toxin/antitoxin (TA) plasmid addiction system. The plasmid was also classified as IncX1 with incompatibility testing. The conjugal transfer and plasmid maintenance regions of pOLA52 therefore seem to represent IncX1 orthologues of the well-characterized IncX2 plasmid R6K. Sequence homology searches in GenBank also suggested a considerably higher prevalence of IncX1 group plasmids than IncX2. The 21 kb 'genetic load' region of pOLA52 was shown to consist of a mosaic, among other things a fragmented Tn3 transposon encoding ampicillin resistance. Most notably the oqxAB and mrkABCDF cassettes were contained within two composite transposons (Tn6010 and Tn6011) that seemed to originate from Klebsiella pneumoniae, thus demonstrating the capability of IncX1 plasmids of facilitating lateral transfer of gene cassettes between different Enterobacteriaceae.
|
1153. |
Wang W,
Seah SY,
( 2008 ) The role of a conserved histidine residue in a pyruvate-specific Class II aldolase. PMID : 18775708 : DOI : 10.1016/j.febslet.2008.08.032 Abstract >>
Histidine 45 in HpaI was replaced with alanine (H45A) and glutamine (H45Q). In the aldol cleavage reaction, kcat values were lowered by 78- and 2059-fold while Km values were increased by 100- and 42-fold in H45A and H45Q, respectively, compared to the wild-type enzyme. Both mutants displayed higher dissociation constants towards the metal cofactor, pyruvate and the transition state analogue, oxalate. Pyruvate proton exchange rates are consequently reduced in H45A and H45Q. pKa for a catalytic base (6.5) is lost in the mutant enzymes and catalysis is dependent on hydroxide ions. The results show that histidine 45 is important for metal cofactor binding and for facilitating C4-OH proton abstraction of the substrate in the reaction mechanism.
|
1154. |
Slade A,
Horrocks AJ,
Lindsay CD,
Dunbar B,
Virden R,
( 1991 ) Site-directed chemical conversion of serine to cysteine in penicillin acylase from Escherichia coli ATCC 11105. Effect on conformation and catalytic activity. PMID : 1849824 : DOI : 10.1111/j.1432-1033.1991.tb15884.x Abstract >>
Penicillin acylase (EC 3.5.1.11) was completely inactivated with equimolar phenylmethane [35S]sulphonyl fluoride (PhMe35SO2F); the stability of the sulphonyl group in the modified protein was determined by measurement of the radioactivity in ultrafiltrates. In 8 M urea, the rate of loss of the sulphonyl group was similar to that observed in PhMeSO2F-inactivated chymotrypsin [Gold, A.M. & Fahrney, D. (1964) Biochemistry 3, 783-791]. Incubation of the PhMeSO2F-inactivated acylase with 0.7 M potassium thioacetate yielded an acetylthiol enzyme which was subsequently converted to a thiol-enzyme during incubation with 10 mM 6-aminopenicillanic acid. 4-Pyridyl-ethylcysteine was released by acid hydrolysis after reaction of the thiol-protein with 4-vinylpyridine. The rates of reaction of thiol-penicillin acylase with iodoacetic acid and 2,2'-dipyridyl disulphide were consistent with the presence of an incompletely accessible cysteinyl sidechain. After carboxymethylating the thiol-enzyme with iodo[2-3H]acetic acid, the label was shown by SDS/PAGE and sequencing analysis to be associated exclusively with the beta-chain NH2-terminal residue, indicating conversion of Ser290 to S-carboxymethyl-cysteine. Near-ultraviolet CD spectra showed the conformation of thiol-penicillin acylase to be indistinguishable from that of the native protein but the catalytic activity was less than 0.02% of that of the normal enzyme. The possibility that Ser290 acts as a nucleophile in catalysis is discussed.
|
1155. |
Boisen N,
Struve C,
Scheutz F,
Krogfelt KA,
Nataro JP,
( 2008 ) New adhesin of enteroaggregative Escherichia coli related to the Afa/Dr/AAF family. PMID : 18443096 : DOI : 10.1128/IAI.01646-07 PMC : PMC2446688 Abstract >>
Enteroaggregative Escherichia coli (EAEC) is an important cause of diarrhea worldwide. We analyzed 17 Danish EAEC strains, isolated in the course of a case control study, for phenotypic and genotypic properties. The strains belonged to at least 14 different serotypes. Using PCR to investigate the prevalence of various putative virulence genes, we found that all but two strains were typical EAEC, as they harbored all or part of the previously described AggR regulon. The majority of the strains harbored genes encoding aggregative adherence fimbriae (AAF). The most common was AAF/I, found in nine strains; eight strains carried no known AAF-related genes. We utilized TnphoA mutagenesis to localize the aggregative adherence (AA) adhesin from one typical EAEC strain, C1010-00, which lacked a known AAF. We identified a TnphoA insertion in a hypothetical Dr-related pilin deposited in GenBank as HdaA. Four additional Danish strains harbored HdaA, and all but one displayed AA to HEp-2 cells. By using PCR primers derived from the pilins and ushers from the three AAF and Hda, we found that 16 of 17 strains exhibited evidence of one of these factors; importantly, the one negative strain also lacked the aggR gene. Cloning of the complete Hda gene cluster and expression in E. coli DH5alpha resulted in AA and complementation of the C1010-00 nonadherent mutant. Four related adhesins have now been found to confer AA in typical EAEC strains; our data suggest that, together, these variants may account for AA in the large majority of strains.
|
1156. |
Tian SF,
Chen BY,
Chu YZ,
Wang S,
( 2008 ) Prevalence of rectal carriage of extended-spectrum beta-lactamase-producing Escherichia coli among elderly people in community settings in China. PMID : 18772941 : DOI : 10.1139/w08-059 Abstract >>
The importance of community-acquired infections due to extended-spectrum beta-lactamase-producing (ESBL) Escherichia coli has been increasingly recognized in recent years. No comprehensive data are available on the prevalence, risk factors, and genotypes of ESBL production in community residents in China. Rectal samples from 270 elderly people were collected in four communities in Shenyang (China). Colonies were screened by double-disk synergy test for ESBL production and then, ESBLs were characterized by PCR and sequencing. The clonal relatedness of all ESBL-producing isolates was determined by pulsed-field gel electrophoresis. Potential risk factors for rectal carriage of ESBL producers were examined by multivariate analysis. The prevalence of rectal carriage of ESBL-producing E. coli was 7.0%. All 19 ESBL-producing isolates produced CTX-M-type ESBLs, including CTX-M-14 (11 strains), CTX-M-22 (3 strains), CTX-M-79 (3 strains), CTX-M-24 (1 strain), and CTX-M-24 and CTX-M-79 together (1 strain). CTX-M-79 ESBL was first detected worldwide. ESBL-producing strains were clonally unrelated. Appearance of ESBL producers is strongly associated with the use of antibiotics in the past 3 months (odds ratio 3.2, 95% CI 1.1-9.0, P = 0.03). Our results show the importance of the intestinal tract as a reservoir for ESBL-producing isolates in community settings in China and that the use of antibiotics in the past 3 months is clearly linked to rectal carriage of ESBL producers.
|
1157. |
Walter EG,
Thomas CM,
Ibbotson JP,
Taylor DE,
( 1991 ) Transcriptional analysis, translational analysis, and sequence of the kilA-tellurite resistance region of plasmid RK2Ter. PMID : 1846856 : DOI : 10.1128/jb.173.3.1111-1119.1991 PMC : PMC207231 Abstract >>
The tellurite resistance (Ter) determinant of the IncP alpha plasmid RK2Ter, a variant of RK2 (also called RP4), is located between the kilA and korA genes involved in plasmid replication control. Transcriptional and translational fusions were constructed between the gene for beta-galactosidase and the kilA and Ter genes by using the transpositional phage mini-Mu. These fusions indicated that the Ter genes are transcribed in the same direction as kilA and that transcription and translation of the cloned kilA gene are occurring and may not be lethal to the bacterial cell even in the absence of korA. The nucleotide sequence of this region was determined, and three open reading frames (ORFs) were identified. The first ORF codes for KilA, a 28-kDa hydrophilic protein. The second ORF, telA, codes for a hydrophilic protein of 42 kDa. The third ORF, telB, codes for a hydrophobic protein of 32 kDa. This protein appears to be located in the inner membrane of the bacterial cell, since fusions of TelB to alkaline phosphatase were obtained by using TnphoA. All three proteins were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after overproduction using the T7 RNA polymerase/promoter system. The same three proteins were produced when Tes and Ter derivatives of RP4 were expressed in an in vitro transcription-translation system. A single Ser-to-Cys missense mutation in telB was found to be responsible for mutation of RK2 to Ter.
|
1158. |
Brockhausen I,
Hu B,
Liu B,
Lau K,
Szarek WA,
Wang L,
Feng L,
( 2008 ) Characterization of two beta-1,3-glucosyltransferases from Escherichia coli serotypes O56 and O152. PMID : 18487334 : DOI : 10.1128/JB.00160-08 PMC : PMC2446995 Abstract >>
The O antigens of outer membrane-bound lipopolysaccharides (LPS) in gram-negative bacteria are oligosaccharides consisting of repeating units with various structures and antigenicities. The O56 and O152 antigens of Escherichia coli both contain a Glc-beta1-3-GlcNAc linkage within the repeating unit. We have cloned and identified the genes (wfaP in O56 and wfgD in O152) within the two O-antigen gene clusters that encode glucosyltransferases involved in the synthesis of this linkage. A synthetic substrate analog of the natural acceptor substrate undecaprenol-pyrophosphate-lipid [GlcNAc-alpha-PO3-PO3-(CH2)11-O-phenyl] was used as an acceptor and UDP-Glc as a donor substrate to demonstrate that both wfgD and wfaP encode glucosyltransferases. Enzyme products from both glucosyltransferases were isolated by high-pressure liquid chromatography and analyzed by nuclear magnetic resonance. The spectra showed the expected Glc-beta1-3-GlcNAc linkage in the products, confirming that both WfaP and WfgD are forms of UDP-Glc: GlcNAc-pyrophosphate-lipid beta-1,3-glucosyltransferases. Both WfaP and WfgD have a DxD sequence, which is proposed to interact with phosphate groups of the nucleotide donor through the coordination of a metal cation, and a short hydrophobic sequence at the C terminus that may help to associate the enzymes with the inner membrane. We showed that the enzymes have similar properties and substrate recognition. They both require a divalent cation (Mn2+ or Mg2+) for activity, are deactivated by detergents, have a broad pH optimum, and require the pyrophosphate-sugar linkage in the acceptor substrate for full activity. Substrates lacking phosphate or pyrophosphate linked to GlcNAc were inactive. The length of the aliphatic chain of acceptor substrates also contributes to the activity.
|
1159. |
Strauch E,
Hammerl JA,
Konietzny A,
Schneiker-Bekel S,
Arnold W,
Goesmann A,
Pühler A,
Beutin L,
( 2008 ) Bacteriophage 2851 is a prototype phage for dissemination of the Shiga toxin variant gene 2c in Escherichia coli O157:H7. PMID : 18824528 : DOI : 10.1128/IAI.00875-08 PMC : PMC2583581 Abstract >>
The production of Shiga toxin (Stx) (verocytotoxin) is a major virulence factor of Escherichia coli O157:H7 strains (Shiga toxin-producing E. coli [STEC] O157). Two types of Shiga toxins, designated Stx1 and Stx2, are produced in STEC O157. Variants of the Stx2 type (Stx2, Stx2c) are associated with high virulences of these strains for humans. A bacteriophage designated 2851 from a human STEC O157 encoding the Stx2c variant was described previously. Nucleotide sequence analysis of the phage 2851 genome revealed 75 predicted coding sequences and indicated a mosaic structure typical for lambdoid phages. Analyses of free phages and K-12 phage 2851 lysogens revealed that upon excision from the bacterial chromosome, the loss of a phage-encoded IS629 element leads to fusion of phage antA and antB genes, with the generation of a recombined antAB gene encoding a strong antirepressor. In wild-type E. coli O157 as well as in K-12 strains, phage 2851 was found to be integrated in the sbcB locus. Additionally, phage 2851 carries an open reading frame which encodes an OspB-like type III effector similar to that found in Shigella spp. Investigation of 39 stx(2c) E. coli O157 strains revealed that all except 1 were positive for most phage 2851-specific genes and possessed a prophage with the same border sequences integrated into the sbcB locus. Phage 2851-specific sequences were absent from most stx(2c)-negative E. coli O157 strains, and we suggest that phage 2851-like phages contributed significantly to the dissemination of the Stx2c variant toxin within this group of E. coli.
|
1160. |
Périchon B,
Bogaerts P,
Lambert T,
Frangeul L,
Courvalin P,
Galimand M,
( 2008 ) Sequence of conjugative plasmid pIP1206 mediating resistance to aminoglycosides by 16S rRNA methylation and to hydrophilic fluoroquinolones by efflux. PMID : 18458128 : DOI : 10.1128/AAC.01540-07 PMC : PMC2443923 Abstract >>
Self-transferable IncFI plasmid pIP1206, isolated from an Escherichia coli clinical isolate, carries two new resistance determinants: qepA, which confers resistance to hydrophylic fluoroquinolones by efflux, and rmtB, which specifies a 16S rRNA methylase conferring high-level aminoglycoside resistance. Analysis of the 168,113-bp sequence (51% G+C) revealed that pIP1206 was composed of several subregions separated by copies of insertion sequences. Of 151 open reading frames, 56 (37%) were also present in pRSB107, isolated from a bacterium in a sewage treatment plant. pIP1206 contained four replication regions (RepFIA, RepFIB, and two partial RepFII regions) and a transfer region 91% identical with that of pAPEC-O1-ColBM, a plasmid isolated from an avian pathogenic E. coli. A putative oriT region was found upstream from the transfer region. The antibiotic resistance genes tet(A), catA1, bla(TEM-1), rmtB, and qepA were clustered in a 33.5-kb fragment delineated by two IS26 elements that also carried a class 1 integron, including the sulI, qacEDelta1, aad4, and dfrA17 genes and Tn10, Tn21, and Tn3-like transposons. The plasmid also possessed a raffinose operon, an arginine deiminase pathway, a putative iron acquisition gene cluster, an S-methylmethionine metabolism operon, two virulence-associated genes, and a type I DNA restriction-modification (R-M) system. Three toxin/antitoxin systems and the R-M system ensured stabilization of the plasmid in the host bacteria. These data suggest that the mosaic structure of pIP1206 could have resulted from recombination between pRSB107 and a pAPEC-O1-ColBM-like plasmid, combined with structural rearrangements associated with acquisition of additional DNA by recombination and of mobile genetic elements by transposition.
|
1161. |
Liu B,
Knirel YA,
Feng L,
Perepelov AV,
Senchenkova SN,
Wang Q,
Reeves PR,
Wang L,
( 2008 ) Structure and genetics of Shigella O antigens. PMID : 18422615 : DOI : 10.1111/j.1574-6976.2008.00114.x Abstract >>
This review covers the O antigens of the 46 serotypes of Shigella, but those of most Shigella flexneri are variants of one basic structure, leaving 34 Shigella distinct O antigens to review, together with their gene clusters. Several of the structures and gene clusters are reported for the first time and this is the first such group for which structures and DNA sequences have been determined for all O antigens. Shigella strains are in effect Escherichia coli with a specific mode of pathogenicity, and 18 of the 34 O antigens are also found in traditional E. coli. Three are very similar to E. coli O antigens and 13 are unique to Shigella strains. The O antigen of Shigella sonnei is quite atypical for E. coli and is thought to have transferred from Plesiomonas. The other 12 O antigens unique to Shigella strains have structures that are typical of E. coli, but there are considerably more anomalies in their gene clusters, probably reflecting recent modification of the structures. Having the complete set of structures and genes opens the way for experimental studies on the role of this diversity in pathogenicity.
|
1162. |
Vizán JL,
Hernández-Chico C,
del Castillo I,
Moreno F,
( 1991 ) The peptide antibiotic microcin B17 induces double-strand cleavage of DNA mediated by E. coli DNA gyrase. PMID : 1846808 : PMC : PMC452668 Abstract >>
Microcin B17 (MccB17) is a bactericidal peptide antibiotic which inhibits DNA replication. Two Escherichia coli MccB17 resistant mutants were isolated and the mutations were shown to map to 83 min of the genetic map. Cloning of the mutations and Tn5 insertional analysis demonstrated that they were located inside gyrB. The approximate location of the mutations within gyrB was determined by constructing hybrid genes, as a previous step to sequencing. Both mutations were shown to consist of a single AT----GC transition at position 2251 of the gene, which produces a Trp751----Arg substitution in the amino acid sequence of the GyrB polypeptide. The inhibitory effect of MccB17 on replicative cell-free extracts was assayed. In this in vitro system, interaction of MccB17 with a component of the extracts induced double-strand cleavage of plasmid DNA. In vivo treatment with MccB17 also induced a well-defined cleavage pattern on chromosomal DNA. These effects were not observed with a MccB17-resistant, gyrB mutant. Altogether, our results indicate that MccB17 blocks DNA gyrase by trapping an enzyme-DNA cleavable complex. Thus, the mode of action of this peptide antibiotic resembles that of quinolones and a variety of antitumour drugs currently used in cancer chemotherapy. MccB17 is the first peptide shown to inhibit a type II DNA topoisomerase.
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1163. |
Oteo J,
Delgado-Iribarren A,
Vega D,
Bautista V,
Rodríguez MC,
Velasco M,
Saavedra JM,
Pérez-Vázquez M,
García-Cobos S,
Martínez-Martínez L,
Campos J,
( 2008 ) Emergence of imipenem resistance in clinical Escherichia coli during therapy. PMID : 18775649 : DOI : 10.1016/j.ijantimicag.2008.06.012 Abstract >>
The molecular epidemiology and the mechanisms of resistance of Escherichia coli isolated from two patients infected by imipenem-resistant strains are reported in this study. From one patient, three closely related consecutive isolates of E. coli were recovered; the first was carbapenem-susceptible but acquired imipenem resistance after treatment with ertapenem, and the third isolate was again imipenem-susceptible. An additional imipenem-resistant isolate was recovered from another patient who received imipenem. The genetic relatedness of the E. coli isolates was determined by pulsed-field gel electrophoresis (PFGE) after digestion with XbaI. Standard polymerase chain reaction (PCR) conditions were used to amplify several beta-lactamase genes coding for carbapenemases, extended-spectrum beta-lactamases (ESBLs) and plasmid-mediated AmpC; the E. coli ampC gene promoter was also amplified and sequenced. Primers OmpF-F/OmpF-R and OmpC-F/OmpC-R were used to amplify the ompF and ompC genes. The outer membrane protein (OMP) profiles were studied by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Imipenem-resistant E. coli isolates did not produce carbapenemases but lacked the two major OMPs OmpF and OmpC and had ampC promoter mutations; in addition, one of the imipenem-resistant isolates produced the CMY-2 cephalosporinase, whilst the other produced the new CTX-M-67 ESBL. Carbapenem resistance in this study was associated with lack of expression of OmpF and OmpC porins. Additional mechanisms of beta-lactam resistance, such as plasmid-mediated AmpC and ESBL production, were also found. Development of carbapenem resistance in a CTX-M-67-producing E. coli is first described in this study.
|
1164. |
Feng L,
Liu B,
Liu Y,
Ratiner YA,
Hu B,
Li D,
Zong X,
Xiong W,
Wang L,
( 2008 ) A genomic islet mediates flagellar phase variation in Escherichia coli strains carrying the flagellin-specifying locus flk. PMID : 18441064 : DOI : 10.1128/JB.01937-07 PMC : PMC2446816 Abstract >>
The occurrence of unilateral flagellar phase variation was previously demonstrated in Escherichia coli strains carrying the non-fliC flagellin-specifying locus flk. In this study, we investigated the mechanism involved in this process. By using sequencing and sequence analysis, the flk region between the chromosomal genes yhaC and rnpB was characterized in all described flk-positive E. coli strains, including the H35 strain identified in this study (the other strains used are H3, H36, H47, and H53 strains), and this region was found to contain a putative integrase gene and flanking direct repeats in addition to the flk flagellin-specifying gene flkA and a fliC repressor gene, flkB, indicating that there is a typical genomic islet (GI), which was designated the flk GI. The horizontal transfer potential of the flk GI was indicated by detection of the excised extrachromosomal circular form of the flk GI. By generating fliC-expressing variants of H3 and H47 strains, unilateral flagellar phase variation in flk-positive strains was shown to be mediated by excision of the flk GI. The function of the proposed integrase gene was confirmed by deletion and a complementation test. The potential integration sites of the flk GI were identified. A general model for flagellar phase variation in flk-positive E. coli strains can be expressed as fliC(off) + flkA(on) --> fliC(on) + flkA(none). This is the first time that a molecular mechanism for flagellar phase variation has been reported for E. coli.
|
1165. |
Arbeloa A,
Bulgin RR,
MacKenzie G,
Shaw RK,
Pallen MJ,
Crepin VF,
Berger CN,
Frankel G,
( 2008 ) Subversion of actin dynamics by EspM effectors of attaching and effacing bacterial pathogens. PMID : 18331467 : DOI : 10.1111/j.1462-5822.2008.01136.x PMC : PMC2610399 Abstract >>
Rho GTPases are common targets of bacterial toxins and type III secretion system effectors. IpgB1 and IpgB2 of Shigella and Map of enteropathogenic (EPEC) and enterohemorrhagic (EHEC) Escherichia coli were recently grouped together on the basis that they share a conserved WxxxE motif. In this study, we characterized six WxxxE effectors from attaching and effacing pathogens: TrcA and EspM1 of EPEC strain B171, EspM1 and EspM2 of EHEC strain Sakai and EspM2 and EspM3 of Citrobacter rodentium. We show that EspM2 triggers formation of global parallel stress fibres, TrcA and EspM1 induce formation of localized parallel stress fibres and EspM3 triggers formation of localized radial stress fibres. Using EspM2 and EspM3 as model effectors, we report that while substituting the conserved Trp with Ala abolished activity, conservative Trp to Tyr or Glu to Asp substitutions did not affect stress-fibre formation. We show, using dominant negative constructs and chemical inhibitors, that the activity of EspM2 and EspM3 is RhoA and ROCK-dependent. Using Rhotekin pull-downs, we have shown that EspM2 and EspM3 activate RhoA; translocation of EspM2 and EspM3 triggered phosphorylation of cofilin. These results suggest that the EspM effectors modulate actin dynamics by activating the RhoA signalling pathway.
|
1166. |
Ren Y,
Liu B,
Cheng J,
Liu F,
Feng L,
Wang L,
( 2008 ) Characterization of Escherichia coli O3 and O21 O antigen gene clusters and development of serogroup-specific PCR assays. PMID : 18700154 : DOI : 10.1016/j.mimet.2008.07.010 Abstract >>
Escherichia coli O3 and O21 are associated with enteroaggregative E. coli (EAEC). EAEC strains are often non-typable using the routine agglutination method due to their aggregative phenotype. Typing of E. coli O3 and O21 may also be impeded by cross-reactions with O152 or O83. In this study, the O antigen gene clusters of E. coli O3 and O21 were characterized, and PCR assays based on O antigen specific genes wzx (encoding O unit flippase) and wzy (encoding O unit polymerase) from each strain were developed. By screening against all 186 known E. coli O serotypes, the PCR assays were shown to be highly specific to O3 and O21 respectively. The sensitivity of the assays was determined to be 1 pg per microl of chromosomal DNA and 2 CFU per 10 g of water samples. The PCR assays were also applied to 658 clinical E. coli isolates, and 100% of detection accuracy was obtained. The PCR assays developed here are suitable for the detection and identification of E. coli O3 and O21 strains in environmental and clinical samples.
|
1167. |
Ding H,
Yang Y,
Lu Q,
Wang Y,
Chen Y,
Deng L,
Wang A,
Deng Q,
Zhang H,
Wang C,
Liu L,
Xu X,
Wang L,
Shen X,
( 2008 ) The prevalence of plasmid-mediated AmpC beta-lactamases among clinical isolates of Escherichia coli and Klebsiella pneumoniae from five children's hospitals in China. PMID : 18449580 : DOI : 10.1007/s10096-008-0532-4 Abstract >>
The purpose of this study was to investigate the prevalence of plasmid-mediated AmpC beta-lactamases in Escherichia coli and Klebsiella pneumoniae from five children's hospitals in China. A total of 494 E. coli and 637 K. pneumoniae isolates were collected from five children's hospitals in China from 2005 to 2006. The isolates with decreased susceptibility to cefoxitin were subjected to confirmation test with 3-aminophenyl boronic acid. Polymerase chain reaction (PCR) amplification of the blaAmpC, blaTEM, blaCTXM, and blaSHV genes and their gene sequencing were performed. Transconjugants were achieved by conjugation experiments. Plasmid-mediated AmpC beta-lactamases were found in 10.1% of K. pneumoniae (64/637) and in 2.0% of E. coli (10/494) strains. The proportion of plasmid-mediated AmpC-producing strains significantly increased from 2005 (2.6%) to 2006 (9.3%) (p<0.001). The DHA-1-producing isolates were the most prevalent type (93.2%, 69/74). The sequences of blaDHA-1 genes were all identical to those from the GenBank. Strains of blaCMY-2 were isolated from five isolates (6.8%), which were all from E. coli. One sequence of blaCMY-2 differs from blaCMY-2 in the GenBank. Eighteen of the 74 (24.3%) AmpC-producing K. pneumoniae and E. coli isolates coproduced an extended-spectrum beta-lactamase (ESBL). Cefoxitin resistance was transferred to 15 of the 74 positive strains (20.3%). Our study has demonstrated the occurrence of plasmid-mediated AmpC beta-lactamases in E. coli and K. pneumoniae in Chinese pediatric patients and DHA-1 type AmpC enzymes had the highest prevalent rate. The CMY-2 AmpC beta-lactamases from the children's hospitals in China in this study are the first reported. Hence, continuous surveillance of the prevalence and evolution of AmpC beta-lactamase is important.
|
1168. |
Bae IK,
Lee YH,
Jeong HJ,
Hong SG,
Lee SH,
Jeong SH,
( 2008 ) A novel bla(CTX-M-14) gene-harboring complex class 1 integron with an In4-like backbone structure from a clinical isolate of Escherichia coli. PMID : 18703304 : DOI : 10.1016/j.diagmicrobio.2008.06.006 Abstract >>
Escherichia coli BDE0502 contained 3 beta-lactamase genes including bla(DHA-1), bla(SHV-12), and bla(CTX-M-14). The bla(CTX-M-14) gene was found on a complex class 1 integron with an In4-like backbone structure. The bla(CTX-M-14) gene was proceeded by a partial copy of ISEcp1 in addition to the complete copy of ISCR1 element.
|
1169. |
Wolfson JJ,
May KL,
Thorpe CM,
Jandhyala DM,
Paton JC,
Paton AW,
( 2008 ) Subtilase cytotoxin activates PERK, IRE1 and ATF6 endoplasmic reticulum stress-signalling pathways. PMID : 18433465 : DOI : 10.1111/j.1462-5822.2008.01164.x PMC : PMC2575110 Abstract >>
Subtilase cytotoxin (SubAB) is the prototype of a new family of AB(5) cytotoxins produced by Shiga toxigenic Escherichia coli. Its cytotoxic activity is due to its capacity to enter cells and specifically cleave the essential endoplasmic reticulum (ER) chaperone BiP (GRP78). In the present study, we have examined its capacity to trigger the three ER stress-signalling pathways in Vero cells. Activation of PKR-like ER kinase was demonstrated by phosphorylation of eIF2alpha, which occurred within 30 min of toxin treatment, and correlated with inhibition of global protein synthesis. Activation of inositol-requiring enzyme 1 was demonstrated by splicing of X-box-binding protein 1 mRNA, while activating transcription factor 6 activation was demonstrated by depletion of the 90 kDa uncleaved form, and appearance of the 50 kDa cleaved form. The rapidity with which ER stress-signalling responses are triggered by exposure of cells to SubAB is consistent with the hypothesis that cleavage by the toxin causes BiP to dissociate from the signalling molecules.
|
1170. |
Oloomi M,
Bouzari S,
( 2008 ) Molecular profile and genetic diversity of cytolethal distending toxin (CDT)-producing Escherichia coli isolates from diarrheal patients. PMID : 18321363 : DOI : 10.1111/j.1600-0463.2008.00910.x Abstract >>
Cytolethal distending toxin (CDT)-producing Escherichia coli strains are considered to be a heterogeneous group of E. coli. In the present investigation, 20 CDT-producing E. coli strains, which had already been shown to be cytotoxic necrotizing factor (cnf) gene positive, were selected by PCR. Since these strains proved to be CDT producers on CHO cells but were partially characterized by PCR, they were subjected to PCR analysis to amplify the complete coding region of cdt genes. Moreover, the genetic relatedness of these strains was examined by pulse field gel electrophoresis (PFGE). To check the extent of homogeneity of these strains at the chromosomal level, tRNA insertion site analysis was performed. The CDT-producing E. coli strains under investigation were shown to be heterogeneous and diverse in regard to their genetic analysis. This observed diversity could be an independent acquisition of virulence genes that might occur through horizontal gene transfer by mobile genetic elements. This conclusion is based on the fact that data shown by tRNA insertion site analysis revealed that there is no common pattern of insertion among these isolates although they do share a common trait of CDT production.
|
1171. |
Osawa T,
Sugiura N,
Shimada H,
Hirooka R,
Tsuji A,
Shirakawa T,
Fukuyama K,
Kimura M,
Kimata K,
Kakuta Y,
( 2009 ) Crystal structure of chondroitin polymerase from Escherichia coli K4. PMID : 18771653 : DOI : 10.1016/j.bbrc.2008.08.121 Abstract >>
Elongation of glycosaminoglycan chains, such as heparan and chondroitin, is catalyzed by bi-functional glycosyltransferases, for which both 3-dimensional structures and reaction mechanisms remain unknown. The bacterial chondroitin polymerase K4CP catalyzes elongation of the chondroitin chain by alternatively transferring the GlcUA and GalNAc moiety from UDP-GlcUA and UDP-GalNAc to the non-reducing ends of the chondroitin chain. Here, we have determined the crystal structure of K4CP in the presence of UDP and UDP-GalNAc as well as with UDP and UDP-GlcUA. The structures consisted of two GT-A fold domains in which the two active sites were 60A apart. UDP-GalNAc and UDP-GlcUA were found at the active sites of the N-terminal and C-terminal domains, respectively. The present K4CP structures have provided the structural basis for further investigating the molecular mechanism of biosynthesis of chondroitin chain.
|
1172. |
Korotkov KV,
Hol WG,
( 2008 ) Structure of the GspK-GspI-GspJ complex from the enterotoxigenic Escherichia coli type 2 secretion system. PMID : 18438417 : DOI : 10.1038/nsmb.1426 Abstract >>
Gram-negative bacteria translocate various proteins including virulence factors across their outer membrane via type 2 secretion systems (T2SSs). T2SSs are thought to contain a pseudopilus, a subcomplex formed by one major and several minor pseudopilins. We report the crystal structure of the complex formed by three minor pseudopilins from enterotoxigenic Escherichia coli. The GspK-GspI-GspJ complex has quasihelical characteristics and an architecture consistent with a localization at the pseudopilus tip. The alpha-domain of GspK has a previously unobserved fold with an unexpected dinuclear metal binding site. The area surrounding its disulfide bridge is conserved and might interact with other T2SS components or with secreted proteins.
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1173. |
Eberle W,
Pastore A,
Sander C,
Rösch P,
( 1991 ) The structure of ColE1 rop in solution. PMID : 1841691 : Abstract >>
The structure of the ColE1 repressor of primer (rop) protein in solution was determined from the proton nuclear magnetic resonance data by a combined use of distance geometry and restrained molecular dynamics calculations. A set of structures was determined with low internal energy and virtually no violations of the experimental distance restraints. Rop forms homodimers: Two helical hairpins are arranged as an antiparallel four helix bundle with a left-handed rope-like twist of the helix axes and with left-handed bundle topology. The very compact packing of the side chains in the helix interfaces of the rop coiled-coil structure may well account for its high stability. Overall, the solution structure is highly similar to the recently determined X-ray structure (Banner, D.W., Kokkinidis, M. and Tsernoglou, D. (1987) J. Mol. Biol., 196, 657-675), although there are minor differences in regions where packing forces appear to influence the crystal structure.
|
1174. |
Kawasaki H,
Koyama T,
Conlon JM,
Yamakura F,
Iwamuro S,
( N/A ) Antimicrobial action of histone H2B in Escherichia coli: evidence for membrane translocation and DNA-binding of a histone H2B fragment after proteolytic cleavage by outer membrane proteinase T. PMID : 18706965 : DOI : 10.1016/j.biochi.2008.07.003 Abstract >>
Previous studies have led to the isolation of histone H2B with antibacterial properties from an extract of the skin of the Schlegel's green tree frog Rhacophorus schlegelii and it is now demonstrated that the intact peptide is released into norepinephrine-stimulated skin secretions. In order to investigate the mechanism of action of this peptide, a maltose-binding protein (MBP)-fused histone H2B (MBP-H2B) conjugate was prepared and subjected to antimicrobial assay. The fusion protein showed bacteriostatic activity against Escherichia coli strain JCM5491 with a minimum inhibitory concentration of 11 microM. The lysate prepared from JCM5491 cells was capable of fragmenting MBP-H2B within the histone H2B region, but the lysate from the outer membrane proteinase T (OmpT) gene-deleted BL21(DE3) cells was not. FITC-labeled MBP-H2B (FITC-MBP-H2B) penetrated into the bacterial cell membrane of JCM5491 and ompT-transformed BL21(DE3) cells, but not into ompT-deleted BL21(DE3) cells. Gel retardation assay using MBP-H2B-deletion mutants indicated that MBP-H2B bound to DNA at a site within the N-terminal region of histone H2B. Consequently, it is proposed that the antimicrobial action of histone H2B involves, at least in part, penetration of an OmpT-produced N-terminal histone H2B fragment into the bacterial cell membrane with subsequent inhibition of cell functions.
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1175. |
Vetting MW,
Park CH,
Hegde SS,
Jacoby GA,
Hooper DC,
Blanchard JS,
( 2008 ) Mechanistic and structural analysis of aminoglycoside N-acetyltransferase AAC(6')-Ib and its bifunctional, fluoroquinolone-active AAC(6')-Ib-cr variant. PMID : 18710261 : DOI : 10.1021/bi800664x PMC : PMC2855648 Abstract >>
Enzymatic modification of aminoglycoside antibiotics mediated by regioselective aminoglycoside N-acetyltransferases is the predominant cause of bacterial resistance to aminoglycosides. A recently discovered bifunctional aminoglycoside acetyltransferase (AAC(6')-Ib variant, AAC(6')-Ib-cr) has been shown to catalyze the acetylation of fluoroquinolones as well as aminoglycosides. We have expressed and purified AAC(6')-Ib-wt and its bifunctional variant AAC(6')-Ib-cr in Escherichia coli and characterized their kinetic and chemical mechanism. Initial velocity and dead-end inhibition studies support an ordered sequential mechanism for the enzyme(s). The three-dimensional structure of AAC(6')-Ib-wt was determined in various complexes with donor and acceptor ligands to resolutions greater than 2.2 A. Observation of the direct, and optimally positioned, interaction between the 6'-NH 2 and Asp115 suggests that Asp115 acts as a general base to accept a proton in the reaction. The structure of AAC(6')-Ib-wt permits the construction of a molecular model of the interactions of fluoroquinolones with the AAC(6')-Ib-cr variant. The model suggests that a major contribution to the fluoroquinolone acetylation activity comes from the Asp179Tyr mutation, where Tyr179 makes pi-stacking interactions with the quinolone ring facilitating quinolone binding. The model also suggests that fluoroquinolones and aminoglycosides have different binding modes. On the basis of kinetic properties, the pH dependence of the kinetic parameters, and structural information, we propose an acid/base-assisted reaction catalyzed by AAC(6')-Ib-wt and the AAC(6')-Ib-cr variant involving a ternary complex.
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1176. |
García-Fernández A,
Chiaretto G,
Bertini A,
Villa L,
Fortini D,
Ricci A,
Carattoli A,
( 2008 ) Multilocus sequence typing of IncI1 plasmids carrying extended-spectrum beta-lactamases in Escherichia coli and Salmonella of human and animal origin. PMID : 18367460 : DOI : 10.1093/jac/dkn131 Abstract >>
Plasmids belonging to incompatibility group I1 (IncI1) are widespread in Enterobacteriaceae and are characterized by the presence of a cluster of genes encoding the type IV pili, contributing to the virulence of Shiga-toxigenic Escherichia coli. Recently, IncI1 plasmids were identified in E. coli and Salmonella strains of animal origin as responsible for the dissemination of beta-lactamase genes. Plasmid multilocus sequence typing (pMLST) was developed to discern naturally occurring IncI1 plasmids in homogeneous groups according to their allele assortment. pMLST was developed by selecting multiple target genes on the available complete IncI1 plasmid DNA sequences. Sixteen plasmids, all assigned to the IncI1 group by the PCR-based replicon typing method, were included in this study. They were analysed for beta-lactamase genes and typed by restriction fragment length polymorphism (RFLP) and pMLST. Sixteen plasmids identified in E. coli and Salmonella isolated from animals and humans in different countries carried bla(CMY-2), bla(CTX-M-15), bla(CTX-M-1), bla(CTX-M-14), bla(TEM-52), bla(SHV-12) or bla(TEM-1) beta-lactamase genes. These plasmids were classified by RFLP in nine different groups corresponding to the nine sequence types determined by pMLST. The pMLST method was suitable for rapid and easy subtyping of IncI1 plasmids. This study demonstrates that the pMLST method can contribute to the epidemiological description of circulation of specific resistance plasmids among beta-lactamase producers isolated from animals and humans.
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1177. |
Adams-Haduch JM,
Paterson DL,
Doi Y,
( 2008 ) Escherichia coli isolate coproducing 16S rRNA Methylase and CTX-M-type extended-spectrum beta-lactamase isolated from an outpatient in the United States. PMID : 18195064 : DOI : 10.1128/AAC.01320-07 PMC : PMC2258542 Abstract >>
N/A
|
1178. |
Wu J,
Rosen BP,
( 1991 ) The ArsR protein is a trans-acting regulatory protein. PMID : 1838573 : DOI : 10.1111/j.1365-2958.1991.tb00779.x Abstract >>
The arsR gene encodes the regulatory protein of the plasmid-encoded arsenical resistance operon. A series of in-frame fusions was constructed between the C-terminally truncated arsR gene and the coding region for the mature form of beta-lactamase (blaM). Fusions containing most of the arsR gene were still inducible by arsenicals. Fusions containing less than 102 residues of the 117-residue ArsR protein were constitutive. When a wild-type arsR gene was placed in trans, the constitutive constructs were again inducible. The results demonstrate that the ArsR protein is a trans-acting regulatory protein which controls its own expression.
|
1179. |
de Boer PA,
Crossley RE,
Hand AR,
Rothfield LI,
( 1991 ) The MinD protein is a membrane ATPase required for the correct placement of the Escherichia coli division site. PMID : 1836760 : PMC : PMC453190 Abstract >>
The proper placement of the cell division site in Escherichia coli requires the site-specific inactivation of potential division sites at the cell poles in a process that is mediated by the MinC, MinD and MinE proteins. During the normal division cycle MinD plays two roles. It activates the MinC-dependent mechanism that is responsible for the inactivation of potential division sites and it also renders the division inhibition system sensitive to the topological specificity factor MinE. MinE suppresses the division block at the normal division site at mid-cell but not all cell poles, thereby ensuring the normal division pattern. In this study the MinD protein was purified to homogeneity and shown to bind ATP and to have ATPase activity. When the putative ATP binding domain of MinD was altered by site-directed mutagenesis, the mutant protein was no longer able to activate the MinC-dependent division inhibition system. Immunoelectron microscopy showed that MinD was located in the inner membrane region of the cell envelope. These results show that MinD is a membrane ATPase and suggest that the ATPase activity plays an essential role in the functions of the MinD protein during the normal division process.
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1180. |
Reznikoff WS,
( 2008 ) Transposon Tn5. PMID : 18680433 : DOI : 10.1146/annurev.genet.42.110807.091656 Abstract >>
Tn5 was one of the first transposons to be identified (10). As a result of Tn5's early discovery and its simple macromolecular requirements for transposition, the Tn5 system has been a very productive tool for studying the molecular mechanism of DNA transposition. These studies are of broad value because they offer insights into DNA transposition in general, because DNA transposition is a useful model with which to understand other types of protein-DNA interactions such as retroviral DNA integration and the DNA cleavage events involved in immunoglobulin gene formation, and because Tn5-derived tools are useful adjuncts in genetic experimentation.
|
1181. |
Sunde M,
Solheim H,
Slettemeås JS,
( 2008 ) Genetic linkage between class 1 integrons with the dfrA12-orfF-aadA2 cassette array and sul3 in Escherichia coli. PMID : 18367349 : DOI : 10.1016/j.vetmic.2008.02.001 Abstract >>
N/A
|
1182. |
Mellmann A,
Bielaszewska M,
Köck R,
Friedrich AW,
Fruth A,
Middendorf B,
Harmsen D,
Schmidt MA,
Karch H,
( 2008 ) Analysis of collection of hemolytic uremic syndrome-associated enterohemorrhagic Escherichia coli. PMID : 18680658 : DOI : 10.3201/eid1408.071082 PMC : PMC2600372 Abstract >>
Multilocus sequence typing of 169 non-O157 enterohemorrhagic Escherichia coli (EHEC) isolated from patients with hemolytic uremic syndrome (HUS) demonstrated 29 different sequence types (STs); 78.1% of these strains clustered in 5 STs. From all STs and serotypes identified, we established a reference panel of EHEC associated with HUS (HUSEC collection).
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1183. |
Ong CL,
Ulett GC,
Mabbett AN,
Beatson SA,
Webb RI,
Monaghan W,
Nimmo GR,
Looke DF,
McEwan AG,
Schembri MA,
( 2008 ) Identification of type 3 fimbriae in uropathogenic Escherichia coli reveals a role in biofilm formation. PMID : 18055599 : DOI : 10.1128/JB.01523-07 PMC : PMC2223576 Abstract >>
Catheter-associated urinary tract infection (CAUTI) is the most common nosocomial infection in the United States. Uropathogenic Escherichia coli (UPEC), the most common cause of CAUTI, can form biofilms on indwelling catheters. Here, we identify and characterize novel factors that affect biofilm formation by UPEC strains that cause CAUTI. Sixty-five CAUTI UPEC isolates were characterized for phenotypic markers of urovirulence, including agglutination and biofilm formation. One isolate, E. coli MS2027, was uniquely proficient at biofilm growth despite the absence of adhesins known to promote this phenotype. Mini-Tn5 mutagenesis of E. coli MS2027 identified several mutants with altered biofilm growth. Mutants containing insertions in genes involved in O antigen synthesis (rmlC and manB) and capsule synthesis (kpsM) possessed enhanced biofilm phenotypes. Three independent mutants deficient in biofilm growth contained an insertion in a gene locus homologous to the type 3 chaperone-usher class fimbrial genes of Klebsiella pneumoniae. These type 3 fimbrial genes (mrkABCDF), which were located on a conjugative plasmid, were cloned from E. coli MS2027 and could complement the biofilm-deficient transconjugants when reintroduced on a plasmid. Primers targeting the mrkB chaperone-encoding gene revealed its presence in CAUTI strains of Citrobacter koseri, Citrobacter freundii, Klebsiella pneumoniae, and Klebsiella oxytoca. All of these mrkB-positive strains caused type 3 fimbria-specific agglutination of tannic acid-treated red blood cells. This is the first description of type 3 fimbriae in E. coli, C. koseri, and C. freundii. Our data suggest that type 3 fimbriae may contribute to biofilm formation by different gram-negative nosocomial pathogens.
|
1184. |
Krahn JM,
Jackson MR,
DeRose EF,
Howell EE,
London RE,
( 2007 ) Crystal structure of a type II dihydrofolate reductase catalytic ternary complex. PMID : 18052202 : DOI : 10.1021/bi701532r PMC : PMC3743094 Abstract >>
Type II dihydrofolate reductase (DHFR) is a plasmid-encoded enzyme that confers resistance to bacterial DHFR-targeted antifolate drugs. It forms a symmetric homotetramer with a central pore which functions as the active site. Its unusual structure, which results in a promiscuous binding surface that accommodates either the dihydrofolate (DHF) substrate or the NADPH cofactor, has constituted a significant limitation to efforts to understand its substrate specificity and reaction mechanism. We describe here the first structure of a ternary R67 DHFR.DHF.NADP+ catalytic complex, resolved to 1.26 A. This structure provides the first clear picture of how this enzyme, which lacks the active site carboxyl residue that is ubiquitous in Type I DHFRs, is able to function. In the catalytic complex, the polar backbone atoms of two symmetry-related I68 residues provide recognition motifs that interact with the carboxamide on the nicotinamide ring, and the N3-O4 amide function on the pteridine ring. This set of interactions orients the aromatic rings of substrate and cofactor in a relative endo geometry in which the reactive centers are held in close proximity. Additionally, a central, hydrogen-bonded network consisting of two pairs of Y69-Q67-Q67'-Y69' residues provides an unusually tight interface, which appears to serve as a "molecular clamp" holding the substrates in place in an orientation conducive to hydride transfer. In addition to providing the first clear insight regarding how this extremely unusual enzyme is able to function, the structure of the ternary complex provides general insights into how a mutationally challenged enzyme, i.e., an enzyme whose evolution is restricted to four-residues-at-a-time active site mutations, overcomes this fundamental limitation.
|
1185. |
Lasaro MA,
Rodrigues JF,
Mathias-Santos C,
Guth BE,
Balan A,
Sbrogio-Almeida ME,
Ferreira LC,
( 2008 ) Genetic diversity of heat-labile toxin expressed by enterotoxigenic Escherichia coli strains isolated from humans. PMID : 18223074 : DOI : 10.1128/JB.00988-07 PMC : PMC2293181 Abstract >>
The natural diversity of the elt operons, encoding the heat-labile toxin LT-I (LT), carried by enterotoxigenic Escherichia coli (ETEC) strains isolated from humans was investigated. For many years, LT was supposed to be represented by a rather conserved toxin, and one derivative, produced by the reference H10407 strain, was intensively studied either as a virulence factor or as a vaccine adjuvant. Amplicons encompassing the two LT-encoding genes (eltA and eltB) of 51 human-derived ETEC strains, either LT(+) (25 strains) only or LT(+)/ST(+) (26 strains), isolated from asymptomatic (24 strains) or diarrheic (27 strains) subjects, were subjected to restriction fragment length polymorphism (RFLP) analysis and DNA sequencing. Seven polymorphic RFLP types of the H10407 strain were detected with six (BsaI, DdeI, HhaI, HincII, HphI, and MspI) restriction enzymes. Additionally, the single-nucleotide polymorphic analysis revealed 50 base changes in the elt operon, including 21 polymorphic sites at eltA and 9 at eltB. Based on the deduced amino acid sequences, 16 LT types were identified, including LT1, expressed by the H10407 strain and 23 other strains belonging to seven different serotypes, and LT2, expressed by 11 strains of six different serotypes. In vitro experiments carried out with purified toxins indicated that no significant differences in GM1-binding affinity could be detected among LT1, LT2, and LT4. However, LT4, but not other toxin types, showed reduced toxic activities measured either in vitro with cultured cells (Y-1 cells) or in vivo in rabbit ligated ileal loops. Collectively, these results indicate that the natural diversity of LTs produced by wild-type ETEC strains isolated from human hosts is considerably larger than previously assumed and may impact the pathogeneses of the strains and the epidemiology of the disease.
|
1186. |
Kazakov T,
Vondenhoff GH,
Datsenko KA,
Novikova M,
Metlitskaya A,
Wanner BL,
Severinov K,
( 2008 ) Escherichia coli peptidase A, B, or N can process translation inhibitor microcin C. PMID : 18223070 : DOI : 10.1128/JB.01956-07 PMC : PMC2293190 Abstract >>
The heptapeptide-nucleotide microcin C (McC) targets aspartyl-tRNA synthetase. Upon its entry into a susceptible cell, McC is processed to release a nonhydrolyzable aspartyl-adenylate that inhibits aspartyl-tRNA synthetase, leading to the cessation of translation and cell growth. Here, we surveyed Escherichia coli cells with singly, doubly, and triply disrupted broad-specificity peptidase genes to show that any of three nonspecific oligopeptidases (PepA, PepB, or PepN) can effectively process McC. We also show that the rate-limiting step of McC processing in vitro is deformylation of the first methionine residue of McC.
|
1187. |
Liu YF,
Yan JJ,
Ko WC,
Tsai SH,
Wu JJ,
( 2008 ) Characterization of carbapenem-non-susceptible Escherichia coli isolates from a university hospital in Taiwan. PMID : 18292097 : DOI : 10.1093/jac/dkn049 Abstract >>
To investigate characteristics of nine carbapenem-non-susceptible (CP-NS) Escherichia coli isolates collected between 1999 and 2005 at a Taiwanese university hospital. Genetic relatedness was analysed by PFGE. beta-Lactamases were characterized by PCR and isoelectric focusing. Outer membrane proteins and transcripts were investigated by SDS-PAGE and northern blotting. Cloning experiments were performed to investigate the role of membrane permeability in carbapenem non-susceptibility. The nine CP-NS isolates were found to produce the CMY-2 AmpC enzyme (n = 8), the CTX-M-14-type extended-spectrum beta-lactamase (ESBL) (n = 1), the SHV-12 ESBL (n = 1) and the IMP-8-type metallo-beta-lactamase (n = 1) alone or in combination. All CP-NS isolates revealed a decrease in the transcription and protein expression of ompC, and susceptibility to carbapenems was restored in one isolate by introducing the cloned ompC gene. PFGE revealed genetic diversity among the nine isolates. All patients with the CP-NS isolates had been treated with carbapenems (six patients) and/or extended-spectrum cephalosporins (five patients) before isolation. Our study suggests that the decreased susceptibility to carbapenems in E. coli in the hospital might arise by the stepwise accumulations of multiple drug-resistance determinants in different clones.
|
1188. |
García-Villegas MR,
De La Vega FM,
Galindo JM,
Segura M,
Buckingham RH,
Guarneros G,
( 1991 ) Peptidyl-tRNA hydrolase is involved in lambda inhibition of host protein synthesis. PMID : 1833189 : PMC : PMC453084 Abstract >>
Escherichia coli rap mutants do not support vegetative growth of bacteriophage lambda and die upon transcription of lambda DNA bar sites. Bacteria harbouring a pth(ts) mutation synthesize thermosensitive peptidyl-tRNA hydrolase (Pth) and die at 42 degrees C from a defect in protein synthesis. We present evidence that both rap and pth(ts) mutations affect the same gene: (i) peptidyl-tRNA hydrolase activity was found to be defective in rap mutants; (ii) at a threshold temperature, pth cells, like rap mutants, prevented lambda growth and were killed by transcription of cloned bar sites; (iii) sequencing a 1600 bp DNA fragment comprising both loci revealed an ORF located within the limits set by a complementation analysis and encoding a putative polypeptide of 21 kDa; (iv) cloning and sequencing of rap and pth(ts) mutant DNAs both revealed single nucleotide transitions from the wild type ORF sequence, resulting in Arg134 to His and Gly101 to Asp changes respectively. Analysis of plasmid-directed proteins identified a polypeptide of approximately 21 kDa; the N-terminal sequence, amino acid composition and isoelectric point of this protein match those expected from the ORF nucleotide sequence. We propose that Pth activity, directly or indirectly, is the target for lambda bar RNA leading to rap cell death.
|
1189. |
Pradel N,
Bertin Y,
Martin C,
Livrelli V,
( 2008 ) Molecular analysis of shiga toxin-producing Escherichia coli strains isolated from hemolytic-uremic syndrome patients and dairy samples in France. PMID : 18245246 : DOI : 10.1128/AEM.02688-07 PMC : PMC2292610 Abstract >>
Shiga toxin-producing Escherichia coli (STEC) has been associated with food-borne diseases ranging from uncomplicated diarrhea to hemolytic-uremic syndrome (HUS). While most outbreaks are associated with E. coli O157:H7, about half of the sporadic cases may be due to non-O157:H7 serotypes. To assess the pathogenicity of STEC isolated from dairy foods in France, 40 strains isolated from 1,130 raw-milk and cheese samples were compared with 15 STEC strains isolated from patients suffering from severe disease. The presence of genes encoding Shiga toxins (stx(1), stx(2), and variants), intimin (eae and variants), adhesins (bfp, efa1), enterohemolysin (ehxA), serine protease (espP), and catalase-peroxidase (katP) was determined by PCR and/or hybridization. Plasmid profiling, ribotyping, and pulsed-field gel electrophoresis (PFGE) were used to further compare the strains at the molecular level. A new stx(2) variant, stx(2-CH013), associated with an O91:H10 clinical isolate was identified. The presence of the stx(2), eae, and katP genes, together with a combination of several stx(2) variants, was clearly associated with human-pathogenic strains. In contrast, dairy food STEC strains were characterized by a predominance of stx(1), with a minority of isolates harboring eae, espP, and/or katP. These associations may help to differentiate less virulent STEC strains from those more likely to cause disease in humans. Only one dairy O5 isolate had a virulence gene panel identical to that of an HUS-associated strain. However, the ribotype and PFGE profiles were not identical. In conclusion, most STEC strains isolated from dairy products in France showed characteristics different from those of strains isolated from patients.
|
1190. |
Miele L,
Strack B,
Kruft V,
Lanka E,
( 1991 ) Gene organization and nucleotide sequence of the primase region of IncP plasmids RP4 and R751. PMID : 1818755 : Abstract >>
The primase genes of RP4 are part of the primase operon located within the Tra1 region of this conjugative plasmid. The operon contains a total of seven transfer genes four of which (traA, B, C, D) are described here. Determination of the nucleotide sequence of the primase region confirmed the existence of an overlapping gene arrangement at the DNA primase locus (traC) with in-phase translational initiation signals. The traC gene encodes two acidic and hydrophilic polypeptide chains of 1061 (TraC1) and 746 (TraC2) amino acids corresponding to molecular masses of 116,721 and 81,647 Da. In contrast to RP4 the IncP beta plasmid R751 specifies four large primase gene products (192, 152, 135 and 83 kDa) crossreacting with anti-RP4 DNA primase serum. As shown by deletion analysis at least the 135 and 83 kDa polypeptides are two separate translational products that by analogy with the RP4 primases, arise from in-phase translational initiation sites. Even the smallest primase gene products TraC2 (RP4) and TraC4 (R751) exhibit primase activity. Nucleotide sequencing of the R751 primase region revealed the existence of three in-phase traC translational initiation signals leading to the expression of gene products with molecular masses of 158,950 Da, 134,476 Da, and 80,759 Da. The 192 kDa primase polypeptide is suggested to be a fusion protein resulting from an in frame translational readthrough of the traD UGA stopcodon. Distinct sequence similarities can be detected between the TraC proteins of RP4 and R751 gene products TraC3 and TraC4 and in addition between the TraD proteins of both plasmids. The R751 traC3 gene contains a stretch of 507 bp which is unrelated to RP4 traC or any other RP4 Tra1 gene.
|
1191. |
Revilla C,
Garcillán-Barcia MP,
Fernández-López R,
Thomson NR,
Sanders M,
Cheung M,
Thomas CM,
de la Cruz F,
( 2008 ) Different pathways to acquiring resistance genes illustrated by the recent evolution of IncW plasmids. PMID : 18268088 : DOI : 10.1128/AAC.00982-07 PMC : PMC2292564 Abstract >>
DNA sequence analysis of five IncW plasmids (R388, pSa, R7K, pIE321, and pIE522) demonstrated that they share a considerable portion of their genomes and allowed us to define the IncW backbone. Among these plasmids, the backbone is stable and seems to have diverged recently, since the overall identity among its members is higher than 95%. The only gene in which significant variation was observed was trwA; the changes in the coding sequence correlated with parallel changes in the corresponding TrwA binding sites at oriT, suggesting a functional connection between both sets of changes. The present IncW plasmid diversity is shaped by the acquisition of antibiotic resistance genes as a consequence of the pressure exerted by antibiotic usage. Sequence comparisons pinpointed the insertion events that differentiated the five plasmids analyzed. Of greatest interest is that a single acquisition of a class I integron platform, into which different gene cassettes were later incorporated, gave rise to plasmids R388, pIE522, and pSa, while plasmids R7K and pIE321 do not contain the integron platform and arose in the antibiotic world because of the insertion of several antibiotic resistance transposons.
|
1192. |
Mocktar C,
Govinden U,
Sturm AW,
Essack SY,
( 2008 ) CMY-20, a novel AmpC-type beta-lactamase from South African clinical Escherichia coli isolates. PMID : 18207348 : DOI : 10.1016/j.diagmicrobio.2007.11.009 Abstract >>
In this study, we report the presence of a novel plasmid-mediated AmpC-type beta-lactamase that was isolated from 3 clinical Escherichia coli isolates at a tertiary teaching hospital in Durban, South Africa. The nucleotide sequence of the genes encoding this novel beta-lactamase was found to be >94% identical to the nucleotide sequences of the plasmid-mediated AmpC-type beta-lactamases originating from Citrobacter freundii. This enzyme differed from CMY-2 by 3 amino acid substitutions and was designated CMY-20.
|
1193. |
Cuthbertson L,
Kimber MS,
Whitfield C,
( 2007 ) Substrate binding by a bacterial ABC transporter involved in polysaccharide export. PMID : 18032609 : DOI : 10.1073/pnas.0705709104 PMC : PMC2148323 Abstract >>
ATP-binding-cassette (ABC) transporters are responsible for the export of a wide variety of cell-surface glycoconjugates in both Gram-positive and Gram-negative bacteria. These include the O-antigenic polysaccharide (O-PS) portion of lipopolysaccharide, a crucial virulence determinant in Gram-negative pathogens. O-PSs are synthesized by one of two fundamentally different pathways. Escherichia coli O serotypes O8 and O9a provide the prototype systems for studying O-PS export via ABC transporters. The transporter is composed of the transmembrane component Wzm and the nucleotide-binding component Wzt. Although the N-terminal domain of Wzt is a conventional ABC protein, the C-terminal domain of Wzt (C-Wzt) is a unique structural element that determines the specificity of the transporter for either the O8 or O9a O-PS. We show here that the two domains of Wzt can function when expressed as separate polypeptides; both are essential for export. In vitro, C-Wzt binds its cognate O-PS by recognizing a residue located at the nonreducing end of the polymer. The crystal structure of C-Wzt(O9a) is reported here and reveals a beta sandwich with an immunoglobulin-like topology that contains the O-PS-binding pocket. Substrate interactions with nucleotide-binding domains have been demonstrated in an ABC exporter previously. However, to our knowledge substrate binding by a discrete, cytoplasmic accessory domain in an extended nucleotide-binding domain polypeptide has not previously been demonstrated. Elucidation of the substrate-recognition system involved in O-PS export provides insight into the mechanism that coordinates polymer biosynthesis, termination, and export.
|
1194. |
Bauer AP,
Ludwig W,
Schleifer KH,
( 2008 ) A novel DNA microarray design for accurate and straightforward identification of Escherichia coli safety and laboratory strains. PMID : 18262744 : DOI : 10.1016/j.syapm.2008.01.001 Abstract >>
Escherichia coli K-12, B, C and W strains and their derivates are declared in biological safety guidelines as risk group 1 organisms as they are unable to colonise the human gut. Differentiation and identification of these safety strains is mainly based on pulsed-field gel electrophoresis (PFGE), phage sensitivity tests or PCR-based methods. However, these methods are either tedious and time consuming (phage sensitivity, PFGE) or based on single specific fragments (PCR) or patterns (PFGE) lacking additional information for further differentiation of the strains. In the current study, subtractive hybridisation techniques were applied to detect specific DNA fragments which were used to design a microarray (chip) for accurate and simple identification of these organisms, and to differentiate them from other E. coli strains. The chip can be used to identify E. coli safety strains and monitor them during ongoing experiments for changes in their genome and culture purity. The hybridisation layout of the microarray was arranged in such a way that the respective lineages of safety strains could be easily identified as distinct letters (K, B, C or W). Differentiation of single strains or subtyping was possible with further probes. In addition, a set of probes targeting genes coding for common virulence factors has been included, both to differentiate safety strains from pathogenic variants and to make sure that no transfer of these genes happens during handling or storage. The reliability of the approach has been tested on a comprehensive selection of E. coli laboratory strains and pathogenic representatives.
|
1195. |
Obarska-Kosinska A,
Taylor JE,
Callow P,
Orlowski J,
Bujnicki JM,
Kneale GG,
( 2008 ) HsdR subunit of the type I restriction-modification enzyme EcoR124I: biophysical characterisation and structural modelling. PMID : 18164032 : DOI : 10.1016/j.jmb.2007.11.024 PMC : PMC2878639 Abstract >>
Type I restriction-modification (RM) systems are large, multifunctional enzymes composed of three different subunits. HsdS and HsdM form a complex in which HsdS recognizes the target DNA sequence, and HsdM carries out methylation of adenosine residues. The HsdR subunit, when associated with the HsdS-HsdM complex, translocates DNA in an ATP-dependent process and cleaves unmethylated DNA at a distance of several thousand base-pairs from the recognition site. The molecular mechanism by which these enzymes translocate the DNA is not fully understood, in part because of the absence of crystal structures. To date, crystal structures have been determined for the individual HsdS and HsdM subunits and models have been built for the HsdM-HsdS complex with the DNA. However, no structure is available for the HsdR subunit. In this work, the gene coding for the HsdR subunit of EcoR124I was re-sequenced, which showed that there was an error in the published sequence. This changed the position of the stop codon and altered the last 17 amino acid residues of the protein sequence. An improved purification procedure was developed to enable HsdR to be purified efficiently for biophysical and structural analysis. Analytical ultracentrifugation shows that HsdR is monomeric in solution, and the frictional ratio of 1.21 indicates that the subunit is globular and fairly compact. Small angle neutron-scattering of the HsdR subunit indicates a radius of gyration of 3.4 nm and a maximum dimension of 10 nm. We constructed a model of the HsdR using protein fold-recognition and homology modelling to model individual domains, and small-angle neutron scattering data as restraints to combine them into a single molecule. The model reveals an ellipsoidal shape of the enzymatic core comprising the N-terminal and central domains, and suggests conformational heterogeneity of the C-terminal region implicated in binding of HsdR to the HsdS-HsdM complex.
|
1196. |
Van Camp BM,
Crow RR,
Peng Y,
Varela MF,
( 2007 ) Amino acids that confer transport of raffinose and maltose sugars in the raffinose permease (RafB) of Escherichia coli as implicated by spontaneous mutations at Val-35, Ser-138, Ser-139, Gly-389 and Ile-391. PMID : 18008022 : DOI : 10.1007/s00232-007-9077-1 PMC : PMC2440673 Abstract >>
In order to identify amino acid residues in the Escherichia coli raffinose-H(+) permease (RafB) that play a role in sugar selection and transport, we first incubated E. coli HS4006 containing plasmid pRU600 (expresses inducible raffinose permease and alpha-galactosidase) on maltose MacConkey indicator plates overnight. Initially, all colonies were white, indicating no fermentation of maltose. Upon further incubation, 100 mutants appeared red. pRU600 DNA was prepared from 55 mutants. Five mutants transferred the phenotype for fermentation of maltose (red). Plasmid DNA from five maltose-positive phenotype transformants was prepared and sequenced, revealing three distinct types of mutations. Two mutants exhibited Val-35-->Ala (MT1); one mutant had Ile-391-->Ser (MT2); and two mutants had Ser-138-->Asp, Ser-139-->Leu and Gly-389-->Ala (MT3). Transport studies of [(3)H]-maltose showed that cells harboring MT1, MT2 and MT3 had greater uptake (P
|
1197. |
Zdziarski J,
Svanborg C,
Wullt B,
Hacker J,
Dobrindt U,
( 2008 ) Molecular basis of commensalism in the urinary tract: low virulence or virulence attenuation? PMID : 18039831 : DOI : 10.1128/IAI.01215-07 PMC : PMC2223460 Abstract >>
In some patients, Escherichia coli strains establish significant bacteriuria without causing symptoms of urinary tract infection (UTI). These asymptomatic-bacteriuria (ABU) strains have been shown to express fewer virulence factors than the uropathogenic E. coli (UPEC) strains that cause severe, symptomatic UTI. Paradoxically, ABU strains carry many typical UPEC virulence genes, and the molecular basis of their low virulence therefore remains unclear. This study examined whether ABU strains might evolve from UPEC by genome loss and virulence gene attenuation. The presence of conserved E. coli K-12 genes was examined using an E. coli K-12 strain MG1655-specific DNA array and the distribution of UPEC virulence-related genes was examined with the E. coli pathoarray. Two groups of strains could be distinguished. Several ABU strains were shown by multilocus sequence typing and by comparative genomic analyses to be related to UPEC but to have smaller genome sizes. There were significant alterations in essential virulence genes, including reductive evolution by point mutations, DNA rearrangements, and deletions. Other strains were unrelated to UPEC and lacked most of the virulence-associated genes. The results suggest that some ABU strains arise from virulent strains by attenuation of virulence genes while others are nonvirulent and resemble commensal strains. We propose that virulence attenuation might constitute a general mechanism for mucosal pathogens to evolve toward commensalism.
|
1198. |
Lindberg S,
Xia Y,
Sondén B,
Göransson M,
Hacker J,
Uhlin BE,
( 2008 ) Regulatory Interactions among adhesin gene systems of uropathogenic Escherichia coli. PMID : 18039830 : DOI : 10.1128/IAI.01010-07 PMC : PMC2223471 Abstract >>
Uropathogenic Escherichia coli strain J96 carries multiple determinants for fimbrial adhesins. The regulatory protein PapB of P fimbriae has previously been implicated in potential coregulatory events. The focB gene of the F1C fimbria determinant is highly homologous to papB; the translated sequences share 81% identity. In this study we investigated the role of PapB and FocB in regulation of the F1C fimbriae. By using gel mobility shift assays, we showed that FocB binds to sequences in both the pap and foc operons in a somewhat different manner than PapB. The results of both in vitro cross-linking and in vivo oligomerization tests indicated that FocB could function in an oligomeric fashion. Furthermore, our results suggest that PapB and FocB can form heterodimers and that these complexes can repress expression of the foc operon. The effect of FocB on expression of type 1 fimbriae was also tested. Taken together, the results that we present expand our knowledge about a regulatory network for different adhesin gene systems in uropathogenic E. coli and suggest a hierarchy for expression of the fimbrial adhesins.
|
1199. |
Morinaga N,
Yahiro K,
Matsuura G,
Moss J,
Noda M,
( 2008 ) Subtilase cytotoxin, produced by Shiga-toxigenic Escherichia coli, transiently inhibits protein synthesis of Vero cells via degradation of BiP and induces cell cycle arrest at G1 by downregulation of cyclin D1. PMID : 18005237 : DOI : 10.1111/j.1462-5822.2007.01094.x PMC : PMC3021990 Abstract >>
Subtilase cytotoxin (SubAB) is a AB(5) type toxin produced by Shiga-toxigenic Escherichia coli, which exhibits cytotoxicity to Vero cells. SubAB B subunit binds to toxin receptors on the cell surface, whereas the A subunit is a subtilase-like serine protease that specifically cleaves chaperone BiP/Grp78. As noted previously, SubAB caused inhibition of protein synthesis. We now show that the inhibition of protein synthesis was transient and occurred as a result of ER stress induced by cleavage of BiP; it was closely associated with phosphorylation of double-stranded RNA-activated protein kinase-like ER kinase (PERK) and eukaryotic initiation factor-2alpha (eIF2alpha). The phosphorylation of PERK and eIF2alpha was maximal at 30-60 min and then returned to the control level. Protein synthesis after treatment of cells with SubAB was suppressed for 2 h and recovered, followed by induction of stress-inducible C/EBP-homologous protein (CHOP). BiP degradation continued, however, even after protein synthesis recovered. SubAB-treated cells showed cell cycle arrest in G1 phase, which may result from cyclin D1 downregulation caused by both SubAB-induced translational inhibition and continuous prolonged proteasomal degradation.
|
1200. |
Bronowski C,
Smith SL,
Yokota K,
Corkill JE,
Martin HM,
Campbell BJ,
Rhodes JM,
Hart CA,
Winstanley C,
( 2008 ) A subset of mucosa-associated Escherichia coli isolates from patients with colon cancer, but not Crohn's disease, share pathogenicity islands with urinary pathogenic E. coli. PMID : 18227261 : DOI : 10.1099/mic.0.2007/013086-0 Abstract >>
Adherent and invasive mucosa-associated Escherichia coli have been implicated in the pathogenesis of colon cancer and inflammatory bowel diseases. It has been reported that such isolates share features of extraintestinal E. coli (ExPEC) and particularly uropathogenic E. coli (UPEC). We used suppression subtractive hybridization (SSH) to subtract the genome of E. coli K-12 from that of a colon cancer mucosal E. coli isolate. Of the subtracted sequences, 53 % were present in the genomes of one or more of three sequenced UPEC strains but absent from the genome of an enterohaemorrhagic E. coli (EHEC) strain. Of the subtracted sequences, 80 % matched at least one UPEC genome, whereas only 4 % were absent from the UPEC genomes but present in the genome of the EHEC strain. A further genomic subtraction against the UPEC strain 536 enriched for sequences matching mobile genetic elements, other ExPEC strains, and other UPEC strains or commensals, rather than strains associated with gastrointestinal disease. We analysed the distribution of selected subtracted sequences and UPEC-associated pathogenicity islands (PAIs) amongst a panel of mucosa-associated E. coli isolated from colonoscopic biopsies of patients with colon cancer, patients with Crohn's disease and controls. This enabled us to identify a group of isolates from colon cancer (30-40 %) carrying multiple genes previously categorized as UPEC-specific and implicated in virulence.
|
1201. |
Chong DC,
Paton JC,
Thorpe CM,
Paton AW,
( 2008 ) Clathrin-dependent trafficking of subtilase cytotoxin, a novel AB5 toxin that targets the endoplasmic reticulum chaperone BiP. PMID : 18042253 : DOI : 10.1111/j.1462-5822.2007.01085.x Abstract >>
Subtilase cytotoxin (SubAB) is the prototype of a new family of AB5 cytotoxins produced by Shiga toxigenic Escherichia coli. Its cytotoxic activity is due to its capacity to enter cells and specifically cleave the endoplasmic reticulum (ER) chaperone BiP. However, its trafficking within target cells has not been investigated previously. In Vero cells, fluorescence colocalization with subcellular markers established that SubAB is trafficked from the cell surface to the ER via a retrograde pathway similar, but not identical, to those of Shiga toxin (Stx) and cholera toxin (Ctx), with their pathways converging at the Golgi. The clathrin inhibitor phenylarsine oxide prevented SubAB entry and BiP cleavage in SubAB-treated Vero, HeLa and N2A cells, while cholesterol depletion did not, demonstrating that, unlike either Stx or Ctx, SubAB internalization is exclusively clathrin-dependent.
|
1202. |
Cook PD,
Holden HM,
( 2008 ) GDP-4-keto-6-deoxy-D-mannose 3-dehydratase, accommodating a sugar substrate in the active site. PMID : 18045869 : DOI : 10.1074/jbc.M708893200 Abstract >>
Colitose is a dideoxysugar found in the O-antigen of the lipopolysaccharide that coats the outer membrane of some Gram-negative bacteria. Four enzymes are required for its production starting from D-mannose-1-phosphate and GTP. The focus of this investigation is GDP-4-keto-6-deoxy-D-mannose 3-dehydratase or ColD, which catalyzes the removal of the C3'-hydroxyl group from GDP-4-keto-6-deoxymannose. The enzyme is pyridoxal 5'-phosphate-dependent, but unlike most of these proteins, the conserved lysine residue that covalently holds the cofactor in the active site is replaced with a histidine residue. Here we describe the three-dimensional structure of ColD, determined to 1.7A resolution, whereby the active site histidine has been replaced with an asparagine residue. For this investigation, crystals of the site-directed mutant protein were grown in the presence of GDP-4-amino-4,6-dideoxy-D-mannose (GDP-perosamine). The electron density map clearly reveals the presence of the sugar analog trapped in the active site as an external aldimine. The active site is positioned between the two subunits of the dimer. Whereas the pyrophosphoryl groups of the ligand are anchored to the protein via Arg-219 and Arg-331, the hydroxyl groups of the hexose only lie within hydrogen bonding distance to ordered water molecules. Interestingly, the hexose moiety of the ligand adopts a boat rather than the typically observed chair conformation. Activity assays demonstrate that this mutant protein cannot catalyze the dehydration step. Additionally, we report data revealing that wild-type ColD is able to catalyze the production of GDP-4-keto-3,6-dideoxymannose using GDP-perosamine instead of GDP-4-keto-6-deoxymannose as a substrate.
|
1203. |
Khachatryan AR,
Besser TE,
Call DR,
( 2008 ) The streptomycin-sulfadiazine-tetracycline antimicrobial resistance element of calf-adapted Escherichia coli is widely distributed among isolates from Washington state cattle. PMID : 18039823 : DOI : 10.1128/AEM.01534-07 PMC : PMC2223241 Abstract >>
Association of specific antimicrobial resistance patterns with unrelated selective traits has long been implicated in the maintenance of antimicrobial resistance in a population. Previously we demonstrated that Escherichia coli strains with a specific resistance pattern (resistant to streptomycin, sulfadiazine, and tetracycline [SSuT]) have a selective advantage in dairy calf intestinal environments and in the presence of a milk supplement commonly fed to the calves. In the present study we identified the sequence of the genetic element that confers the SSuT phenotype and show that this element is present in a genetically diverse group of E. coli isolates, as assessed by macrorestriction digestion and pulsed-field gel electrophoresis. This element was also found in E. coli isolates from 18 different cattle farms in Washington State. Using in vitro competition experiments we further demonstrated that SSuT strains from 17 of 18 farms were able to outcompete pansusceptible strains. In a separate set of experiments, we were able to transfer the antimicrobial resistance phenotype by electroporation to a laboratory strain of E. coli (DH10B), making that new strain more competitive during in vitro competition with the parental DH10B strain. These data indicate that a relatively large genetic element conferring the SSuT phenotype is widely distributed in E. coli from cattle in Washington State. Furthermore, our results indicate that this element is responsible for maintenance of these traits owing to linkage to genetic traits that confer a selective advantage in the intestinal lumens of dairy calves.
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1204. |
Enne VI,
Cassar C,
Sprigings K,
Woodward MJ,
Bennett PM,
( 2008 ) A high prevalence of antimicrobial resistant Escherichia coli isolated from pigs and a low prevalence of antimicrobial resistant E. coli from cattle and sheep in Great Britain at slaughter. PMID : 18053066 : DOI : 10.1111/j.1574-6968.2007.00991.x Abstract >>
The incidence of antimicrobial resistance and expressed and unexpressed resistance genes among commensal Escherichia coli isolated from healthy farm animals at slaughter in Great Britain was investigated. The prevalence of antimicrobial resistance among the isolates varied according to the animal species; of 836 isolates from cattle tested only 5.7% were resistant to one or more antimicrobials, while only 3.0% of 836 isolates from sheep were resistant to one or more agents. However, 92.1% of 2480 isolates from pigs were resistant to at least one antimicrobial. Among isolates from pigs, resistance to some antimicrobials such as tetracycline (78.7%), sulphonamide (66.9%) and streptomycin (37.5%) was found to be common, but relatively rare to other agents such as amikacin (0.1%), ceftazidime (0.1%) and coamoxiclav (0.2%). The isolates had a diverse range of resistance gene profiles, with tet(B), sul2 and strAB identified most frequently. Seven out of 615 isolates investigated carried unexpressed resistance genes. One trimethoprim-susceptible isolate carried a complete dfrA17 gene but lacked a promoter for it. However, in the remaining six streptomycin-susceptible isolates, one of which carried strAB while the others carried aadA, no mutations or deletions in gene or promoter sequences were identified to account for susceptibility. The data indicate that antimicrobial resistance in E. coli of animal origin is due to a broad range of acquired genes.
|
1205. |
Parsons Y,
Hall RM,
Stokes HW,
( 1991 ) A new trimethoprim resistance gene, dhfrX, in the In7 integron of plasmid pDGO100. PMID : 1804022 : DOI : 10.1128/aac.35.11.2436 PMC : PMC245401 Abstract >>
A new trimethoprim resistance determinant, designated dhfrX, was identified in the In7 integron of pDGO100. The sequence of the dhfrX dihydrofolate reductase is up to 28% identical to the sequences of several known dihydrofolate reductase proteins. The dhfrX gene is adjacent to the second 3'-conserved segment of the In7 integron, but the first 77 bases of this segment are not present.
|
1206. |
Uhlich GA,
Sinclair JR,
Warren NG,
Chmielecki WA,
Fratamico P,
( 2008 ) Characterization of Shiga toxin-producing Escherichia coli isolates associated with two multistate food-borne outbreaks that occurred in 2006. PMID : 18083883 : DOI : 10.1128/AEM.01618-07 PMC : PMC2258581 Abstract >>
Shiga toxin-producing Escherichia coli isolates from two 2006 outbreaks were compared to other O157:H7 isolates for virulence genotype, biofilm formation, and stress responses. Spinach- and lettuce-related-outbreak strains had similar pulsed-field gel electrophoresis patterns, and all carried both stx2 and stx2c variant genes. Cooperative biofilm formation involving an E. coli O157:H7 strain and a non-O157:H7 strain was also demonstrated.
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1207. |
Nomura N,
Masai H,
Inuzuka M,
Miyazaki C,
Ohtsubo E,
Itoh T,
Sasamoto S,
Matsui M,
Ishizaki R,
Arai K,
( 1991 ) Identification of eleven single-strand initiation sequences (ssi) for priming of DNA replication in the F, R6K, R100 and ColE2 plasmids. PMID : 1761225 : DOI : 10.1016/0378-1119(91)90482-q Abstract >>
Based on the ability to complement the poor growth of an M13 phage derivative lacking the complementary strand origin, eleven single-strand initiation sequences (ssi) for DNA replication are identified in the F, R6K, R100 and ColE2 plasmids. Six of them were from F, two from near the gamma and alpha origins (ori) of R6K, two from the vicinity of the basic replicon of R100 and one from near the ori of ColE2. They can be classified into two groups based on the morphology of the plaques and the length of nucleotide (nt) sequences required for ssi activity; one group that gives rise to larger and clearer plaques and can be reduced to nearly 100 nt (seven out of eleven), and another that generates smaller and less clear plaques and requires more than 200 nt for full activity (four out of eleven). Sequence homology is detected among some members from both groups. The possible biological roles of the ssi are discussed.
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1208. |
Bai L,
Schüller S,
Whale A,
Mousnier A,
Marches O,
Wang L,
Ooka T,
Heuschkel R,
Torrente F,
Kaper JB,
Gomes TA,
Xu J,
Phillips AD,
Frankel G,
( 2008 ) Enteropathogenic Escherichia coli O125:H6 triggers attaching and effacing lesions on human intestinal biopsy specimens independently of Nck and TccP/TccP2. PMID : 17984209 : DOI : 10.1128/IAI.01199-07 PMC : PMC2223649 Abstract >>
Typical enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) employ either Nck, TccP/TccP2, or Nck and TccP/TccP2 pathways to activate the neuronal Wiskott-Aldrich syndrome protein (N-WASP) and to trigger actin polymerization in cultured cells. This phenotype is used as a marker for the pathogenic potential of EPEC and EHEC strains. In this paper we report that EPEC O125:H6, which represents a large category of strains, lacks the ability to utilize either Nck or TccP/TccP2 and hence triggers actin polymerization in vitro only inefficiently. However, we show that infection of human intestinal biopsies with EPEC O125:H6 results in formation of typical attaching and effacing lesions. Expression of TccP in EPEC O125:H6, which harbors an EHEC O157-like Tir, resulted in efficient actin polymerization in vitro and enhanced colonization of human intestinal in vitro organ cultures with detectable N-WASP and electron-dense material at the site of bacterial adhesion. These results show the existence of a natural category of EPEC that colonizes the gut mucosa using Nck- and TccP-independent mechanisms. Importantly, the results highlight yet again the fact that conclusions made on the basis of in vitro cell culture models cannot be extrapolated wholesale to infection of mucosal surfaces and that the ability to induce actin polymerization on cultured cells should not be used as a definitive marker for EPEC and EHEC virulence.
|
1209. |
Nicolas-Chanoine MH,
Blanco J,
Leflon-Guibout V,
Demarty R,
Alonso MP,
Caniça MM,
Park YJ,
Lavigne JP,
Pitout J,
Johnson JR,
( 2008 ) Intercontinental emergence of Escherichia coli clone O25:H4-ST131 producing CTX-M-15. PMID : 18077311 : DOI : 10.1093/jac/dkm464 Abstract >>
Concomitant with the recent emergence of CTX-M-type extended-spectrum beta-lactamases (ESBLs), Escherichia coli has become the enterobacterial species most affected by ESBLs. Multiple locales are encountering CTX-M-positive E. coli, including specifically CTX-M-15. To gain insights into the mechanism underlying this phenomenon, we assessed clonality and diversity of virulence profiles within an international collection of CTX-M-15-positive E. coli. Forty-one ESBL-positive E. coli isolates from eight countries and three continents (Europe, Asia and North America) were selected for study based on suspected clonality. Phylogenetic group, ERIC2 PCR profile, O H serotype, AmpC variant and antibiotic susceptibility were determined. Multilocus sequence typing (MLST) and PFGE provided additional discrimination. Virulence potential was inferred by detection of 46 virulence factor (VF) genes. Thirty-six (88%) of the 41 E. coli isolates exhibited the same set of core characteristics: phylogenetic group B2, ERIC2 PCR profile 1, serotype O25:H4, AmpC EC6, ciprofloxacin resistance and MLST profile ST131. By PFGE, the 36 isolates constituted one large cluster at the 68% similarity level; this comprised 17 PFGE groups (defined at 85% similarity), some of which included strains from different countries. The 36 isolates exhibited highly (91% to 100%) similar VF profiles. We describe a broadly disseminated, CTX-M-15-positive and virulent E. coli clonal group with highly homogeneous virulence genotypes and subgroups exhibiting highly similar PFGE profiles, suggesting recent emergence. Understanding how this clone has emerged and successfully disseminated within the hospital and community, including across national boundaries, should be a public health priority.
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1210. |
Lee JE,
Reed J,
Shields MS,
Spiegel KM,
Farrell LD,
Sheridan PP,
( 2007 ) Phylogenetic analysis of Shiga toxin 1 and Shiga toxin 2 genes associated with disease outbreaks. PMID : 18053224 : DOI : 10.1186/1471-2180-7-109 PMC : PMC2211750 Abstract >>
Shiga toxins 1 and 2 (Stx1 and Stx2) are bacteriophage-encoded proteins that have been associated with hemorrhagic colitis, hemolytic uremic syndrome and other severe disease conditions. Stx1 and Stx2 are genetically and immunologically distinct but share the same compound toxin structure, method of entry and enzymatic function. Phylogenetic analysis was performed using Stx1 and Stx2 amino acid and nucleotide sequences from 41 strains of Escherichia coli, along with known stx sequences available from GenBank. The analysis confirmed the Stx1 and Stx2 divergence, and showed that there is generally more sequence variation among stx2 genes than stx1. The phylograms showed generally flat topologies among our strains' stx1 and stx2 genes. In the stx2 gene, 39.5% of the amino acid sites display very low nonsynonymous to synonymous substitution ratios. The stx1 and stx2 genes used in this phylogenetic study show sequence conservation with no significant divergence with respect to place or time. These data could indicate that Shiga toxins are experiencing purifying selection.
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1211. |
Kachroo AH,
Kancherla AK,
Singh NS,
Varshney U,
Mahadevan S,
( 2007 ) Mutations that alter the regulation of the chb operon of Escherichia coli allow utilization of cellobiose. PMID : 18028317 : DOI : 10.1111/j.1365-2958.2007.05999.x Abstract >>
Wild-type strains of Escherichia coli are normally unable to metabolize cellobiose. However, cellobiose-positive (Cel(+)) mutants arise upon prolonged incubation on media containing cellobiose as the sole carbon source. We show that the Cel(+) derivatives carry two classes of mutations that act concertedly to alter the regulation of the chb operon involved in the utilization of N,N'-diacetylchitobiose. These consist of mutations that abrogate negative regulation by the repressor NagC as well as single base-pair changes in the transcriptional regulator chbR that translate into single-amino-acid substitutions. Introduction of chbR from two Cel(+) mutants resulted in activation of transcription from the chb promoter at a higher level in the presence of cellobiose, in reporter strains carrying disruptions of the chromosomal chbR and nagC. These transformants also showed a Cel(+) phenotype on MacConkey cellobiose medium, suggesting that the wild-type permease and phospho-beta-glucosidase, upon induction, could recognize, transport and cleave cellobiose respectively. This was confirmed by expressing the wild-type genes encoding the permease and phospho-beta-glucosidase under a heterologous promoter. Biochemical characterization of one of the chbR mutants, chbRN238S, showed that the mutant regulator makes stronger contact with the target DNA sequence within the chb promoter and has enhanced recognition of cellobiose 6-phosphate as an inducer compared with the wild-type regulator.
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1212. |
Zhao L,
Chen X,
Xu X,
Song G,
Liu X,
( 2009 ) Analysis of the AIDA-I gene sequence and prevalence in Escherichia coli isolates from pigs with post-weaning diarrhoea and oedema disease. PMID : 18077196 : DOI : 10.1016/j.tvjl.2007.10.021 Abstract >>
Three hundred and twenty-four strains of Escherichia coli isolated from weaned pigs with diarrhoea or oedema disease in Eastern China were screened by multiplex PCR for the presence of the gene encoding adhesin involved in diffuse adhesion I (AIDA-I). Two AIDA-I positive strains were subjected to analysis of the nucleotide sequence of the complete orfA and orfB of the AIDA gene. The AIDA-I positive E. coli isolates were also assessed for five fimbriae (F4, F5, F6, F18 and F41) by monoclonal antibodies and for toxin genes (STa, STb, LT, EAST1, Stx2e) by PCR. Twenty-one (6.5%) of the isolates possessed AIDA-I genes. Of these isolates, two carried AIDA-I genes as the only demonstrated virulence factors, and the remaining isolates carried other virulence factor genes. Comparing the AIDA-I sequence from porcine and human sources, a high homology of orfA both in porcine E. coli and human E. coli was observed. However, each orfB of the two porcine E. coli isolates was 3864 nucleotides long compared with 3861 for the E. coli 2787 orfB, and showed 96.5% homology to E. coli 2787. The data indicated (1) that AIDA-I may be an occasional virulence factor in post-weaning diarrhoea and oedema disease in pigs, (2) that it has the potential to transfer between porcine and human E. coli, and (3) that there is a genetic diversity in orfB between human and porcine E. coli.
|
1213. |
Cook PD,
Holden HM,
( 2007 ) A structural study of GDP-4-keto-6-deoxy-D-mannose-3-dehydratase: caught in the act of geminal diamine formation. PMID : 17997582 : DOI : 10.1021/bi701686s PMC : PMC2533848 Abstract >>
Di- and trideoxysugars are an important class of carbohydrates synthesized by certain plants, fungi, and bacteria. Colitose, for example, is a 3,6-dideoxysugar found in the O-antigens of Gram-negative bacteria such as Escherichia coli, Salmonella enterica, Yersinia pseudotuberculosis, and Vibrio cholerae, among others. These types of dideoxysugars are thought to serve as antigenic determinants and to play key roles in bacterial defense and survival. Four enzymes are required for the biochemical synthesis of colitose starting from mannose-1-phosphate. The focus of this investigation, GDP-4-keto-6-deoxy-d-mannose-3-dehydratase (ColD), catalyzes the third step in the pathway, namely the PLP-dependent removal of the C3'-hydroxyl group from GDP-4-keto-6-deoxymannose. Whereas most PLP-dependent enzymes contain an active site lysine, ColD utilizes a histidine as its catalytic acid/base. The ping-pong mechanism of the enzyme first involves the conversion of PLP to PMP followed by the dehydration step. Here we present the three-dimensional structure of a site-directed mutant form of ColD whereby the active site histidine has been replaced with a lysine. The electron density reveals that the geminal diamine, a tetrahedral intermediate in the formation of PMP from PLP, has been trapped within the active site region. Functional assays further demonstrate that this mutant form of ColD cannot catalyze the dehydration reaction.
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1214. |
Nicklasson M,
Sjöling A,
Lebens M,
Tobias J,
Janzon A,
Brive L,
Svennerholm AM,
( 2008 ) Mutations in the periplasmic chaperone leading to loss of surface expression of the colonization factor CS6 in enterotoxigenic Escherichia coli (ETEC) clinical isolates. PMID : 18037262 : DOI : 10.1016/j.micpath.2007.06.009 Abstract >>
Enterotoxigenic Escherichia coli (ETEC) cause diarrhoea by adhesion to human enterocytes by one or more colonization factors (CFs) and secretion of heat-labile (LT) and/or heat-stable (ST) enterotoxins. Expression of coli surface antigen 6 (CS6) on the bacterial surface, usually associated with ETEC strains that produce ST alone or in combination with LT, is rarely found in strains expressing only LT. However, a number of LT-only strains which are genotypically positive but phenotypically negative for CS6 have been identified. In this study, eight such strains from India and Guinea-Bissau belonging to different clones were analysed. The CS6 operon cssABCD was transcribed but protein analyses suggested that the structural subunits CssA and CssB of CS6 were absent in the periplasm. Most strains contained truncating mutations within the periplasmic chaperone-encoding gene cssC and protein modelling indicated that this severely affected the substrate-binding capacity of the chaperone. A single-nucleotide polymorphism (SNP) (A-->T) in the 5'-untranslated region of cssC distinguished the eight strains from ETEC strains that do express CS6 on the surface and may be a potential marker for ETEC strains containing phenotypically silent cssABCD. The study emphasizes the importance of using both genotypic and phenotypic methods in epidemiological studies of ETEC, e.g. for vaccine development.
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1215. |
Leyton DL,
Adams LM,
Kelly M,
Sloan J,
Tauschek M,
Robins-Browne RM,
Hartland EL,
( 2007 ) Contribution of a novel gene, rpeA, encoding a putative autotransporter adhesin to intestinal colonization by rabbit-specific enteropathogenic Escherichia coli. PMID : 17620350 : DOI : 10.1128/IAI.00972-06 PMC : PMC1951200 Abstract >>
Rabbit-specific enteropathogenic Escherichia coli (REPEC) is an attaching and effacing pathogen of young rabbits. Using signature-tagged mutagenesis, we identified several known colonization factors of REPEC as well as a gene predicted to encode a novel autotransporter protein. This novel gene was termed rpeA for REPEC plasmid-encoded autotransporter.
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1216. |
Nikkila H,
Gennis RB,
Sligar SG,
( 1991 ) Cloning and expression of the gene encoding the soluble cytochrome b562 of Escherichia coli. PMID : 1761034 : DOI : 10.1111/j.1432-1033.1991.tb16377.x Abstract >>
The gene for the soluble cytochrome b562 from Escherichia coli B has been cloned on a SalI fragment. The analysis of the gene reveals the presence of a leader sequence in front of the sequence encoding the mature protein. Expression of cytochrome b562 using the lac-promoter produced the protein to a level of 3-5% of total protein. This over-production enables employment of a simple, high-yield purification protocol to obtain homogeneous cytochrome b562. Spectroscopic and N-terminal sequence analyses of the purified protein demonstrate that it is identical to the chromosomally expressed cytochrome b562 purified and characterized from E. coli B [Itagaki, E. & Hager, L.P. (1966) J. Biol. Chem. 241, 3687-3695]. It is demonstrated that the genomic sequence codes for a classic N-terminal signal sequence and that mature cytochrome b562 is translocated to the periplasmic space.
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1217. |
Koh TH,
Sng LH,
Wang G,
Hsu LY,
Lin RT,
Tee NW,
( 2007 ) Emerging problems with plasmid-mediated DHA and CMY AmpC beta-lactamases in Enterobacteriaceae in Singapore. PMID : 17587551 : DOI : 10.1016/j.ijantimicag.2007.04.014 Abstract >>
N/A
|
1218. |
Gomez-Duarte OG,
Chattopadhyay S,
Weissman SJ,
Giron JA,
Kaper JB,
Sokurenko EV,
( 2007 ) Genetic diversity of the gene cluster encoding longus, a type IV pilus of enterotoxigenic Escherichia coli. PMID : 17951389 : DOI : 10.1128/JB.00722-07 PMC : PMC2168600 Abstract >>
Enterotoxigenic Escherichia coli (ETEC) strains produce a type IV pilus named Longus. We identified a 16-gene cluster involved in the biosynthesis of Longus that has 57 to 95% identity at the protein level to CFA/III, another type IV pilus of ETEC. Alleles of the Longus structural subunit gene lngA demonstrate a diversity of 12 to 19% at the protein level with strong positive selection for point replacements and horizontal transfer.
|
1219. |
Mammeri H,
Poirel L,
Nordmann P,
( 2007 ) Extension of the hydrolysis spectrum of AmpC beta-lactamase of Escherichia coli due to amino acid insertion in the H-10 helix. PMID : 17586561 : DOI : 10.1093/jac/dkm227 Abstract >>
To characterize the naturally occurring expanded-spectrum beta-lactamase from an Escherichia coli clinical isolate and to compare it with a wild-type beta-lactamase. The chromosome-borne ampC genes from E. coli BER and E. coli EC2 were PCR amplified, sequenced and cloned into an expression vector. Antimicrobial susceptibilities of the parental isolate and the recombinant strains were determined by agar dilution methods. Kinetic parameters were determined from purified AmpC BER and AmpC EC2. AmpC BER was overexpressed in its original clinical isolate because of mutations in the promoter region of its gene at positions -42 and -18. The analysis of the ampC coding sequence revealed a 6 bp insertion when compared with the wild-type sequence leading to the tandem duplication of two alanine residues inside the H-10 helix. AmpC BER-producing recombinants were resistant to ceftazidime, had reduced susceptibility to other oxyiminocephalosporins (cefotaxime and cefepime), but had a greater susceptibility to cefoxitin when compared with the recombinant expressing the wild-type beta-lactamase AmpC EC2. The affinity of AmpC BER for cephalosporins and imipenem was increased, whereas the hydrolysis rate was decreased for all these compounds. In addition, the IC50 values of clavulanic acid and tazobactam for AmpC BER were increased. This work sheds new light on structure-function relationships of expanded-spectrum AmpC beta-lactamases.
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1220. |
Smajs D,
Strouhal M,
Matejková P,
Cejková D,
Cursino L,
Chartone-Souza E,
Smarda J,
Nascimento AM,
( 2008 ) Complete sequence of low-copy-number plasmid MccC7-H22 of probiotic Escherichia coli H22 and the prevalence of mcc genes among human E. coli. PMID : 17936903 : DOI : 10.1016/j.plasmid.2007.08.002 Abstract >>
The complete sequence of the plasmid MccC7-H22 encoding microcin C7, isolated from probiotic E. coli H22, was determined and analyzed. DNA of pMccC7-H22 comprises 32,014 bp and contains 39 predicted ORFs. Two main gene clusters, i.e., genes involved in plasmid replication and maintenance and genes encoding microcin C7 synthesis, are separated by several ORFs homologous to ORFs present in IS (insertion sequence) elements and transposons. Additional 14 ORFs code for proteins with similarities to known proteins (4 ORFs) or for hypothetical proteins with unknown function (10 ORFs). The differences in G+C content of individual ORFs and gene clusters of pMccC7-H22 indicate a mosaic structure for the plasmid, resulting from recombination events. Real-time PCR quantification was applied to measure the copy number of pMccC7-H22. Escherichia coli H22 carries approximately 5 copies of pMccC7-H22 per chromosome and thus pMccC7-H22 belongs to the group of relatively low-copy-number plasmids. Following 360 generations, all bacterial colonies (out of 100 tested) synthesized microcin C7 indicating that pMccC7-H22 is stably maintained in E. coli H22. Screening of 105 E. coli strains isolated from human fecal samples revealed 2 (1.9%) strains that produced microcin C7.
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1221. |
Plainvert C,
Bidet P,
Peigne C,
Barbe V,
Médigue C,
Denamur E,
Bingen E,
Bonacorsi S,
( 2007 ) A new O-antigen gene cluster has a key role in the virulence of the Escherichia coli meningitis clone O45:K1:H7. PMID : 17905975 : DOI : 10.1128/JB.01013-07 PMC : PMC2168960 Abstract >>
A new highly pathogenic clone of Escherichia coli meningitis strains harboring the unusual serogroup O45 has recently emerged in France. To gain insight into the pathogenicity of this new clone, we investigated the possible role of antigen O45 in the virulence of strain S88 (O45:K1:H7), representative of this emerging clone. We first showed that the S88 O-antigen gene cluster sequence differs from that of O45 in the reference strain E. coli 96-3285, suggesting that the two O45 polysaccharides, while probably sharing a community of epitopes, represent two different antigens. The unique functional organization of the two O-antigen gene clusters and the low DNA sequence homology of the orthologous genes suggest that the two loci originated from a common ancestor and have since undergone multiple recombination events. Phylogenetic analysis based on the flanking gene gnd sequences indicates that the S88 antigen O45 (O45(S88)) gene cluster may have been acquired, at least in part, from another member of the Enterobacteriaceae. Mutagenesis of the O45(S88) antigen gene cluster was used for functional analysis of the loci and revealed the crucial role of the O polysaccharide in S88 virulence in a neonatal rat meningitis model. We also developed a PCR method to specifically identify the O45(S88) antigen gene cluster. Together, our findings suggest that horizontal acquisition of a new O-antigen gene cluster, at least partly from another species, may have been a key event in the emergence and virulence of the E. coli O45:K1:H7 clone in France.
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1222. |
Wang W,
Perepelov AV,
Feng L,
Shevelev SD,
Wang Q,
Senchenkova SN,
Han W,
Li Y,
Shashkov AS,
Knirel YA,
Reeves PR,
Wang L,
( 2007 ) A group of Escherichia coli and Salmonella enterica O antigens sharing a common backbone structure. PMID : 17600060 : DOI : 10.1099/mic.0.2007/004192-0 Abstract >>
The O-antigen moiety of the LPS is one of the most variable cell surface components of the Gram-negative bacterial outer membrane. Variation is due to the presence of different sugars and sugar linkages. Here, it is reported that a group of Escherichia coli O serogroups (O17, O44, O73, O77 and O106), and the Salmonella enterica serogroup O : 6,14 (H), share a common four-sugar backbone O-subunit structure, and possess almost identical O-antigen gene clusters. Whereas the E. coli O77 antigen does not have any substitutions, the other O antigens in this group differ by the addition of one or two glucose side branches at various positions of the backbone. The O-antigen gene clusters for all members of the group encode only the proteins required for biosynthesis of the common four-sugar backbone. The identification of three genes within a putative prophage in the E. coli O44 genome is also reported; these genes are presumably involved in the glucosylation of the basic tetrasaccharide unit. This was confirmed by deletion of one of the genes, which encodes a putative glucosyltransferase. Structural analysis of the O antigen produced by the mutant strain demonstrated the absence of glucosylation. An O-antigen structure shared by five E. coli and one S. enterica serogroups, all of which have a long evolutionary history, suggests that the common backbone may be important for the survival of E. coli strains in the environment, or for their pathogenicity.
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1223. |
Navarro F,
Mesa RJ,
Miró E,
Gómez L,
Mirelis B,
Coll P,
( 2007 ) Evidence for convergent evolution of CTX-M-14 ESBL in Escherichia coli and its prevalence. PMID : 17559406 : DOI : 10.1111/j.1574-6968.2007.00791.x Abstract >>
A total of 2440 Escherichia coli strains isolated in 2003 at the Hospital de la Santa Creu i Sant Pau were evaluated for the presence of extended-spectrum beta-lactamases. Two different nucleotide sequences that encode the same beta-lactamase, CTX-M-14, were detected when the bla(CTX-M-14)-genes of 35 E. coli isolates were analysed. Thirty-two of the 35 had the previously described sequence of the bla(CTX-M-14) (AF252622), named bla(CTX-M-14a), and the remaining three isolates showed a nucleotide sequence identical to that of the bla(CTX-M-9) gene except for one nucleotide, named bla(CTX-M-14b). Characterisation of the regions surrounding the bla(CTX-M-14a) showed the ISEcp1 and the IS903 upstream and downstream, respectively, of the bla gene, whereas the regions surrounding the bla(CTX-M-14b) contained the genetic environment described for the bla(CTX-M-9) gene, the In60. Characterisation by hybridisation showed that the bla(CTX-M-14a) was present in IncK plasmids, whereas the bla(CTX-M-14b) was found in the HI2 Inc group. The CTX-M-14 ESBL in E. coli isolates is the result of the convergence of two different genes.
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1224. |
Van der Auwera G,
Baute J,
Bauwens M,
Peck I,
Piette D,
Pycke M,
Asselman P,
Depicker A,
( 2008 ) Development and application of novel constructs to score C:G-to-T:A transitions and homologous recombination in Arabidopsis. PMID : 17921342 : DOI : 10.1104/pp.107.105213 PMC : PMC2230553 Abstract >>
We report on the development of five missense mutants and one recombination substrate of the beta-glucuronidase (GUS)-encoding gene of Escherichia coli and their use for detecting mutation and recombination events in transgenic Arabidopsis (Arabidopsis thaliana) plants by reactivation of GUS activity in clonal sectors. The missense mutants were designed to find C:G-to-T:A transitions in a symmetrical sequence context and are in that respect complementary to previously published GUS point mutants. Small peptide tags (hemagglutinin tag and Strep tag II) and green fluorescent protein were translationally fused to GUS, which offers possibilities to check for mutant GUS production levels. We show that spontaneous mutation and recombination events took place. Mutagenic treatment of the plants with ethyl methanesulfonate and ultraviolet-C increased the number of mutations, validating the use of these constructs to measure mutation and recombination frequencies in plants exposed to biotic or abiotic stress conditions, or in response to different genetic backgrounds. Plants were also subjected to heavy metals, methyl jasmonate, salicylic acid, and heat stress, for which no effect could be seen. Together with an ethyl methanesulfonate mutation induction level much higher than previously described, the need is illustrated for many available scoring systems in parallel. Because all GUS missense mutants were cloned in a bacterial expression vector, they can also be used to score mutation events in E. coli.
|
1225. |
Wang Y,
Xu Y,
Perepelov AV,
Qi Y,
Knirel YA,
Wang L,
Feng L,
( 2007 ) Biochemical characterization of dTDP-D-Qui4N and dTDP-D-Qui4NAc biosynthetic pathways in Shigella dysenteriae type 7 and Escherichia coli O7. PMID : 17905981 : DOI : 10.1128/JB.00777-07 PMC : PMC2168959 Abstract >>
O-antigen variation due to the presence of different types of sugars and sugar linkages is important for the survival of bacteria threatened by host immune systems. The O antigens of Shigella dysenteriae type 7 and Escherichia coli O7 contain 4-(N-acetylglycyl)amino-4,6-dideoxy-d-glucose (d-Qui4NGlyAc) and 4-acetamido-4,6-dideoxy-d-glucose (d-Qui4NAc), respectively, which are sugars not often found in studied polysaccharides. In this study, we characterized the biosynthetic pathways for dTDP-d-Qui4N and dTDP-d-Qui4NAc (the nucleotide-activated precursors of d-Qui4NGlyAc and d-Qui4NAc in O antigens). Predicted genes involved in the synthesis of the two sugars were cloned, and the gene products were overexpressed and purified as His-tagged fusion proteins. In vitro enzymatic reactions were carried out using the purified proteins, and the reaction products were analyzed by capillary electrophoresis, electrospray ionization-mass spectrometry, and nuclear magnetic resonance spectroscopy. It is shown that in S. dysenteriae type 7 and E. coli O7, dTDP-d-Qui4N is synthesized from alpha-d-glucose-1-phosphate in three reaction steps catalyzed by glucose-1-phosphate thymidyltransferase (RmlA), dTDP-d-glucose 4,6-dehydratase (RmlB), and dTDP-4-keto-6-deoxy-d-glucose aminotransferase (VioA). An additional acetyltransferase (VioB) catalyzes the conversion of dTDP-d-Qui4N into dTDP-d-Qui4NAc in E. coli O7. Kinetic parameters and some other properties of VioA and VioB are described and differences between VioA proteins from S. dysenteriae type 7 (VioA(D7)) and E. coli O7 (VioA(O7)) discussed. To our knowledge, this is the first time that functions of VioA and VioB have been biochemically characterized. This study provides valuable enzyme sources for the production of dTDP-d-Qui4N and dTDP-d-Qui4NAc, which are potentially useful in the pharmaceutical industry for drug development.
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1226. |
Klontz EH,
Tomich AD,
Günther S,
Lemkul JA,
Deredge D,
Silverstein Z,
Shaw JF,
McElheny C,
Doi Y,
Wintrode PL,
MacKerell AD,
Sluis-Cremer N,
Sundberg EJ,
( 2017 ) Structure and Dynamics of FosA-Mediated Fosfomycin Resistance in Klebsiella pneumoniae and Escherichia coli. PMID : 28874374 : DOI : 10.1128/AAC.01572-17 PMC : PMC5655077 Abstract >>
Fosfomycin exhibits broad-spectrum antibacterial activity and is being reevaluated for the treatment of extensively drug-resistant pathogens. Its activity in Gram-negative organisms, however, can be compromised by expression of FosA, a metal-dependent transferase that catalyzes the conjugation of glutathione to fosfomycin, rendering the antibiotic inactive. In this study, we solved the crystal structures of two of the most clinically relevant FosA enzymes: plasmid-encoded FosA3 from Escherichia coli and chromosomally encoded FosA from Klebsiella pneumoniae (FosAKP). The structure, molecular dynamics, catalytic activity, and fosfomycin resistance of FosA3 and FosAKP were also compared to those of FosA from Pseudomonas aeruginosa (FosAPA), for which prior crystal structures exist. E. coli TOP10 transformants expressing FosA3 and FosAKP conferred significantly greater fosfomycin resistance (MIC, >1,024 �gg/ml) than those expressing FosAPA (MIC, 16 �gg/ml), which could be explained in part by the higher catalytic efficiencies of the FosA3 and FosAKP enzymes. Interestingly, these differences in enzyme activity could not be attributed to structural differences at their active sites. Instead, molecular dynamics simulations and hydrogen-deuterium exchange experiments with FosAKP revealed dynamic interconnectivity between its active sites and a loop structure that extends from the active site of each monomer and traverses the dimer interface. This dimer interface loop is longer and more extended in FosAKP and FosA3 than in FosAPA, and kinetic analyses of FosAKP and FosAPA loop-swapped chimeric enzymes highlighted its importance in FosA activity. Collectively, these data yield novel insights into fosfomycin resistance that could be leveraged to develop new strategies to inhibit FosA and potentiate fosfomycin activity.
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1227. |
Varney KM,
Inman KG,
Farfan M,
Dudley E,
Fletcher J,
Weber DJ,
Nataro JP,
Velarde JJ,
( 2007 ) Solution structure of the novel dispersin protein of enteroaggregative Escherichia coli. PMID : 17986189 : DOI : 10.1111/j.1365-2958.2007.05985.x Abstract >>
Enteroaggregative Escherichia coli (EAEC), increasingly recognized as an important cause of infant and travelers' diarrhoea, exhibits an aggregative, stacked-brick pattern of adherence to epithelial cells. Adherence is mediated by aggregative adherence fimbriae (AAFs), which are encoded on the pAA virulence plasmid. We recently described a highly prevalent pAA plasmid-borne gene, aap, which encodes a protein (nicknamed dispersin) that is secreted to the bacterial cell surface. Dispersin-null mutants display a unique hyper-aggregating phenotype, accompanied by collapse of AAF pili onto the bacterial cell surface. To study the mechanism of this effect, we solved the structure of dispersin from EAEC strain 042 using solution NMR, revealing a stable beta-sandwich with a conserved net positive surface charge of +3 to +4 among 23 dispersin alleles. Experimental data suggest that dispersin binds non-covalently to lipopolysaccharide on the surface of the bacterium. We also show that the AAF organelles contribute positive charge to the bacterial surface, suggesting that dispersin's role in fimbrial function is to overcome electrostatic attraction between AAF and the bacterial surface.
|
1228. |
Wang GQ,
Wu CM,
Du XD,
Shen ZQ,
Song LH,
Chen X,
Shen JZ,
( 2008 ) Characterization of integrons-mediated antimicrobial resistance among Escherichia coli strains isolated from bovine mastitis. PMID : 17897793 : DOI : 10.1016/j.vetmic.2007.08.003 Abstract >>
To assess the prevalence of antimicrobial resistance and class I integrons in Escherichia coli strains (n=58) isolated from bovine mastitis in Inner Mongolia, antimicrobial susceptibility and the presence of various types of integrons were characterized. Most isolates were susceptible to amikacin, colistin, ceftazidime, gentamicin and kanamycin, while those also exhibited high resistant incidence rates to ampicillin, amoxicillin, sulfadiazine and sulfamethoxydiazine. The integrase gene of integrons was amplified by PCR using degenerate primers. The integrons were confirmed by restriction fragment length polymorphism (RFLP) analysis of positive PCR products. Neither class II nor class III integron was detected, while 56.90% (n=33) of the isolates were positive for the presence of intI1 gene. Sequencing analysis of gene cassettes revealed that seven gene cassettes were found, which encoded resistance to trimethoprim (dfrA1 and dfrA17), aminoglycosides (aacA4, aadA1 and aadA5) and chloramphenicol (catB3), respectively. Of them, the gene cassette array dfrA17-aadA5 was found most prevalent (62.96%). The percentage of positive-integron among the isolates whose resistant profile was relatively broad (n> or =7) is 100.00%, while the one in narrow-profile isolates (n=2-6) is 30.56%. The correlation analysis revealed the incidence of integrons among the isolates were highly related to the resistant profile, indicating integrons play an important role in the dissemination and spread of the antimicrobial resistant strains.
|
1229. |
Bidet P,
Mahjoub-Messai F,
Blanco J,
Blanco J,
Dehem M,
Aujard Y,
Bingen E,
Bonacorsi S,
( 2007 ) Combined multilocus sequence typing and O serogrouping distinguishes Escherichia coli subtypes associated with infant urosepsis and/or meningitis. PMID : 17570118 : DOI : 10.1086/518897 Abstract >>
The genetic relatedness of 223 invasive Escherichia coli strains that cause either meningitis or urosepsis without meningitis in young infants was determined by multilocus sequence typing (MLST), ribotyping, and phylogenetic polymerase chain reaction grouping. We also determined the serotypes and virulence genotypes (on the basis of 11 virulence genes). The strains belonged to 29 sequence type complexes (STc), 20 ribotypes, 26 O serogroups, and 39 virulence genotypes. MLST combined with O serogrouping identified 49 subtypes, or "sequence O types." Some sequence O types were almost exclusively associated with either urosepsis (STc27(O2), STc27(O6), and STc29(O2)) or meningitis (STc29(O18)). In contrast, STc29(O45) was equally frequent in these 2 infection sites. Similarly, several virulence genotypes were specifically associated with one of these syndromes. These results point to the existence of specialized invasive subtypes that cause urosepsis or meningitis in infants and identify a new dually virulent invasive clone.
|
1230. |
Sevim A,
Ozgumus OB,
Celik-Sevim E,
Alpay-Karaoglu S,
Sandalli C,
( 2007 ) Molecular characterization of antibiotic resistant Escherichia coli strains isolated from tap and spring waters in a coastal region in Turkey. PMID : 17978796 : Abstract >>
A hundred and seventeen antibiotic-resistant Escherichia coli strains were isolated from public tap and spring waters which were polluted by fecal coliforms. There were no significant differences between two water sources as to the coliform pollution level (p> 0.05). All E. coli isolates were detected to be resistant to one or more antibiotics tested. Nearly 42% of the isolates showed multiresistant phenotype. Three (2.5%) of these isolates contained class 1 integron. Sequencing analysis of variable regions of the class 1 integrons showed two gene cassette arrays, dfr1-aadA1 and dhfrA17-aadA5. Resistance to ampicillin, tetracycline or trimethoprim-sulfamethoxazole was transferable according to the results of conjugation experiments. The rate of tetracycline resistance was 15%. tet(A)-mediated tetracycline resistance was widespread among tetracycline-resistant E. coli isolates. Genotyping by BOX-polymerase chain reaction (BOX-PCR) showed that some of the strains were epidemiologically related. This is the first report on the prevalence and characterization of class 1 integron-containing E. coli isolates of environmental origin in Turkey.
|
1231. |
Moura A,
Henriques I,
Ribeiro R,
Correia A,
( 2007 ) Prevalence and characterization of integrons from bacteria isolated from a slaughterhouse wastewater treatment plant. PMID : 17913715 : DOI : 10.1093/jac/dkm340 Abstract >>
To investigate the presence and distribution of integron-carrying bacteria from a slaughterhouse wastewater treatment plant (WWTP). Enterobacteriaceae and aeromonads were isolated at different stages of the wastewater treatment process and screened for the presence of integrase genes by dot-blot hybridization. Integrase-positive strains were characterized in terms of phylogenetic affiliation, genetic content of integrons and antimicrobial resistance profiles. Plasmid location of some integrons was established by Southern-blot hybridization. Strains containing integron-carrying plasmids were selected for mating experiments. Integrase genes were present in all samples, including the final effluent. The global prevalence was determined to be 35%, higher than in other aquatic environments. Forty-two integrase-positive isolates were further characterized. Nine distinct cassette arrays were found, containing genes encoding resistance to beta-lactams (bla(OXA-30)), aminoglycosides (aadA1, aadA2, aadA13, aadB), streptothricin (sat1, sat2), trimethoprim (dfrA1, dfrA12), a putative esterase (estX) and a protein with unknown function (orfF). Gene cassette arrays aadA1, dfrAI-aadA1 and estX-sat2-aadA1 were common to aeromonads and Enterobacteriaceae. The class 2 integron containing an estX-sat2-aadA1 cassette array was detected for the first time in Aeromonas sp. Nearly 12% (5 out of 43) of intI genes were located in plasmids. intI genes from isolates MM.1.3 and MM.1.5 were successfully conjugated into Escherichia coli at frequencies of 3.79 x 10(-5) and 5.46 x 10(-5) per recipient cell, respectively. Our data support the hypothesis that WWTPs constitute a potential hot spot for horizontal gene transfer and for selection of antimicrobial resistance genes among aquatic bacteria. Moreover, water discharges represent a possible risk for dissemination of undesirable genetic traits.
|
1232. |
Beutin L,
Miko A,
Krause G,
Pries K,
Haby S,
Steege K,
Albrecht N,
( 2007 ) Identification of human-pathogenic strains of Shiga toxin-producing Escherichia coli from food by a combination of serotyping and molecular typing of Shiga toxin genes. PMID : 17557838 : DOI : 10.1128/AEM.00873-07 PMC : PMC1951031 Abstract >>
We examined 219 Shiga toxin-producing Escherichia coli (STEC) strains from meat, milk, and cheese samples collected in Germany between 2005 and 2006. All strains were investigated for their serotypes and for genetic variants of Shiga toxins 1 and 2 (Stx1 and Stx2). stx(1) or variant genes were detected in 88 (40.2%) strains and stx(2) and variants in 177 (80.8%) strains. Typing of stx genes was performed by stx-specific PCRs and by analysis of restriction fragment length polymorphisms (RFLP) of PCR products. Major genotypes of the Stx1 (stx(1), stx(1c), and stx(1d)) and the Stx2 (stx(2), stx(2d), stx(2-O118), stx(2e), and stx(2g)) families were detected, and multiple types of stx genes coexisted frequently in STEC strains. Only 1.8% of the STEC strains from food belonged to the classical enterohemorrhagic E. coli (EHEC) types O26:H11, O103:H2, and O157:H7, and only 5.0% of the STEC strains from food were positive for the eae gene, which is a virulence trait of classical EHEC. In contrast, 95 (43.4%) of the food-borne STEC strains carried stx(2) and/or mucus-activatable stx(2d) genes, an indicator for potential high virulence of STEC for humans. Most of these strains belonged to serotypes associated with severe illness in humans, such as O22:H8, O91:H21, O113:H21, O174:H2, and O174:H21. stx(2) and stx(2d) STEC strains were found frequently in milk and beef products. Other stx types were associated more frequently with pork (stx(2e)), lamb, and wildlife meat (stx(1c)). The combination of serotyping and stx genotyping was found useful for identification and for assignment of food-borne STEC to groups with potential lower and higher levels of virulence for humans.
|
1233. |
Hümbelin M,
Suri B,
Rao DN,
Hornby DP,
Eberle H,
Pripfl T,
Kenel S,
Bickle TA,
( 1988 ) Type III DNA restriction and modification systems EcoP1 and EcoP15. Nucleotide sequence of the EcoP1 operon, the EcoP15 mod gene and some EcoP1 mod mutants. PMID : 2837577 : DOI : 10.1016/0022-2836(88)90330-0 Abstract >>
This paper presents the nucleotide sequence of the mod-res operon of phage P1, which encodes the two structural genes for the EcoP1 type III restriction and modification system. We have also sequenced the mod gene of the allelic EcoP15 system. The mod gene product is responsible for binding the system-specific DNA recognition sequences in both restriction and modification; it also catalyses the modification reaction. A comparison of the two mod gene product sequences shows that they have conserved amino and carboxyl ends but have completely different sequences in the middle of the molecules. Two alleles of the EcoP1 mod gene that are defective in modification but not in restriction were also sequenced. The mutations in both alleles lie within the non-conserved regions.
|
1234. |
Ruhe ZC,
Nguyen JY,
Xiong J,
Koskiniemi S,
Beck CM,
Perkins BR,
Low DA,
Hayes CS,
( 2017 ) CdiA Effectors Use Modular Receptor-Binding Domains To Recognize Target Bacteria. PMID : 28351921 : DOI : 10.1128/mBio.00290-17 PMC : PMC5371414 Abstract >>
Contact-dependent growth inhibition (CDI) systems encode CdiA effectors, which bind to specific receptors on neighboring bacteria and deliver C-terminal toxin domains to suppress target cell growth. Two classes of CdiA effectors that bind distinct cell surface receptors have been identified, but the molecular basis of receptor specificity is not understood. Alignment of BamA-specific CdiAEC93 from Escherichia coli EC93 and OmpC-specific CdiAEC536 from E. coli 536 suggests that the receptor-binding domain resides within a central region that varies between the two effectors. In support of this hypothesis, we find that CdiAEC93 fragments containing residues Arg1358 to Phe1646 bind specifically to purified BamA. Moreover, chimeric CdiAEC93 that carries the corresponding sequence from CdiAEC536 is endowed with OmpC-binding activity, demonstrating that this region dictates receptor specificity. A survey of E. coli CdiA proteins reveals two additional effector classes, which presumably recognize distinct receptors. Using a genetic approach, we identify the outer membrane nucleoside transporter Tsx as the receptor for a third class of CdiA effectors. Thus, CDI systems exploit multiple outer membrane proteins to identify and engage target cells. These results underscore the modularity of CdiA proteins and suggest that novel effectors can be constructed through genetic recombination to interchange different receptor-binding domains and toxic payloads.IMPORTANCE CdiB/CdiA two-partner secretion proteins mediate interbacterial competition through the delivery of polymorphic toxin domains. This process, known as contact-dependent growth inhibition (CDI), requires stable interactions between the CdiA effector protein and specific receptors on the surface of target bacteria. Here, we localize the receptor-binding domain to the central region of E. coli CdiA. Receptor-binding domains vary between CdiA proteins, and E. coli strains collectively encode at least four distinct effector classes. Further, we show that receptor specificity can be altered by exchanging receptor-binding regions, demonstrating the modularity of this domain. We propose that novel CdiA effectors are naturally generated through genetic recombination to interchange different receptor-binding domains and toxin payloads.
|
1235. |
Shaikh N,
Holt NJ,
Johnson JR,
Tarr PI,
( 2007 ) Fim operon variation in the emergence of Enterohemorrhagic Escherichia coli: an evolutionary and functional analysis. PMID : 17559392 : DOI : 10.1111/j.1574-6968.2007.00781.x Abstract >>
Fim operons were examined to illuminate the emergence of Escherichia coli O157:H7 from the less-virulent E. coli O55:H7. A fim invertible element deletion occurred only after O157:H7 descended from O55:H7, and after sorbitol nonfermenting O157 diverged. Type 1 pili nonexpression correlates with this deletion in all enterohemorrhagic E. coli (EHEC) tested. An N135K FimH mutation in the two most evolved O157:H7 clusters is not found in other EHEC. These data refine the evolutionary history of an emerging pathogen.
|
1236. |
Creuzburg K,
Schmidt H,
( 2007 ) Molecular characterization and distribution of genes encoding members of the type III effector nleA family among pathogenic Escherichia coli strains. PMID : 17553972 : DOI : 10.1128/JCM.00038-07 PMC : PMC1951211 Abstract >>
In this study, we investigated the occurrence of the previously described gene nleA(4795) and variants of nleA, putatively encoding non-locus-of-enterocyte-effacement-encoded type III effector proteins with functions that are unknown. nleA variants were detected in 150 out of 170 enteropathogenic Escherichia coli strains and enterohemorrhagic E. coli strains, two of them being eae negative. Besides the known variants nleA(4795), Z6024, and the espI-like gene, 11 novel nleA variants with different lengths and sequence identities at the deduced amino acid level (between 71% and 96%) have been identified. Whereas most of the serogroups associated with more severe disease were quite homogenous with respect to the presence of a particular nleA variant, other serogroups were not. Moreover, Southern blot hybridization revealed that certain strains carry two copies of nleA in their chromosome, frequently encoding different variants. In most cases, the open reading frame of one of the copies was disrupted, usually by an insertion element. Furthermore, transmission of the type III effector-encoding gene could be shown by transduction of nleA-carrying bacteriophages to a laboratory E. coli strain.
|
1237. |
Seiffert SN,
Carattoli A,
Schwendener S,
Collaud A,
Endimiani A,
Perreten V,
( 2017 ) Plasmids Carrying blaCMY -2/4 in Escherichia coli from Poultry, Poultry Meat, and Humans Belong to a Novel IncK Subgroup Designated IncK2. PMID : 28360894 : DOI : 10.3389/fmicb.2017.00407 PMC : PMC5350095 Abstract >>
The blaCMY -2/4-carrying IncB/O/K-like plasmids of seven Escherichia coli strains from poultry, poultry meat and human urine samples were examined using comparative analysis of whole plasmid sequences. The incompatibility group was determined by analysis of the incRNAI region and conjugation assays with strains containing the IncK and IncB/O reference plasmids. Strains were additionally characterized using MLST and MIC determination. The complete DNA sequences of all plasmids showed an average nucleotide identity of 91.3%. Plasmids were detected in E. coli sequence type (ST) 131, ST38, ST420, ST1431, ST1564 and belonged to a new plasmid variant (IncK2) within the IncK and IncB/O groups. Notably, one E. coli from poultry meat and one from human contained the same plasmid. The presence of a common recently recognized IncK2 plasmid in diverse E. coli from human urine isolates and poultry meat production suggests that the IncK2 plasmids originated from a common progenitor and have the capability to spread to genetically diverse E. coli in different reservoirs. This discovery is alarming and stresses the need of rapidly introducing strict hygiene measures throughout the food chain, limiting the spread of such plasmids in the human settings.
|
1238. |
Båga M,
Norgren M,
Normark S,
( 1987 ) Biogenesis of E. coli Pap pili: papH, a minor pilin subunit involved in cell anchoring and length modulation. PMID : 2882856 : DOI : 10.1016/0092-8674(87)90565-4 Abstract >>
The biogenesis of Escherichia coli Pap pili, encoded by the pap gene cluster, was studied. A novel gene, papH, was identified and found to encode a weakly expressed pilin-like protein. PapH was dispensable for digalactoside-specific binding and for formation of Pap pili. However, in papH deletion mutants 50%-70% of total pilus antigen was found free of the cells. We present evidence showing coregulation of papH and the adjacent gene, papA, which encodes the major pilin subunit. A decrease in the PapA to PapH ratio resulted in a large fraction of cells producing shortened pili, whereas overproduction of PapA relative to PapH resulted in cells with lengthened pili. The data show that PapH has roles in anchoring the pilus to the cell and in modulating pilus length.
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1239. |
Bitar I,
Piazza A,
Gaiarsa S,
Villa L,
Pedroni P,
Oliva E,
Nucleo E,
Pagani L,
Carattoli A,
Migliavacca R,
( 2017 ) ST405 NDM-5 producing Escherichia coli in Northern Italy: the first two clinical cases. PMID : 28159670 : DOI : 10.1016/j.cmi.2017.01.020 Abstract >>
N/A
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1240. |
Shinoda S,
Matsuoka H,
Tsuchie T,
Miyoshi S,
Yamamoto S,
Taniguchi H,
Mizuguchi Y,
( 1991 ) Purification and characterization of a lecithin-dependent haemolysin from Escherichia coli transformed by a Vibrio parahaemolyticus gene. PMID : 1791426 : DOI : 10.1099/00221287-137-12-2705 Abstract >>
Lecithin-dependent haemolysin (LDH) of Vibrio parahaemolyticus was purified from Escherichia coli C600 transformed with a plasmid (pHL591) ligated with a 1.5 kb DNA fragment of V. parahaemolyticus. The final preparation comprised two LDH proteins with different molecular masses which were immunologically cross-reactive and had the same enzymic activity. The LDH was a phospholipase hydrolysing both fatty acid esters of phospholipid, i.e. it hydrolysed phosphatidylcholine (PC) to lysophosphatidylcholine (LPC) and then LPC to glycerophosphorylcholine (GPC). From this point of view, LDH should be classified as a phospholipase B. Phospholipase B, however, does not usually show haemolytic activity, because the intermediate (LPC), which is the actual haemolytic agent, is immediately hydrolysed to the final product (GPC). On the other hand, LPC formed by LDH action was comparatively stable, because the rates of the two reactions catalysed by LDH, PC to LPC and LPC to GPC, are almost the same. This is the reason that LDH shows haemolytic activity. Therefore, LDH of V. parahaemolyticus is an atypical phospholipase to be designated as phospholipase A2/lysophospholipase.
|
1241. |
Inamoto S,
Yoshioka Y,
Ohtsubo E,
( 1988 ) Identification and characterization of the products from the traJ and traY genes of plasmid R100. PMID : 2836369 : DOI : 10.1128/jb.170.6.2749-2757.1988 PMC : PMC211198 Abstract >>
The nucleotide sequence of part of the tra region of R100 including traJ and traY was determined, and the products of several tra genes were identified. The nucleotide sequence of traJ, encoding a protein of 223 amino acids, showed poor homology with the corresponding segments of other plasmids related to R100, but the deduced amino acid sequences showed low but significant homology. The first four amino acids at the N-terminal region of the TraJ protein were not essential for positive regulation of expression of traY, the first gene of the traYZ operon. The nucleotide sequence of traY shows that this gene may use TTG as the initiation codon and that it encodes a protein of 75 amino acids. Analysis of the traY gene product, which was obtained as the fusion protein with beta-galactosidase, showed that the N-terminal region of the product has an amino acid sequence identical to that deduced from the assigned frame but lacks formylmethionine. traY of plasmid F, which encodes a larger protein than the TraY protein of R100, is thought to use ATG as an initiation codon. However, a TTG initiation codon was found in the preceding region of the previously assigned traY coding frame of F. Interestingly, when translation of traY of F was initiated from TTG, the amino acid sequence homologous to the TraY protein of R100 appeared in tandem in the TraY protein of F. This may suggest that traY of F has undergone duplication of a gene like the traY gene of R100.
|
1242. |
Pansegrau W,
Miele L,
Lurz R,
Lanka E,
( 1987 ) Nucleotide sequence of the kanamycin resistance determinant of plasmid RP4: homology to other aminoglycoside 3'-phosphotransferases. PMID : 2832861 : Abstract >>
The kanamycin resistance determinant of the broad-host-range plasmid RP4 encodes an aminoglycoside 3'-phosphotransferase of type I. The nucleotide sequence of the kanamycin resistance gene (Kmr) and the right end of the insertion element IS8 of plasmid RP4 has been determined. The gene (816 bp) is located between IS8 and the region (Tra 1) encoding plasmid factors mediating bacterial conjugation. Kmr and Tra 1 are transcribed toward each other. The nucleotide sequence has been compared to five related aphA genes originating from gram-negative and gram-positive organisms and from antibiotic producers. Among these that of Tn903 shares the highest degree of similarity (60%) with the RP4 gene. Significant similarities were also detected between the amino acid sequences of the six enzymes. The C-terminal domains of six different aminoglycoside 3'-phosphotransferases (APH(3'] are highly conserved. They are substantially similar to segments of a variety of enzymes using ATP as cofactor. The role of the C-terminal sequences of APH(3') as potential domains for ATP recognition and binding is discussed.
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1243. |
Singh T,
Das S,
Ramachandran VG,
Wani S,
Shah D,
Maroof KA,
Sharma A,
( 2017 ) Distribution of Integrons and Phylogenetic Groups among Enteropathogenic Escherichia coli Isolates from Children <5 Years of Age in Delhi, India. PMID : 28443072 : DOI : 10.3389/fmicb.2017.00561 PMC : PMC5385330 Abstract >>
Integrons by means of horizontal gene transfer carry multidrug resistance genes (MDR) among bacteria, including E. coli. The aim of this study was to determine the antibiotic resistance profiles and the genes associated with them, to gain insights in the distribution of phylogroups, prevalence, and characterization of class 1, 2 and 3 integrons among Enteropathogenic E. coli (EPEC) isolates, from children upto 5 years of age from Delhi and National Capital Region (NCR), India. A total of 120 E. coli isolates, including 80 from diarrheagenic E. coli (cases) and 40 from healthy isolates (controls) were recruited in this study. After isolation of E. coli, screening for EPEC was done by conventional multiplex PCR. Antibiotic suseptibility test was performed using disk diffusion method and further confirmed by minimum inhibitory concentration (MICs) by E-test. The presence and characterization of integrons and antimicrobial resistance genes were performed by PCR and DNA sequencing. Phylogeny determination was carried out by quadruplex PCR. EPEC strains were found in 64 of the 80 diarrheagenic cases, out of which 38 were MDR. In the 40 healthy controls, 23 were found to be EPEC strain, out of which only 2 were MDR. Amongst 80 diarrheagenic cases, class 1 integron were observed in 43 isolates, class 2 integron in 12 isolates and 9 isolates were found with co-existence of both. Similarly, in healthy controls; class 1 integron in 9 and class 2 integron in 7 isolates were observed with co-existence in 3 isolates. None of the isolates included class 3 integron. The dfr was the most commonly identified gene cassette within the integron-positive isolates. Phylogenetic studies showed considerable representation of phylogroup B2 in both diarrheagenic cases and healthy controls. This study reiterates the importance of class 1 integron predominantly for acquisition of antibiotic resistance genes among EPEC isolates. Furthermore, it also ascertains the possible association between multidrug resistance and presence of integrons. Approximately 91% of isolates were easily assigned to their respective phylogroups. Assessment of the relationship between antibiotic resistance and dominant phylogroups detected was also attempted. This study also highlights the increased burden of antimicrobial resistance in healthy controls.
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1244. |
Zahrl D,
Wagner A,
Tscherner M,
Koraimann G,
( 2007 ) GroEL plays a central role in stress-induced negative regulation of bacterial conjugation by promoting proteolytic degradation of the activator protein TraJ. PMID : 17586648 : DOI : 10.1128/JB.00005-07 PMC : PMC1952051 Abstract >>
Transcription of DNA transfer genes is a prerequisite for conjugative DNA transfer of F-like plasmids. Transfer gene expression is sensed by the donor cell and is regulated by a complex network of plasmid- and host-encoded factors. In this study we analyzed the effect of induction of the heat shock regulon on transfer gene expression and DNA transfer in Escherichia coli. Raising the growth temperature from 22 degrees C to 43 degrees C transiently reduced transfer gene expression to undetectable levels and reduced conjugative transfer by 2 to 3 orders of magnitude. In contrast, when host cells carried the temperature-sensitive groEL44 allele, heat shock-mediated repression was alleviated. These data implied that the chaperonin GroEL was involved in negative regulation after heat shock. Investigation of the role of GroEL in this regulatory process revealed that, in groEL(Ts) cells, TraJ, the plasmid-encoded master activator of type IV secretion (T4S) system genes, was less susceptible to proteolysis and had a prolonged half-life compared to isogenic wild-type E. coli cells. This result suggested a direct role for GroEL in proteolysis of TraJ, down-regulation of T4S system gene expression, and conjugation after heat shock. Strong support for this novel role for GroEL in regulation of bacterial conjugation was the finding that GroEL specifically interacted with TraJ in vivo. Our results further suggested that in wild-type cells this interaction was followed by rapid degradation of TraJ whereas in groEL(Ts) cells TraJ remained trapped in the temperature-sensitive GroEL protein and thus was not amenable to proteolysis.
|
1245. |
Fong DH,
Burk DL,
Blanchet J,
Yan AY,
Berghuis AM,
( 2017 ) Structural Basis for Kinase-Mediated Macrolide Antibiotic Resistance. PMID : 28416110 : DOI : 10.1016/j.str.2017.03.007 Abstract >>
The macrolides are a class of antibiotic, characterized by a large macrocyclic lactone ring that can be inactivated by macrolide phosphotransferase enzymes. We present structures for MPH(2')-I and MPH(2')-II in the apo state, and in complex with GTP analogs and six different macrolides. These represent the first structures from the two main classes of macrolide phosphotransferases. The structures show that the enzymes are related to the aminoglycoside phosphotransferases, but are distinguished from them by the presence of a large interdomain linker that contributes to an expanded antibiotic binding pocket. This pocket is largely hydrophobic, with a negatively charged patch located at a conserved aspartate residue, rationalizing the broad-spectrum resistance conferred by the enzymes. Complementary mutation studies provide insights into factors governing substrate specificity. A comparison with macrolides bound to their natural target, the 50S ribosome, suggests avenues for next-generation antibiotic development.
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1246. |
Hii JH,
Gyles C,
Morooka T,
Karmali MA,
Clarke R,
De Grandis S,
Brunton JL,
( 1991 ) Development of verotoxin 2- and verotoxin 2 variant (VT2v)-specific oligonucleotide probes on the basis of the nucleotide sequence of the B cistron of VT2v from Escherichia coli E32511 and B2F1. PMID : 1757536 : PMC : PMC270418 Abstract >>
We and others have noted that there are serological differences between verotoxin 2 (VT2) (also known as Shiga-like toxin II) produced by Escherichia coli C600(933W) and the VT2 variant (VT2v) produced by strain E32511. Recent reports have described nucleotide sequence differences between the VT2v B subunit cistron of E32511 and B2F1 and that of VT2. We have confirmed the sequence differences and have used them to design oligonucleotide probes which differentiate the B subunit cistron of VT2v from that of VT2. Isolates of VT-producing E. coli obtained from human as well as food and veterinary sources were classified according to the toxin phenotype by using a toxin neutralization assay with VT2-specific monoclonal antibody and VT2v-specific polyclonal antisera. Using the oligonucleotide probes in colony hybridization, we detected 35 of 35 VT2 producers and 16 of 16 VT2v producers. One VT2 producer was falsely identified as containing the VT2v gene. The E32511 strain in our collection hybridized only with the VT2-specific probe. Southern hybridization of radiolabeled oligonucleotide probes showed that strains carried zero to one copy of the VT2 gene and zero to two copies of the VT2v gene. We conclude that colony hybridization with the VT2- and VT2-specific probes is highly predictive of the toxin phenotypes in the clinical isolates described in this study.
|
1247. |
Rea D,
Fülöp V,
Bugg TD,
Roper DI,
( 2007 ) Structure and mechanism of HpcH: a metal ion dependent class II aldolase from the homoprotocatechuate degradation pathway of Escherichia coli. PMID : 17881002 : DOI : 10.1016/j.jmb.2007.06.048 Abstract >>
Microorganisms are adept at degrading chemically resistant aromatic compounds. One of the longest and most well characterized aromatic catabolic pathways is the 4-hydroxyphenylacetic acid degradation pathway of Escherichia coli. The final step involves the conversion of 4-hydroxy-2-oxo-heptane-1,7-dioate into pyruvate and succinic semialdehyde. This reaction is catalyzed by 4-hydroxy-2-oxo-heptane-1,7-dioate aldolase (HpcH), a member of the divalent metal ion dependent class II aldolase enzymes that have great biosynthetic potential. We have solved the crystal structure of HpcH in the apo form, and with magnesium and the substrate analogue oxamate bound, to 1.6 A and 2.0 A, respectively. Comparison with similar structures of the homologous 2-dehydro-3-deoxygalactarate aldolase, coupled with site-directed mutagenesis data, implicate histidine 45 and arginine 70 as key catalytic residues.
|
1248. |
Izumi A,
Rea D,
Adachi T,
Unzai S,
Park SY,
Roper DI,
Tame JR,
( 2007 ) Structure and mechanism of HpcG, a hydratase in the homoprotocatechuate degradation pathway of Escherichia coli. PMID : 17559873 : DOI : 10.1016/j.jmb.2007.05.006 Abstract >>
HpcG catalyses the hydration of a carbon-carbon double bond without the aid of any cofactor other than a simple divalent metal ion such as Mg(2+). Since the substrate has a nearby carbonyl group, it is believed that it first isomerises to form a pair of conjugated double bonds in the enol tautomer before Michael addition of water. Previous chemical studies of the reaction, and that of the related enzyme MhpD, have failed to provide a clear picture of the mechanism. The substrate itself is unstable, preventing co-crystallisation or soaking of crystals, but oxalate is a strong competitive inhibitor. We have solved the crystal structure of the protein in the apo form, and with magnesium and oxalate bound. Modelling substrate into the active site suggests the attacking water molecule is not part of the metal coordination shell, in contrast to a previous proposal. Our model suggests that geometrically strained cis isomer intermediates do not lie on the reaction pathway, and that separate groups are involved in the isomerisation and hydration steps.
|
1249. |
Pugsley AP,
( 1987 ) Nucleotide sequencing of the structural gene for colicin N reveals homology between the catalytic, C-terminal domains of colicins A and N. PMID : 2834623 : DOI : 10.1111/j.1365-2958.1987.tb01938.x Abstract >>
An 1800 bp fragment of DNA from a natural ColN plasmid (pCHAP4) encompassing the colicin N structural gene (cna) and its regulatory region was subjected to nucleotide sequencing and deletion analysis. The region of DNA immediately upstream from cna contains two tandemly-arranged and overlapping potential LexA binding sites (SOS boxes), in line with the previous demonstration that cna expression is repressed by LexA protein. Deletion of the LexA binding site allowed efficient transcription of cna from an upstream lacZ promoter, whereas its presence reduced lacZ-promoted cna expression to varying extents depending on the proximity of lacZp and the SOS boxes. The molecular weight of colicin N, as deduced from the nucleotide sequence, is 41,696, which is close to the experimentally determined molecular weight of 39,000. Colicin N has a glycine-rich amino terminus similar to that found in many other colicins. Part of the glycine-rich domain of colicin N could be replaced by an unrelated sequence devoid of glycine residues without affecting either colicin release or activity. The carboxy-terminal half of colicin N exhibits significant homology to the C-terminus of colicin A. The latter colicin forms pores in the cytoplasmic membrane of Escherichia coli, thereby depolarizing the membrane and causing cell death. The C-terminus of colicin A is endowed with this catalytic activity. Although colicin N was previously found to cause lysis of Escherichia coli cells, a more detailed investigation revealed that it too depolarizes the Escherichia coli cytoplasmic membrane and that lysis is a secondary effect.
|
1250. |
Dodd HM,
Bennett PM,
( 1987 ) The R46 site-specific recombination system is a homologue of the Tn3 and gamma delta (Tn1000) cointegrate resolution system. PMID : 2832517 : DOI : 10.1099/00221287-133-8-2031 Abstract >>
The nucleotide sequence of the R46 site-specific recombination system has been determined. The organization of the recombination gene (perR46) and the site at which it acts (per site), together with the extensive sequence homology displayed with the tnpR genes and res sites of the transposons Tn3 and gamma delta (Tn1000), suggests that they have been derived from a Tn3-like element. These site-specific recombination functions of R46 play a role in plasmid maintenance.
|
1251. |
Moran RA,
Anantham S,
Holt KE,
Hall RM,
( 2017 ) Prediction of antibiotic resistance from antibiotic resistance genes detected in antibiotic-resistant commensal Escherichia coli using PCR or WGS. PMID : 28039273 : DOI : 10.1093/jac/dkw511 Abstract >>
To assess the effectiveness of bioinformatic detection of resistance genes in whole-genome sequences in correctly predicting resistance phenotypes. Genomes of a collection of well-characterized commensal Escherichia coli were sequenced using Illumina HiSeq technology and assembled with SPAdes. Antibiotic resistance genes identified by PCR, SRST2 analysis of reads and ResFinder analysis of SPAdes assemblies were compared with known resistance phenotypes. Generally, the antibiotic resistance genes detected using bioinformatic methods were concordant, but only ARG-ANNOT included sat2 . However, the presence or absence of genes was not always predictive of the phenotype. In one strain, trimethoprim resistance was due to a known mutation in the chromosomal folA gene. In cases where the copy number was low, the aadA5 gene downstream of dfrA17 did not confer streptomycin or spectinomycin resistance. Resistance genes were found in the genomes that were not detected previously by PCRs targeting a limited gene set and gene cassettes in class 1 or class 2 integrons. In one isolate, the aadA1 gene cassette in the estX - aadA1 cassettes pair was outside an integron context and was not expressed. The qnrS1 gene, conferring reduced susceptibility to fluoroquinolones, and the bla CMY-2 gene, encoding an ESBL, were each detected in a single isolate and mphA (macrolide resistance) was present in six isolates surrounded by IS 26 and IS 6100 . WGS analysis detected more genes than PCR. Some were not expressed, causing inconsistencies with the experimentally determined phenotype. An unpredicted chromosomal folA mutation causing trimethoprim resistance was found.
|
1252. |
Almakki A,
Maure A,
Pantel A,
Romano-Bertrand S,
Masnou A,
Marchandin H,
Jumas-Bilak E,
Licznar-Fajardo P,
( 2017 ) NDM-5-producing Escherichia coli in an urban river in Montpellier, France. PMID : 28435018 : DOI : 10.1016/j.ijantimicag.2017.04.003 Abstract >>
N/A
|
1253. |
Shinagawa H,
Makino K,
Amemura M,
Kimura S,
Iwasaki H,
Nakata A,
( 1988 ) Structure and regulation of the Escherichia coli ruv operon involved in DNA repair and recombination. PMID : 2842314 : DOI : 10.1128/jb.170.9.4322-4329.1988 PMC : PMC211445 Abstract >>
The ruv gene of Escherichia coli, which is involved in DNA repair and recombination, was cloned on a plasmid vector. The DNA of the ruv region was sequenced; it had two open reading frames in tandem that could code for 22- and 37-kilodalton proteins. The proteins encoded by these open reading frames were identified by the maxicell method. The two genes were aligned in the same orientation and regulated by the SOS system, so the two genes probably constitute an operon. The distal one complemented the ruv mutations. Transcription of the operon was studied both in vivo and in vitro. Two transcription initiation sites were identified upstream of the coding frames, and the transcription from both sites was repressed by the LexA repressor. A DNA sequence that is homologous to the SOS box and bound by LexA protein was found in the regulatory region of the operon. The amino acid sequence of Ruv protein deduced from the DNA sequence shows a high degree of homology to the consensus sequence shared by ATP-binding proteins.
|
1254. |
Alonso CA,
Michael GB,
Li J,
Somalo S,
Simón C,
Wang Y,
Kaspar H,
Kadlec K,
Torres C,
Schwarz S,
( 2017 ) Analysis of blaSHV-12-carrying Escherichia coli clones and plasmids from human, animal and food sources. PMID : 28333184 : DOI : 10.1093/jac/dkx024 Abstract >>
This study aimed at characterizing 23 Escherichia coli isolates from various sources and their respective bla SHV-12 -carrying plasmids and sequencing one of these plasmids completely. Isolates were typed by XbaI-PFGE, MLST and PCR-based phylotyping. Transformed bla SHV-12 -carrying plasmids were examined by replicon typing, S1-nuclease, conjugation, EcoRI-HindIII-BamHI digests and plasmid MLST. Co-located resistance genes and integrons as well as the bla SHV-12 genetic environment were analysed by PCR and sequencing. One IncI1 plasmid was sequenced completely using HiSeq 2500 and gap closure by PCRs and Sanger sequencing. Among the 23 SHV-12-positive E. coli , some isolates from different sources showed the same characteristics: ST23/phylogroup A (human, dog, livestock), ST57/D (wild bird, chicken meat) and ST117/D (chicken meat, chicken). All bla SHV-12 genes were horizontally transferable via 30-120 kb plasmids of incompatibility groups IncI1 (n = 17), IncK (n = 3), IncF (n = 1), IncX3 (n = 1) and a non-typeable plasmid. IncK plasmids, indistinguishable in size and restriction patterns, were found in isolates from different sources (ST57/D, meat; ST131/B2, meat; ST57/B1, dog). The IncI1- bla SHV-12 -carrying plasmids were mostly assigned to plasmid ST (pST) 26 and pST3. Three plasmids showed novel pSTs (pST214, pST215). The majority of the IncI1 transformants exhibited resistance to �]-lactams, chloramphenicol and streptomycin (in relation with a class 1 integron containing an estX - psp - aadA2 - cmlA1 - aadA1 - qacI gene cassette array), and to tetracycline. A novel bla SHV-12 environment was detected and whole plasmid sequencing revealed a Tn 21 -derived- bla SHV12 -�GTn 1721 resistance complex. Results from this study suggest that the dissemination of bla SHV-12 genes occurs by vertical (clonal) and horizontal transfer, the latter mainly mediated through IncI1 multidrug-resistance plasmids.
|
1255. |
Pickett CL,
Weinstein DL,
Holmes RK,
( 1987 ) Genetics of type IIa heat-labile enterotoxin of Escherichia coli: operon fusions, nucleotide sequence, and hybridization studies. PMID : 2822667 : DOI : 10.1128/jb.169.11.5180-5187.1987 PMC : PMC213924 Abstract >>
Operon fusions for the Escherichia coli heat-labile enterotoxin type IIa (LT-IIa) operon were isolated and characterized. The LT-IIa genes are organized in a transcriptional unit similar to those of cholera toxin (CT) and the closely related E. coli heat-labile toxin type I (LT-I, with subtypes LTh-I and LTp-I). The nucleotide sequence of the LT-IIa genes was determined and compared with the sequences of LTh-I and CT. The A subunit gene of LT-IIa was found to be 57% homologous with the A subunit gene of LTh-I and 55% homologous with the A gene of CT. Most of the homology derived from the region of the A gene which encodes the A1 fragment. The B gene of LT-IIa was not homologous with the B gene of LTh-I or CT. DNA probes containing various portions of the LT-IIa genes and adjacent sequences were used for hybridization studies with restriction endonuclease fragments of DNA from a collection of LT-II-producing strains. These studies showed that a probe containing much of the A subunit gene hybridized well to DNA from the various strains, but a probe for the B subunit gene did not.
|
1256. |
Ouellette M,
Bissonnette L,
Roy PH,
( 1987 ) Precise insertion of antibiotic resistance determinants into Tn21-like transposons: nucleotide sequence of the OXA-1 beta-lactamase gene. PMID : 2823258 : DOI : 10.1073/pnas.84.21.7378 PMC : PMC299299 Abstract >>
Several plasmid-encoded beta-lactamases are on multiresistance transposable elements. The OXA-1 beta-lactamase gene is part of Tn2603, which is borne on the R plasmid RGN238. We report here the complete nucleotide sequence of the OXA-1 beta-lactamase gene and flanking sequences. The OXA-1 gene shows a greater than 50% sequence divergence from the OXA-2 gene, yet there is significant functional similarity at the peptide level. Analysis of 5' and 3' flanking sequences shows that Tn2603 differs from its probable precursor, Tn21, by a precise 1004-base-pair insertion, containing the OXA-1 structural gene, at the target sequence AAAGTT, which is located between the Tn21 streptomycin/spectinomycin (aadA) promoter and its structural gene. A 5- for 6-base repeat of the target sequence is found at the end of the insertion. The same precise insertion and repeat of the target sequence are found for the OXA-2 gene from R46. The 5' flanking regions of two other genes, the trimethoprim-resistance gene from R388 and the gentamicin resistance (aadB) gene from pDGO100, are greater than 98% homologous to the 5' flanking sequences of the OXA-1, OXA-2, and aadA genes until they diverge at the target sequence. From the available sequence data a recombinational hot spot is defined at the nucleotide level 5' of the aadA gene of Tn21, and a second potential hot spot is proposed 3' of this gene.
|
1257. |
Wang J,
Guo ZW,
Zhi CP,
Yang T,
Zhao JJ,
Chen XJ,
Zeng L,
Lv LC,
Zeng ZL,
Liu JH,
( 2017 ) Impact of plasmid-borne oqxAB on the development of fluoroquinolone resistance and bacterial fitness in Escherichia coli. PMID : 28160469 : DOI : 10.1093/jac/dkw576 Abstract >>
To investigate the impact of plasmid-borne oqxAB genes on the development of fluoroquinolone resistance, mutations and bacterial fitness in Escherichia coli . MICs and mutation prevention concentrations were compared among E. coli strain TOP10 and two corresponding transformants harbouring the OqxAB-encoding plasmids. Mutants were selected by serial passages with the 0.5-fold MIC of ciprofloxacin, and were randomly selected to determine mutations. Bacterial fitness was evaluated by competition assays in vitro and in vivo . The oqxAB -carrying plasmids contributed to a 4-8-fold increase in the ciprofloxacin MIC and increased the ciprofloxacin mutation prevention concentration by 8-16-fold. The MIC of ciprofloxacin for the two transformants increased faster than that of E. coli TOP10 by serial passaging. Novel mutations in gyrB (A468P or F458V) were first observed. Mutations in gyrA were distributed at codons 87 and 83 in the two transformants, whereas mutation A119E in gyrA dominated in the TOP10 mutants. Although the two oqxAB -bearing plasmids caused a decrease in fitness in vitro , their fitness increased when combined with more than one chromosomal mutation, and clear biological benefits were observed in vivo . The mutations in gyrB were associated with a fitness cost, which could be compensated for by additional mutations. The novel mutation gyrA �GS83 significantly reduced biological fitness both in vitro and in vivo , and was thus quickly replaced by more beneficial mutations in the population. The possession of plasmid-borne oqxAB may facilitate the evolution of fluoroquinolone resistance, and the fitness cost of OqxAB-encoding plasmids could be compensated by additional chromosomal mutations.
|
1258. |
Chang K,
Aaronson W,
Sutton A,
Finn CW,
Lindner W,
Kotsatos M,
Vann WF,
Silver RP,
Abeijon C,
( 1987 ) Purification, properties, and genetic location of Escherichia coli cytidine 5'-monophosphate N-acetylneuraminic acid synthetase. PMID : 2826425 : Abstract >>
N-Acetylneuraminic acid cytidylyltransferase (EC 2.7.7.43) (CMP-NeuAc synthetase) catalyzes the formation of cytidine monophosphate N-acetylneuraminic acid. We have purified CMP-NeuAc synthetase from an Escherichia coli O18:K1 cytoplasmic fraction to apparent homogeneity by ion exchange chromatography and affinity chromatography on CDP-ethanolamine linked to agarose. The enzyme has a specific activity of 2.1 mumol/mg/min and migrates as a single protein and activity band on nondenaturing polyacrylamide gel electrophoresis. The enzyme has a requirement for Mg2+ or Mn2+ and exhibits optimal activity between pH 9.0 and 10. The apparent Michaelis constants for the CTP and NeuAc are 0.31 and 4 mM, respectively. The CTP analogues 5-mercuri-CTP and CTP-2',3'-dialdehyde are inhibitors. The purified CMP-N-acetylneuraminic acid synthetase has a molecular weight of approximately 50,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The gene encoding CMP-N-acetylneuraminic acid synthetase is located on a 3.3-kilobase HindIII fragment. The purified enzyme appears to be identical to the 50,000 Mr polypeptide encoded by this gene based on insertion mutations that result in the loss of detectable enzymatic activity. The amino-terminal sequence of the purified protein was used to locate the start codon for the CMP-NeuAc synthetase gene. Both the enzyme and the 50,000 Mr polypeptide have the same NH2-terminal amino acid sequence. Antibodies prepared to a peptide derived from the NH2-terminal amino acid sequence bind to purified CMP-NeuAc synthetase.
|
1259. |
Turner AK,
Grinsted J,
( 1987 ) DNA sequence of the transposase gene of the new category of class II transposon, Tn2501. PMID : 2827105 : DOI : 10.1093/nar/15.23.10049 PMC : PMC306554 Abstract >>
N/A
|
1260. |
Liu Z,
Wang Y,
Walsh TR,
Liu D,
Shen Z,
Zhang R,
Yin W,
Yao H,
Li J,
Shen J,
( 2017 ) Plasmid-Mediated Novel blaNDM-17 Gene Encoding a Carbapenemase with Enhanced Activity in a Sequence Type 48 Escherichia coli Strain. PMID : 28242668 : DOI : 10.1128/AAC.02233-16 PMC : PMC5404555 Abstract >>
Carbapenem-resistant Enterobacteriaceae (CRE) have spread worldwide, leaving very few treatment options available. New Delhi metallo-beta-lactamase (NDM) is the main carbapenemase mediating CRE resistance and is of increasing concern. NDM-positive Enterobacteriaceae of human origin are frequently identified; however, the emergence of NDM, and particularly novel variants, in bacteria of food animal origin has never been reported. Here, we characterize a novel NDM variant (assigned NDM-17) identified in a �]-lactam-resistant sequence type 48 (ST48) Escherichia coli strain that was isolated from a chicken in China. Compared to NDM-1, NDM-17 had three amino acid substitutions (V88L, M154L, and E170K) that confer significantly enhanced carbapenemase activity. Compared to NDM-5, NDM-17 had only one amino acid substitution (E170K) and slightly increased isolate resistance to carbapenem, as indicated by increased MIC values. The gene encoding NDM-17 (blaNDM-17) was located on an IncX3 plasmid, which was readily transferrable to recipient E. coli strain J53 by conjugation, suggesting the possibility of the rapid dissemination of blaNDM-17 Enzyme kinetics showed that NDM-17 could hydrolyze all �]-lactams tested, except for aztreonam, and had a significantly higher affinity for all �]-lactams tested than did NDM-5. The emergence of this novel NDM variant could pose a threat to public health because of its transferability and enhanced carbapenemase activity.
|
1261. |
Brisson-Noël A,
Arthur M,
Courvalin P,
( 1988 ) Evidence for natural gene transfer from gram-positive cocci to Escherichia coli. PMID : 2832378 : DOI : 10.1128/jb.170.4.1739-1745.1988 PMC : PMC211025 Abstract >>
High-level resistance to macrolide-lincosamide-streptogramin type B (MLS) antibiotics in Escherichia coli BM2570 is due to the presence on the conjugative plasmid pIP1527 of the MLS resistance determinant ermBC, which is almost identical to the erm genes previously described in plasmid pAM77 from Streptococcus sanguis (ermAM) and in transposon Tn917 from Enterococcus faecalis (ermB). This gene and its regulatory region are located downstream from the insertion sequence IS1. The 23S rRNA methylase encoded by pIP1527 differs by three and six amino acids from those encoded by Tn917 and pAM77, respectively. Unlike the streptococcal elements which confer the inducible MLS phenotype, the ermBC gene is expressed constitutively in E. coli and Bacillus subtilis, due to several mutations in the regulatory region. Transcription of the ermBC gene starts from three different sites following three overlapping promoters which function in both E. coli and B. subtilis. Promoters P2 and P3 are located within the region homologous to pAM77 and Tn917, and P1 is a hybrid promoter constituted by -35 and -10 sequences located at the end of IS15 and in the streptococcal region, respectively. These results constitute evidence for the recent in vivo transfer from Streptococcus spp. to E. coli. This transfer could have been mediated by transposons such as Tn917 or Tn1545 from Streptococcus pneumoniae, which also bears an MLS determinant that is homologous to ermB. We speculate that the insertion sequences IS15 and IS1 could have played a role in the expression and dissemination of ermBC, which has been found in numerous strains of enterobacteria.
|
1262. |
Sun J,
Fang LX,
Wu Z,
Deng H,
Yang RS,
Li XP,
Li SM,
Liao XP,
Feng Y,
Liu YH,
( 2017 ) Genetic Analysis of the IncX4 Plasmids: Implications for a Unique Pattern in the mcr-1 Acquisition. PMID : 28336940 : DOI : 10.1038/s41598-017-00095-x PMC : PMC5428312 Abstract >>
IncX4 plasmids are associated with the dissemination of the mcr-1 genes in Enterobacteriaceae. We screened IncX4 plasmids among 2,470 isolates of Enterobacteriaceae and determined the mcr-1 positive isolates. Forty-three isolates were observed to carry IncX4 type plasmid, among which 13 were identified to carry mcr-1 gene. Three representative mcr-1-positive IncX4 plasmids were selected for high-throughput sequencing. Comparative genomics showed that the mcr-1-carrying IncX4 plasmids exhibit remarkable similarity in the backbone, and the major distinction lies in the region containing mcr-1. The major variable regions of all the IncX4 plasmids were fully characterized by PCR-RFLP. The results revealed that the mcr-1 was located on the Variable Region I of IncX4 plasmids in 11 E. coli isolates. Among them, nine E. coli strains possess an epidemic pCSZ4-like IncX4 plasmid containing mcr-1. ISApl1 was presumably involved in the transposition of the mcr-1 cassette and then was lost. Similar genetic contexts were found in different plasmids, even the E. coli chromosome, implying the acquisition of mcr-1 by a unique common mechanism.
|
1263. |
Morlon J,
Chartier M,
Bidaud M,
Lazdunski C,
( 1988 ) The complete nucleotide sequence of the colicinogenic plasmid ColA. High extent of homology with ColE1. PMID : 2832701 : DOI : 10.1007/bf00330599 Abstract >>
The complete nucleotide sequence of the colicinogenic plasmid ColA has been determined. The plasmid DNA consists of 6720 bp (molecular weight 4.48 X 10(6]. Fifteen putative biological functions have been identified using the functional map previously determined. These include 11 genes and 3 DNA sites. Nine genes encode proteins of which 3 have been fully characterized. The replication region of ColA coding for RNAI and RNAII is highly homologous to that of ColE1 and Clo DF13. The same holds true for the site-specific recombination region containing palindromic symmetry and involved in stable maintenance of the plasmids. A high percentage of homology has been detected for putative mobility proteins encoded by ColA and ColE1. The exclusion proteins are also highly homologous.
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1264. |
Ronecker HJ,
Rak B,
( 1987 ) Genetic organization of insertion element IS2 based on a revised nucleotide sequence. PMID : 2830172 : DOI : 10.1016/0378-1119(87)90337-4 Abstract >>
We identified a transposable element resident in the chromosome of Escherichia coli K-12 strain HB101. This is an approx. 4400-bp-long transposon flanked by two copies of insertion sequence (IS) 1 element in direct orientation. One of the IS1 elements was found to be integrated into an IS2 element between IS2 bp 139 and bp 140 with the large moiety of IS2 within the transposon. The sequence of this part of IS2 differs from the published sequence of galOP-308::IS2 at a number of positions. Restriction analysis of the published allele, however, indicated that both alleles may in fact be identical. Since six of the eight differences found alter open reading frames, the revised sequence presents a new outlook for the potential genetic organization of IS2.
|
1265. |
Li R,
Xie M,
Zhang J,
Yang Z,
Liu L,
Liu X,
Zheng Z,
Chan EW,
Chen S,
( 2017 ) Genetic characterization of mcr-1-bearing plasmids to depict molecular mechanisms underlying dissemination of the colistin resistance determinant. PMID : 28073961 : DOI : 10.1093/jac/dkw411 Abstract >>
To analyse and compare mcr-1-bearing plasmids from animal Escherichia coli isolates, and to investigate potential mechanisms underlying dissemination of mcr-1. Ninety-seven ESBL-producing E. coli strains isolated from pig farms in China were screened for the mcr-1 gene. Fifteen mcr-1-positive strains were subjected to molecular characterization and bioinformatic analysis of the mcr-1-bearing plasmids that they harboured. Three major types of mcr-1-bearing plasmids were recovered: IncX4 (?33 kb), IncI2 (?60 kb) and IncHI2 (?216-280 kb), among which the IncX4 and IncI2 plasmids were found to harbour the mcr-1 gene only, whereas multiple resistance elements including blaCTX-M, blaCMY, blaTEM, fosA, qnrS, floR and oqxAB were detected, in various combinations, alongside mcr-1 in the IncHI2 plasmids. The profiles of mcr-1-bearing plasmids in the test strains were highly variable, with coexistence of two mcr-1-bearing plasmids being common. However, the MIC of colistin was not affected by the number of mcr-1-carrying plasmids harboured. Comparative analysis of the plasmids showed that they contained an mcr-1 gene cassette with varied structures (mcr-1-orf, ISApl1-mcr-1-orf and Tn6330), with the IncHI2 type being the most active in acquiring foreign resistance genes. A novel transposon, Tn6330, with the structure ISApl1-mcr-1-orf-ISApl1 was found to be the key element mediating translocation of mcr-1 into various plasmid backbones through formation of a circular intermediate. The mcr-1 gene can be disseminated via multiple mobile elements including Tn6330, its circular intermediate and plasmids harbouring such elements. It is often co-transmitted with other resistance determinants through IncHI2 plasmids. The functional mechanism of Tn6330, a typical composite transposon harbouring mcr-1, should be further investigated.
|
1266. |
Vakulenko S,
Kálmán M,
Horváth B,
Simoncsits A,
( 1987 ) The nucleotide sequence of an aminoglycoside 3'-phosphotransferase gene from E. coli. PMID : 2823223 : DOI : 10.1093/nar/15.19.8111 PMC : PMC306333 Abstract >>
N/A
|
1267. |
Al-Jardani A,
Al-Abri SS,
Sonnevend ?,
Pál T,
Ghazawi A,
Darwish D,
Villa L,
Carattoli A,
Hashmey R,
Aldeesi Z,
Jamal W,
Rotimi V,
( 2017 ) Characterization of NDM-7 Carbapenemase-Producing Escherichia coli Isolates in the Arabian Peninsula. PMID : 28156193 : DOI : 10.1089/mdr.2016.0216 Abstract >>
The purpose of this study was to characterize the New Delhi metallo-beta lactamase (NDM)-7-producing Enterobacteriaceae isolated in the Arabian Peninsula. Enterobacteriaceae identified to carry blaNDM-7 in a collection of 157 NDM-producing isolates from Kuwait, Oman, Saudi Arabia, and the United Arab Emirates (UAE) were investigated for their antibiotic and disinfectant susceptibility, and resistance gene content. The virulence profile, phylogenetic and sequence types of the isolates were also determined. The plasmids carrying the blaNDM-7 were transferred, and their complete nucleotide sequence was determined. Four NDM-7-producing Escherichia coli isolated in Kuwait, Oman, and the UAE, respectively, were identified. They were clonally unrelated, carried a few virulence determinants only, and belonged to clonal complexes CC10 and CC23, or ST448. They were all multi-drug resistant but remained susceptible to fosfomycin, tigecycline, and colistin. In all isolates, blaNDM-7 was located on IncX3 type plasmids of a variable size, not harboring any further resistance genes. The plasmids exhibited a high degree of similarity to each other and to pKpN01-NDM7 from Canada, with various size deletions and insertions. Our findings show that IncX3 type plasmids play an important role in the spread of the currently rare NDM-7 variant in the Arabian Peninsula. This association of blaNDM-7 with the IncX3-type plasmid is particularly worrisome, as this type of plasmid was proved to spread other carbapenemases in various species of Enterobacteriaceae worldwide at a high efficiency.
|
1268. |
Schramm E,
Olschläger T,
Tröger W,
Braun V,
( 1988 ) Sequence, expression and localization of the immunity protein for colicin B. PMID : 2830463 : DOI : 10.1007/bf00338410 Abstract >>
Cells of Escherichia coli containing the cbi locus on plasmids are immune to colicin B which kills cells by dissipating the membrane potential through pore formation in the cytoplasmic membrane. The nucleotide sequence of the cbi region was determined. It contains an open reading frame for a polypeptide consisting of 175 amino acids. The amino acid sequence is homologous to the primary structure of the colicin A immunity protein. This, and the strong homology between the pore-forming domains of colicins A and B suggests a common evolutionary origin for both colicins. The immunity protein could be identified following strong overexpression of cbi. The electrophoretically determined molecular weight of 20,000 was close to the calculated molecular weight of 20,185. The protein contains four large hydrophobic regions. The immunity protein was localized in the membrane fraction and was mainly contained in the cytoplasmic membrane. It is proposed that the immunity protein inactivates the colicin in the cytoplasmic membrane.
|
1269. |
Kozlowski M,
Thatte V,
Lau PC,
Visentin LP,
Iyer VN,
( 1987 ) Isolation and structure of the replicon of the promiscuous plasmid pCU1. PMID : 2828186 : DOI : 10.1016/0378-1119(87)90377-5 Abstract >>
Evidence is presented to indicate that a PvuII fragment of approx. 2 kb isolated from the 39-kb IncN-group plasmid pCU-1 contains all plasmid-borne determinants for stable maintenance as an extrachromosomal element in Escherichia coli K-12. The fragment was sequenced. The features of this sequence include a group of 13 direct tandem repeats of 37 bp and a second group of two other direct repeats of 30 bp flanking a third partial member of this group. In addition, for a 19-bp sequence that overlaps a member of this second group, there are inverted repeats that straddle the members of the first group. There are three open reading frames within the fragment. We compare features of this sequence with that of other plasmid replicons and draw attention to similar and to dissimilar features.
|
1270. |
DiRusso CC,
( 1988 ) Nucleotide sequence of the fadR gene, a multifunctional regulator of fatty acid metabolism in Escherichia coli. PMID : 2843809 : DOI : 10.1093/nar/16.16.7995 PMC : PMC338505 Abstract >>
The Escherichia coli fadR gene is a multifunctional regulator of fatty acid and acetate metabolism. In the present work the nucleotide sequence of the 1.3 kb DNA fragment which encodes FadR has been determined. The coding sequence of the fadR gene is 714 nucleotides long and is preceded by a typical E. coli ribosome binding site and is followed by a sequence predicted to be sufficient for factor-independent chain termination. Primer extension experiments demonstrated that the transcription of the fadR gene initiates with an adenine nucleotide 33 nucleotides upstream from the predicted start of translation. The derived fadR peptide has a calculated molecular weight of 26,972. This is in reasonable agreement with the apparent molecular weight of 29,000 previously estimated on the basis of maxi-cell analysis of plasmid encoded proteins. There is a segment of twenty amino acids within the predicted peptide which resembles the DNA recognition and binding site of many transcriptional regulatory proteins.
|
1271. |
Schwartz E,
Kröger M,
Rak B,
( 1988 ) IS150: distribution, nucleotide sequence and phylogenetic relationships of a new E. coli insertion element. PMID : 2841644 : DOI : 10.1093/nar/16.14.6789 PMC : PMC338333 Abstract >>
Recently we identified the new insertion (IS) sequence IS150 in various strains of Escherichia coli K-12. We have screened other strains of E. coli and Salmonella typhimurium for the presence of homologous sequences. The strains of E. coli K-12 and W tested contain one or more copies of homology to IS150. We have also determined the complete nucleotide sequence of a copy of IS150 inserted into IS1. Comparison of nucleotide and deduced amino acid sequences of IS150, IS2, IS3, IS51, IS600 and IS629 reveals significant homologies suggesting that these elements are members of a family of phylogenetically related insertion sequences.
|
1272. |
Tsuchimoto S,
Ohtsubo H,
Ohtsubo E,
( 1988 ) Two genes, pemK and pemI, responsible for stable maintenance of resistance plasmid R100. PMID : 2832364 : DOI : 10.1128/jb.170.4.1461-1466.1988 PMC : PMC210989 Abstract >>
Plasmid R100 was found to have two genes, designated pemK and pemI, that were responsible for its stable inheritance during cell division. They are located near the region that is essential for autonomous replication. Under conditions that inhibit replication of R100 derivatives, the plasmid containing these pem genes gave only a few segregants in viable cells and increased the number of nonviable cells in the population, suggesting that a product from the pem region stabilized the plasmid by killing plasmid-free segregants. Inactivation of one of the two translational open reading frames in the pem region caused the loss of the killing function, and thus, the open reading frame is a gene designated pemK, which encodes the killing factor. The coexistence of the pem+ plasmid with a high-copy-number plasmid carrying the other open reading frame inhibited stabilization, and thus, the second open reading frame is a gene designated pemI, which encodes the inhibitor which might control the killing function of pemK. It is likely that the two open reading frames were transcribed from a promoter. There were no significant homologies in DNA sequences between the pem gene of R100 and the genes previously shown to be responsible for the stable inheritance of the other plasmids.
|
1273. |
Béjar S,
Bouché F,
Bouché JP,
( 1988 ) Cell division inhibition gene dicB is regulated by a locus similar to lambdoid bacteriophage immunity loci. PMID : 2836697 : DOI : 10.1007/bf00322439 Abstract >>
A mutation (dicA1) of a repressor gene located in the terminus region of the Escherichia coli chromosome has previously been shown to lead to temperature-dependent inhibition of division, and to be complemented by plasmids carrying either dicA or an adjacent gene dicC. In this study, operon fusions in the region coding for the division inhibition gene dicB have been used to show that temperature sensitivity does not result from high temperature inactivation of the dicA repressor. Sequence comparisons indicate that dicA and dicC are similar to genes c2 and cro respectively of bacteriophage P22, and carry similarly organized tandem operators, indicating a common evolutionary origin for dicAC and P22 immC. Nevertheless, the consensus half-operator sequence of dicAC, TGTTA-GYYA, differs significantly from that of P22 immC (ATT-TAAGAN). An analysis of the in vivo control of promoters dicAp, dicBp and dicCp placed upstream of malQ shows that the dicAC system is functionally similar to that of an immunity region, with the possible exception of an absence of pairwise cooperative binding. Our results also indicate that the dicA1 mutation causes a switch to permanent control by dicC at all temperatures.
|
1274. |
Nakamura H,
Murakami H,
Yamato I,
Anraku Y,
( 1988 ) Nucleotide sequence of the cybB gene encoding cytochrome b561 in Escherichia coli K12. PMID : 2836696 : DOI : 10.1007/bf00322437 Abstract >>
The complete nucleotide sequence of the Escherichia coli cybB gene for diheme cytochrome b561 and its flanking region was determined. The cybB gene comprises 525 nucleotides and encodes a 175 amino acid polypeptide with a molecular weight of 20,160. From its deduced amino acid sequence, cytochrome b561 is predicted to be very hydrophobic (polarity 33.7%) and to have three membrane spanning regions. Histidines, canonical ligand residues for protohemes, are localized in these regions, and the heme pockets are thought to be in the cytoplasmic membrane. No significant homology of the primary structure of cytochrome b561 with those of other bacterial b-type cytochromes was observed.
|
1275. |
Fee BE,
Dempsey WB,
( 1988 ) Nucleotide sequence of gene X of antibiotic resistance plasmid R100. PMID : 2837741 : DOI : 10.1093/nar/16.10.4726 PMC : PMC336665 Abstract >>
N/A
|
1276. |
Rhen M,
Väisänen-Rhen V,
Saraste M,
Korhonen TK,
( 1986 ) Organization of genes expressing the blood-group-M-specific hemagglutinin of Escherichia coli: identification and nucleotide sequence of the M-agglutinin subunit gene. PMID : 2883087 : DOI : 10.1016/0378-1119(86)90371-9 Abstract >>
The organization of genes encoding the blood group M-specific hemagglutinin (M-agglutinin) of Escherichia coli strain IH11165 was studied with a cloned 6.5-kb DNA segment. This DNA segment contains at least five genes which code for the polypeptides of 12.5, 30, 80, 18.5 and 21 kDa. The 30-, 80- and 21-kDa polypeptides are synthesized as precursors that are approximately 2 kDa larger. The 21-kDa polypeptide was identified as the M-agglutinin subunit by its reactivity with anti-M-agglutinin serum. Nucleotide sequence analysis of the corresponding gene showed that the M-agglutinin precursor had a 24-amino acid (aa) signal sequence, while the mature protein is 146 aa residues long. Although the organization of the M-agglutinin gene cluster resembles those of other E. coli adhesins, there is no significant sequence homology between the M-agglutinin subunit and the subunits of the other potentially related proteins in E. coli.
|
1277. |
Blanco C,
( 1987 ) Transcriptional and translational signals of the uidA gene in Escherichia coli K12. PMID : 2823062 : DOI : 10.1007/bf00328145 Abstract >>
The expression of uidA is negatively controlled by the products of the uidR and uxuR genes and is sensitive to catabolite repression. The locations of the transcriptional and translational signals of uidA were determined using lac gene fusions and S1 mapping experiments. The promoter structure of uidA resembles that of a promoter activated by cAMP receptor protein (CRP); putative control regions are located at positions -10 and -35 (relative to the transcription start site), are separated by more than 17 bp and exhibit poor homology with the normally recognized consensus sequences. Moreover, 80 bp separate the promoter from the translational signals. No CRP binding site was detected in the promoter region of uidA. Two operator sites, 01 and 02, were identified: 01 has a greater affinity for the UidR repressor, whereas 02 has a greater affinity for the UxuR repressor, but the two repressor molecules are able to bind at both the 01 and 02 sites. Analysis of two operator constitutive mutations allowed the location of one of the two UidR repressor binding sites; it contains palindromic units spanning the TaqI site of the uidA control region.
|
1278. |
Thomas CM,
Ibbotson JP,
Wang NY,
Smith CA,
Tipping R,
Loader NM,
( 1988 ) Gene regulation on broad host range plasmid RK2: identification of three novel operons whose transcription is repressed by both KorA and KorC. PMID : 2838814 : DOI : 10.1093/nar/16.12.5345 PMC : PMC336771 Abstract >>
The product of the korA gene of broad host range plasmid RK2 is a key transcriptional repressor which regulates not only the expression of the essential replication gene trfA but also its own expression and that of the kilA operon. It has previously been proposed that korA also encodes a positive activator of transcription of the korC gene, which may act as a transcriptional antiterminator. Here we show that the action of korA in relation to korC can be explained entirely through the korA protein's property as a transcriptional repressor. The limited ability of the previously cloned korC gene to suppress kilC on its own is shown to be due to the fact that korC in RK2 is transcribed from the bla promoter of Tn1 which was deleted in the original korC clones. We demonstrate that korA is a second repressor along with korC of three operons, one of which encodes kilC, the other two not having been described previously and serving an as yet unknown function. We have designated these operons kcrA, B and C for KorC-regulated. Putative kilC is designated kcrC. The homology between the expression signals of these operons suggests that they have arisen by duplication. This is confirmed in the case of kcrA and B by the existence of considerable homology between the products of the first ORFs in each of these operons.
|
1279. |
Boll EJ,
Marti R,
Hasman H,
Overballe-Petersen S,
Stegger M,
Ng K,
Knøchel S,
Krogfelt KA,
Hummerjohann J,
Struve C,
( 2017 ) Turn Up the Heat-Food and Clinical Escherichia coli Isolates Feature Two Transferrable Loci of Heat Resistance. PMID : 28439262 : DOI : 10.3389/fmicb.2017.00579 PMC : PMC5383660 Abstract >>
Heat treatment is a widely used process to reduce bacterial loads in the food industry or to decontaminate surfaces, e.g., in hospital settings. However, there are situations where lower temperatures must be employed, for instance in case of food production such as raw milk cheese or for decontamination of medical devices such as thermo-labile flexible endoscopes. A recently identified locus of heat resistance (LHR) has been shown to be present in and confer heat resistance to a variety of Enterobacteriaceae, including Escherichia coli isolates from food production settings and clinical ESBL-producing E. coli isolates. Here, we describe the presence of two distinct LHR variants within a particularly heat resistant E. coli raw milk cheese isolate. We demonstrate for the first time in this species the presence of one of these LHRs on a plasmid, designated pFAM21805, also encoding type 3 fimbriae and three bacteriocins and corresponding self-immunity proteins. The plasmid was highly transferable to other E. coli strains, including Shiga-toxin-producing strains, and conferred LHR-dependent heat resistance as well as type 3 fimbriae-dependent biofilm formation capabilities. Selection for and acquisition of this "survival" plasmid by pathogenic organisms, e.g., in food production environments, may pose great concern and emphasizes the need to screen for the presence of LHR genes in isolates.
|
1280. |
Cookson AL,
Biggs PJ,
Marshall JC,
Reynolds A,
Collis RM,
French NP,
Brightwell G,
( 2017 ) Culture independent analysis using gnd as a target gene to assess Escherichia coli diversity and community structure. PMID : 28404985 : DOI : 10.1038/s41598-017-00890-6 PMC : PMC5429811 Abstract >>
Current culture methods to investigate changes in Escherichia coli community structure are often slow and laborious. Genes such as gnd (6-phosphogluconate dehydrogenase) have a highly variable nucleotide sequence and may provide a target for E. coli microbiome analysis using culture-independent methods. Metabarcoded PCR primers were used to generate separate libraries from calf faecal samples for high throughput sequencing. Although a total of 348 separate gnd sequence types (gSTs) were identified, 188 were likely to be due to sequencing errors. Of the remaining 160 gSTs, 92 did not match those in a database of 319 separate gnd sequences. 'Animal' was the main determinant of E. coli diversity with limited impact of sample type or DNA extraction method on intra-host E. coli community variation from faeces and recto-anal mucosal swab samples. This culture-independent study has addressed the difficulties of quantifying bacterial intra-species diversity and revealed that, whilst individual animals may harbour >50 separate E. coli strains, communities are dominated by <10 strains alongside a large pool of subdominant strains present at low abundances. This method will be useful for characterising the diversity and population structure of E. coli in experimental studies designed to assess the impact of interventions on the gut microbiome.
|
1281. |
Connell N,
Han Z,
Moreno F,
Kolter R,
( 1987 ) An E. coli promoter induced by the cessation of growth. PMID : 2835580 : DOI : 10.1111/j.1365-2958.1987.tb00512.x Abstract >>
The production of the bacterial DNA replication inhibitor Microcin B17 is induced as cultures enter stationary phase. Using S1 nuclease protection assays we have shown that this induction is the result of increased levels of transcription initiation from a promoter located upstream from mcbA, the structural gene for Microcin B17. Upstream from the start site of transcription there is a rather typical -35 region. However, there is no good homology to the consensus -10 region. While most of the cell's transcription is shut off as a result of the cessation of growth, transcription from the mcbA promoter continues for several hours in stationary phase. A single-copy gene fusion between mcbA and lacZ was used to monitor the response of the promoter to different nutritional conditions and in different host backgrounds altered in metabolic regulatory loci. Starvation for nitrogen, phosphate or carbon sources all induced transcription from the promoter. Levels of transcription were reduced in ompR backgrounds. In contrast, mutations in other global regulatory loci, fnr, relA and cya had little or no effect.
|
1282. |
Hou X,
Perepelov AV,
Guo X,
Senchenkova SN,
Shashkov AS,
Liu B,
Knirel YA,
Wang L,
( 2017 ) A gene cluster at an unusual chromosomal location responsible for the novel O-antigen synthesis in Escherichia coli O62 by the ABC transporter-dependent pathway. PMID : 28402541 : DOI : 10.1093/glycob/cwx030 Abstract >>
The O-antigen is a part of the outer membrane of Gram-negative bacteria and is related to bacterial virulence. It is one of the most variable cell constituents, and its structural diversity is almost entirely due to genetic variation of the O-antigen gene cluster. In this study, the O-antigen structure of Escherichia coli O62 was elucidated by chemical analysis and nuclear magnetic resonance spectroscopy, but showing not consistent with the O-antigen gene cluster between conserved genes galF and gnd reported earlier. The complete genome of E. coli O62 was then sequenced and analyzed, and another O-antigen gene cluster was found and characterized that correlated perfectly with the established O-antigen structure. A deletion and complementation experiment confirmed the functionality of the novel gene cluster and demonstrated that the O62-antigen is synthesized by the ABC transporter-dependent system. To our knowledge, this is the first report that the O-antigen gene cluster is positioned at a novel locus in E. coli. Comparative analysis indicated that E. coli O62 likely originated from E. coli O68 via an IS event resulting in the repression of the O68-antigen synthesis, followed by the acquisition of a novel O-antigen gene cluster from Enterobacter aerogenes.
|
1283. |
Jiang W,
Men S,
Kong L,
Ma S,
Yang Y,
Wang Y,
Yuan Q,
Cheng G,
Zou W,
Wang H,
( 2017 ) Prevalence of Plasmid-Mediated Fosfomycin Resistance Gene fosA3 Among CTX-M-Producing Escherichia coli Isolates from Chickens in China. PMID : 28379732 : DOI : 10.1089/fpd.2016.2230 Abstract >>
The aim of this study was to investigate the prevalence of fosfomycin resistance gene fosA3 and characterize plasmids harboring fosA3 among CTX-M-producing Escherichia coli from chickens in China. A total of 234 CTX-M-producing E. coli isolates collected from chickens from 2014 to 2016 were screened for the presence of plasmid-mediated fosfomycin resistance genes (fosA, fosA3, and fosC2). Clonal relatedness of fosA3-positive isolates was determined by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). The genetic environment of fosA3 was analyzed by polymerase chain reaction (PCR) mapping. Plasmids were studied by using conjugation experiments, PCR-based replicon typing and plasmid MLST. Sixty-four (27.4%) fosA3-positive E. coli isolates were identified in this study. The gene blaCTX-M-55 (31/64) was predominant among these strains, followed by blaCTX-M-14 (18/64) and blaCTX-M-65 (14/64). Various PFGE patterns and sequence types (STs) indicated that these isolates were clonally unrelated. Seven different genetic environments of fosA3 were identified and two new combinations (ISEcp1-blaCTX-M-65-�GIS903D-IS26-fosA3-orf1-orf2-�Gorf3-IS26 and IS26-ISEcp1-blaCTX-M-3-orf477-blaTEM-1-IS26-fosA3-orf1-orf2-�Gorf3-IS26) were discovered for the first time. Conjugation experiments were successful for 47 isolates and 33 transconjugants harbored a single plasmid. Plasmids carrying fosA3 belonged to incompatibility group IncFII (17/33), IncI1 (2/33), IncHI2 (3/33), and IncB/O (1/33). F33:A-:B- plasmids carrying blaCTX-M-55, IncHI2/ST3 plasmids carrying blaCTX-M-65, and F2:A-:B-plasmids carrying blaCTX-M-55 were found in E. coli isolates from different provinces. Our results revealed a considerable prevalence of fosA3 gene among CTX-M-producing E. coli with clonal diversity from chickens in China. The transmission of different kinds of plasmids is responsible for the dissemination of fosA3 in chicken farms in China.
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1284. |
Knowles M,
Stinson S,
Lambert D,
Carrillo C,
Koziol A,
Gauthier M,
Blais B,
( 2016 ) Genomic Tools for Customized Recovery and Detection of Foodborne Shiga Toxigenic Escherichia coli. PMID : 28221970 : DOI : 10.4315/0362-028X.JFP-16-220 Abstract >>
Genomic antimicrobial resistance (AMR) prediction tools have the potential to support foodborne illness outbreak investigations through their application in the analysis of bacterial genomes from causative strains. The AMR marker profile of a strain of interest, initially identified in outbreak-associated clinical samples, may serve as the basis for customization of selective enrichment media, facilitating its recovery from samples in a food safety investigation. Different possibilities for AMR analyses include the use of comprehensive AMR gene databases such as the Comprehensive Antibiotic Resistance Database, which can be mined with in-house bioinformatics alignment tools (e.g., Antimicrobial Resistance Marker Identifier), or publicly available tools based on clinically relevant acquired AMR gene databases (e.g., ResFinder). In combination with a previously reported pipeline (SigSeekr) designed to identify specific DNA sequences associated with a particular strain for its rapid identification by PCR, it should be possible to deploy custom recovery and identification tools for the efficient detection of priority pathogens such as Shiga toxigenic Escherichia coli (STEC) outbreak strains within the time frame of an active investigation. Using a laboratory STEC strain as a model, trimethoprim resistance identified by both Antimicrobial Resistance Marker Identifier and ResFinder was used as the basis for its selective recovery against a background of commensal E. coli bacteria in ground beef samples. Enrichment in modified tryptic soy broth containing trimethoprim greatly enhanced the recovery of low numbers of model strain cells inoculated in ground beef samples, as verified by the enumeration of colonies on plating media using a strain-specific PCR method to determine the recovery efficiency for the target strain. We discuss the relative merits of different AMR marker prediction tools for this purpose and describe how such tools can be utilized to good effect in a typical outbreak investigation scenario.
|
1285. |
Navarra G,
Zihlmann P,
Jakob RP,
Stangier K,
Preston RC,
Rabbani S,
Smiesko M,
Wagner B,
Maier T,
Ernst B,
( 2017 ) Carbohydrate-Lectin Interactions: An Unexpected Contribution to Affinity. PMID : 28076665 : DOI : 10.1002/cbic.201600615 Abstract >>
Uropathogenic E. coli exploit PapG-II adhesin for infecting host cells of the kidney; the expression of PapG-II at the tip of bacterial pili correlates with the onset of pyelonephritis in humans, a potentially life-threatening condition. It was envisaged that blocking PapG-II (and thus bacterial adhesion) would provide a viable therapeutic alternative to conventional antibiotic treatment. In our search for potent PapG-II antagonists, we observed an increase in affinity when tetrasaccharide 1, the natural ligand of PapG-II in human kidneys, was elongated to hexasaccharide 2, even though the additional Sia�\(2-3)Gal extension is not in direct contact with the lectin. ITC studies suggest that the increased affinity results from partial desolvation of nonbinding regions of the hexasaccharide; this is ultimately responsible for perturbation of the outer hydration layers. Our results are in agreement with previous observations and suggest a general mechanism for modulating carbohydrate-protein interactions based on nonbinding regions of the ligand.
|
1286. |
Francoz E,
Dassa E,
( 1988 ) 3' end of the malEFG operon in E.coli: localization of the transcription termination site. PMID : 2836810 : DOI : 10.1093/nar/16.9.4097 PMC : PMC336577 Abstract >>
The nucleotide sequence of a 981 bp's HincII-PvuII DNA fragment containing the 3' end of the malEFG operon in E. coli was determined. This sequence displayed a putative Rho-independent transcription termination site localized 87 bp's after the stop codon of malG. When cloned into plasmid pKG1800, the HincII-PvuII fragment containing this structure acted as a strong transcription termination signal. By S1 mapping, we demonstrated that the 3' end of the malEFG transcript coincided with the putative transcription termination site. One short open reading frames orf1 (123 bp) and and the beginning of another one orf2 were localized after malG. The transcription termination site is localized within orf1. Consequently malG is the last gene of the malEFG operon. orf2 corresponds exactly to the 5' part of the xylE gene reported independently (Davis & Henderson, 1987) as the gene coding for the XylE protein, the xylose-proton symport of Escherichia coli.
|
1287. |
Pruthvishree BS,
Vinodh Kumar OR,
Sinha DK,
Malik YPS,
Dubal ZB,
Desingu PA,
Shivakumar M,
Krishnaswamy N,
Singh BR,
( 2017 ) Spatial molecular epidemiology of carbapenem-resistant and New Delhi metallo beta-lactamase (blaNDM)-producing Escherichia coli in the piglets of organized farms in India. PMID : 28345184 : DOI : 10.1111/jam.13455 Abstract >>
A cross-sectional study was conducted in 10 government-organized pig farms between 2014 and 2016 representing seven states of India to understand the epidemiology of carbapenem resistance in the Escherichia coli. In this study, fecal sample (n = 673) from non-diarrheic (n = 501) and diarrheic (n = 172) piglets were processed for isolation of carbapenem resistant E. coli. Of 673, E. coli isolate (n = 112) was genotyped for confirming the carbapenem resistance and associated virulence factors. Of the 112 isolates, 23 were phenotypically resistant to carbapenem and 8 were carrying the New Delhi metallo beta-lactamase (blaNDM) gene. The carbapenem-resistant isolates also produced extended spectrum beta-lactamases and were multidrug resistant. The PCR-based pathotyping revealed the presence of stx1, stx2, eae and hlyA genes. The enterobacterial repetitive intergenic consensus PCR dendrogram analysis of the isolates yielded three distinct clusters. The statistical analysis revealed no association between carriages of carbapenem-resistant E. coli in different breed of piglets however, location, sex, health status of piglets and age showed significant difference. The spatial analysis with SaTScan helped in identification of carbapenem-resistant clusters. The presence of carbapenem resistant E. coli isolates with virulence genes in the piglet poses a potential public health risk through possible access and spread via the food chain and environment. Efflux pump may also play an important role in carbapenem resistance in piglet E. coli isolates. Furthermore, identification of risk factors in relation to spatial clusters will help in designing preventive strategies for reducing the risk of spread of carbapenem resistant bacteria. 1. Piglets harbor carbapenem resistant E. coli and have great public health significance. 2. Apart from carbapenemase, efflux pump is also important for carbapenem resistance. 3. This is the first report of blaNDM in the piglets from India.
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1288. |
Ishiguro N,
Sato G,
( 1988 ) Nucleotide sequence of insertion sequence IS3411, which flanks the citrate utilization determinant of transposon Tn3411. PMID : 2832386 : DOI : 10.1128/jb.170.4.1902-1906.1988 PMC : PMC211048 Abstract >>
The nucleotide sequences of insertion sequences IS3411L (left) and IS3411R (right), present as direct terminal repeats in the citrate utilization of citrate utilization transposon Tn3411, and of IS3411 (generated by intramolecular recombination between IS3411L and IS3411R) were determined. The three IS3411 elements (IS3411R, IS3411L, and IS3411) were 1,309 base pairs long and identical in DNA sequence. IS3411 had 27-base-pair terminal inverted repeats with three bases mismatched and one long open reading frame (240 amino acids) that was proposed to be a transposase. Three polypeptides of 29,000, 27,000, and about 10,000 molecular weight, determined by IS3411, were identified in minicells. Since Tn3411 generates a 3-base-pair repeat upon integration, the nucleotide sequences of IS3411 were compared with those of IS3.
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1289. |
Rabbani S,
Krammer EM,
Roos G,
Zalewski A,
Preston R,
Eid S,
Zihlmann P,
Prévost M,
Lensink MF,
Thompson A,
Ernst B,
Bouckaert J,
( 2017 ) Mutation of Tyr137 of the universal Escherichia coli fimbrial adhesin FimH relaxes the tyrosine gate prior to mannose binding. PMID : 28250938 : DOI : 10.1107/S2052252516016675 PMC : PMC5331462 Abstract >>
The most prevalent diseases manifested by Escherichia coli are acute and recurrent bladder infections and chronic inflammatory bowel diseases such as Crohn's disease. E. coli clinical isolates express the FimH adhesin, which consists of a mannose-specific lectin domain connected via a pilin domain to the tip of type 1 pili. Although the isolated FimH lectin domain has affinities in the nanomolar range for all high-mannosidic glycans, differentiation between these glycans is based on their capacity to form predominantly hydrophobic interactions within the tyrosine gate at the entrance to the binding pocket. In this study, novel crystal structures of tyrosine-gate mutants of FimH, ligand-free or in complex with heptyl �\-d-O-mannopyranoside or 4-biphenyl �\-d-O-mannopyranoside, are combined with quantum-mechanical calculations and molecular-dynamics simulations. In the Y48A FimH crystal structure, a large increase in the dynamics of the alkyl chain of heptyl �\-d-O-mannopyranoside attempts to compensate for the absence of the aromatic ring; however, the highly energetic and stringent mannose-binding pocket of wild-type FimH is largely maintained. The Y137A mutation, on the other hand, is the most detrimental to FimH affinity and specificity: (i) in the absence of ligand the FimH C-terminal residue Thr158 intrudes into the mannose-binding pocket and (ii) ethylenediaminetetraacetic acid interacts strongly with Glu50, Thr53 and Asn136, in spite of multiple dialysis and purification steps. Upon mutation, pre-ligand-binding relaxation of the backbone dihedral angles at position 137 in the tyrosine gate and their coupling to Tyr48 via the interiorly located Ile52 form the basis of the loss of affinity of the FimH adhesin in the Y137A mutant.
|
1290. |
Price C,
Lingner J,
Bickle TA,
Firman K,
Glover SW,
( 1989 ) Basis for changes in DNA recognition by the EcoR124 and EcoR124/3 type I DNA restriction and modification enzymes. PMID : 2784505 : DOI : 10.1016/0022-2836(89)90369-0 Abstract >>
EcoR124 and EcoR124/3 are type I DNA restriction and modification systems. The EcoR124/3 system arose from the EcoR124 system some 15 years ago and at the electron microscopic DNA heteroduplex level the genes for both systems are still apparently identical. We have shown that the DNA sequences recognized by the two systems are GAA(N6)RTCG for EcoR124 and GAA(N7)RTCG for EcoR124/3. The sequences thus differ only in the length of the non-specific spacer. This difference nevertheless places the two specific domains of the EcoR124/3 recognition sequence 0.34 nm further apart and rotates them 36 degrees with respect to those of EcoR124, which implies major structural differences in the proteins recognizing these sequences. We have now determined the nucleotide sequences of the hsdS and hsdM genes of both systems and of the hsdR gene of EcoR124/3. The hsdS gene products provide DNA sequence specificity in both restriction and modification, the hsdM gene products are necessary for modification and all three hsd gene products are required for restriction. The only difference that we have detected between the two systems is that a 12 base-pair sequence towards the middle of the hsdS gene is repeated twice in the EcoR124 gene and three times in the EcoR124/3 gene. We have deleted one of the repeats in the EcoR124/3 gene and shown that this changes the specificity to that of EcoR124. Thus, the extra four amino acids in the middle of the EcoR124/3 hsdS gene product, which in an alpha-helical configuration would extend 0.6 nm, are sufficient to explain the differences in sequence recognition. We suggest that the EcoR124/3 system was generated by an unequal crossing over and argue that this kind of specificity change should not be rare in Nature.
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1291. |
Okubo T,
Matushita M,
Ohara Y,
Matsuo J,
Oguri S,
Fukumoto T,
Hayasaka K,
Akizawa K,
Shibuya H,
Shimizu C,
Yamaguchi H,
( 2017 ) Ciliates promote the transfer of a plasmid encoding blaNDM-5 from Escherichia coli, isolated from a hospital in Japan, to other human pathogens. PMID : 28167346 : DOI : 10.1016/j.ijantimicag.2017.01.003 Abstract >>
N/A
|
1292. |
Hall RM,
Vockler C,
( 1987 ) The region of the IncN plasmid R46 coding for resistance to beta-lactam antibiotics, streptomycin/spectinomycin and sulphonamides is closely related to antibiotic resistance segments found in IncW plasmids and in Tn21-like transposons. PMID : 2821509 : DOI : 10.1093/nar/15.18.7491 PMC : PMC306263 Abstract >>
The nucleotide sequence of a 2.5 kb segment of the pKM101 (R46) genome has been determined. The 1.3 kb from a BamHI site at 153 to base 1440 differs by only 2 bases from a part of the published sequence of the aadB (gentamicin resistance) gene region including the coding region for the N-terminal 70 amino acids of the predicted aadB product. The same sequence has been found 5'-to the dhfrII gene of R388 and to the aadA gene of Tn21 (R538-1). Three open reading frames are located in this region, two on the same strand as the resistance genes and one on the complementary strand. The latter predicts a polypeptide of 337 amino acids, whose N-terminal segment is 40% homologous to the predicted product of an open reading frame of 179 amino acids located next to the dhfrI gene of Tn7. The oxa2 (oxacillin resistance) gene predicts a long polypeptide commencing with (the N-terminal) 70 amino acids of the aadB product. A similar arrangement is found in the aadA gene of R538-1. The N-terminal segment of an aadA gene is located 3'- to oxa2, separated by 36 bases. Sequences surrounding the BamHI site are identical to sequences 5'- to the tnpM gene of Tn21 and homology ceases where homology between Tn21 and Tn501 commences. The possibility that this antibiotic resistance segment is a discrete mobile DNA element is discussed.
|
1293. |
López-Alonso JP,
Fabbretti A,
Kaminishi T,
Iturrioz I,
Brandi L,
Gil-Carton D,
Gualerzi CO,
Fucini P,
Connell SR,
( 2017 ) Structure of a 30S pre-initiation complex stalled by GE81112 reveals structural parallels in bacterial and eukaryotic protein synthesis initiation pathways. PMID : 27986852 : DOI : 10.1093/nar/gkw1251 PMC : PMC5389724 Abstract >>
In bacteria, the start site and the reading frame of the messenger RNA are selected by the small ribosomal subunit (30S) when the start codon, typically an AUG, is decoded in the P-site by the initiator tRNA in a process guided and controlled by three initiation factors. This process can be efficiently inhibited by GE81112, a natural tetrapeptide antibiotic that is highly specific toward bacteria. Here GE81112 was used to stabilize the 30S pre-initiation complex and obtain its structure by cryo-electron microscopy. The results obtained reveal the occurrence of changes in both the ribosome conformation and initiator tRNA position that may play a critical role in controlling translational fidelity. Furthermore, the structure highlights similarities with the early steps of initiation in eukaryotes suggesting that shared structural features guide initiation in all kingdoms of life.
|
1294. |
Olschläger T,
Braun V,
( 1987 ) Sequence, expression, and localization of the immunity protein for colicin M. PMID : 2820942 : DOI : 10.1128/jb.169.10.4765-4769.1987 PMC : PMC213852 Abstract >>
Escherichia coli strains carrying the cmi locus on plasmids are immune against colicin M, which primarily inhibits murein biosynthesis, followed by lysis of cells. The nucleotide sequence of the cmi region was determined. It contains an open reading frame for a polypeptide with a molecular weight of 19,227. However, the major protein band observed on polyacrylamide gels after transcription and translation in an in vitro system or in minicells had an apparent molecular weight between 15,000 and 16,000. The nucleotide sequence contained internal ATG codons, two of which could serve for the synthesis of polypeptides with molecular weights of 15,349 and 15,996, respectively. A subclone with a DNA fragment that encoded these two shorter polypeptides exhibited full immunity. The colicin M immunity protein was found in the cytoplasmic membrane. The colicin M activity and immunity genes were transcribed in opposite directions. Both properties are typical of the channel-forming colicins and are in contrast to the colicins with endonuclease activities. However, colicin M does not form channels and exhibits no structural similarity to channel-forming colicins.
|
1295. |
Stibitz S,
Davies JE,
( 1987 ) Tn602: a naturally occurring relative of Tn903 with direct repeats. PMID : 2819910 : Abstract >>
We report the characterization of Tn602, a transposon encoding resistance to kanamycin and related aminoglycosides present on the R-plasmid pGD10. Tn602 is highly homologous to the previously characterized Tn903, present on the R-plasmid R6, in that it consists of a gene for aminoglycoside-phosphotransferase-3'-I (homologous to that of Tn903) flanked by copies of an IS-element homologous to IS903. Tn602 differs from Tn903 in the following respects: the flanking IS-elements (IS602) are in direct rather than inverted orientation as in Tn903; the fusion points between the IS-elements and the central region are different from those in Tn903; and several sequence changes, detected by the loss and acquisition of restriction sites, show the two repeats of IS602 to be nonidentical and different from IS903, IS102, and IS903.B. These structural details suggest that Tn602 and Tn903 evolved separately from related modules.
|
1296. |
Casu B,
Smart J,
Hancock MA,
Smith M,
Sygusch J,
Baron C,
( 2016 ) Structural Analysis and Inhibition of TraE from the pKM101 Type IV Secretion System. PMID : 27634044 : DOI : 10.1074/jbc.M116.753327 PMC : PMC5095433 Abstract >>
Gram-negative bacteria use type IV secretion systems (T4SSs) for a variety of macromolecular transport processes that include the exchange of genetic material. The pKM101 plasmid encodes a T4SS similar to the well-studied model systems from Agrobacterium tumefaciens and Brucella suis Here, we studied the structure and function of TraE, a homolog of VirB8 that is an essential component of all T4SSs. Analysis by X-ray crystallography revealed a structure that is similar to other VirB8 homologs but displayed an altered dimerization interface. The dimerization interface observed in the X-ray structure was corroborated using the bacterial two-hybrid assay, biochemical characterization of the purified protein, and in vivo complementation, demonstrating that there are different modes of dimerization among VirB8 homologs. Analysis of interactions using the bacterial two-hybrid and cross-linking assays showed that TraE and its homologs from Agrobacterium, Brucella, and Helicobacter pylori form heterodimers. They also interact with heterologous VirB10 proteins, indicating a significant degree of plasticity in the protein-protein interactions of VirB8-like proteins. To further assess common features of VirB8-like proteins, we tested a series of small molecules derived from inhibitors of Brucella VirB8 dimerization. These molecules bound to TraE in vitro, docking predicted that they bind to a structurally conserved surface groove of the protein, and some of them inhibited pKM101 plasmid transfer. VirB8-like proteins thus share functionally important sites, and these can be exploited for the design of specific inhibitors of T4SS function.
|
1297. |
Ali T,
Ur Rahman S,
Zhang L,
Shahid M,
Zhang S,
Liu G,
Gao J,
Han B,
( 2016 ) ESBL-Producing Escherichia coli from Cows Suffering Mastitis in China Contain Clinical Class 1 Integrons with CTX-M Linked to ISCR1. PMID : 27965653 : DOI : 10.3389/fmicb.2016.01931 PMC : PMC5127808 Abstract >>
The prevalence of pathogenic multi-drug resistant (MDR) extended-spectrum �]-lactamase (ESBL)-producing Escherichia coli is rapidly increasing, becoming a global concern. In a veterinary context, ESBL-producing E. coli are mostly reported in poultry and pigs. Here, we report on the prevalence and characterize ESBL-producing E. coli isolated from diverse dairy farms in China. Overall, 36 (23.53%) out of 153 E. coli isolates from mastitic milk samples (n = 1252) were confirmed as ESBL-producers by double-disc synergy testing and PCR. Nucleotide analysis of PCR amplicons revealed that blaCTX-M was the predominant ESBL gene detected in 28 (77.78%) isolates, with blaCTX-M-15 being the major (78.57%) allele encoding for ESBLs. Also, 20 (55.56%) and 6 (16.67%) of the ESBL isolates were carrying blaTEM and blaSHV genes, respectively, in singlet or in combination. The majority of these isolates belonged to phylo-group A (69.44%) and D (16.67%). Strikingly, all these isolates were found to be MDR showing high resistance to cephalosporins including the fourth generation cefepime and common non �]-lactams. Additionally, class 1 integrons (intI1) were found in 30 (83.33%) isolates. Analysis of the class 1 integrons variable regions indicated that they were carrying up to five different gene cassettes conferring resistance to various drugs with a predominant combination of dfrA17-aadA5 genes in tandem, conferring resistance to aminoglycosides and trimethoprim. However, no ESBL encoding genes were found in the cassettes. Interestingly, 22 (66.11%) of the ESBL isolates were also carrying insertion sequence common region 1 (ISCR1) which was found to be associated with most of the CTX-M genes. Altogether, the current study reports on the high prevalence of ESBL-positive E. coli, particularly CTX-M-15, carrying clinical class 1 integrons and ISCR1 elements are likely indicative of their rapid and wider dissemination, posing threats to veterinary and public health. To the best of our knowledge, this is the first comprehensive study to report on the alarming high occurrence of ESBL-producing E. coli from mastitic cows in China.
|
1298. |
Göldner A,
Graus H,
Högenauer G,
( 1987 ) The origin of transfer of P307. PMID : 2827206 : Abstract >>
The DNA fragment carrying the oriT region from the enterotoxin plasmid P307 was isolated and its polynucleotide sequence was determined. Using Southern hybridization assays with a synthetic oligonucleotide probe, the oriT region was identified on a 7.9-kb EcoRI fragment from P307. By ligating the fragment with the cloning vector pUC119, plasmid pAG10 was obtained. The physical map of the insert was determined and oriT was located on a 540-bp BglII/SalI fragment. After this fragment was subcloned into sequencing phages, the polynucleotide sequence was established. Part of the sequence proved to be almost identical to segments of the oriT regions of the plasmids F and R1; another neighboring region was very different among all three sequences. The polynucleotide sequence proximal to traM is highly similar to that of F but different from that of R1.
|
1299. |
Li R,
Xie M,
Lv J,
Wai-Chi Chan E,
Chen S,
( 2017 ) Complete genetic analysis of plasmids carrying mcr-1 and other resistance genes in an Escherichia coli isolate of animal origin. PMID : 27999050 : DOI : 10.1093/jac/dkw509 Abstract >>
To investigate the genetic features of three plasmids recovered from an MCR-1 and ESBL-producing Escherichia coli strain, HYEC7, and characterize the transmission mechanism of mcr-1 . The genetic profiles of three plasmids were determined by PCR, S1-PFGE, Southern hybridization and WGS analysis. The ability of the mcr-1 -bearing plasmid to undergo conjugation was also assessed. The mcr-1 -bearing transposon Tn 6330 was characterized by PCR and DNA sequencing. Complete sequences of three plasmids were obtained. A non-conjugative phage P7-like plasmid, pHYEC7- mcr1 , was found to harbour the mcr-1 -bearing transposon Tn 6330 , which could be excised from the plasmid by generating a circular intermediate harbouring mcr-1 and the IS Apl1 element. The insertion of the circular intermediate into another plasmid, pHYEC7-IncHI2, could form pHNSHP45-2, the original IncHI2-type mcr-1 -carrying plasmid that was reported. The third plasmid, pHYEC7-110, harboured two replicons, IncX1 and IncFIB, and comprised multiple antimicrobial resistance mobile elements, some of which were shared by pHYEC7-IncHI2. The Tn 6330 element located in the phage-like plasmid pHYEC7- mcr1 could be excised from the plasmid and formed a circular intermediate that could be integrated into plasmids containing the IS Apl1 element. This phenomenon indicated that Tn 6330 is a key element responsible for widespread dissemination of mcr-1 among various types of plasmids and bacterial chromosomes. The dissemination rate of such an element may be further enhanced upon translocation into phage-like vectors, which may also be transmitted via transduction events.
|
1300. |
Shen Z,
Ding B,
Bi Y,
Wu S,
Xu S,
Xu X,
Guo Q,
Wang M,
( 2017 ) CTX-M-190, a Novel �]-Lactamase Resistant to Tazobactam and Sulbactam, Identified in an Escherichia coli Clinical Isolate. PMID : 27821452 : DOI : 10.1128/AAC.01848-16 PMC : PMC5192109 Abstract >>
A novel �]-lactamase, CTX-M-190, derived from CTX-M-55 by a single substitution of Ser for Thr at position 133 (Ser133Thr), was identified in a natural Escherichia coli clinical isolate. CTX-M-190 exhibited potent hydrolytic activity against cefotaxime, with a kcat/Km ratio of 14.5 �gM-1 s-1, and was highly resistant to inhibition by the �]-lactamase inhibitors tazobactam and sulbactam, whose 50% inhibitory concentrations were 77- and 55-fold higher, respectively, for CTX-M-190 than for CTX-M-55. blaCTX-M-190 was located within the genetic platform ISEcp1-blaCTX-M-orf477, which was harbored by a 70-kb IncI1 plasmid.
|
1301. |
Fang L,
Li X,
Li L,
Li S,
Liao X,
Sun J,
Liu Y,
( 2016 ) Co-spread of metal and antibiotic resistance within ST3-IncHI2 plasmids from E. coli isolates of food-producing animals. PMID : 27143648 : DOI : 10.1038/srep25312 PMC : PMC4855149 Abstract >>
Concerns have been raised in recent years regarding co-selection for antibiotic resistance among bacteria exposed to heavy metals, particularly copper and zinc, used as growth promoters for some livestock species. In this study, 25 IncHI2 plasmids harboring oqxAB (20/25)/blaCTX-M (18/25) were found with sizes ranging from ?260 to ?350 kb and 22 belonged to the ST3-IncHI2 group. In addition to blaCTX-M and oqxAB, pcoA-E (5/25) and silE-P (5/25), as well as aac(6')-Ib-cr (18/25), floR (16/25), rmtB (6/25), qnrS1(3/25) and fosA3 (2/25), were also identified on these IncHI2 plasmids. The plasmids carried pco and sil contributed to increasing in the MICs of CuSO4 and AgNO3. The genetic context surrounding the two operons was well conserved except some variations within the pco operon. The ~32 kb region containing the two operons identified in the IncHI2 plasmids was also found in chromosomes of different Enterobacteriaceae species. Further, phylogenetic analysis of this structure showed that Tn7-like transposon might play an important role in cross-genus transfer of the sil and pco operons among Enterobacteriaceae. In conclusion, co-existence of the pco and sil operons, and oqxAB/blaCTX-M as well as other antibiotic resistance genes on IncHI2 plasmids may promote the development of multidrug-resistant bacteria.
|
1302. |
Geldart K,
Forkus B,
McChesney E,
McCue M,
Kaznessis YN,
( 2016 ) pMPES: A Modular Peptide Expression System for the Delivery of Antimicrobial Peptides to the Site of Gastrointestinal Infections Using Probiotics. PMID : 27782051 : DOI : 10.3390/ph9040060 PMC : PMC5198035 Abstract >>
Antimicrobial peptides are a promising alternative to traditional antibiotics, but their utility is limited by high production costs and poor bioavailability pro?les. Bacterial production and delivery of antimicrobial peptides (AMPs) directly at the site of infection may offer a path for effective therapeutic application. In this study, we have developed a vector that can be used for the production and secretion of seven antimicrobial peptides from both Escherichia coli MC1061 F' and probiotic E.coli Nissle 1917. The vector pMPES (Modular Peptide Expression System) employs the Microcin V (MccV) secretion system and a powerful synthetic promoter to drive AMP production. Herein, we demonstrate the capacity of pMPES to produce inhibitory levels of MccV, Microcin L (MccL), Microcin N (McnN), Enterocin A (EntA), Enterocin P (EntP), Hiracin JM79 (HirJM79) and Enterocin B (EntB). To our knowledge, this is the ?rst demonstration of such a broadly-applicable secretion system for AMP production. This type of modular expression system could expedite the development of sorely needed antimicrobial technologies.
|
1303. |
Bajar BT,
Wang ES,
Zhang S,
Lin MZ,
Chu J,
( 2016 ) A Guide to Fluorescent Protein FRET Pairs. PMID : 27649177 : DOI : 10.3390/s16091488 PMC : PMC5038762 Abstract >>
F?rster or fluorescence resonance energy transfer (FRET) technology and genetically encoded FRET biosensors provide a powerful tool for visualizing signaling molecules in live cells with high spatiotemporal resolution. Fluorescent proteins (FPs) are most commonly used as both donor and acceptor fluorophores in FRET biosensors, especially since FPs are genetically encodable and live-cell compatible. In this review, we will provide an overview of methods to measure FRET changes in biological contexts, discuss the palette of FP FRET pairs developed and their relative strengths and weaknesses, and note important factors to consider when using FPs for FRET studies.
|
1304. |
Paiva MC,
Reis MP,
Costa PS,
Dias MF,
Bleicher L,
Scholte LLS,
Nardi RMD,
Nascimento AMA,
( 2017 ) Identification of new bacteria harboring qnrS and aac(6')-Ib/cr and mutations possibly involved in fluoroquinolone resistance in raw sewage and activated sludge samples from a full-scale WWTP. PMID : 27984803 : DOI : 10.1016/j.watres.2016.11.056 Abstract >>
Wastewater treatment plants (WWTPs) harbor bacteria and antimicrobial resistance genes, favoring gene exchange events and resistance dissemination. Here, a culture-based and metagenomic survey of qnrA, qnrB, qnrS, and aac(6')-Ib genes from raw sewage (RS) and activated sludge (AS) of a full-scale municipal WWTP was performed. A total of 96 bacterial isolates were recovered from nalidixic acid-enrichment cultures. Bacteria harboring the aac(6')-Ib gene predominated in RS, whereas qnrS-positive isolates were specific to AS. Novel qnrS- and aac(6')-Ib-cr positive species were identified: Morganella morganii, Providencia rettgeri, and Pseudomonas guangdongensis (qnrS), and Alcaligenes faecalis and P. rettgeri (aac(6')-Ib-cr). Analysis of qnrS and aac(6')-Ib sequences from isolates and clone libraries suggested that the diversity of qnrS is wider than that of aac(6')-Ib. A large number of amino acid mutations were observed in the QnrS and AAC(6')-Ib proteins at previously undetected positions, whose structural implications are not clear. An accumulation of mutations at the C72, Q73, L74, A75 and M76 positions of QnrS, and D181 of AAC(6')-Ib might be important for resistance. These findings add significant information on bacteria harboring qnrS and aac(6')-Ib genes, and the presence of novel mutations that may eventually emerge in clinical isolates.
|
1305. |
Farkas A,
Cr?ciuna? C,
Chiriac C,
Szekeres E,
Coman C,
Butiuc-Keul A,
( 2016 ) Exploring the Role of Coliform Bacteria in Class 1 Integron Carriage and Biofilm Formation During Drinking Water Treatment. PMID : 27079455 : DOI : 10.1007/s00248-016-0758-0 Abstract >>
This study investigates the role of coliforms in the carriage of class 1 integron and biocide resistance genes in a drinking water treatment plant and explores the relationship between the carriage of such genes and the biofouling abilities of the strain. The high incidence of class 1 integron and biocide resistance genes (33.3 % of the isolates) highlights the inherent risk of genetic contamination posed by coliform populations during drinking water treatment. The association between the presence of intI1 gene and qac gene cassettes, especially qacH, was greater in biofilm cells. In coliforms recovered from biofilms, a higher frequency of class 1 integron elements and higher diversity of genetic patterns occurred, compared to planktonic cells. The coliform isolates under the study proved to mostly carry non-classical class 1 integrons lacking the typical qacE�G1/sul1 genes or a complete tni module, but bearing the qacH gene. No link was found between the carriage of integron genes and the biofouling degree of the strain, neither in aerobic or in anaerobic conditions. Coliform bacteria isolated from established biofilms rather adhere in oxygen depleted environments, while the colonization ability of planktonic cells is not significantly affected by oxygen availability.
|
1306. |
Lee WC,
Matthews S,
Garnett JA,
( 2016 ) Crystal structure and analysis of HdaB: The enteroaggregative Escherichia coli AAF/IV pilus tip protein. PMID : 27400770 : DOI : 10.1002/pro.2982 PMC : PMC5029526 Abstract >>
Enteroaggregative Escherichia coli is the primary cause of pediatric diarrhea in developing countries. They utilize aggregative adherence fimbriae (AAFs) to promote initial adherence to the host intestinal mucosa, promote the formation of biofilms, and mediate host invasion. Five AAFs have been identified to date and AAF/IV is amongst the most prevalent found in clinical isolates. Here we present the X-ray crystal structure of the AAF/IV tip protein HdaB at 2.0 ? resolution. It shares high structural homology with members of the Afa/Dr superfamily of fimbriae, which are involved in host invasion. We highlight surface exposed residues that share sequence homology and propose that these may function in invasion and also non-conserved regions that could mediate HdaB specific adhesive functions.
|
1307. |
Tapader R,
Bose D,
Basu P,
Mondal M,
Mondal A,
Chatterjee NS,
Dutta P,
Basu S,
Bhadra RK,
Pal A,
( 2016 ) Role in proinflammatory response of YghJ, a secreted metalloprotease from neonatal septicemic Escherichia coli. PMID : 27389679 : DOI : 10.1016/j.ijmm.2016.06.003 Abstract >>
Neonatal sepsis is the invasion of microbial pathogens into blood stream and is associated with a systemic inflammatory response with production and release of a wide range of inflammatory mediators. The increased serum levels of cytokines were found to correlate with the severity and mortality in course of sepsis. There have been no reports on the role of microbial proteases in stimulation of proinflammatory response in neonatal sepsis. We have identified YghJ, a secreted metalloprotease from a neonatal septicemic Escherichia coli (NSEC) isolate. The protease was partially purified from culture supernatant by successive anion and gel filtration chromatography. MS/MS peptide sequencing of the protease showed homology with YghJ. YghJ was cloned, expressed and purified in pBAD TOPO expression vector. YghJ was found to be proteolytically active against Methoxysuccinyl Ala-Ala-Pro-Met-p-nitroanilide oligopeptide substrate, but not against casein and gelatin. YghJ showed optimal activity at pH 7-8 and at temperatures 37-40�XC. YghJ showed clear changes in cellular morphologies of Int407, HT-29 and HEK293 cells. YghJ stimulated the secretion of cytokines IL-1�\, IL-1�] and TNF-�\ in murine macrophages (RAW 264.7) and IL-8 from human intestinal epithelial cells (HT-29). YghJ also down-regulated the production of anti-inflammatory cytokines such as IL-10. YghJ is present in both septicemic (78%) and fecal E. coli isolates (54%). However, expression and secretion of YghJ is significantly higher among the septicemic (89%) than the fecal isolates (33%). This is the first study to show the role of a microbial protease, YghJ in triggering proinflammatory response in NSEC.
|
1308. |
Parul S,
Bist B,
Sharma B,
Jain U,
Yadav JK,
( 2016 ) A study on association of virulence determinants of verotoxic Escherichia coli isolated from cattle calves. PMID : 27651684 : DOI : 10.14202/vetworld.2016.915-918 PMC : PMC5021845 Abstract >>
The present study was conducted to find the association among virulence determinants of verotoxic Escherichia coli (VTEC) isolated from cattle calf feces. A total of 216 cattle calf fecal samples were collected aseptically and processed under required conditions for the isolation of E. coli. The isolates were further subjected to multiplex polymerase chain reaction (mPCR) for the detection of virulent genes. All the VTEC isolates were serotyped at the Central Research Institute, Kasauli, Himachal Pradesh. The VTEC isolates were observed for the enterohemolysin production on washed sheep blood agar (wSBA). A total of 177 presumptive E. coli were isolated from 216 calf fecal samples revealing an overall prevalence of E. coli to be 81.94%. A total of 32 (14.81%) isolates were detected as VTEC through mPCR. The prevalence of verotoxin genes vt1, vt2, and combination of vt1+vt2 in the VTEC isolates was found to be 12 (37.5%), 14 (43.75%), and 6 (18.75%), respectively. Other virulent genes eaeA and hlyA were found in 6 and 11 VTEC strains with prevalence values of 18.75% and 34.37%, respectively. A total of 13 different O serogroups were revealed in serotyping of 32 VTEC isolates. Out of 32 VTEC strains, only 26 (81.25%) were enterohemolytic on wSBA as they produced the characteristic small, turbid zone of hemolysis around the streaking line. Although enterohemolysin production has been attributed to the presence of hlyA gene, only 11 of 26 enterohemolysin producing VTEC were found to be harboring the hlyA gene (11/26) 42.03%. The present study concludes that there might be an association between the presence of verotoxin genes and enterohemolysin production in VTEC group of E. coli.
|
1309. |
Matsumura Y,
Pitout JD,
Gomi R,
Matsuda T,
Noguchi T,
Yamamoto M,
Peirano G,
DeVinney R,
Bradford PA,
Motyl MR,
Tanaka M,
Nagao M,
Takakura S,
Ichiyama S,
( 2016 ) Global Escherichia coli Sequence Type 131 Clade with blaCTX-M-27 Gene. PMID : 27767006 : DOI : 10.3201/eid2211.160519 PMC : PMC5088012 Abstract >>
The Escherichia coli sequence type (ST) 131 C2/H30Rx clade with the blaCTX-M-15 gene had been most responsible for the global dissemination of extended-spectrum �]-lactamase (ESBL)-producing E. coli. ST131 C1/H30R with blaCTX-M-27 emerged among ESBL-producing E. coli in Japan during the late 2000s. To investigate the possible expansion of a single clade, we performed whole-genome sequencing for 43 Japan and 10 global ST131 isolates with blaCTX-M-27 (n = 16), blaCTX-M-14 (n = 16), blaCTX-M-15 (n = 13), and others (n = 8). We also included 8 ST131 genomes available in public databases. Core genome-based analysis of 61 isolates showed that ST131 with blaCTX-M-27 from 5 countries formed a distinct cluster within the C1/H30R clade, named C1-M27 clade. Accessory genome analysis identified a unique prophage-like region, supporting C1-M27 as a distinct clade. Our findings indicate that the increase of ESBL-producing E. coli in Japan is due mainly to emergence of the C1-M27 clade.
|
1310. |
Moran RA,
Hall RM,
( 2017 ) Analysis of pCERC7, a small antibiotic resistance plasmid from a commensal ST131 Escherichia coli, defines a diverse group of plasmids that include various segments adjacent to a multimer resolution site and encode the same NikA relaxase accessory protein enabling mobilisation. PMID : 27826018 : DOI : 10.1016/j.plasmid.2016.11.001 Abstract >>
The ampicillin resistance plasmid pCERC7, carrying transposon Tn2 with an IS4 insertion, was detected in the draft genome of a commensal Escherichia coli isolate. The genome data also revealed that this isolate belongs to ST131, clade B. pCERC7 is 9712bp comprised of a 3319bp backbone, Tn2::IS4 (6388bp) and 5bp of target site duplication, and was present at a copy number of 40. pCERC7 is related to several plasmids composed of only the backbone, or the backbone with the Tn2 insertion in the same position. These plasmids have been found previously in Escherichia coli or Salmonella enterica recovered in several different countries from as early as the 1970s. This group was named the NTP16 group after the best studied example. pCERC7 was annotated using available information about plasmids in this group and additional analyses. The backbone includes genes for RNA I and RNA II to initiate replication and the Tn2 interrupts a gene found here to encode a protein 66% identical to the Rom regulatory protein of ColE1. NTP16 family plasmids include a gene, previously designated mobA, that was found to encode a homologue (53% identical) of the NikA relaxase accessory protein of the conjugative IncI1 plasmid R64, which is known to bind to the R64 oriT. However, a nikB relaxase gene is not present, indicating that a relaxase must be supplied in trans for mobilisation by R64 to occur, as demonstrated previously for NTP16. Hence, MobA of NTP16 and relatives was renamed NikA. Upstream of nikA, we found a region closely related to the oriT of R64. pCERC7 and all members of the NTP16 family also include a multimer resolution site, nmr, similar to the cer site of ColE1. The backbone of the NTP16 family also includes genes for a demonstrated toxin-antitoxin system, LsoAB. Several more distantly related groups of plasmids that include a very closely related nmr-nikA-oriT segment (99.4-93.7% DNA identity) were identified in the GenBank non-redundant DNA database. All use an RNA I/RNA II-Rom system for replication initiation, but each contains a unique fragment adjacent to the nmr site. The segment of the NTP16/pCERC7 group that encodes the LsoAB toxin-antitoxin system is replaced by a different segment in other family groups. The point at which the sequences diverge is between the XerC and XerD sites of the dif site at one end of nmr, suggesting that the evolution of this broad group of plasmids involves XerC/XerD recombination.
|
1311. |
da Silva KC,
Cunha MP,
Cerdeira L,
de Oliveira MG,
de Oliveira MC,
Gomes CR,
Lincopan N,
Knöbl T,
Moreno AM,
( 2017 ) High-virulence CMY-2- and CTX-M-2-producing avian pathogenic Escherichia coli strains isolated from commercial turkeys. PMID : 27773543 : DOI : 10.1016/j.diagmicrobio.2016.10.001 Abstract >>
This study reports the high-virulence phylogenetic backgrounds of CMY-2- and CTX-M-2-producing avian pathogenic Escherichia coli strains isolated from turkeys sent to slaughter and condemned by airsacculitis in Brazil. Among 300 air sac samples, seven E. coli strains produced plasmid-mediated CMY-2-type AmpC, of which three carried also the blaCTX-M-2 Extended Spectrum Beta-Lactamase encoding gene. Interestingly, the transfer of the blaCMY-2 gene was positive for three E. coli strains, being associated with the presence of IncI1 plasmids. The complete sequence of the representative pJB10 plasmid revealed that the blaCMY-2 gene was within a transposon-like element in the classical genetic environment consisting of tnpA-blaCMY-2-blc-sugE structure. This plasmid with 94-kb belonged to the sequence type (ST) 12 among IncI1 plasmids, which has been associated with the worldwide spread of blaCMY-2 among Salmonella enterica and E. coli. Furthermore, to the best of our knowledge, this is the first complete sequence of a CMY-2-encoding plasmid derived from an Escherichia coli isolated from food-producing animals in Latin America.
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1312. |
Wen Y,
Pu X,
Zheng W,
Hu G,
( 2016 ) High Prevalence of Plasmid-Mediated Quinolone Resistance and IncQ Plasmids Carrying qnrS2 Gene in Bacteria from Rivers near Hospitals and Aquaculture in China. PMID : 27427763 : DOI : 10.1371/journal.pone.0159418 PMC : PMC4948828 Abstract >>
Effluents from hospital and aquaculture are considered important sources of quinolone resistance. However, little information is available on the impact of this effluent on nearby rivers. In this study, 188 ciprofloxacin-resistant bacterial isolates obtained from rivers near hospitals and aquaculture were screened for plasmid-mediated quinolone resistance (PMQR) genes. Species identification, antibiotic susceptibility testing, and PMQR gene transferability assessment were conducted for PMQR-positive bacteria. Representative qnrS2-encoding plasmids were subsequently sequenced using a primer-walking approach. In total, 44 isolates (23.4%) were positive for qnr genes (16 qnrB2, 3 qnrS1, and 25 qnrS2) and 32 isolates (17.0%) were positive for aac(6')-Ib-cr. Other PMQR genes were not detected. The qnrB2 and aac(6')-Ib-cr genes had a higher prevalence in aquaculture samples than in hospital samples, and were significantly associated with Enterobacteriaceae (p < 0.05). In contrast, the prevalence of qnrS2 was not site-related, but was significantly associated with Aeromonas spp. (p < 0.05). All PMQR isolates were resistant to three or more classes of antibiotics. Eleven qnrS2-harboring plasmids from Aeromonas spp., including a novel conjugative plasmid pHP18, were selected for sequencing. These plasmids were small in size (6,388-16,197 bp) and belonged to the IncQ or IncU plasmid family, with qnrS2 being part of a mobile insertion cassette. Taken together, our findings suggest that aquaculture is a possible source for aac(6')-Ib-cr and qnrB2 dissemination, and demonstrate the ubiquity of qnrS2 in aquatic environments. Finally, Aeromonas spp. served as vectors for qnrS2 with the help of IncQ-type plasmids.
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1313. |
Ferdous M,
Friedrich AW,
Grundmann H,
de Boer RF,
Croughs PD,
Islam MA,
Kluytmans-van den Bergh MF,
Kooistra-Smid AM,
Rossen JW,
( 2016 ) Molecular characterization and phylogeny of Shiga toxin-producing Escherichia coli isolates obtained from two Dutch regions using whole genome sequencing. PMID : 27058887 : DOI : 10.1016/j.cmi.2016.03.028 Abstract >>
Shiga toxin-producing Escherichia coli (STEC) is one of the major causes of human gastrointestinal disease and has been implicated in sporadic cases and outbreaks of diarrhoea, haemorrhagic colitis and haemolytic uremic syndrome worldwide. In this study, we determined the molecular characteristics and phylogenetic relationship of STEC isolates, and their genetic diversity was compared to that of other E. coli populations. Whole genome sequencing was performed on 132 clinical STEC isolates obtained from the faeces of 129 Dutch patients with gastrointestinal complaints. STEC isolates of this study belonged to 44 different sequence types (STs), 42 serogenotypes and 14 stx subtype combinations. Antibiotic resistance genes were more frequently present in stx1-positive isolates compared to stx2 and stx1 + stx2-positive isolates. The iha, mchB, mchC, mchF, subA, ireA, senB, saa and sigA genes were significantly more frequently present in eae-negative than in eae-positive STEC isolates. Presence of virulence genes encoding type III secretion proteins and adhesins was associated with isolates obtained from patients with bloody diarrhoea. Core genome phylogenetic analysis showed that isolates clustered according to their ST or serogenotypes irrespective of stx subtypes. Isolates obtained from patients with bloody diarrhoea were from diverse phylogenetic backgrounds. Some STEC isolates shared common ancestors with non-STEC isolates. Whole genome sequencing is a powerful tool for clinical microbiology, allowing high-resolution molecular typing, population structure analysis and detailed molecular characterization of strains. STEC isolates of a substantial genetic diversity and of distinct phylogenetic groups were observed in this study.
|
1314. |
Harmer CJ,
Partridge SR,
Hall RM,
( 2016 ) pDGO100, a type 1 IncC plasmid from 1981 carrying ARI-A and a Tn1696-like transposon in a novel integrating element. PMID : 27318267 : DOI : 10.1016/j.plasmid.2016.06.002 Abstract >>
Most A/C plasmids sequenced to date were recovered in the last two decades. To gain insight into the evolution of this group, the IncC plasmid pDGO100, found in a multiply antibiotic-resistant Escherichia coli strain isolated in 1981, was sequenced. pDGO100 belongs to the type 1 lineage and carries an ARI-A antibiotic resistance island but not an ARI-B island. The A/C2 backbone of pDGO100 has a deletion in the rhs1 gene previously found in pRMH760 and differs by only six single base pair substitutions from pRMH760, recovered at the same hospital 16years later. This confirms that the separation of type 1 and type 2 IncC plasmids is long standing. The ARI-A islands are also closely related, but pRMH760 contains Tn4352B in tniA of Tn402, while in pDGO100, Tn4352 has inserted into merA of pDUmer. pDGO100 also carries an additional 46kb insertion that includes a Tn1696-like transposon with the dfrB3 gene cassette. This insertion was identified as a novel integrating element, with an int gene at one end, and also includes the fec iron uptake operon that has been acquired from the E. coli chromosome. Related integrating elements carrying the same int gene were found in A/C2, IncHI1, and IncHI2 plasmids, and in the chromosomes of Enterobacter cloacae, Klebsiella oxytoca, and Cronobacter sakazakii isolates. In the Enterobacteriaceae chromosomes, these integrating elements appear to target a gene encoding a radical SAM superfamily protein. In the A/C2, IncHI1, and IncHI2 plasmids, genes encoding a phosphoadenosine phosphosulfate reductase were interrupted. The extremities of the integrating element are highly conserved, whilst the internal gene content varies. The detection of integrative elements in plasmids demonstrates an increased range of locations into which this type of mobile element can integrate and insertion in plasmids is likely to assist their spread.
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1315. |
Sonnevend ?,
Ghazawi A,
Alqahtani M,
Shibl A,
Jamal W,
Hashmey R,
Pal T,
( 2016 ) Plasmid-mediated colistin resistance in Escherichia coli from the Arabian Peninsula. PMID : 27566913 : DOI : 10.1016/j.ijid.2016.07.007 Abstract >>
Searching for the presence of the mcr-1 gene in colistin resistant Enterobacteriaceae in countries of the Arabian Peninsula. Seventy-five independent, colistin resistant Enterobacteriaceae strains isolated from clinical cases in Bahrain, Kuwait, Oman, Saudi Arabia and the United Arab Emirates were tested by PCR for the mcr-1 gene. mcr-1 positive strains were genotyped, and their antibiotic susceptibility was established. The mcr-1 containing plasmids were mobilized into Escherichia coli K-12 and their sequence was determined. Four E. coli isolates (two from Bahrain, one from Saudi Arabia and one from the United Arab Emirates) were identified carrying the mcr-1 gene on conjugative plasmids. They belonged to global multidrug resistant E. coli clones, i.e. ST648, ST224, ST68 and ST131, respectively. One strain carried the blaNDM-1 carbapenemase gene. Three strains carried mcr-1 on IncI2 type plasmids, one of them also harboring a blaCTX-M-64 gene. In the fourth strain mcr-1 was located on a 240kb IncHI2 plasmid co-harboring 13 other resistance genes. This is the first report on the presence of the plasmid-coded mcr-1 gene in a variety of multi-resistant clinical isolates from the Arabian Peninsula indicating that several commonly used antibiotics can potentially facilitate the spread of mcr-1 carrying strains, or directly, mcr-1 containing plasmids.
|
1316. |
Wailan AM,
Sidjabat HE,
Yam WK,
Alikhan NF,
Petty NK,
Sartor AL,
Williamson DA,
Forde BM,
Schembri MA,
Beatson SA,
Paterson DL,
Walsh TR,
Partridge SR,
( 2016 ) Mechanisms Involved in Acquisition of blaNDM Genes by IncA/C2 and IncFIIY Plasmids. PMID : 27114281 : DOI : 10.1128/AAC.00368-16 PMC : PMC4914633 Abstract >>
blaNDM genes confer carbapenem resistance and have been identified on transferable plasmids belonging to different incompatibility (Inc) groups. Here we present the complete sequences of four plasmids carrying a blaNDM gene, pKP1-NDM-1, pEC2-NDM-3, pECL3-NDM-1, and pEC4-NDM-6, from four clinical samples originating from four different patients. Different plasmids carry segments that align to different parts of the blaNDM region found on Acinetobacter plasmids. pKP1-NDM-1 and pEC2-NDM-3, from Klebsiella pneumoniae and Escherichia coli, respectively, were identified as type 1 IncA/C2 plasmids with almost identical backbones. Different regions carrying blaNDM are inserted in different locations in the antibiotic resistance island known as ARI-A, and ISCR1 may have been involved in the acquisition of blaNDM-3 by pEC2-NDM-3. pECL3-NDM-1 and pEC4-NDM-6, from Enterobacter cloacae and E. coli, respectively, have similar IncFIIY backbones, but different regions carrying blaNDM are found in different locations. Tn3-derived inverted-repeat transposable elements (TIME) appear to have been involved in the acquisition of blaNDM-6 by pEC4-NDM-6 and the rmtC 16S rRNA methylase gene by IncFIIY plasmids. Characterization of these plasmids further demonstrates that even very closely related plasmids may have acquired blaNDM genes by different mechanisms. These findings also illustrate the complex relationships between antimicrobial resistance genes, transposable elements, and plasmids and provide insights into the possible routes for transmission of blaNDM genes among species of the Enterobacteriaceae family.
|
1317. |
Zurita J,
Ortega-Paredes D,
Barba P,
( 2016 ) First Description of Shigella sonnei Harboring blaCTX-M-55 Outside Asia. PMID : 27558432 : DOI : 10.4014/jmb.1605.05069 Abstract >>
Shigella sonnei harboring blaCTX-M-55 was isolated outside of Asia for the first time. The blaCTX-M-55 gene was found to be downstream of ISEcp-1 and located in a ~130 kb conjugative plasmid belonging to the I1 incompatibility group. The strain was recovered from a 7-year-old Ecuadorian girl with watery diarrhea who had not travelled abroad. Recent local data describe the emergence of blaCTX-M-55 and other variants typically found in Asia in the Andean Region, suggesting that increased travel of humans and trade relationships with Asian countries are influencing the current Ecuadorian bacterial resistance situation.
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1318. |
Wang S,
Liu X,
Xu X,
Zhao Y,
Yang D,
Han X,
Tian M,
Ding C,
Peng D,
Yu S,
( 2016 ) Escherichia coli type III secretion system 2 (ETT2) is widely distributed in avian pathogenic Escherichia coli isolates from Eastern China. PMID : 27103184 : DOI : 10.1017/S0950268816000820 Abstract >>
Pathogens utilize type III secretion systems to deliver effector proteins, which facilitate bacterial infections. The Escherichia coli type III secretion system 2 (ETT2) which plays a crucial role in bacterial virulence, is present in the majority of E. coli strains, although ETT2 has undergone widespread mutational attrition. We investigated the distribution and characteristics of ETT2 in avian pathogenic E. coli (APEC) isolates and identified five different ETT2 isoforms, including intact ETT2, in 57�P6% (141/245) of the isolates. The ETT2 locus was present in the predominant APEC serotypes O78, O2 and O1. All of the ETT2 loci in the serotype O78 isolates were degenerate, whereas an intact ETT2 locus was mostly present in O1 and O2 serotype strains, which belong to phylogenetic groups B2 and D, respectively. Interestingly, a putative second type III secretion-associated locus (eip locus) was present only in the isolates with an intact ETT2. Moreover, ETT2 was more widely distributed in APEC isolates and exhibited more isoforms compared to ETT2 in human extraintestinal pathogenic E. coli, suggesting that APEC might be a potential risk to human health. However, there was no distinct correlation between ETT2 and other virulence factors in APEC.
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1319. |
Zhang W,
Ba P,
Liu K,
Jin Y,
Wang H,
Guo Q,
Sun H,
Xu J,
Xiong Y,
Xu Y,
Bai X,
Zhao A,
( 2016 ) Genetic Diversity of Intimin Gene of Atypical Enteropathogenic Escherichia coli Isolated from Human, Animals and Raw Meats in China. PMID : 27031337 : DOI : 10.1371/journal.pone.0152571 PMC : PMC4816571 Abstract >>
Atypical enteropathogenic Escherichia coli (aEPEC) is considered to be an emerging enteropathogen that is more prevalent than typical EPEC in developing and developed countries. The major adherence factor, intimin, an outer membrane protein encoded by eae, plays a pivotal role in the pathogenesis of aEPEC. This study investigated the distribution and polymorphisms of intimin subtypes of 143 aEPEC strains from diarrheal patients, healthy carriers, animals, and raw meats in China. These aEPEC strains belonged to more than 71 different serotypes, which comprised 52 O serogroups and 24 H types. Sixty-eight different eae genotypes and 19 intimin subtypes were detected. Eighteen, eight, seven, and five intimin subtypes were identified from 86 diarrheal patients, 14 healthy carriers, 19 animals, and 24 raw meats strains, respectively. Intimin �]1 was the most prevalent subtype in strains from diarrheal patients (34.88%) and animals (47.37%). There was a statistically significant difference in the distribution of eae-�]1 between diarrheal patients and healthy carriers (P = 0.004). Intimin-�c was more predominant among raw meat strains (50%) than among diarrheal patients strains (12.79%, P = 0.0003), healthy carrier strains (7.14%, P = 0.007), or animal strains (15.79%, P = 0.020). The two predominant subtypes (eae-�]1 and eae-�c) had considerable polymorphisms with no significant differences among the four sources. PFGE analysis revealed 119 distinct patterns and the strains were clustered into 11 groups with similarity indices ranging from 63% to 100%. These results suggest that in China, aEPEC strains from different sources are highly heterogeneous. Animals and raw meats are important sources of genetically diverse intimin-harboring aEPEC, which might serve as important transmission vehicles of these bacteria.
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1320. |
Li A,
Yang Y,
Miao M,
Chavda KD,
Mediavilla JR,
Xie X,
Feng P,
Tang YW,
Kreiswirth BN,
Chen L,
Du H,
( 2016 ) Complete Sequences of mcr-1-Harboring Plasmids from Extended-Spectrum-�]-Lactamase- and Carbapenemase-Producing Enterobacteriaceae. PMID : 27090180 : DOI : 10.1128/AAC.00550-16 PMC : PMC4914624 Abstract >>
Here we completely sequenced four mcr-1-haboring plasmids, isolated from two extended-spectrum-�]-lactamase (ESBL)-producing Escherichia coli and two carbapenemase-producing Klebsiella pneumoniae clinical isolates. The mcr-1-harboring plasmids from an E. coli sequence type 2448 (ST2448) isolate and two K. pneumoniae ST25 isolates were identical (all pMCR1-IncX4), belonging to the IncX4 incompatibility group, while the plasmid from an E. coli ST2085 isolate (pMCR1-IncI2) belongs to the IncI2 group. A nearly identical 2.6-kb mcr-1-pap2 element was found to be shared by all mcr-1-carrying plasmids.
|
1321. |
Bai X,
Zhang W,
Tang X,
Xin Y,
Xu Y,
Sun H,
Luo X,
Pu J,
Xu J,
Xiong Y,
Lu S,
( 2016 ) Shiga Toxin-Producing Escherichia coli in Plateau Pika (Ochotona curzoniae) on the Qinghai-Tibetan Plateau, China. PMID : 27047483 : DOI : 10.3389/fmicb.2016.00375 PMC : PMC4802371 Abstract >>
Shiga toxin-producing Escherichia coli (STEC) are an emerging group of zoonotic pathogens. Ruminants are the natural reservoir of STEC. In this study we determined the prevalence and characteristics of the STEC in plateau pika (Ochotona curzoniae) on the Qinghai-Tibetan Plateau, China. A total of 1116 pika samples, including 294 intestinal contents samples, 317 fecal samples, and 505 intestinal contents samples, were collected from May to August in the years 2012, 2013, and 2015, respectively. Twenty-one samples (1.88%) yielded at least one STEC isolate; in total, 22 STEC isolates were recovered. Thirteen different O serogroups and 14 serotypes were identified. One stx 1 subtype (stx 1a) and three stx 2 subtypes (stx 2a, stx 2b, and stx 2d) were present in the STEC isolates. Fifteen, fourteen, and three STEC isolates harbored the virulence genes ehxA, subA, and astA, respectively. Adherence-associated genes iha and saa were, respectively, present in 72.73 and 68.18% of the STEC isolates. Twenty antibiotics were active against all the STEC isolates; all strains were resistant to penicillin G, and some to cephalothin or streptomycin. The 22 STEC isolates were divided into 16 pulsed-field gel electrophoresis patterns and 12 sequence types. Plateau pikas may play a role in the ongoing circulation of STEC in the Qinghai-Tibetan plateau. This study provides the first report on STEC in plateau pikas and new information about STEC reservoirs in wildlife. Based on the serotypes, virulence gene profiles and multi-locus sequence typing (MLST) analysis, the majority of these pika STECs may pose a low public health risk.
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1322. |
Xavier BB,
Lammens C,
Butaye P,
Goossens H,
Malhotra-Kumar S,
( 2016 ) Complete sequence of an IncFII plasmid harbouring the colistin resistance gene mcr-1 isolated from Belgian pig farms. PMID : 27261261 : DOI : 10.1093/jac/dkw191 Abstract >>
N/A
|
1323. |
Hinenoya A,
Yasuda N,
Hibino T,
Shima A,
Nagita A,
Tsukamoto T,
Yamasaki S,
( 2017 ) Isolation and Characterization of an Escherichia albertii Strain Producing Three Different Toxins from a Child with Diarrhea. PMID : 27580579 : DOI : 10.7883/yoken.JJID.2016.186 Abstract >>
Here, we report a bacterium-isolated as the sole pathogen from a child with diarrhea-harboring eae and 2 different cytolethal distending toxin genes (cdt) that are homologous to Escherichia coli cdt-I and cdt-II. The bacterium was originally identified as atypical E. coli by conventional biochemical testing, but was finally identified as E. albertii by multilocus sequence analysis, which is the only method that can currently differentiate E. albertii from E. coli. The Shiga toxin 2f (stx2f) genes were also detected in the strain. Production of these 3 toxins was confirmed by western blotting and/or a cytotoxicity assay using eukaryotic cell lines. This is the first report showing the biological activity of CDT-I, CDT-II, and Stx2f in E. albertii.
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1324. |
Zhuge X,
Tang F,
Zhu H,
Mao X,
Wang S,
Wu Z,
Lu C,
Dai J,
Fan H,
( 2016 ) AutA and AutR, Two Novel Global Transcriptional Regulators, Facilitate Avian Pathogenic Escherichia coli Infection. PMID : 27113849 : DOI : 10.1038/srep25085 PMC : PMC4844996 Abstract >>
Bacteria can change its lifestyle during inhabiting in host niches where they survive and replicate by rapidly altering gene expression pattern to accommodate the new environment. In this study, two novel regulators in avian pathogenic Escherichia coli (APEC) were identified and designated as AutA and AutR. RT-PCR and �]-galactosidase assay results showed that AutA and AutR co-regulated the expression of adhesin UpaB in APEC strain DE205B. Electrophoretic mobility shift assay showed that AutA and AutR could directly bind the upaB promoter DNA. In vitro transcription assay indicated that AutA could activate the upaB transcription, while AutR inhibited the upaB transcription due to directly suppressing the activating effect of AutA on UpaB expression. Transcriptome analysis showed that AutA and AutR coherently affected the expression of hundreds of genes. Our study confirmed that AutA and AutR co-regulated the expression of DE205B K1 capsule and acid resistance systems in E. coli acid fitness island (AFI). Moreover, phenotypic heterogeneity in expression of K1 capsule and acid resistance systems in AFI during host-pathogen interaction was associated with the regulation of AutA and AutR. Collectively speaking, our studies presented that AutA and AutR are involved in APEC adaptive lifestyle change to facilitate its infection.
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1325. |
Zhu YQ,
Zhao JY,
Xu C,
Zhao H,
Jia N,
Li YN,
( 2016 ) Identification of an NDM-5-producing Escherichia coli Sequence Type 167 in a Neonatal Patient in China. PMID : 27406405 : DOI : 10.1038/srep29934 PMC : PMC4942816 Abstract >>
Emergence of New Delhi metallo-�]-lactamase-producing Enterobacteriaceae has become a challenging threat to public health. Two carbapenem-resistant Escherichia coli, strain QD28 and QD29, were recovered from the aspirating sputum of a neonate and the urine of an adult in a Chinese hospital in 2013. Molecular typing revealed that both isolates belonged to the sequence type 167, but they were clonally diverse. Both isolates exhibited resistance to carbapenems, cephalosporins, ciprofloxacin, gentamicin, piperacillin-tazobactam and trimethoprim-sulfamethoxazole. In addition, strain QD28 was also resistant to aztreonam, and strain QD29 was resistant to amikacin, fosfomycin and minocycline. Antimicrobial resistance gene screening revealed that strain QD28 harbored aac(6')-Ib, blaCTX-M-14, blaNDM-5, blaTEM-1 and sul1 genes, and strain QD29 harbored aac(6')-Ib, blaCTX-M-3, blaNDM-5, blaTEM-1, rmtB, sul1 and sul2 genes. The blaNDM-5 gene was found to be located on a 46-kb plasmid in two isolates, and further sequence analysis showed that this plasmid was highly similar to the previously reported IncX3 plasmid pNDM-MGR194 in India. This is the first identification of blaNDM-5-carrying E. coli in the neonatal infection.
|
1326. |
Ross TK,
Achberger EC,
Braymer HD,
( 1989 ) Nucleotide sequence of the McrB region of Escherichia coli K-12 and evidence for two independent translational initiation sites at the mcrB locus. PMID : 2649480 : DOI : 10.1128/jb.171.4.1974-1981.1989 PMC : PMC209847 Abstract >>
The McrB restriction system of Escherichia coli K-12 is responsible for the biological inactivation of foreign DNA that contains 5-methylcytosine residues (E. A. Raleigh and G. Wilson, Proc. Natl. Acad. Sci. USA 83:9070-9074, 1986). Within the McrB region of the chromosome is the mcrB gene, which encodes a protein of 51 kilodaltons (kDa) (T. K. Ross, E. C. Achberger, and H. D. Braymer, Gene 61:277-289, 1987), and the mcrC gene, the product of which is 39 kDa (T. K. Ross, E. C. Achberger, and H. D. Braymer, Mol. Gen. Genet., in press). The nucleotide sequence of a 2,695-base-pair segment encompassing the McrB region was determined. The deduced amino acid sequence was used to identify two open reading frames specifying peptides of 455 and 348 amino acids, corresponding to the products of the mcrB and mcrC genes, respectively. A single-nucleotide overlap was found to exist between the termination codon of the mcrB gene and the proposed initiation codon of the mcrC gene. The presence of an additional peptide of 33 kDa in strains containing various recombinant plasmids with portions of the McrB region has been reported by Ross et al. (Gene 61:277-289, 1987). The analysis of frameshift and deletion mutants of one such hybrid plasmid, pRAB-13, provided evidence for a second translational initiation site within the McrB open reading frame. The proposed start codon for translation of the 33-kDa peptide lies 481 nucleotides downstream from the initiation codon for the 51-kDa mcrB gene product. The 33-kDa peptide may play a regulatory role in the McrB restriction of DNA containing 5-methylcytosine.
|
1327. |
Zhang R,
Sun B,
Wang Y,
Lei L,
Schwarz S,
Wu C,
( 2016 ) Characterization of a cfr-Carrying Plasmid from Porcine Escherichia coli That Closely Resembles Plasmid pEA3 from the Plant Pathogen Erwinia amylovora. PMID : 26525796 : DOI : 10.1128/AAC.02114-15 PMC : PMC4704220 Abstract >>
The multiresistance gene cfr was found in two porcine Escherichia coli isolates, one harboring it on the conjugative 33,885-bp plasmid pFSEC-01, the other harboring it in the chromosomal DNA. Sequence analysis of pFSEC-01 revealed that a 6,769-bp fragment containing the cfr gene bracketed by two IS26 elements was inserted into a plasmid closely related to pEA3 from the plant pathogen Erwinia amylovora, suggesting that pFSEC-01 may be transferred between different bacterial genera of both animal and plant origin.
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1328. |
Lefurgy ST,
Malashkevich VN,
Aguilan JT,
Nieves E,
Mundorff EC,
Biju B,
Noel MA,
Toro R,
Baiwir D,
Papp-Wallace KM,
Almo SC,
Frere JM,
Bou G,
Bonomo RA,
( 2016 ) Analysis of the Structure and Function of FOX-4 Cephamycinase. PMID : 26525784 : DOI : 10.1128/AAC.01887-15 PMC : PMC4750714 Abstract >>
Class C �]-lactamases poorly hydrolyze cephamycins (e.g., cefoxitin, cefotetan, and moxalactam). In the past 2 decades, a new family of plasmid-based AmpC �]-lactamases conferring resistance to cefoxitin, the FOX family, has grown to include nine unique members descended from the Aeromonas caviae chromosomal AmpC. To understand the basis for the unique cephamycinase activity in the FOX family, we determined the first X-ray crystal structures of FOX-4, apo enzyme and the acyl-enzyme with its namesake compound, cefoxitin, using the Y150F deacylation-deficient variant. Notably, recombinant expression of N-terminally tagged FOX-4 also yielded an inactive adenylylated enzyme form not previously observed in �]-lactamases. The posttranslational modification (PTM), which occurs on the active site Ser64, would not seem to provide a selective advantage, yet might present an opportunity for the design of novel antibacterial drugs. Substantial ligand-induced changes in the enzyme are seen in the acyl-enzyme complex, particularly the R2 loop and helix H10 (P289 to N297), with movement of F293 by 10.3 ?. Taken together, this study provides the first picture of this highly proficient class C cephamycinase, uncovers a novel PTM, and suggests a possible cephamycin resistance mechanism involving repositioning of the substrate due to the presence of S153P, N289P, and N346I substitutions in the ligand binding pocket.
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1329. |
Kato K,
Ishii R,
Hirano S,
Ishitani R,
Nureki O,
( 2015 ) Structural Basis for the Catalytic Mechanism of DncV, Bacterial Homolog of Cyclic GMP-AMP Synthase. PMID : 25865248 : DOI : 10.1016/j.str.2015.01.023 Abstract >>
Cyclic dinucleotides (CDNs) play key roles as second messengers and signaling molecules in bacteria and metazoans. The newly identified dinucleotide cyclase in Vibrio cholerae (DncV) produces three different CDNs containing two 3'-5' phosphodiester bonds, and its predominant product is cyclic GMP-AMP, whereas mammalian cyclic GMP-AMP synthase (cGAS) produces only cyclic GMP-AMP containing mixed 2'-5' phosphodiester bonds. We report the crystal structures of V. cholerae and Escherichia coli DncV in complex with various nucleotides in the pre-reaction states. The high-resolution structures revealed that DncV preferably recognizes ATP and GTP as acceptor and donor nucleotides, respectively, in the first nucleotidyl transfer reaction. Considering the recently reported intermediate structures, our pre-reaction state structures provide the precise mechanism of 3'-5' linked cyclic AMP-GMP production in bacteria. A comparison with cGAS in the pre-reaction states suggests that the orientation of the acceptor nucleotide primarily determines the distinct linkage specificities between DncV and cGAS.
|
1330. |
Saul D,
Spiers AJ,
McAnulty J,
Gibbs MG,
Bergquist PL,
Hill DF,
( 1989 ) Nucleotide sequence and replication characteristics of RepFIB, a basic replicon of IncF plasmids. PMID : 2651415 : DOI : 10.1128/jb.171.5.2697-2707.1989 PMC : PMC209954 Abstract >>
A second autonomous replicon of P307, RepFIB, has been isolated that has significant homology with other replicons in IncFI group plasmids. Eleven homologous repeats of 21 base pairs are present on the sequence and flank an open reading frame capable of coding for a protein of about Mr = 40,000. This protein was identified by maxicell analysis of cloned RepFIB. A series of deletion mutations of RepFIB were inserted into a DNA polymerase I-dependent vector and examined for their replication proficiency in a polA1 strain. These experiments defined a minimal replication region of 1.6 kilobases which includes the three repeats immediately upstream and downstream of the open reading frame. Deletion of a second set of repeats further downstream doubled the copy number of a chimeric plasmid replicating under RepFIB control. It was concluded that these repeats control the copy number of the replicon. Incompatibility tests showed that all three sets of repeats could express incompatibility with a resident RepFIB plasmid.
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1331. |
Ghaderpour A,
Ho WS,
Chew LL,
Bong CW,
Chong VC,
Thong KL,
Chai LC,
( 2015 ) Diverse and abundant multi-drug resistant E. coli in Matang mangrove estuaries, Malaysia. PMID : 26483759 : DOI : 10.3389/fmicb.2015.00977 PMC : PMC4586456 Abstract >>
E.coli, an important vector distributing antimicrobial resistance in the environment, was found to be multi-drug resistant, abundant, and genetically diverse in the Matang mangrove estuaries, Malaysia. One-third (34%) of the estuarine E. coli was multi-drug resistant. The highest antibiotic resistance prevalence was observed for aminoglycosides (83%) and beta-lactams (37%). Phylogenetic groups A and B1, being the most predominant E. coli, demonstrated the highest antibiotic resistant level and prevalence of integrons (integron I, 21%; integron II, 3%). Detection of phylogenetic group B23 downstream of fishing villages indicates human fecal contamination as a source of E. coli pollution. Enteroaggregative E. coli (1%) were also detected immediately downstream of the fishing village. The results indicated multi-drug resistance among E. coli circulating in Matang estuaries, which could be reflective of anthropogenic activities and aggravated by bacterial and antibiotic discharges from village lack of a sewerage system, aquaculture farms and upstream animal husbandry.
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1332. |
Lee CS,
Li JJ,
Doi Y,
( 2015 ) Complete sequence of conjugative IncA/C plasmid encoding CMY-2 �]-lactamase and RmtE 16S rRNA methyltransferase. PMID : 25896689 : DOI : 10.1128/AAC.00852-15 PMC : PMC4468717 Abstract >>
N/A
|
1333. |
Wang D,
Huang X,
Chen J,
Mou Y,
Li H,
Yang L,
( 2015 ) Characterization of genetic structures of the QepA3 gene in clinical isolates of Enterobacteriaceae. PMID : 26528280 : DOI : 10.3389/fmicb.2015.01147 PMC : PMC4606065 Abstract >>
QepA is one of the genes that confer quinolone resistance in bacteria. The aim of this study was to analyze the genetic structures of plasmids that carry a qepA3, a recently discovered allele of qepA in Enterobacteriaceae clinical isolates. 656 non-redundant Enterobacteriaceae clinical isolates were screened for the qepA3 gene and five isolates were identified to carry the gene. Plasmids were isolated from these isolates and were found to increase antibiotic resistance once the plasmids were transferred to Escherichia coli. These plasmids were subcloned and sequenced to analyze the genetic structures surrounding the qepA3 gene. The results showed that the five plasmids had different genetic structures; two of the qepA3-containning isolates had either the bla CTX-M-14 or bla TEM-12 gene instead of the bla TEM-1 gene. The structures of both pKP3764 and pECL3786 have not been previously described. In comparison with pHPA, there were a number of changes in DNA sequences up- and down-stream of the qepA3 gene. These findings provide better understanding of the genetic variations in qepA3 and would be useful for diagnosis and control of quinolone resistance in clinical settings.
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1334. |
Bajaj P,
Kanaujia PK,
Singh NS,
Sharma S,
Kumar S,
Virdi JS,
( 2016 ) Quinolone co-resistance in ESBL- or AmpC-producing Escherichia coli from an Indian urban aquatic environment and their public health implications. PMID : 26498967 : DOI : 10.1007/s11356-015-5609-x Abstract >>
Quinolone and �]-lactam antibiotics constitute major mainstay of treatment against infections caused by pathogenic Escherichia coli. Presence of E. coli strains expressing co-resistance to both these antibiotic classes in urban aquatic environments which are consistently being used for various anthropogenic activities represents a serious public health concern. From a heterogeneous collection of 61 E. coli strains isolated from the river Yamuna traversing through the National Capital Territory of Delhi (India), those harboring blaCTX-M-15 (n = 10) or blaCMY-42 (n = 2) were investigated for co-resistance to quinolones and the molecular mechanisms thereof. Resistance was primarily attributed to amino acid substitutions in the quinolone resistance-determining regions (QRDRs) of GyrA (S83L �� D87N) and ParC (S80I �� E84K). One of the E. coli strains, viz., IPE, also carried substitutions in GyrB and ParE at positions Ser492��Asn and Ser458��Ala, respectively. The phenotypically susceptible strains nevertheless carried plasmid-mediated quinolone resistance (PMQR) gene, viz., qnrS, which showed co-transfer to the recipient quinolone-sensitive E. coli J53 along with the genes encoding �]-lactamases and led to increase in minimal inhibitory concentrations of quinolone antibiotics. To the best of our knowledge, this represents first report of molecular characterization of quinolone co-resistance in E. coli harboring genes for ESBLs or AmpC �]-lactamases from a natural aquatic environment of India. The study warrants true appreciation of the potential of urban aquatic environments in the emergence and spread of multi-drug resistance and underscores the need to characterize resistance genetic elements vis-?-vis their public health implications, irrespective of apparent phenotypic resistance.
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1335. |
Hagelueken G,
Hoffmann J,
Schubert E,
Duthie FG,
Florin N,
Konrad L,
Imhof D,
Behrmann E,
Morgner N,
Schiemann O,
( 2016 ) Studies on the X-Ray and Solution Structure of FeoB from Escherichia coli BL21. PMID : 27332122 : DOI : 10.1016/j.bpj.2016.05.018 PMC : PMC4919421 Abstract >>
The ferrous iron transporter FeoB is an important factor in the iron metabolism of many bacteria. Although several structural studies have been performed on its cytosolic GTPase domain (NFeoB), the full-length structure of FeoB remains elusive. Based on a crystal packing analysis that was performed on crystals of NFeoB, a trimeric structure of the FeoB channel was proposed, where the transport pore runs along the trimer axis. Because this trimer has not been observed in some subsequently solved structures of NFeoB homologs, it remains unclear whether or not the trimer is indeed functionally relevant. Here, pulsed electron-electron double resonance spectroscopy, negative stain electron microscopy, and native mass spectrometry are used to analyze the oligomeric state of different soluble and full-length FeoB constructs. The results show that the full-length protein is predominantly monomeric, whereas dimers and trimers are formed to a small percentage. Furthermore, the solution structure of the switch I region is analyzed by pulsed electron-electron double resonance spectroscopy and a new, to our knowledge, crystal structure of NFeoB from Escherichia coli BL21 is presented.
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1336. |
Wang S,
Bao Y,
Meng Q,
Xia Y,
Zhao Y,
Wang Y,
Tang F,
ZhuGe X,
Yu S,
Han X,
Dai J,
Lu C,
( 2015 ) IbeR facilitates stress-resistance, invasion and pathogenicity of avian pathogenic Escherichia coli. PMID : 25768126 : DOI : 10.1371/journal.pone.0119698 PMC : PMC4359115 Abstract >>
Systemic infections by avian pathogenic Escherichia coli (APEC) are economically devastating to poultry industries worldwide. IbeR, located on genomic island GimA, was shown to serve as an RpoS-like regulator in rpoS gene mutation neonatal meningitis E. coli (NMEC) RS218. However, the role of IbeR in pathogenicity of APEC carrying active RpoS has not yet been investigated. We showed that the APEC IbeR could elicit antibodies in infected ducks, suggesting that IbeR might be involved in APEC pathogenicity. To investigate the function of IbeR in APEC pathogenesis, mutant and complementation strains were constructed and characterized. Inactivation of ibeR led to attenuated virulence and reduced invasion capacity towards DF-1 cells, brains and cerebrospinal fluid (CSF) in vitro and in vivo. Bactericidal assays demonstrated that the mutant strain had impaired resistance to environmental stress and specific pathogen-free (SPF) chicken serum. These virulence-related phenotypes were restored by genetic complementation. Quantitative real-time reverse transcription PCR revealed that IbeR controlled expression of stress-resistance genes and virulence genes, which might led to the associated virulence phenotype.
|
1337. |
Baede VO,
Wagenaar JA,
Broens EM,
Duim B,
Dohmen W,
Nijsse R,
Timmerman AJ,
Hordijk J,
( 2015 ) Longitudinal study of extended-spectrum-�]-lactamase- and AmpC-producing Enterobacteriaceae in household dogs. PMID : 25779568 : DOI : 10.1128/AAC.04576-14 PMC : PMC4432141 Abstract >>
A longitudinal study was performed to (i) investigate the continuity of shedding of extended-spectrum-beta-lactamase (ESBL)-producing Enterobacteriaceae in dogs without clinical signs, (ii) identify dominant plasmid-mediated ESBL genes, and (iii) quantify ESBL-producing Enterobacteriaceae in feces. Fecal samples from 38 dogs were collected monthly for 6 months. Additional samples were collected from 7 included dogs on a weekly basis for 6 weeks. Numbers of CFU per gram of feces for non-wild-type Enterobacteriaceae were determined by using MacConkey agar supplemented with 1 mg/liter cefotaxime (MCC), and those for total Enterobacteriaceae were determined by using MacConkey agar. Cefotaxime-resistant isolates were screened by PCR and sequence analysis for the presence of bla(CTX-M), bla(CMY), bla(SHV), bla(OXA), and bla(TEM) gene families. Bacterial species were identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis. PCR-negative isolates were tested by a double-disk synergy test for enhanced AmpC expression. A total of 259 samples were screened, and 126 samples were culture positive on MCC, resulting in 352 isolates, 327 of which were Escherichia coli. Nine dogs were continuously positive during this study, and 6 dogs were continuously negative. Monthly or weekly shifts in fecal shedding were observed for 23 dogs. Genotyping showed a large variety of ESBL genes and gene combinations at single and multiple consecutive sampling moments. The ESBL genes bla(CTX-M-1), bla(CTX-M-14), bla(CTX-M-15), bla(SHV-12), and bla(CMY-2) were most frequently found. The mean number of CFU of non-wild-type Enterobacteriaceae was 6.11 �� 10(8) CFU/g feces. This study showed an abundance of ESBL-producing Enterobacteriaceae in dogs in the Netherlands, mostly in high concentrations. Fecal shedding was shown to be highly dynamic over time, which is important to consider when studying ESBL epidemiology.
|
1338. |
Loh S,
Cram D,
Skurray R,
( 1989 ) Nucleotide sequence of the leading region adjacent to the origin of transfer on plasmid F and its conservation among conjugative plasmids. PMID : 2693941 : DOI : 10.1007/bf00261174 Abstract >>
The leading region of the Escherichia coli K12 F plasmid is the first segment of DNA to be transferred into the recipient cell during conjugal transfer. We report the nucleotide sequence of the 64.20-66.77F portion of the leading region immediately adjacent to the origin of transfer, oriT. The 2582 bp region encodes three open reading frames, ORF95, ORF169 and ORF273; the product of ORF273, is equivalent in size and map location to the 35 kDa protein, 6d, previously described (Cram et al. 1984). S1 nuclease analyses of mRNA transcripts have identified a potential promoter for ORF95 and ORF273 and indicated that these ORFs are transcribed as a single transcript; in contrast, ORF169 appears to be transcribed from two overlapping promoters on the complementary DNA strand. The products of ORF95 and ORF273 are mainly hydrophilic and are probably located in the cytoplasm. ORF273 shares some homology with DNA-binding proteins. There is a signal peptide sequence at the NH2-terminus of ORF169 and the mature form of ORF169 probably resides in the periplasm due to its hydrophilic nature. Both ORF273 and ORF169 are well conserved among conjugative F-like and a few non-F-like plasmids. On the other hand, ORF95 sequences are only present on some of these plasmids. Several primosome and integration host factor recognition sites are present implicating this region in DNA metabolism and/or replication functions.
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1339. |
Paul-Satyaseela M,
Murali S,
Thirunavukkarasu B,
Naraharirao MH,
Jambulingam M,
( 2016 ) Characterization of Antibiotic Resistance Profiles of Ocular Enterobacteriaceae Isolates. PMID : 27141313 : DOI : 10.1556/1886.2015.00047 PMC : PMC4838984 Abstract >>
Emergence of extended-spectrum �]-lactamase (ESBL) and fluoroquinolone resistance among ocular Enterobacteriaceae is increasing in higher frequency. Therefore, studies are being carried out to understand their multidrug resistance pattern. A total of 101 Enterobacteriaceae isolates recovered from various ocular diseases in a tertiary eye care center at Chennai, India during the period of January 2011 to June 2014 were studied. Forty one randomly chosen isolates were subjected to antibiotic susceptibility by minimum inhibitory concentration (MIC) and genotypic analysis. Of them, 16 were ESBL producers, one was carbapenemase producer and four were resistant to ertapenem which could be due to porin loss associated with AmpC production, and 17 were resistant to fluoroquinolones. Sixteen isolates harbored ESBL genes in which 14 had more than one gene and none of them were positive for blaNDM-1 gene. QNR genes were detected in 18 isolates. ESBL producers were predominantly isolated from conjunctiva. A high degree of ESBL production and fluoroquinolone resistance is seen among the genus Klebsiella sp. Hence, monitoring the rate of ESBL prevalence plays a vital role in the administration of appropriate intravitreal antibiotics to save the vision and also to reduce the development of drug resistance in ocular pathogens.
|
1340. |
Zurfluh K,
Klumpp J,
Nüesch-Inderbinen M,
Stephan R,
( 2016 ) Full-Length Nucleotide Sequences of mcr-1-Harboring Plasmids Isolated from Extended-Spectrum-�]-Lactamase-Producing Escherichia coli Isolates of Different Origins. PMID : 27324774 : DOI : 10.1128/AAC.00935-16 PMC : PMC4997865 Abstract >>
Here, we present the full sequences of three mcr-1-carrying plasmids isolated from extended-spectrum-�]-lactamase (ESBL)-producing Escherichia coli The plasmids belong to three different replicon types and are 34,640 bp, 209,401 bp, and 247,885 bp in size. We describe for the first time a composite transposon containing mcr-1 localized on a multidrug-resistant (MDR) IncHI2 plasmid harboring additional determinants of resistance to six different classes of antibiotics, including the ESBL gene blaCTX-M-1, and heavy metal resistance.
|
1341. |
Zhi C,
Lv L,
Yu LF,
Doi Y,
Liu JH,
( 2016 ) Dissemination of the mcr-1 colistin resistance gene. PMID : 26973307 : DOI : 10.1016/S1473-3099(16)00063-3 Abstract >>
N/A
|
1342. |
Na JH,
Cha SS,
( 2016 ) Structural basis for the extended substrate spectrum of AmpC BER and structure-guided discovery of the inhibition activity of citrate against the class C �]-lactamases AmpC BER and CMY-10. PMID : 27487828 : DOI : 10.1107/S2059798316011311 Abstract >>
AmpC BER is an extended substrate spectrum class C �]-lactamase with a two-amino-acid insertion in the R2 loop compared with AmpC EC2. The crystal structures of AmpC BER (S64A mutant) and AmpC EC2 were determined. Structural comparison of the two proteins revealed that the insertion increases the conformational flexibility of the R2 loop. Two citrate molecules originating from the crystallization solution were observed in the active site of the S64A mutant. One citrate molecule makes extensive interactions with active-site residues that are highly conserved among class C �]-lactamases, whereas the other one is weakly bound. Based on this structural observation, it is demonstrated that citrate, a primary metabolite that is widely used as a food additive, is a competitive inhibitor of two class C �]-lactamases (AmpC BER and CMY-10). Consequently, the data indicate enhancement of the flexibility of the R2 loop as an operative strategy for molecular evolution of extended-spectrum class C �]-lactamases, and also suggest that the citrate scaffold is recognized by the active sites of class C �]-lactamases.
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1343. |
Tsuboi S,
Yamamura S,
Imai A,
Iwasaki K,
( 2016 ) Unexpected Diversity of pepA Genes Encoding Leucine Aminopeptidases in Sediments from a Freshwater Lake. PMID : 26936797 : DOI : 10.1264/jsme2.ME15117 PMC : PMC4791116 Abstract >>
We herein designed novel PCR primers for universal detection of the pepA gene, which encodes the representative leucine aminopeptidase gene, and investigated the genetic characteristics and diversity of pepA genes in sediments of hypereutrophic Lake Kasumigaura, Japan. Most of the amino acid sequences deduced from the obtained clones (369 out of 370) were related to PepA-like protein sequences in the M17 family of proteins. The developed primers broadly detected pepA-like clones associated with diverse bacterial phyla-Alpha-, Beta-, Gamma-, and Deltaproteobacteria, Acidobacteria, Actinobacteria, Aquificae, Chlamydiae, Chloroflexi, Cyanobacteria, Firmicutes, Nitrospirae, Planctomycetes, and Spirochetes as well as the archaeal phylum Thaumarchaeota, indicating that prokaryotes in aquatic environments possessing leucine aminopeptidase are more diverse than previously reported. Moreover, prokaryotes related to the obtained pepA-like clones appeared to be r- and K-strategists, which was in contrast to our previous findings showing that the neutral metalloprotease gene clones obtained were related to the r-strategist genus Bacillus. Our results suggest that an unprecedented diversity of prokaryotes with a combination of different proteases participate in sedimentary proteolysis.
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1344. |
Last NB,
Kolmakova-Partensky L,
Shane T,
Miller C,
( 2016 ) Mechanistic signs of double-barreled structure in a fluoride ion channel. PMID : 27449280 : DOI : 10.7554/eLife.18767 PMC : PMC4969038 Abstract >>
The Fluc family of F(-) ion channels protects prokaryotes and lower eukaryotes from the toxicity of environmental F(-). In bacteria, these channels are built as dual-topology dimers whereby the two subunits assemble in antiparallel transmembrane orientation. Recent crystal structures suggested that Fluc channels contain two separate ion-conduction pathways, each with two F(-) binding sites, but no functional correlates of this unusual architecture have been reported. Experiments here fill this gap by examining the consequences of mutating two conserved F(-)-coordinating phenylalanine residues. Substitution of each phenylalanine specifically extinguishes its associated F(-) binding site in crystal structures and concomitantly inhibits F(-) permeation. Functional analysis of concatemeric channels, which permit mutagenic manipulation of individual pores, show that each pore can be separately inactivated without blocking F(-) conduction through its symmetry-related twin. The results strongly support dual-pathway architecture of Fluc channels.
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1345. |
Perez-Casal JF,
Gammie AE,
Crosa JH,
( 1989 ) Nucleotide sequence analysis and expression of the minimum REPI replication region and incompatibility determinants of pColV-K30. PMID : 2703470 : DOI : 10.1128/jb.171.4.2195-2201.1989 PMC : PMC209877 Abstract >>
We sequenced the minimum REPI replication region and the incompatibility determinants of pColV-K30. The minimum replication region contains an open reading frame which corresponds to a 35-kilodalton (kDa) protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis with maxicells transformed with a number of deletion derivatives demonstrated that this replication region encodes a 39-kDa protein and also established the direction of transcription of the RepI protein gene. The 39-kDa polypeptide was identified as the trans-acting factor essential for replication of REPI-containing plasmids. A translated region of the nucleotide sequence of the RepI protein gene showed homology with the helix-turn-helix binding domains of a number of DNA-binding proteins and also with other plasmid replication proteins. Further nucleotide analysis of the REPI region revealed the presence of direct and inverted repeat sequences in the incE, incF, and ori regions. The REPI ori also contained a perfect DnaA-binding site in addition to a high frequency of occurrence of the DNA adenine methylation (dam) site 5'GATC3'.
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1346. |
Beutin L,
Delannoy S,
Fach P,
( 2016 ) Genetic Analysis and Detection of fliC H1 and fliC H12 Genes Coding for Serologically Closely Related Flagellar Antigens in Human and Animal Pathogenic Escherichia coli. PMID : 26913025 : DOI : 10.3389/fmicb.2016.00135 PMC : PMC4753304 Abstract >>
The E. coli flagellar types H1 and H12 show a high serological cross-reactivity and molecular serotyping appears an advantageous method to establish a clear discrimination between these flagellar types. Analysis of fliC H1 and fliC H12 gene sequences showed that they were 97.5% identical at the nucleotide level. Because of this high degree of homology we developed a two-step real-time PCR detection procedure for reliable discrimination of H1 and H12 flagellar types in E. coli. In the first step, a real-time PCR assay for common detection of both fliC H1 and fliC H12 genes is used, followed in a second step by real-time PCR assays for specific detection of fliC H1 and fliC H12, respectively. The real-time PCR for common detection of fliC H1 and fliC H12 demonstrated 100% sensitivity and specificity as it reacted with all tested E. coli H1 and H12 strains and not with any of the reference strains encoding all the other 51 flagellar antigens. The fliC H1 and fliC H12 gene specific assays detected all E. coli H1 and all E. coli H12 strains, respectively (100% sensitivity). However, both assays showed cross-reactions with some flagellar type reference strains different from H1 and H12. The real-time PCR assays developed in this study can be used in combination for the detection and identification of E. coli H1 and H12 strains isolated from different sources.
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1347. |
Hansen KH,
Bortolaia V,
Nielsen CA,
Nielsen JB,
Schønning K,
Agersø Y,
Guardabassi L,
( 2016 ) Host-Specific Patterns of Genetic Diversity among IncI1-I�^ and IncK Plasmids Encoding CMY-2 �]-Lactamase in Escherichia coli Isolates from Humans, Poultry Meat, Poultry, and Dogs in Denmark. PMID : 27235431 : DOI : 10.1128/AEM.00495-16 PMC : PMC4984282 Abstract >>
CMY-2 is the most common plasmid-mediated AmpC �]-lactamase in Escherichia coli isolates of human and animal origin. The aim of this study was to elucidate the epidemiology of CMY-2-producing E. coli in Denmark. Strain and plasmid relatedness was studied in 93 CMY-2-producing clinical and commensal E. coli isolates collected from 2006 to 2012 from humans, retail poultry meat, broilers, and dogs. Multilocus sequence typing (MLST), antimicrobial susceptibility testing, and conjugation were performed in conjunction with plasmid replicon typing, plasmid multilocus sequence typing (pMLST), restriction fragment length polymorphism (RFLP), and sequencing of selected blaCMY-2-harboring plasmids. MLST revealed high strain diversity, with few E. coli lineages occurring in multiple host species and sample types. blaCMY-2 was detected on plasmids in 83 (89%) isolates. Most (75%) of the plasmids were conjugative and did not (96%) cotransfer resistance to antimicrobials other than cephalosporins. The main replicon types identified were IncI1-I�^ (55%) and IncK (39%). Isolates from different host species mainly carried distinct plasmid subtypes. Seven of the 18 human isolates harbored IncI1-I�^/sequence type 2 (ST2), IncI1-I�^/ST12, or IncK plasmids highly similar to those found among animal isolates, even though highly related human and animal plasmids differed by nonsynonymous single nucleotide polymorphisms (SNPs) or insertion sequence elements. This study clearly demonstrates that the epidemiology of CMY-2 can be understood only by thorough plasmid characterization. To date, the spread of this �]-lactam resistance determinant in Denmark is mainly associated with IncK and IncI1-I�^ plasmids that are generally distributed according to host-specific patterns. These baseline data will be useful to assess the consequences of the increasing human exposure to CMY-2-producing E. coli via animal sources. CMY-2 is the most common plasmid-mediated AmpC �]-lactamase in Escherichia coli This �]-lactamase is poorly inhibited by clavulanic acid and confers resistance to cephamycins, third-generation cephalosporins, and aztreonam. Furthermore, resistance to carbapenems has been reported in E. coli as a result of production of plasmid-encoded CMY-2 �]-lactamase in combination with decreased outer membrane permeability. The gene encoding CMY-2 generally resides on transferable plasmids belonging to different incompatibility groups. The prevalence of CMY-2-mediated cephalosporin resistance in E. coli varies significantly depending on the geographical region and host. This study demonstrates that the epidemiology of CMY-2 can be understood only by thorough plasmid characterization. To date, the spread of this �]-lactam resistance determinant in Denmark is mainly associated with IncK and IncI1-I�^ plasmids, which are generally distributed according to host-specific patterns. These data will be useful to assess the consequences of the increasing human exposure to CMY-2-producing E. coli via animal sources.
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1348. |
Perreten V,
Strauss C,
Collaud A,
Gerber D,
( 2016 ) Colistin Resistance Gene mcr-1 in Avian-Pathogenic Escherichia coli in South Africa. PMID : 27161625 : DOI : 10.1128/AAC.00548-16 PMC : PMC4914693 Abstract >>
N/A
|
1349. |
Power ML,
Samuel A,
Smith JJ,
Stark JS,
Gillings MR,
Gordon DM,
( 2016 ) Escherichia coli out in the cold: Dissemination of human-derived bacteria into the Antarctic microbiome. PMID : 27179324 : DOI : 10.1016/j.envpol.2016.04.013 Abstract >>
Discharge of untreated sewage into Antarctic environments presents a risk of introducing non-native microorganisms, but until now, adverse consequences have not been conclusively identified. Here we show that sewage disposal introduces human derived Escherichia coli carrying mobile genetic elements and virulence traits with the potential to affect the diversity and evolution of native Antarctic microbial communities. We compared E. coli recovered from environmental and animal sources in Antarctica to a reference collection of E. coli from humans and non-Antarctic animals. The distribution of phylogenetic groups and frequency of 11 virulence factors amongst the Antarctic isolates were characteristic of E. coli strains more commonly associated with humans. The rapidly emerging E. coli ST131 and ST95 clones were found amongst the Antarctic isolates, and ST95 was the predominant E. coli recovered from Weddell seals. Class 1 integrons were found in 15% of the Antarctic E. coli with 4 of 5 identified gene cassette arrays containing antibiotic resistance genes matching those common in clinical contexts. Disposing untreated sewage into the Antarctic environment does disseminate non-native microorganisms, but the extent of this impact and implications for Antarctic ecosystem health are, as yet, poorly understood.
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1350. |
Asli A,
Alexander JA,
Vuckovic M,
Maiti SN,
Parr TR,
Brown ED,
Malouin F,
Strynadka NC,
Wright GD,
King AM,
King DT,
French S,
Brouillette E,
( 2016 ) Structural and Kinetic Characterization of Diazabicyclooctanes as Dual Inhibitors of Both Serine-�]-Lactamases and Penicillin-Binding Proteins. PMID : 26731698 : DOI : 10.1021/acschembio.5b00944 Abstract >>
Avibactam is a diazabicyclooctane �]-lactamase inhibitor possessing outstanding but incomplete efficacy against multidrug-resistant Gram-negative pathogens in combination with �]-lactam antibiotics. Significant pharmaceutical investment in generating derivatives of avibactam warrants a thorough characterization of their activity. We show here through structural and kinetic analysis that select diazabicyclooctane derivatives display effective but varied inhibition of two clinically important �]-lactamases (CTX-M-15 and OXA-48). Furthermore, these derivatives exhibit considerable antimicrobial activity (MIC ? 2 �gg/mL) against clinical isolates of Pseudomonas aeruginosa, Escherichia coli, and Enterobacter spp. Imaging of cell phenotype along with structural and biochemical experiments unambiguously demonstrate that this activity, in E. coli, is a result of targeting penicillin-binding protein 2. Our results suggest that structure-activity relationship studies for the purpose of drug discovery must consider both �]-lactamases and penicillin-binding proteins as targets. We believe that this approach will yield next-generation combination or monotherapies with an expanded spectrum of activity against currently untreatable Gram-negative pathogens.
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1351. |
Siqueira AK,
Michael GB,
Domingos DF,
Ferraz MM,
Ribeiro MG,
Schwarz S,
Leite DS,
( 2016 ) Diversity of class 1 and 2 integrons detected in Escherichia coli isolates from diseased and apparently healthy dogs. PMID : 27302904 : DOI : 10.1016/j.vetmic.2016.05.005 Abstract >>
Escherichia coli is one of the major pathogens causing urinary tract infections (UTIs) and pyometra in dogs. The aims of this study were to investigate canine E. coli isolates for the presence of class 1 and 2 integrons by PCR/sequencing and to characterize these isolates and their integron-carrying plasmids. Isolates were characterized by phylotyping, XbaI-macrorestriction analysis and plasmid transfer experiments. Plasmids were analyzed by S1 nuclease-PFGE, replicon typing, conjugation and restriction analysis. Antimicrobial resistance was investigated by antimicrobial susceptibility testing and PCR/sequencing. From 158 E. coli of dogs suffering from UTIs (n=51) and pyometra (n=52) or being apparently healthy (n=55), 13 isolates harboured class 1 (n=10) or class 2 integrons (n=3). They were distributed among the phylogenetic groups A (3/13), B1 (6/13), B2 (3/13) and D (1/13). Two isolates showed indistinguishable XbaI-patterns, but differed in the remaining characteristics. Another two isolates (UTI or apparently healthy) displayed different XbaI-patterns, but harboured similar plasmids. Integrons were found on plasmids of incompatibility groups IncF, IncF-IncFIC, IncFIB-IncHI2, IncFIB-IncN, IncFIC or IncHI2 and three of them were conjugative. Resistances to aminoglycosides, sulphonamides and trimethoprim were commonly detected. Class 1 integrons carried the gene cassette arrays dfrA12-orfF-aadA28, �GdfrA17-aadA5, dfrA29, aadA7, aadA29 or dfrA12-orfF-aadA2-cmlA1-aadA1. Class 2 integrons carried the array dfrA1-sat2-aadA30. Two extended-spectrum �]-lactamase genes (blaCTX-M-2) and one AmpC �]-lactamase gene (blaCMY-2) were also detected on plasmids. These findings indicate the potential risk of the dissemination and persistence of E. coli and/or integron-carrying plasmids in companion animals.
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1352. |
Mo SS,
Slettemeås JS,
Berg ES,
Norström M,
Sunde M,
( 2016 ) Plasmid and Host Strain Characteristics of Escherichia coli Resistant to Extended-Spectrum Cephalosporins in the Norwegian Broiler Production. PMID : 27111852 : DOI : 10.1371/journal.pone.0154019 PMC : PMC4844124 Abstract >>
Escherichia coli resistant to extended-spectrum cephalosporins have been detected in the Norwegian broiler production, despite the fact that antimicrobial agents are rarely used. The genetic mechanism responsible for cephalosporin resistance is mainly attributed to the presence of the blaCMY-2 gene encoding a plasmid-mediated AmpC-beta-lactamase (pAmpC). The aim of this study was to characterize and compare blaCMY-2 containing Escherichia coli isolated from the intestinal flora of broilers and retail chicken meat (fillets) to identify possible successful clones and/or resistance plasmids widespread in the Norwegian broiler production. Methods used included PCR based phylotyping, conjugation experiments, plasmid replicon typing, pulsed-field gel electrophoresis, multiple locus variable-number tandem-repeats analysis and whole genome sequencing. The nucleotide sequence of an IncK plasmid carrying blaCMY-2 was determined. Intestinal isolates displayed a higher degree of genetic diversity than meat isolates. A cluster of genetically related isolates belonging to ST38, phylogroup D, carrying blaCMY-2 containing IncK plasmids was identified. Furthermore, genes encoding plasmid stability systems (relBE/stbDE and pndAC) were identified on the IncK plasmid. Single nucleotide polymorphism (SNP) analysis of a subset of isolates confirmed a close genetic relationship within the two most prevalent STs. The IncK plasmids within these two STs also shared a high degree of similarity. Cephalosporin-resistant E. coli with the same genetic characteristics have been identified in the broiler production in other European countries, and the IncK plasmid characterized in this study showed close homology to a plasmid isolated from retail chicken meat in the Netherlands. The results indicate that both clonal expansion and horizontal transfer of blaCMY-2 containing plasmids contribute to dissemination of cephalosporin resistant E. coli in the broiler production. The presence of plasmid stability systems may explain why the IncK plasmid containing blaCMY-2 is maintained and disseminated in the Norwegian broiler production in absence of selection pressure from the use of antimicrobial agents.
|
1353. |
Chen CY,
Nguyen LH,
Cottrell BJ,
Irwin PL,
Uhlich GA,
( 2016 ) Multiple mechanisms responsible for strong Congo-red-binding variants of Escherichia coli O157:H7 strains. PMID : 26702633 : DOI : 10.1093/femspd/ftv123 Abstract >>
High variability in the expression of csgD-dependent, biofilm-forming and adhesive properties is common among Shiga toxin-producing Escherichia coli. Although many strains of serotype O157:H7 form little biofilm, conversion to stronger biofilm phenotypes has been observed. In this study, we screened different strains of serotype O157:H7 for the emergence of strong Congo-red (CR) affinity/biofilm-forming properties and investigated the underlying genetic mechanisms. Two major mechanisms which conferred stronger biofilm phenotypes were identified: mutations (insertion, deletion, single nucleotide change) in rcsB region and stx-prophage excision from the mlrA site. Restoration of the native mlrA gene (due to prophage excision) resulted in strong biofilm properties to all variants. Whereas RcsB mutants showed weaker CR affinity and biofilm properties, it provided more possibilities for phenotypic presentations through heterogenic sequence mutations.
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1354. |
Ziegelin G,
Fürste JP,
Lanka E,
( 1989 ) TraJ protein of plasmid RP4 binds to a 19-base pair invert sequence repetition within the transfer origin. PMID : 2663846 : Abstract >>
Transfer of plasmid RP4 during bacterial conjugation requires the plasmid-encoded TraJ protein, which binds to the transfer origin (F?rste, J. P., Pansegrau, W., Ziegelin, G., Kr?ger, M., and Lanka, E. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 1771-1775). As indicated by traJ mutants, the TraJ protein is a constituent of the relaxosome, the initiation complex of transfer DNA replication. The traJ gene maps adjacent to the transfer origin (oriT). The structural gene consists of a 372-base pair sequence encoding a polypeptide of 122 amino acids (13,282 Da). TraJ was purified from an Escherichia coli strain overproducing the protein. DNA footprinting experiments involving DNase I demonstrated that the purified protein binds to the right arm of a 19-base pair inverted repeat within oriT. Hydroxyl radical footprints of the DNA-protein complex revealed that TraJ protein is bound to only one side of the DNA helix.
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1355. |
Yasueda H,
Horii T,
Itoh T,
( 1989 ) Structural and functional organization of ColE2 and ColE3 replicons. PMID : 2651878 : DOI : 10.1007/bf00339719 Abstract >>
The complete nucleotide sequences of the 1.5 kb regions of ColE2 and ColE3 plasmids containing the segments sufficient for autonomous replication have been determined. They are quite homologous (greater than 90%), indicating that these two plasmids share common mechanisms of initiation of replication and its regulation. An open reading frame with a coding capacity for a protein of about 300 amino acids is present in both ColE2 and ColE3 and it actually specifies the Rep (for replication) protein, which is the plasmid specific trans-acting factor required for autonomous replication. The amino acid sequences of the Rep proteins of ColE2 and ColE3 are quite homologous (greater than 90%). The cis-acting sites (origins) where replication initiates in the presence of the trans-acting factors consist of 32 bp for ColE2 and 33bp for ColE3. They are the smallest of all the prokaryotic replication origins so far reported. They are nonhomologous only at two positions, one of which, a deletion of a single nucleotide in ColE2 (or an insertion in ColE3), determines the plasmid specificity in interaction of the origins with the Rep proteins. Both plasmids carry a region with an identical nucleotide sequence and the one in ColE2, the IncA region, has been shown to express incompatibility against both ColE2 and ColE3. These results indicate that these plasmids share a common IncA determinant. A possibility that a small anti-sense RNA is involved in copy number control and incompatibility (IncA function) was suggested.
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1356. |
Hanafusa T,
Sakai A,
Tominaga A,
Enomoto M,
( 1989 ) Isolation and characterization of Escherichia coli hag operator mutants whose hag48 expression has become repressible by a Salmonella H1 repressor. PMID : 2659972 : DOI : 10.1007/bf00332229 Abstract >>
The expression of an Escherichia coli K12 flagellin gene, hagA48, is insensitive to the Salmonella H1 repressor (rh1+). By selecting merodiploid cells H2-rh1on-off/F'hag48 for motility in the presence of anti-H48 serum, mutants which had escaped from inhibition by the serum because of repression of their hag48 expression by rh1+ were isolated. Their nucleotide sequences were examined in the region containing the promoter, the position of which was confirmed by S1 nuclease analysis of the transcriptional initiation site. The two independently isolated mutants had the same heptamer insertion AGACGAT at a site overlapping with the promoter sequence, creating a putative operator sequence homologous to Salmonella H1, but not to H2. Other candidates for operator mutants had reduced flagellar synthesis because of mutations between the transcriptional and translational initiation sites or in the structural gene. The sequence analysis also revealed a repetitive extragenic palindrome (REP) consensus sequence and a transcriptional terminator of hag48 in a small, functionally unknown open reading frame (ORF).
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1357. |
Lanza VF,
de Toro M,
Garcillán-Barcia MP,
Mora A,
Blanco J,
Coque TM,
de la Cruz F,
( 2014 ) Plasmid flux in Escherichia coli ST131 sublineages, analyzed by plasmid constellation network (PLACNET), a new method for plasmid reconstruction from whole genome sequences. PMID : 25522143 : DOI : 10.1371/journal.pgen.1004766 PMC : PMC4270462 Abstract >>
Bacterial whole genome sequence (WGS) methods are rapidly overtaking classical sequence analysis. Many bacterial sequencing projects focus on mobilome changes, since macroevolutionary events, such as the acquisition or loss of mobile genetic elements, mainly plasmids, play essential roles in adaptive evolution. Existing WGS analysis protocols do not assort contigs between plasmids and the main chromosome, thus hampering full analysis of plasmid sequences. We developed a method (called plasmid constellation networks or PLACNET) that identifies, visualizes and analyzes plasmids in WGS projects by creating a network of contig interactions, thus allowing comprehensive plasmid analysis within WGS datasets. The workflow of the method is based on three types of data: assembly information (including scaffold links and coverage), comparison to reference sequences and plasmid-diagnostic sequence features. The resulting network is pruned by expert analysis, to eliminate confounding data, and implemented in a Cytoscape-based graphic representation. To demonstrate PLACNET sensitivity and efficacy, the plasmidome of the Escherichia coli lineage ST131 was analyzed. ST131 is a globally spread clonal group of extraintestinal pathogenic E. coli (ExPEC), comprising different sublineages with ability to acquire and spread antibiotic resistance and virulence genes via plasmids. Results show that plasmids flux in the evolution of this lineage, which is wide open for plasmid exchange. MOBF12/IncF plasmids were pervasive, adding just by themselves more than 350 protein families to the ST131 pangenome. Nearly 50% of the most frequent �^-proteobacterial plasmid groups were found to be present in our limited sample of ten analyzed ST131 genomes, which represent the main ST131 sublineages.
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1358. |
Yan X,
Fratamico PM,
Bono JL,
Baranzoni GM,
Chen CY,
( BMC microbiology ) PMID : 25887577 : DOI : 10.1186/s12866-015-0413-9 PMC : PMC4393859 Abstract >>
Various H-serotypes of the Shiga toxin-producing Escherichia coli (STEC) O104, including H4, H7, H21, and H��, have been associated with sporadic cases of illness and have caused food-borne outbreaks globally. In the U.S., STEC O104:H21 caused an outbreak associated with milk in 1994. However, there is little known on the evolutionary origins of STEC O104 strains, and how genotypic diversity contributes to pathogenic potential of various O104 H-antigen serotypes isolated from different ecological niches and/or geographical regions. Two STEC O104:H21 (milk outbreak strain) and O104:H7 (cattle isolate) strains were shot-gun sequenced, and the genomes were closed. The intimin (eae) gene, involved in the attaching-effacing phenotype of diarrheagenic E. coli, was not found in either strain. Examining various O104 genome sequences, we found that two complete" left and right end portions of the locus of enterocyte effacement (LEE) pathogenicity island were present in 13 O104 strains; however, the central portion of LEE was missing, where the eae gene is located. In O104:H4 strains, the missing central portion of the LEE locus was replaced by a pathogenicity island carrying the aidA (adhesin involved in diffuse adherence) gene and antibiotic resistance genes commonly carried on plasmids. Enteroaggregative E. coli-specific virulence genes and European outbreak O104:H4-specific stx2-encoding Escherichia P13374 or Escherichia TL-2011c bacteriophages were missing in some of the O104:H4 genome sequences available from public databases. Most of the genomic variations in the strains examined were due to the presence of different mobile genetic elements, including prophages and genomic island regions. The presence of plasmids carrying virulence-associated genes may play a role in the pathogenic potential of O104 strains.
|
1359. |
Ho PL,
Liu MC,
Lo WU,
Lai EL,
Lau TC,
Law OK,
Chow KH,
( 2015 ) Prevalence and characterization of hybrid blaCTX-M among Escherichia coli isolates from livestock and other animals. PMID : 25861872 : DOI : 10.1016/j.diagmicrobio.2015.02.010 Abstract >>
This study investigated 248 extended-spectrum �]-lactamase-producing Escherichia coli isolates from 2012 to 2013 for hybrid blaCTX-M genes. blaCTX-M genes were detected in 228 isolates of which 14 isolates were hybrid blaCTX-M positive (6 blaCTX-M-123, 6 blaCTX-M-64, and 2 blaCTX-M-132). The 14 hybrid blaCTX-M-carrying isolates (8 from chickens, 2 each from pigs and cattle, 1 each from dog and rodent) were genetically diverse. All but 2 hybrid blaCTX-M were carried on IncI1 (5 blaCTX-M-123) and IncI2 (6 blaCTX-M-64 and one blaCTX-M-132) plasmids. Our IncI1 and IncI2 plasmids had pHNAH4-1-like and pHN1122-1-like restriction fragment length polymorphism patterns, respectively. Genetic relatedness of the plasmids to pHNAH4-1 and pHN1122-1 were confirmed by complete sequencing of 3 plasmids, pCTXM123_C0996, pCTXM64_C0967, and pCTXM132_P0421. Plasmids closely related to pHNAH4-1 and pHN1122-1 and carrying different blaCTX-M alleles have been reported from multiple geographic areas in China previously. The findings highlighted the wide dissemination of hybrid blaCTX-M variants in different parts of China.
|
1360. |
Howland CJ,
Rees CE,
Barth PT,
Wilkins BM,
( 1989 ) The ssb gene of plasmid ColIb-P9. PMID : 2651402 : DOI : 10.1128/jb.171.5.2466-2473.1989 PMC : PMC209922 Abstract >>
The IncI1 plasmid ColIb-P9 was found to carry a single-stranded DNA-binding (SSB) protein gene (ssb) that maps about 11 kilobase pairs from the origin of transfer in the region transferred early during bacterial conjugation. The cloned gene was able to suppress the UV and temperature sensitivity of an ssb-1 strain of Escherichia coli K-12. The nucleotide sequence of the ColIb ssb gene was determined, giving a predicted molecular weight of 19,110 for the SSB protein. Sequence data show that ColIb ssb is very similar to the ssb gene on plasmid F, which is also known to map in the leader region. High-level expression of ssb on ColIb required derepression of the transfer (tra) genes and the activity of the positive regulatory system controlling these genes, suggesting that the SSB protein contributes to the conjugative processing of DNA. A mutant of ColIbdrd-1 carrying a Tn903-derived insertion in ssb was constructed, but it was unaffected in the ability to generate plasmid transconjugants and it was maintained apparently stably in donor cells both following mating and during vegetative growth. Hence, no biological role of ColIb SSB protein was detected. However, unlike the parental plasmid, such ColIb ssb mutants conferred a marked Psi+ (plasmid-mediated SOS inhibition) phenotype on recA441 and recA730 strains, implying a functional relationship between SSB and Psi proteins.
|
1361. |
Genilloud O,
Moreno F,
Kolter R,
( 1989 ) DNA sequence, products, and transcriptional pattern of the genes involved in production of the DNA replication inhibitor microcin B17. PMID : 2644225 : DOI : 10.1128/jb.171.2.1126-1135.1989 PMC : PMC209710 Abstract >>
The 3.8-kilobase segment of plasmid DNA that contains the genes required for production of the DNA replication inhibitor microcin B17 was sequenced. The sequence contains four open reading frames which were shown to be translated in vivo by the construction of fusions to lacZ. The location of these open reading frames fits well with the location of the four microcin B17 production genes, mcbABCD, identified previously through genetic complementation. The products of the four genes have been identified, and the observed molecular weights of the proteins agree with those predicted from the nucleotide sequence. The transcription of these genes was studied by using fusions to lacZ and physical mapping of mRNA start sites. Three promoters were identified in this region. The major promoter for all the genes is a growth phase-regulated OmpR-dependent promoter located upstream of mcbA. A second promoter is located within mcbC and is responsible for a low-level basal expression of mcbD. A third promoter, located within mcbD, promotes transcription in the reverse direction starting within mcbD and extending through mcbC. The resulting mRNA appears to be an untranslated antisense transcript that could play a regulatory role in the expression of these genes.
|
1362. |
Xu G,
An W,
Wang H,
Zhang X,
( 2015 ) Prevalence and characteristics of extended-spectrum �]-lactamase genes in Escherichia coli isolated from piglets with post-weaning diarrhea in Heilongjiang province, China. PMID : 26500640 : DOI : 10.3389/fmicb.2015.01103 PMC : PMC4597763 Abstract >>
The purpose of this study was to investigate the prevalence of extended spectrum �]-lactamase (ESBL) genes in Escherichia coli isolated from post-weaning diarrhea (PWD) piglets in Heilongjiang province, China. Of 458 E. coli isolated from 589 fecal samples from PWD piglets, a total of 198 isolates were confirmed as ESBL producers by the double-disk synergy test (DDST). Polymerase chain reaction (PCR) and sequencing were performed to identify genes for ESBL, plasmid-mediated quinolone resistance (PMQR), and integrons. Of the 198 isolates, bla CTX-M and bla TEM were detected in 191 and 149 isolates, respectively. Sequencing revealed that 10 bla CTX-M subtypes were detected, and bla CTX-M-14 was the most prevalent, followed by bla CTX-M-55 and bla CTX-M-65. Of the 149 TEM-positive strains, four were bla TEM-52 and the rest were bla TEM-1. Among the 198 ESBL-positive isolates, 173 isolates were found to harbor at least one PMQR gene, with oqxAB, qnrS, qnrB, qepA, and aac(6')-Ib-cr being detected alone or in combination in 125, 114, 26, 24, and 45 strains, respectively. One hundred and fifty-five ESBL-positive isolates were also positive for class I integron (int1), and eight different gene cassette arrays were confirmed in 110 isolates by restriction fragment length polymorphism (RFLP) and DNA sequencing analyses, with predominance of dfrA17-aadA5, dfrA12-orfF-aadA2, and dfrA1-aadA1 arrays. To the best of our knowledge, this is the first report of the bla TEM-52 gene in pig E. coli isolates in China and this is also the first description of the coexistence of the qnrB, qnrS, aac(6')-Ib-cr, qepA, and oqxAB genes in one E. coli strain.
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1363. |
Johnson TJ,
Hargreaves M,
Shaw K,
Snippes P,
Lynfield R,
Aziz M,
Price LB,
( 2015 ) Complete Genome Sequence of a Carbapenem-Resistant Extraintestinal Pathogenic Escherichia coli Strain Belonging to the Sequence Type 131 H30R Subclade. PMID : 25858844 : DOI : 10.1128/genomeA.00272-15 PMC : PMC4392156 Abstract >>
Here, we report the completed genome sequence of a carbapenem-resistant extraintestinal pathogenic Escherichia coli sequence type 131 (ST131) isolate, MNCRE44. The isolate was obtained in 2012 in Minnesota, USA, from a sputum sample from a hospitalized patient with multiple comorbidities, and it belongs to the H30R sublineage.
|
1364. |
Blyn LB,
Braaten BA,
White-Ziegler CA,
Rolfson DH,
Low DA,
( 1989 ) Phase-variation of pyelonephritis-associated pili in Escherichia coli: evidence for transcriptional regulation. PMID : 2656260 : PMC : PMC400848 Abstract >>
The regulation of pyelonephritis-associated pili (pap) pilin gene transcription has been examined using two operons (pap-17 and pap-21) isolated from the pyelonephritogenic Escherichia coli strain C1212. DNA sequence analysis and E. coli minicell analysis were used to map two genes (papB and papI) within the pilin regulatory regions of both pap-17 and pap-21, and the protein products of these genes were identified. Pilin transcription, initiated at the papBA promoter, was monitored by constructing single copy operon fusions with lacZYA in E. coli K-12. Inoculation of E. coli (pap'-lac) strains onto solid M9 minimal medium containing glycerol and the Lac indicator X-gal (M9-Glycerol) yielded both Lac+ and Lac- colony phenotypes. The Lac+ ("phase on') and Lac- ("phase off') phenotypes were heritable since reinoculation of M9-Glycerol with bacteria picked from Lac+ colonies gave rise to a much higher fraction of Lac+ colonies than reinoculation of M9-Glycerol with bacteria picked from Lac- colonies. Measurement of phase transition rates for E. coli (pap17'-lac) inoculated onto M9-Glycerol showed that the Lac(-)----Lac+ transition frequency (1.57 X 10(-4)/cell/generation) was reduced 35-fold when cells were inoculated onto minimal medium containing glucose (M9-Glucose). However, the Lac+----Lac-transition frequency obtained using M9-Glycerol (2.60 X 10(-2)/cell/generation) was 1.4-fold lower compared to results obtained with M9-Glucose. In contrast, lowering the incubation temperature of E. coli (pap17'-lac) cultures from 37 degrees C to 23 degrees C caused all cells to shift to the Lac- state.(ABSTRACT TRUNCATED AT 250 WORDS)
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1365. |
Hedegaard L,
Klemm P,
( 1989 ) Type 1 fimbriae of Escherichia coli as carriers of heterologous antigenic sequences. PMID : 2576014 : DOI : 10.1016/0378-1119(89)90471-x Abstract >>
A strategy has been designed for the construction of recombinant bacterial strains which eventually may become useful as live vaccines and which may also be relevant for the preparation of conventional vaccines. The approach used is the fusion of small antigenic peptide sequences into specific segments of a protein whose location on the bacterial surface ensures that the recombinant organism is able to present the inserted antigen to the host (animal or human) infected by the bacterium. The chosen surface protein is a naturally occurring polymer of Escherichia coli, viz., type 1 fimbriae. The results obtained show that fusion of such foreign sequences into selected points of the structural protein of the fimbriae results in the production of functionally normal type 1 fimbriae. Furthermore, hybrid fimbriae carrying such small epitope sequences can be recognized by antibodies directed against the foreign parent protein. This observation is an important prerequisite for the eventual design of useful vaccines. The analysis of the fimbrial protein and its potential as a carrier of foreign peptides from hepatitis B surface antigen, foot-and-mouth disease virus and poliovirus indicated that there may be several positions in the protein which may turn out to be relevant for this purpose and be important fusion sites.
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1366. |
Chihomvu P,
Stegmann P,
Pillay M,
( 2015 ) Characterization and structure prediction of partial length protein sequences of pcoA, pcoR and chrB genes from heavy metal resistant bacteria from the Klip River, South Africa. PMID : 25837632 : DOI : 10.3390/ijms16047352 PMC : PMC4425021 Abstract >>
The Klip River has suffered from severe anthropogenic effects from industrial activities such as mining. Long-term exposure to heavy metal pollution has led to the development of heavy metal resistant strains of Pseudomonas sp. KR23, Lysinibacillus sp. KR25, and E. coli KR29. The objectives of this study were to characterize the genetics of copper and chromate resistance of the isolates. Copper and chromate resistance determinants were cloned and sequenced. Open reading frames (ORFs) related to the genes CopA and CopR were identified in E. coli KR29, PcoA in Lysinibacillus sp. KR25 and none related to chromate resistance were detected. The 3D-models predicted by I-TASSER disclose that the PcoA proteins consist of �]-sheets, which form a part of the cupredoxin domain of the CopA copper resistance family of genes. The model for PcoR_29 revealed the presence of a helix turn helix; this forms part of a DNA binding protein, which is part of a heavy metal transcriptional regulator. The bacterial strains were cured using ethidium bromide. The genes encoding for heavy metal resistance and antibiotic resistance were found to be located on the chromosome for both Pseudomonas sp. (KR23) and E. coli (KR29). For Lysinibacillus (KR25) the heavy metal resistance determinants are suspected to be located on a mobile genetic element, which was not detected using gel electrophoresis.
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1367. |
Mercer RG,
Zheng J,
Garcia-Hernandez R,
Ruan L,
Gänzle MG,
McMullen LM,
( 2015 ) Genetic determinants of heat resistance in Escherichia coli. PMID : 26441869 : DOI : 10.3389/fmicb.2015.00932 PMC : PMC4563881 Abstract >>
Escherichia coli AW1.7 is a heat resistant food isolate and the occurrence of pathogenic strains with comparable heat resistance may pose a risk to food safety. To identify the genetic determinants of heat resistance, 29 strains of E. coli that differed in their of heat resistance were analyzed by comparative genomics. Strains were classified as highly heat resistant strains, exhibiting a D60-value of more than 6 min; moderately heat resistant strains, exhibiting a D60-value of more than 1 min; or as heat sensitive. A ~14 kb genomic island containing 16 predicted open reading frames encoding putative heat shock proteins and proteases was identified only in highly heat resistant strains. The genomic island was termed the locus of heat resistance (LHR). This putative operon is flanked by mobile elements and possesses >99% sequence identity to genomic islands contributing to heat resistance in Cronobacter sakazakii and Klebsiella pneumoniae. An additional 41 LHR sequences with >87% sequence identity were identified in 11 different species of �]- and �^-proteobacteria. Cloning of the full length LHR conferred high heat resistance to the heat sensitive E. coli AW1.7�GpHR1 and DH5�\. The presence of the LHR correlates perfectly to heat resistance in several species of Enterobacteriaceae and occurs at a frequency of 2% of all E. coli genomes, including pathogenic strains. This study suggests the LHR has been laterally exchanged among the �]- and �^-proteobacteria and is a reliable indicator of high heat resistance in E. coli.
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1368. |
Krishnan S,
Chang AC,
Hodges J,
Couraud PO,
Romero IA,
Weksler B,
Nicholson BA,
Nolan LK,
Prasadarao NV,
( 2015 ) Serotype O18 avian pathogenic and neonatal meningitis Escherichia coli strains employ similar pathogenic strategies for the onset of meningitis. PMID : 26407066 : DOI : 10.1080/21505594.2015.1091914 PMC : PMC4826105 Abstract >>
Neonatal meningitis Escherichia coli K1 (NMEC) are thought to be transmitted from mothers to newborns during delivery or by nosocomial infections. However, the source of E. coli K1 causing these infections is not clear. Avian pathogenic E. coli (APEC) have the potential to cause infection in humans while human E. coli have potential to cause colibacillosis in poultry, suggesting that these strains may lack host specificity. APEC strains are capable of causing meningitis in newborn rats; however, it is unclear whether these bacteria use similar mechanisms to that of NMEC to establish disease. Using four representative APEC and NMEC strains that belong to serotype O18, we demonstrate that these strains survive in human serum similar to that of the prototypic NMEC strain E44, a derivative of RS218. These bacteria also bind and enter both macrophages and human cerebral microvascular endothelial cells (HCMEC/D3) with similar frequency as that of E44. The amino acid sequences of the outer membrane protein A (OmpA), an important virulence factor in the pathogenesis of meningitis, are identical within these representative APEC and NMEC strains. Further, these strains also require Fc�^RI-�\ chain (CD64) and Ecgp96 as receptors for OmpA in macrophages and HCMEC/D3, respectively, to bind and enter these cells. APEC and NMEC strains induce meningitis in newborn mice with varying degree of pathology in the brains as assessed by neutrophil recruitment and neuronal apoptosis. Together, these results suggest that serotype O18 APEC strains utilize similar pathogenic mechanisms as those of NMEC strains in causing meningitis.
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1369. |
Kar D,
Bandyopadhyay S,
Bhattacharyya D,
Samanta I,
Mahanti A,
Nanda PK,
Mondal B,
Dandapat P,
Das AK,
Dutta TK,
Bandyopadhyay S,
Singh RK,
( 2015 ) Molecular and phylogenetic characterization of multidrug resistant extended spectrum beta-lactamase producing Escherichia coli isolated from poultry and cattle in Odisha, India. PMID : 25445661 : DOI : 10.1016/j.meegid.2014.11.003 Abstract >>
The present study was undertaken to determine the occurrence and characterization of extended spectrum beta-lactamase (ESBL) producing Escherichia coli isolated from cattle and poultry in Odisha, India. Of 316 E. coli isolated from 305 samples (170 fecal samples from poultry and 135 milk samples from cattle), a total of 18 E. coli isolates were confirmed as ESBL producers by combination disc method and ESBL E-test. The isolates were resistant to oxyimino cephalosporins and monobactam as revealed by disc diffusion assay and determination of minimum inhibitory concentration. Resistance against other antibiotics was frequently noted as well. Further, beta-lactamase genes viz., blaSHV, blaCTXM, blaTEM and blaampC were detected in 17, 13, 9 and 2 isolates, respectively in PCR. Of the 18 ESBL strains, 16 were positive for class I integron (int1), nine of them carried sulphonamide resistance gene (sul1) and one harbored quinolone resistance gene (qnrB). Virulence markers for extraintestinal pathogenic E. coli like astA, tsh and iucD were also present in 4, 3 and 3 isolates, respectively. All the PCR amplified products were cloned and subjected to sequencing for homology analysis and data were submitted to gene bank. Sequence analysis of the amplified variable regions of class 1 integron of four representative isolates revealed the presence of aadA2 and dfrA12 gene cassettes conferring resistance to aminoglycosides and trimethoprim, respectively. Most of the ESBL producing strains emerged as single lineage through phylogenetic analysis by RAPD and ERIC PCR. This is the first ever systemic study on multidrug resistant ESBL producing E. coli in food producing animals from India.
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1370. |
Wailan AM,
Paterson DL,
Caffery M,
Sowden D,
Sidjabat HE,
( 2015 ) Draft Genome Sequence of NDM-5-Producing Escherichia coli Sequence Type 648 and Genetic Context of blaNDM-5 in Australia. PMID : 25858833 : DOI : 10.1128/genomeA.00194-15 PMC : PMC4392145 Abstract >>
We report here the draft genome sequence of uropathogenic Escherichia coli sequence type 648 (ST648) possessing blaNDM-5 from a 55-year-old female in Australia with a history of travel to India. The plasmid-mediated blaNDM-5 was in a genetic context nearly identical to that of the GenBank entry of an IncX3 blaNDM-5 plasmid previously reported from India (Klebsiella pneumoniae MGR-K194).
|
1371. |
Tijet N,
Richardson D,
MacMullin G,
Patel SN,
Melano RG,
( 2015 ) Characterization of multiple NDM-1-producing Enterobacteriaceae isolates from the same patient. PMID : 25845877 : DOI : 10.1128/AAC.04862-14 PMC : PMC4432124 Abstract >>
A male patient was admitted to a community hospital in Ontario, Canada, with an infected sacral ulcer after returning from India, where he was hospitalized. Carbapenem-resistant Escherichia coli (isolated from blood cultures), Enterobacter cloacae, and Providencia stuartii (from urine samples), all positive for bla(NDM-1), were recovered. Comparative NDM-1 plasmid analysis suggests both lateral plasmid transfer and independent acquisition of the bla(NDM-1) gene in these clinical isolates.
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1372. |
Scholz P,
Haring V,
Wittmann-Liebold B,
Ashman K,
Bagdasarian M,
Scherzinger E,
( 1989 ) Complete nucleotide sequence and gene organization of the broad-host-range plasmid RSF1010. PMID : 2653965 : DOI : 10.1016/0378-1119(89)90273-4 Abstract >>
We present the complete nucleotide sequence of RSF1010, a naturally occurring broad-host-range plasmid belonging to the Escherichia coli incompatibility group Q and encoding resistance to streptomycin and sulfonamides. A molecule of RSF1010 DNA consists of 8684 bp and has a G + C content of 61%. Analysis of the distribution of translation start and stop codons in the sequence has revealed the existence of more than 40 open reading frames potentially capable of encoding polypeptides of 60 or more amino acids. To date, products of eleven such potential RSF1010 genes have been identified through the application of controlled expression vector systems, and for eight of them, the reading frame has been confirmed by N- and/or C-terminal amino acid sequence determinations on the purified proteins. The sequencing results are discussed in relation to the systems of replication, host range, conjugal mobilization and antibiotic resistance determinants associated with the RSF1010 plasmid.
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1373. |
Rety S,
Deschamps P,
Leulliot N,
( 2015 ) Structure of Escherichia coli tryptophanase purified from an alkaline-stressed bacterial culture. PMID : 26527264 : DOI : 10.1107/S2053230X15017549 PMC : PMC4631586 Abstract >>
Tryptophanase is a bacterial enzyme involved in the degradation of tryptophan to indole, pyruvate and ammonia, which are compounds that are essential for bacterial survival. Tryptophanase is often overexpressed in stressed cultures. Large amounts of endogenous tryptophanase were purified from Escherichia coli BL21 strain overexpressing another recombinant protein. Tryptophanase was crystallized in space group P6522 in the apo form without pyridoxal 5'-phosphate bound in the active site.
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1374. |
Eaton T,
James R,
( 1989 ) Complete nucleotide sequence of the colicin E9 (cei) gene. PMID : 2646600 : DOI : 10.1093/nar/17.4.1761 PMC : PMC331837 Abstract >>
N/A
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1375. |
Lee JY,
Choi MJ,
Choi HJ,
Ko KS,
( 2016 ) Preservation of Acquired Colistin Resistance in Gram-Negative Bacteria. PMID : 26459897 : DOI : 10.1128/AAC.01574-15 PMC : PMC4704156 Abstract >>
Colistin-resistant mutants were obtained from 17 colistin-susceptible strains of Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Escherichia coli. The stability of colistin resistance in these mutants was investigated. Three of four colistin-resistant P. aeruginosa mutants recovered colistin susceptibility in colistin-free medium; however, colistin-susceptible revertants were obtained from only one strain each of A. baumannii and E. coli. No susceptible revertants were obtained from K. pneumoniae mutants.
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1376. |
Guzman-Verduzco LM,
Kupersztoch YM,
( 1989 ) Rectification of two Escherichia coli heat-stable enterotoxin allele sequences and lack of biological effect of changing the carboxy-terminal tyrosine to histidine. PMID : 2643580 : PMC : PMC313148 Abstract >>
Resequencing estA3, an allele of the methanol-soluble heat-stable enterotoxin of Escherichia coli showed that the proline triplet 19 is in fact an alanine codon; thus, estA alleles 3 and 4 were shown to be identical. Resequencing has also shown that the carboxy terminus of another allele, estA2, is not the previously inferred histidine triplet but the same tyrosine codon reported for all other estA alleles. The improperly inferred histidine codon was used in constructions to fuse estA2 to the B subunit of the heat-labile enterotoxin gene, and the fused gene products as well as three amino acid insertional mutants containing histidine-72 were not efficiently secreted. We show that the defective secretion is not due to histidine as a carboxy-terminal residue, since site-directed mutagenesis of wild-type tyrosine-72 to histidine did not influence the localization of the activity of the methanol-soluble heat-stable enterotoxin.
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1377. |
Gutiérrez D,
Pardo M,
Montero D,
Oñate A,
Farfán MJ,
Ruiz-Pérez F,
Del Canto F,
Vidal R,
( 2015 ) TleA, a Tsh-like autotransporter identified in a human enterotoxigenic Escherichia coli strain. PMID : 25712927 : DOI : 10.1128/IAI.02976-14 PMC : PMC4399053 Abstract >>
Enterotoxigenic Escherichia coli (ETEC), a leading cause of acute diarrhea, colonizes the intestine by means of adhesins. However, 15 to 50% of clinical isolates are negative for known adhesins, making it difficult to identify antigens for broad-coverage vaccines. The ETEC strain 1766a, obtained from a child with watery diarrhea in Chile, harbors the colonization factor CS23 but is negative for other known adhesins. One clone, derived from an ETEC 1766a genomic library (clone G10), did not produce CS23 yet was capable of adhering to Caco-2 cells. The goal of this study was to identify the gene responsible for this capacity. Random transposon-based mutagenesis allowed the identification of a 4,110-bp gene that codes for a homologue of the temperature-sensitive hemagglutinin (Tsh) autotransporter described in avian E. coli strains (97% identity, 90% coverage) and that is called TleA (Tsh-like ETEC autotransporter) herein. An isogenic ETEC 1766a strain with a tleA mutation showed an adhesion level similar to that of the wild-type strain, suggesting that the gene does not direct attachment to Caco-2 cells. However, expression of tleA conferred the capacity for adherence to nonadherent E. coli HB101. This effect coincided with the detection of TleA on the surface of nonpermeabilized bacteria, while, conversely, ETEC 1766a seems to secrete most of the produced autotransporter to the medium. On the other hand, TleA was capable of degrading bovine submaxillary mucin and leukocyte surface glycoproteins CD45 and P-selectin glycoprotein ligand 1 (PSGL-1). These results suggest that TleA promotes colonization of the intestinal epithelium and that it may modulate the host immune response.
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1378. |
De Greve H,
Van Melderen L,
Loris R,
Sterckx YG,
Jové T,
Shkumatov AV,
Garcia-Pino A,
Geerts L,
De Kerpel M,
Lah J,
( 2016 ) A unique hetero-hexadecameric architecture displayed by the Escherichia coli O157 PaaA2-ParE2 antitoxin-toxin complex. PMID : 26996937 : DOI : 10.1016/j.jmb.2016.03.007 Abstract >>
Many bacterial pathogens modulate their metabolic activity, virulence and pathogenicity through so-called "toxin-antitoxin" (TA) modules. The genome of the human pathogen Escherichia coli O157 contains two three-component TA modules related to the known parDE module. Here, we show that the toxin EcParE2 maps in a branch of the RelE/ParE toxin superfamily that is distinct from the branches that contain verified gyrase and ribosome inhibitors. The structure of EcParE2 closely resembles that of Caulobacter crescentus ParE but shows a distinct pattern of conserved surface residues, in agreement with its apparent inability to interact with GyrA. The antitoxin EcPaaA2 is characterized by two �\-helices (H1 and H2) that serve as molecular recognition elements to wrap itself around EcParE2. Both EcPaaA2 H1 and H2 are required to sustain a high-affinity interaction with EcParE2 and for the inhibition of EcParE2-mediated killing in vivo. Furthermore, evidence demonstrates that EcPaaA2 H2, but not H1, determines specificity for EcParE2. The initially formed EcPaaA2-EcParE2 heterodimer then assembles into a hetero-hexadecamer, which is stable in solution and is formed in a highly cooperative manner. Together these findings provide novel data on quaternary structure, TA interactions and activity of a hitherto poorly characterized family of TA modules.
|
1379. |
Ohtsubo H,
Ohtsubo E,
( 1978 ) Nucleotide sequence of an insertion element, IS1. PMID : 273224 : DOI : 10.1073/pnas.75.2.615 PMC : PMC411306 Abstract >>
PSM2, PSM1, and PSM15 are small plasmids derived from R100 by spontaneous deletions at either end of the insertion sequence IS1. These plasmids were used to identify regions neighboring IS1 as well as the IS1 DNA itself, by cleavage with EcoR1, HindIII, Hae III, Hpa II, Hha I, Hinf, and AIu I. The nucleotide sequencing results demonstrate that IS1 contains 768 bases. About 30 bases at the ends of IS1 were found to be repeated in an inverted order. The deletions occurring at the ends of IS1 were found to be due to illegitimate recombination. The hypothesis that RNA polymerase could play an important role in such recombination phenomena is discussed based on the nucleotide sequences surrounding the recombinational hot spots.
|
1380. |
Valat C,
Goldstone RJ,
Hirchaud E,
Haenni M,
Smith DG,
Madec JY,
( 2016 ) Draft Genome Sequences of Enterohemorrhagic Escherichia coli Encoding Extended-Spectrum Beta-Lactamases. PMID : 26868385 : DOI : 10.1128/genomeA.01633-15 PMC : PMC4751309 Abstract >>
Extended-spectrum beta-lactamases (ESBLs) have rarely been observed among Shiga toxigenic Escherichia coli (STEC), and, to our best knowledge, only three ESBL-positive isolates of the enterohemorrhagic E. coli (EHEC) subpathotype have been reported. Here, we present the first draft genome sequences of two ESBL-positive EHEC isolates belonging to serotypes O111:H8 and O151:H16.
|
1381. |
Koraimann G,
Högenauer G,
( 1989 ) A stable core region of the tra operon mRNA of plasmid R1-19. PMID : 2564189 : DOI : 10.1093/nar/17.4.1283 PMC : PMC331803 Abstract >>
The degradation of the polycistronic tra-mRNA of the resistance plasmid R1-19 leads to the accumulation of a well defined series of stable mRNA species. The majority of the most stable mRNAs contains the message for the traA gene only. The differently sized stable mRNAs possess a common 3'terminus within the traL gene but vary at their 5' ends. The 3'terminus probably results from protection against exoribonucleases by a secondary structural feature. We propose that the 5' ends are generated by endoribonucleolytic cleavage. The stability of this part of the tra-mRNA exceeds 30 minutes and probably increases the rate of expression of the traA gene product propilin, the precursor of the sex pilus subunit. The expression of propilin and its processing into a protein of the molecular weight of mature pilin is demonstrated with the isolated gene. The sequence of the so far unknown genes traL and traE of R1-19 is presented.
|
1382. |
Roosendaal B,
de Graaf FK,
( 1989 ) The nucleotide sequence of the fanD gene encoding the large outer membrane protein involved in the biosynthesis of K99 fimbriae. PMID : 2564179 : DOI : 10.1093/nar/17.3.1263 PMC : PMC331763 Abstract >>
N/A
|
1383. |
Kolappan S,
Ng D,
Yang G,
Harn T,
Craig L,
( 2015 ) Crystal Structure of the Minor Pilin CofB, the Initiator of CFA/III Pilus Assembly in Enterotoxigenic Escherichia coli. PMID : 26324721 : DOI : 10.1074/jbc.M115.676106 PMC : PMC4646235 Abstract >>
Type IV pili are extracellular polymers of the major pilin subunit. These subunits are held together in the pilus filament by hydrophobic interactions among their N-terminal �\-helices, which also anchor the pilin subunits in the inner membrane prior to pilus assembly. Type IV pilus assembly involves a conserved group of proteins that span the envelope of Gram-negative bacteria. Among these is a set of minor pilins, so named because they share their hydrophobic N-terminal polymerization/membrane anchor segment with the major pilins but are much less abundant. Minor pilins influence pilus assembly and retraction, but their precise functions are not well defined. The Type IV pilus systems of enterotoxigenic Escherichia coli and Vibrio cholerae are among the simplest of Type IV pilus systems and possess only a single minor pilin. Here we show that the enterotoxigenic E. coli minor pilins CofB and LngB are required for assembly of their respective Type IV pili, CFA/III and Longus. Low levels of the minor pilins are optimal for pilus assembly, and CofB can be detected in the pilus fraction. We solved the 2.0 ? crystal structure of N-terminally truncated CofB, revealing a pilin-like protein with an extended C-terminal region composed of two discrete domains connected by flexible linkers. The C-terminal region is required for CofB to initiate pilus assembly. We propose a model for CofB-initiated pilus assembly with implications for understanding filament growth in more complex Type IV pilus systems as well as the related Type II secretion system.
|
1384. |
McGann P,
Snesrud E,
Maybank R,
Corey B,
Ong AC,
Clifford R,
Hinkle M,
Whitman T,
Lesho E,
Schaecher KE,
( 2016 ) Escherichia coli Harboring mcr-1 and blaCTX-M on a Novel IncF Plasmid: First Report of mcr-1 in the United States. PMID : 27230792 : DOI : 10.1128/AAC.01103-16 PMC : PMC4914657 Abstract >>
N/A
|
1385. |
Akutsu A,
Masaki H,
Ohta T,
( 1989 ) Molecular structure and immunity specificity of colicin E6, an evolutionary intermediate between E-group colicins and cloacin DF13. PMID : 2687234 : DOI : 10.1128/jb.171.12.6430-6436.1989 PMC : PMC210531 Abstract >>
The primary structure of a 3.1-kilobase E6 or E3 segment carrying colicin and related genes was determined. Plasmid ColE6-CT14 showed striking homology to ColE3-CA38 throughout this segment, including homology to the secondary immunity gene, immE8, downstream of the E6 or E3 immunity gene. The ColE3-CA38 and ColE6-CT14 sequences, however, contained an exceptional hot spot region encoding both the colicin-active domain (RNase region) and the immunity protein, reflecting their different immunity specificities. On the other hand, some chimeric plasmids were constructed through homologous recombination between colicin E3 and cloacin DF13 operons. The resulting plasmids were deduced to produce chimeric colicins with a colicin E3-type N-terminal part, a cloacin DF13-type C-terminal-active domain, and the DF13 immunity protein. The killing spectra of the chimeric colicins and the immunities of the plasmids were identical to those of colicin E6 and ColE6-CT14, respectively, showing that the colicin E6 immunity specificity is completely equivalent to that of cloacin DF13. Nevertheless, colicin E6 has been found to show a sequence diversity from cloacin DF13 almost to the same extent as that from colicin E3 in their RNase and immunity regions, indicating that only a small number of amino acids defines the immunity specificity for discrimination between colicins E3 and E6 (or cloacin DF13).
|
1386. |
Imuta N,
Ooka T,
Seto K,
Kawahara R,
Koriyama T,
Kojyo T,
Iguchi A,
Tokuda K,
Kawamura H,
Yoshiie K,
Ogura Y,
Hayashi T,
Nishi J,
( 2016 ) Phylogenetic Analysis of Enteroaggregative Escherichia coli (EAEC) Isolates from Japan Reveals Emergence of CTX-M-14-Producing EAEC O25:H4 Clones Related to Sequence Type 131. PMID : 27252465 : DOI : 10.1128/JCM.00711-16 PMC : PMC4963495 Abstract >>
Enteroaggregative Escherichia coli (EAEC) causes acute or persistent diarrhea. The aggR gene is widely used as a marker for typical EAEC. The heterogeneity of EAEC is well known; however, there are few reports on the phylogenetic relationships of EAEC. Recently, CTX-M extended-spectrum �]-lactamase (ESBL)-producing EAEC strains have been reported worldwide. To characterize EAEC strains in Japan, we investigated the population structure of EAEC. A total of 167 aggR-positive strains isolated from stool specimens from diarrheal patients in Kagoshima (139 strains) and Osaka (28 strains), Japan, between 1992 and 2010 were examined for the prevalence of EAEC virulence markers, the blaCTX-M gene, and the capacity to form biofilms. Multilocus sequence typing was also conducted. EAEC strains were widely distributed across four major E. coli phylogroups. Strains of O111:H21/clonal group 40 (CG40) (30 strains), O126:H27/CG200 (13 strains), and O86a:H27/CG3570 (11 strains) in phylogroup B1 are the historical EAEC clones in Japan, and they exhibited strong biofilm formation. Twenty-nine strains of EAEC O25:H4/CG131 were identified in phylogroup B2, 79% of which produced CTX-M-14. This clone has emerged since 2003. The clone harbored plasmid-encoded EAEC virulence genes but not chromosomal virulence genes and had lower biofilm-forming capacity than historical EAEC strains. This clone most likely emerged from a pandemic uropathogenic O25:H4/sequence type 131 clone by acquiring an EAEC virulence plasmid from canonical EAEC. Surveillance of the horizontal transfer of both virulence and ESBL genes among E. coli strains is important for preventing a worldwide increase in antimicrobial drug resistance.
|
1387. |
Burkinshaw BJ,
Deng W,
Lameignère E,
Wasney GA,
Zhu H,
Worrall LJ,
Finlay BB,
Strynadka NC,
( 2015 ) Structural analysis of a specialized type III secretion system peptidoglycan-cleaving enzyme. PMID : 25678709 : DOI : 10.1074/jbc.M115.639013 PMC : PMC4400350 Abstract >>
The Gram-negative bacterium enteropathogenic Escherichia coli uses a syringe-like type III secretion system (T3SS) to inject virulence or "effector" proteins into the cytoplasm of host intestinal epithelial cells. To assemble, the T3SS must traverse both bacterial membranes, as well as the peptidoglycan layer. Peptidoglycan is made of repeating N-acetylmuramic acid and N-acetylglucosamine disaccharides cross-linked by pentapeptides to form a tight mesh barrier. Assembly of many macromolecular machines requires a dedicated peptidoglycan lytic enzyme (PG-lytic enzyme) to locally clear peptidoglycan. Here we have solved the first structure of a T3SS-associated PG-lytic enzyme, EtgA from enteropathogenic E. coli. Unexpectedly, the active site of EtgA has features in common with both lytic transglycosylases and hen egg white lysozyme. Most notably, the �]-hairpin region resembles that of lysozyme and contains an aspartate that aligns with lysozyme Asp-52 (a residue critical for catalysis), a conservation not observed in other previously characterized lytic transglycosylase families to which the conserved T3SS enzymes had been presumed to belong. Mutation of the EtgA catalytic glutamate, Glu-42, conserved across lytic transglycosylases and hen egg white lysozyme, and this differentiating aspartate diminishes type III secretion in vivo, supporting its essential role in clearing the peptidoglycan for T3SS assembly. Finally, we show that EtgA forms a 1:1 complex with the building block of the polymerized T3SS inner rod component, EscI, and that this interaction enhances PG-lytic activity of EtgA in vitro, collectively providing the necessary strict localization and regulation of the lytic activity to prevent overall cell lysis.
|
1388. |
Zhu-Ge XK,
Pan ZH,
Tang F,
Mao X,
Hu L,
Wang SH,
Xu B,
Lu CP,
Fan HJ,
Dai JJ,
( 2015 ) The effects of upaB deletion and the double/triple deletion of upaB, aatA, and aatB genes on pathogenicity of avian pathogenic Escherichia coli. PMID : 26278540 : DOI : 10.1007/s00253-015-6925-2 Abstract >>
Autotransporters (ATs) are associated with pathogenesis of Avian Pathogenic Escherichia coli (APEC). The molecular characterization of APEC ATs can provide insights about their relevance to APEC pathogenesis. Here, we characterized a conventional autotransporter UpaB in APEC DE205B genome. The upaB existed in 41.9 % of 236 APEC isolates and was predominantly associated with ECOR B2 and D. Our studies showed that UpaB mediates the DE205B adhesion in DF-1 cells, and enhances autoaggregation and biofilm formation of fimbria-negative E. coli AAEC189 (MG1655�Gfim) in vitro. Deletion of upaB of DE205B attenuates the virulence in duck model and early colonization in the duck lungs during APEC systemic infection. Furthermore, double and triple deletion of upaB, aatA, and aatB genes cumulatively attenuated DE205B adhesion in DF-1 cells, accompanying with decreased 50 % lethal dose (LD50) in duck model and the early colonization in the duck lungs. However, DE205B�GupaB/�GaatA/�GaatB might "compensate" the influence of gene deletion by upregulating the expression of fimbrial adhesin genes yqiL, yadN, and vacuolating autotransporter vat during early colonization of APEC. Finally, we demonstrated that vaccination with recombinant UpaB, AatA, and AatB proteins conferred protection against colisepticemia caused by DE205B infection in duck model.
|
1389. |
Jan AT,
Azam M,
Choi I,
Ali A,
Haq QM,
( N/A ) Analysis for the presence of determinants involved in the transport of mercury across bacterial membrane from polluted water bodies of India. PMID : 26887227 : DOI : 10.1016/j.bjm.2015.11.023 PMC : PMC4827696 Abstract >>
Mercury, which is ubiquitous and recalcitrant to biodegradation processes, threatens human health by escaping to the environment via various natural and anthropogenic activities. Non-biodegradability of mercury pollutants has necessitated the development and implementation of economic alternatives with promising potential to remove metals from the environment. Enhancement of microbial based remediation strategies through genetic engineering approaches provides one such alternative with a promising future. In this study, bacterial isolates inhabiting polluted sites were screened for tolerance to varying concentrations of mercuric chloride. Following identification, several Pseudomonas and Klebsiella species were found to exhibit the highest tolerance to both organic and inorganic mercury. Screened bacterial isolates were examined for their genetic make-up in terms of the presence of genes (merP and merT) involved in the transport of mercury across the membrane either alone or in combination to deal with the toxic mercury. Gene sequence analysis revealed that the merP gene showed 86-99% homology, while the merT gene showed >98% homology with previously reported sequences. By exploring the genes involved in imparting metal resistance to bacteria, this study will serve to highlight the credentials that are particularly advantageous for their practical application to remediation of mercury from the environment.
|
1390. |
Baranzoni GM,
Fratamico PM,
Gangiredla J,
Patel I,
Bagi LK,
Delannoy S,
Fach P,
Boccia F,
Anastasio A,
Pepe T,
( 2016 ) Characterization of Shiga Toxin Subtypes and Virulence Genes in Porcine Shiga Toxin-Producing Escherichia coli. PMID : 27148249 : DOI : 10.3389/fmicb.2016.00574 PMC : PMC4838603 Abstract >>
Similar to ruminants, swine have been shown to be a reservoir for Shiga toxin-producing Escherichia coli (STEC), and pork products have been linked with outbreaks associated with STEC O157 and O111:H-. STEC strains, isolated in a previous study from fecal samples of late-finisher pigs, belonged to a total of 56 serotypes, including O15:H27, O91:H14, and other serogroups previously associated with human illness. The isolates were tested by polymerase chain reaction (PCR) and a high-throughput real-time PCR system to determine the Shiga toxin (Stx) subtype and virulence-associated and putative virulence-associated genes they carried. Select STEC strains were further analyzed using a Minimal Signature E. coli Array Strip. As expected, stx 2e (81%) was the most common Stx variant, followed by stx 1a (14%), stx 2d (3%), and stx 1c (1%). The STEC serogroups that carried stx 2d were O15:H27, O159:H16 and O159:H-. Similar to stx 2a and stx 2c, the stx 2d variant is associated with development of hemorrhagic colitis and hemolytic uremic syndrome, and reports on the presence of this variant in STEC strains isolated from swine are lacking. Moreover, the genes encoding heat stable toxin (estIa) and enteroaggregative E. coli heat stable enterotoxin-1 (astA) were commonly found in 50 and 44% of isolates, respectively. The hemolysin genes, hlyA and ehxA, were both detected in 7% of the swine STEC strains. Although the eae gene was not found, other genes involved in host cell adhesion, including lpfAO113 and paa were detected in more than 50% of swine STEC strains, and a number of strains also carried iha, lpfAO26, lpfAO157, fedA, orfA, and orfB. The present work provides new insights on the distribution of virulence factors among swine STEC strains and shows that swine may carry Stx1a-, Stx2e-, or Stx2d-producing E. coli with virulence gene profiles associated with human infections.
|
1391. |
Yugendran T,
Harish BN,
( 2016 ) High incidence of plasmid-mediated quinolone resistance genes among ciprofloxacin-resistant clinical isolates of Enterobacteriaceae at a tertiary care hospital in Puducherry, India. PMID : 27168994 : DOI : 10.7717/peerj.1995 PMC : PMC4860338 Abstract >>
Background. Plasmid-mediated quinolone resistance (PMQR) has received considerable attention recently. Data analysis in Jawaharlal Institute of Postgraduate Medical Education & Research (JIPMER) revealed 75% of the Enterobacteriaceae isolates to be ciprofloxacin-resistant in 2012. Few reports regarding the prevalence of PMQR are available from India. Hence, the present study was carried out to ascertain the prevalence of PMQR genes among clinical isolates of ciprofloxacin-resistant Enterobacteriaceae in JIPMER. Methods. The study included 642 ciprofloxacin-resistant clinical Enterobacteriaceae isolates. JIPMER hospital's annual consumption data for fluoroquinolones were retrieved from the Department of Pharmacy. The test isolates were screened for the presence of qnr A, B, D, S and aac(6')-Ib-cr genes. PMQR-positive isolates alone were tested for the presence of class I (intI1) and class II (intI2) integrons. Randomly selected PCR amplicons were sequenced and analysed using MEGA software. A total of 30 PMQR strains chosen at random were assessed for the transferability of the PMQR genes. Results. A majority of the strains exhibited high MIC values with 106 strains exhibiting MIC values >256 ?g/mL. The aac(6')-Ib-cr gene had the highest prevalence at 64% (414) while, qnrB and qnrS genes were present in 15% (97) and 10% (64) of the isolates respectively. None of the strains were positive for qnrA and qnrD. All PMQR-positive isolates were screened for class I (intI1) and class II (intI2) integrons. Class I integron was found to be predominant among the test isolates with a few of them carrying both the classes of integrons. Transferability of PMQR genes to transconjugants was identified. Conclusion. The incidence of PMQR genes in the tertiary-care setup of the JIPMER hospital was found to be high which could be probably due to the increased prescription of fluoroquinolones. Thus, there is a need for rational usage of fluoroquinolones.
|
1392. |
Mulvey MR,
Loewen PC,
( 1989 ) Nucleotide sequence of katF of Escherichia coli suggests KatF protein is a novel sigma transcription factor. PMID : 2690013 : DOI : 10.1093/nar/17.23.9979 PMC : PMC335226 Abstract >>
The katF gene of Escherichia coli has been sequenced revealing a 1086 base pair open reading frame from which the sequence of a 362 amino acid protein has been deduced. The direction of transcription of katF was confirmed by expression of the gene cloned in both directions behind a T7 promoter. The KatF protein expressed in vitro migrates with an apparent size of 42 kDa. Comparison of the katF sequence to the sequence of rpoD, which encodes the sigma subunit of RNA polymerase, revealed a 181 bp region with 65% homology and a 38 bp segment that was 87% homologous. A 62 amino acid region of the predicted KatF protein sequence was found to be 85% homologous to the corresponding sequence of sigma 70, including a segment implicated in core polymerase binding. Homology was also observed with the heat shock regulatory protein encoded by htpR.
|
1393. |
Hoschützky H,
Lottspeich F,
Jann K,
( 1989 ) Isolation and characterization of the alpha-galactosyl-1,4-beta-galactosyl-specific adhesin (P adhesin) from fimbriated Escherichia coli. PMID : 2562836 : PMC : PMC313043 Abstract >>
The alpha-galactosyl-1,4-beta-galactosyl-specific adhesin (P adhesin) was isolated from the fimbria-adhesin complex (FAC) of recombinant Escherichia coli strains expressing the F7(1), F8, or F13 fimbrial antigens. Separation into fimbriae and adhesin was achieved by heating the FAC to 80 degrees C in the presence of Zwittergent 3-16. After removal of the fimbriae by precipitation with lithium chloride, the adhesin was purified by anion-exchange fast protein liquid chromatography in the presence of 4 M urea. The purified adhesins from the three strains had pIs of 4.8 to 5.0 and molecular weights of approximately 35,000. The fimbrillins were smaller, their molecular weights being different with different F antigens. The amino-terminal amino acid sequence of the F7(1)- and F13-derived adhesins were different, that of the F13-derived adhesin being identical to that extrapolated from the DNA sequence of the papG gene (B. Lund, G. Lindberg, B.-I. Marklund, and S. Normark, Proc. Natl. Acad. Sci. USA 84:5898-5902). An antiadhesive monoclonal antibody which reacted with the three P adhesins was prepared. The FAC and the purified adhesins but not the fimbriae from which the adhesins had been removed agglutinated erythrocytes and galactose-galactose-coated latex beads. The adhesion of erythrocytes to the surface-fixed adhesins could be specifically inhibited with alpha-galactosyl-1,4-beta-galactosyl-1,4-glucosyl. The results indicate that the P adhesin(s) of uropathogenic E. coli represents a group of related proteins with conserved receptor recognition domains. The F13-derived P adhesin is the PapG protein postulated by Normark and his colleagues (Lund et al., Proc. Natl. Acad. Sci. USA 84:5898-5902; B. Lund, F. Lindberg, and S. Normark, J. Bacteriol. 170:1887-1894).
|
1394. |
Caron J,
Coffield LM,
Scott JR,
( 1989 ) A plasmid-encoded regulatory gene, rns, required for expression of the CS1 and CS2 adhesins of enterotoxigenic Escherichia coli. PMID : 2563591 : DOI : 10.1073/pnas.86.3.963 PMC : PMC286599 Abstract >>
To be virulent, enterotoxigenic Escherichia coli (ETEC) must produce a toxin and a pilus-like structure that mediates specific attachment to host tissue. Expression of two of these specific adherence structures, CS1 and CS2, requires the presence of a plasmid in an ETEC strain of a particular serotype and biotype. We show here that this plasmid does not contain the structural gene for a pilin protein, as previously believed. Instead we have identified a plasmid-encoded gene called rns that is required for expression of CS1 or CS2 colonization factor antigens and for adhesion. The rns gene, defined by two separately isolated insertion mutations, produces a 26-kDa protein when transcribed and translated in vitro. At the protein level the rns gene product is homologous to AraC, a positive regulator of the arabinose operon of enteric bacteria, and to RhaR and RhaS, which regulate the rhamnose operon of E. coli. The homology of the Rns protein to AraC is localized to regions that are believed to bind to DNA. Moreover, the sequence of one of these homologous regions is consistent with a DNA binding helix-turn-helix motif. The average G + C content of E. coli DNA is 50%; yet the rns gene contains only 28% G + C, suggesting that it was acquired from some other organism.
|
1395. |
Nichols KB,
Totsika M,
Moriel DG,
Lo AW,
Yang J,
Wurpel DJ,
Rossiter AE,
Strugnell RA,
Henderson IR,
Ulett GC,
Beatson SA,
Schembri MA,
( 2016 ) Molecular Characterization of the Vacuolating Autotransporter Toxin in Uropathogenic Escherichia coli. PMID : 26858103 : DOI : 10.1128/JB.00791-15 PMC : PMC4859599 Abstract >>
The vacuolating autotransporter toxin (Vat) contributes to uropathogenic Escherichia coli (UPEC) fitness during systemic infection. Here, we characterized Vat and investigated its regulation in UPEC. We assessed the prevalence of vat in a collection of 45 UPEC urosepsis strains and showed that it was present in 31 (68%) of the isolates. The isolates containing the vat gene corresponded to three major E. coli sequence types (ST12, ST73, and ST95), and these strains secreted the Vat protein. Further analysis of the vat genomic locus identified a conserved gene located directly downstream of vat that encodes a putative MarR-like transcriptional regulator; we termed this gene vatX The vat-vatX genes were present in the UPEC reference strain CFT073, and reverse transcriptase PCR (RT-PCR) revealed that the two genes are cotranscribed. Overexpression of vatX in CFT073 led to a 3-fold increase in vat gene transcription. The vat promoter region contained three putative nucleation sites for the global transcriptional regulator histone-like nucleoid structuring protein (H-NS); thus, the hns gene was mutated in CFT073 (to generate CFT073 hns). Western blot analysis using a Vat-specific antibody revealed a significant increase in Vat expression in CFT073 hns compared to that in wild-type CFT073. Direct H-NS binding to the vat promoter region was demonstrated using purified H-NS in combination with electrophoresis mobility shift assays. Finally, Vat-specific antibodies were detected in plasma samples from urosepsis patients infected by vat-containing UPEC strains, demonstrating that Vat is expressed during infection. Overall, this study has demonstrated that Vat is a highly prevalent and tightly regulated immunogenic serine protease autotransporter protein of Enterobacteriaceae (SPATE) secreted by UPEC during infection. Uropathogenic Escherichia coli (UPEC) is the major cause of hospital- and community-acquired urinary tract infections. The vacuolating autotransporter toxin (Vat) is a cytotoxin known to contribute to UPEC fitness during murine sepsis infection. In this study, Vat was found to be highly conserved and prevalent among a collection of urosepsis clinical isolates and was expressed at human core body temperature. Regulation of vat was demonstrated to be directly repressed by the global transcriptional regulator H-NS and upregulated by the downstream gene vatX (encoding a new MarR-type transcriptional regulator). Additionally, increased Vat-specific IgG titers were detected in plasma from corresponding urosepsis patients infected with vat-positive isolates. Hence, Vat is a highly conserved and tightly regulated urosepsis-associated virulence factor.
|
1396. |
Kim JS,
Kim MJ,
Kim SJ,
Shin E,
Oh KH,
Kim SG,
Chung GT,
Yoo CK,
Seo KW,
Kim J,
( 2016 ) First description of CTX-M-3 extended-spectrum �]-lactamase in an outbreak strain of Shiga toxin-producing Escherichia coli O103:H2. PMID : 26921938 : DOI : 10.1016/j.ijantimicag.2015.12.014 Abstract >>
N/A
|
1397. |
Ovchinnikova OG,
Mallette E,
Koizumi A,
Lowary TL,
Kimber MS,
Whitfield C,
( 2016 ) Bacterial �]-Kdo glycosyltransferases represent a new glycosyltransferase family (GT99). PMID : 27199480 : DOI : 10.1073/pnas.1603146113 PMC : PMC4896679 Abstract >>
Kdo (3-deoxy-d-manno-oct-2-ulosonic acid) is an eight-carbon sugar mostly confined to Gram-negative bacteria. It is often involved in attaching surface polysaccharides to their lipid anchors. �\-Kdo provides a bridge between lipid A and the core oligosaccharide in all bacterial LPSs, whereas an oligosaccharide of �]-Kdo residues links "group 2" capsular polysaccharides to (lyso)phosphatidylglycerol. �]-Kdo is also found in a small number of other bacterial polysaccharides. The structure and function of the prototypical cytidine monophosphate-Kdo-dependent �\-Kdo glycosyltransferase from LPS assembly is well characterized. In contrast, the �]-Kdo counterparts were not identified as glycosyltransferase enzymes by bioinformatics tools and were not represented among the 98 currently recognized glycosyltransferase families in the Carbohydrate-Active Enzymes database. We report the crystallographic structure and function of a prototype �]-Kdo GT from WbbB, a modular protein participating in LPS O-antigen synthesis in Raoultella terrigena The �]-Kdo GT has dual Rossmann-fold motifs typical of GT-B enzymes, but extensive deletions, insertions, and rearrangements result in a unique architecture that makes it a prototype for a new GT family (GT99). The cytidine monophosphate-binding site in the C-terminal �\/�] domain closely resembles the corresponding site in bacterial sialyltransferases, suggesting an evolutionary connection that is not immediately evident from the overall fold or sequence similarities.
|
1398. |
Baron S,
Jouy E,
Touzain F,
Bougeard S,
Larvor E,
de Boisseson C,
Amelot M,
Keita A,
Kempf I,
( 2016 ) Impact of the administration of a third-generation cephalosporin (3GC) to one-day-old chicks on the persistence of 3GC-resistant Escherichia coli in intestinal flora: An in vivo experiment. PMID : 26931388 : DOI : 10.1016/j.vetmic.2016.01.020 Abstract >>
The aim of the experiment was to evaluate under controlled conditions the impact on the excretion of 3GC-resistant Escherichia coli of the injection of one-day-old chicks with ceftiofur, a third-generation cephalosporin (3GC). Three isolators containing specific-pathogen-free chicks were used. In the first one, 20 birds were injected with ceftiofur then ten of them were orally inoculated with a weak inoculum of a 3GC-resistant E. coli field isolate containing an IncI1/ST3 plasmid encoding a blaCTX-M-1 beta-lactamase. The other chicks were kept as contact birds. None of the 20 birds in the second isolator were injected with ceftiofur, but ten of them were similarly inoculated with the 3GC-resistant strain and the others kept as contact birds. A third isolator contained ten non-injected, non-inoculated chicks. Fecal samples were collected regularly over one month and the E. coli isolated on non-supplemented media were characterized by antimicrobial agar dilution, detection of selected resistance genes and determination of phylogenetic group by PCR. The titers of 3GC-resistant E. coli in individual fecal samples were evaluated by culturing on 3GC-supplemented media. Results showed that the inoculated strain rapidly and abundantly colonized the inoculated and contact birds. The ceftiofur injection resulted in significantly higher percentages of 3GC-resistant E. coli isolates among the analyzed E. coli. No transfer of the 3GC-encoding plasmid to other isolates could be evidenced. In conclusion, these results highlight the dramatic capacity of 3GC-resistant E. coli to colonize and persist in chicks, and the selecting pressure imposed by the off-label use of ceftiofur.
|
1399. |
Chen YY,
Gänzle MG,
( 2016 ) Influence of cyclopropane fatty acids on heat, high pressure, acid and oxidative resistance in Escherichia coli. PMID : 26828814 : DOI : 10.1016/j.ijfoodmicro.2016.01.017 Abstract >>
Heat and high pressure resistant strains of Escherichia coli are a challenge to food safety. This study investigated effects of cyclopropane fatty acids (CFAs) on stress tolerance in the heat- and pressure-resistant strain E. coli AW1.7 and the sensitive strain E. coli MG1655. The role of CFAs was explored by disruption of cfa coding for CFA synthase with an in-frame, unmarked deletion method. Both wild-type strains consumed all the unsaturated fatty acids (C16:1 and C18:1) that were mostly converted to CFAs and a low proportion to saturated fatty acid (C16:0). Moreover, E. coli AW1.7 contained a higher proportion of membrane C19:0 cyclopropane fatty acid than E. coli MG1655 (P<0.05). The �Gcfa mutant strains did not produce CFAs, and the corresponding substrates C16:1 and C18:1 accumulated in membrane lipids. The deletion of cfa did not alter resistance to H2O2 but increased the lethality of heat, high pressure and acid treatments in E. coli AW1.7, and E. coli MG1655. E. coli AW1.7 and its �Gcfa mutant were more resistant to pressure and heat but less resistant to acid stress than E. coli MG1655. Heat resistance of wild-type strains and their �Gcfa mutant was also assessed in beef patties grilled to an internal temperature of 71 �XC. After treatment, cell counts of wild type strains were higher than those of the �Gcfa mutant strains. In conclusion, CFA synthesis in E. coli increases heat, high pressure and acid resistance, and increases heat resistance in food. This knowledge on mechanisms of stress resistance will facilitate the design of intervention methods for improved pathogen control in food production.
|
1400. |
Roos U,
Harkness RE,
Braun V,
( 1989 ) Assembly of colicin genes from a few DNA fragments. Nucleotide sequence of colicin D. PMID : 2677603 : DOI : 10.1111/j.1365-2958.1989.tb00238.x Abstract >>
The nucleotide sequence of a 2.4 kb Dral-EcoRV fragment of pColD-CA23 DNA was determined. The segment of DNA contained the colicin D structural gene (cda) and the colicin D immunity gene (cdi). From the nucleotide sequence it was deduced that colicin D had a molecular weight of 74,683 D and that the immunity protein had a molecular weight of 10,057 D. The amino-terminal portion of colicin D was found to be 96% homologous with the same region of colicin B. Both colicins share the same cell-surface receptor, FepA, and require the TonB protein for uptake. A putative TonB box pentapeptide sequence was identified in the amino terminus of the colicin D protein sequence. Since colicin D inhibits protein synthesis, it was unexpected that no homology was found between the carboxy-terminal part of this colicin and that of the protein synthesis inhibiting colicin E3 and cloacin DF13. This could indicate that colicin D does not function in the same manner as the latter two bacteriocins. The observed homology with colicin B supports the domain structure concept of colicin organization. The structural organization of the colicin operon is discussed. The extensive amino-terminal homology between colicins D and B, and the strong carboxy-terminal homology between colicins B, A, and N suggest an evolutionary assembly of colicin genes from a few DNA fragments which encode the functional domains responsible for colicin activity and uptake.
|
1401. |
Dwarakanath P,
Visweswariah SS,
Subrahmanyam YV,
Shanthi G,
Jagannatha HM,
Balganesh TS,
( 1989 ) Cloning and hyperexpression of a gene encoding the heat-stable toxin of Escherichia coli. PMID : 2680769 : DOI : 10.1016/0378-1119(89)90182-0 Abstract >>
A gene (st) coding for heat-stable toxin (STh) was identified from a plasmid of a locally isolated enterotoxigenic Escherichia coli strain. The gene was cloned and its nucleotide (nt) sequence was determined. Comparison of this nt sequence with that of another st gene reported earlier, showed a single nt substitution within the structural gene for ST. This change resulted in the replacement of proline at position 19 by alanine in the STh of the locally isolated strain. The st gene was hyperexpressed using the phage T7 or the tac promoter vector systems. A 20-fold increase in STh yield was obtained in minimal medium culture supernatants following induction of the T7 promoter. There was no significant accumulation of the precursor peptide within the periplasm of the induced cell, indicating efficient processing under conditions of enhanced transcription of the gene. The yield of STh was monitored using a competitive ELISA, which was found to be a simple and sensitive assay for determining STh concentrations. A rapid and efficient isolation procedure for STh has been developed.
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1402. |
Malhotra-Kumar S,
Xavier BB,
Das AJ,
Lammens C,
Hoang HT,
Pham NT,
Goossens H,
( 2016 ) Colistin-resistant Escherichia coli harbouring mcr-1 isolated from food animals in Hanoi, Vietnam. PMID : 26774248 : DOI : 10.1016/S1473-3099(16)00014-1 Abstract >>
N/A
|
1403. |
Malhotra-Kumar S,
Xavier BB,
Das AJ,
Lammens C,
Butaye P,
Goossens H,
( 2016 ) Colistin resistance gene mcr-1 harboured on a multidrug resistant plasmid. PMID : 26774247 : DOI : 10.1016/S1473-3099(16)00012-8 Abstract >>
N/A
|
1404. |
Kawahara K,
Oki H,
Fukakusa S,
Yoshida T,
Imai T,
Maruno T,
Kobayashi Y,
Motooka D,
Iida T,
Ohkubo T,
Nakamura S,
( 2016 ) Homo-trimeric Structure of the Type IVb Minor Pilin CofB Suggests Mechanism of CFA/III Pilus Assembly in Human Enterotoxigenic Escherichia coli. PMID : 26876601 : DOI : 10.1016/j.jmb.2016.02.003 Abstract >>
In gram-negative bacteria, the assembly of type IV pilus (T4P) and the evolutionally related pseudopilus of type II secretion system involves specialized structural proteins called pilins and pseudopilins, respectively, and is dynamically regulated to promote bacterial pathogenesis. Previous studies have suggested that a structural "tip"-like hetero-complex formed through the interaction of at least three minor (pseudo) pilins plays an important role in this process, while some members of the pathogenic type IVb subfamily are known to have only one such minor pilin subunit whose function is still unknown. Here, we determined the crystal structure of the type IVb minor pilin CofB of colonization factor antigen/III from human enterotoxigenic Escherichia coli at 1.88-? resolution. The crystal structure, in conjunction with physicochemical analysis in solution, reveals a symmetrical homo-trimeric arrangement distinct from the hetero-complexes of minor (pseudo) pilins observed in other T4P and type II secretion systems. Each CofB monomer adopts a unique three-domain architecture, in which the C-terminal �]-sheet-rich lectin domain can effectively initiate trimer association of its pilin-like N-terminal domain through extensive hydrophobic interactions followed by domain swapping at the central hinge-like domain. Deletion of cofB produces a phenotype with no detectable pili formation on the cell surface, while molecular modeling indicates that the characteristic homo-trimeric structure of CofB is well situated at the pilus tip of colonization factor antigen/III formed by the major pilin CofA, suggesting a role for the minor pilin in the efficient initiation of T4P assembly.
|
1405. |
Moran RA,
Holt KE,
Hall RM,
( N/A ) pCERC3 from a commensal ST95 Escherichia coli: A ColV virulence-multiresistance plasmid carrying a sul3-associated class 1 integron. PMID : 26855083 : DOI : 10.1016/j.plasmid.2016.02.002 Abstract >>
The rare sulphonamide resistance gene sul3 was found in the commensal Escherichia coli ST95 strain 22.1-R1 that was isolated in 2010 from the faeces of a healthy Australian adult. The genome of 22.1-R1 was sequenced and a 144,344bp RepFII/FIB plasmid, pCERC3, carrying sul3 was assembled. The sul3 gene is part of a class 1 integron featuring a sul3-containing conserved segment (sul3-CS) that replaced the classic sul1-containing 3'-conserved segment (3'-CS) usually seen in class 1 integrons. The integron contained the cassette array dfrA12-orfF-aadA2-cmlA1-aadA1-qacH, conferring resistance to trimethoprim, streptomycin, spectinomycin, chloramphenicol and quaternary ammonium compound. Two additional antibiotic resistance genes, blaTEM (ampicillin resistance) and tetA(B) (tetracycline) were adjacent to the integron, forming a single resistance region. In pCERC3, the sul3-type class 1 integron was flanked by sequence derived from the tnp and mer modules of Tn21 and was in the same location as In2, the sul1-containing In5-type class 1 integron of Tn21. At one end the sequence extends into Tn2670-derived sequence and then into sequence derived from the plasmid NR1 (R100). Examination of the sequences of eleven more complete sul3-containing plasmids in GenBank confirmed the relationship between sul3-associated integrons and Tn21/Tn2670/NR1. This suggests that the events that formed sul3-associated class 1 integrons occurred within the Tn21/Tn2670 context, most likely in NR1 or a related plasmid. The backbone of pCERC3 is most closely related to the backbones of ColV virulence plasmids and contains a complete ColV operon as well as several virulence associated genes and gene clusters. Hence, pCERC3 is both an antibiotic resistance and virulence plasmid.
|
1406. |
Golomidova AK,
Kulikov EE,
Prokhorov NS,
Guerrero-Ferreira RС,
Knirel YA,
Kostryukova ES,
Tarasyan KK,
Letarov AV,
( 2016 ) Branched Lateral Tail Fiber Organization in T5-Like Bacteriophages DT57C and DT571/2 is Revealed by Genetic and Functional Analysis. PMID : 26805872 : DOI : 10.3390/v8010026 PMC : PMC4728585 Abstract >>
The T5-like siphoviruses DT57C and DT571/2, isolated from horse feces, are very closely related to each other, and most of their structural proteins are also nearly identical to T5 phage. Their LTFs (L-shaped tail fibers), however, are composed of two proteins, LtfA and LtfB, instead of the single Ltf of bacteriophage T5. In silico and mutant analysis suggests a possible branched structure of DT57C and DT571/2 LTFs, where the LtfB protein is connected to the phage tail via the LtfA protein and with both proteins carrying receptor recognition domains. Such adhesin arrangement has not been previously recognized in siphoviruses. The LtfA proteins of our phages are found to recognize different host O-antigen types: E. coli O22-like for DT57C phage and E. coli O87 for DT571/2. LtfB proteins are identical in both phages and recognize another host receptor, most probably lipopolysaccharide (LPS) of E. coli O81 type. In these two bacteriophages, LTF function is essential to penetrate the shield of the host's O-antigens. We also demonstrate that LTF-mediated adsorption becomes superfluous when the non-specific cell protection by O-antigen is missing, allowing the phages to bind directly to their common secondary receptor, the outer membrane protein BtuB. The LTF independent adsorption was also demonstrated on an O22-like host mutant missing O-antigen O-acetylation, thus showing the biological value of this O-antigen modification for cell protection against phages.
|
1407. |
Huang Y,
Shan XF,
Deng H,
Huang YJ,
Mu XP,
Huang AL,
Long QX,
( 2015 ) Epidemiology, Antimicrobial Resistance and �]-lactamase Genotypic Features of Enteropathogenic Escherichia coli Isolated from Children with Diarrhea in Southern China. PMID : 25672408 : DOI : 10.7883/yoken.JJID.2014.120 Abstract >>
The main objective of this study was to investigate the epidemiology, drug resistance and �]-lactamase genotype distribution of enteropathogenic Escherichia coli (EPEC) isolated from pediatric patients with diarrhea in southern China. The prevalence of EPEC in children with diarrhea was 3.53%. The commonest serotypes were O55:K59 and O126:K71, and the typical EPEC were more prevalent than atypical EPEC (51 vs 7). Isolates from this region were most commonly found to be resistant to ampicillin and cotrimoxazole, followed by chloramphenicol, ceftriaxone, and ceftazidime. More than 96% of the strains were susceptible to cefoperazone/sulbactam and imipenem. The most common �]-lactamase genotypes identified in 58 strains were blaCTX-M-1 (60.3%), blaTEM (56.9%), blaCTX-M-9 (27.6%), and blaSHV (15.5%). Among 58 isolates, 22 strains were found to harbor one �]-lactamase gene, and the proportions of resistance to ampicillin, cotrimoxazole, chloramphenicol, ceftriaxone, and ceftazidime, were 81.8%, 63.6%, 40.9%, 18.2%, and 9.1%, respectively. A further 30 strains carrying multiple �]-lactamase genes had increased resistance to the above antimicrobial agents (100%, 83.3%, 70.0%, 60.0%, and 30.0%, respectively). In contrast, antibiotic resistance in the last 6 strains without a detectable �]-lactamase gene was substantially reduced. Drug resistance may be associated with the �]-lactamase gene number, with a greater the number of �]-lactamase genes resulting in higher antibiotic resistance.
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1408. |
Chen M,
Shpirt AM,
Guo X,
Shashkov AS,
Zhuang Y,
Wang L,
Knirel YA,
Liu B,
( 2015 ) Identification serologically, chemically and genetically of two Escherichia coli strains as candidates for new O serogroups. PMID : 26297000 : DOI : 10.1099/mic.0.000136 Abstract >>
Escherichia coli strains are normally identified by the combination of their O and H (and sometimes K) antigens, and serotyping based on the antigens is believed to be crucial for clinical detection and epidemiological investigation. Two E. coli strains, G5413 and G5287, were isolated from faecal samples of female patients with diarrhoea and were not agglutinated with any antisera that cover the well-known O serogroups of E. coli. We elucidated the O-polysaccharide (OPS) structures and analysed the O-antigen gene clusters of these bacteria. The OPS structure of G5413 established by monosaccharide analysis and NMR spectroscopy was found to be unique amongst known bacterial polysaccharide structures. The O-antigen gene cluster of this strain was sequenced and did not match sequence data with any of the 184 O serogroups that have been recognized internationally. Gene functions were tentatively assigned and were appropriate to the OPS structure. Based on these data, we suggest G5413 as a candidate for a new E. coli O serogroup. Both the OPS structure and O-antigen gene cluster of G5287 were identical to those of E. coli L-19, a candidate for another new O serogroup characterized by us recently. Recognition of these two provisional O serogroups increases the number of known O-antigen forms of E. coli to 186.
|
1409. |
Son HM,
Duc HM,
Honjoh K,
Miyamoto T,
( 2015 ) Identification of the newly identified subtilase cytotoxin-encoding gene (subAB2-2) among clinical Shiga toxin-producing Escherichia coli isolates. PMID : 26588258 : DOI : 10.1139/cjm-2015-0519 Abstract >>
Subtilase cytotoxin (SubAB) is an important virulence factor of eae-negative Shiga toxin-producing Escherichia coli (STEC). Three variants of SubAB-encoding genes have been reported in the literature; however, the newly described subAB variant (subAB2-2) was found only in STEC strains from deer meat, sheep, and some wild animals. In this study, subAB variants were detected by PCR and DNA sequencing in 5 out of 12 (41.6%) eae-negative STEC strains isolated from patients. Most subAB-positive STEC strains (80%) harbored the subAB1 gene. The subAB2-2 gene was detected for the first time in the clinical STEC O128:H2 strain. Other virulence genes including stx1a, stx1c, stx2b, ehxA, and tia were also detected in this strain. The DNA sequence analyses of the subAB1 and subAB2-2 genes of the clinical STEC strains showed 99% and 100% identity to those of the reference strains 98NK2 and LM27558stx2, respectively. This is the first report on the detection of the subAB2-2 gene in a clinical STEC isolate.
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1410. |
Du H,
Chen L,
Tang YW,
Kreiswirth BN,
( 2016 ) Emergence of the mcr-1 colistin resistance gene in carbapenem-resistant Enterobacteriaceae. PMID : 26842776 : DOI : 10.1016/S1473-3099(16)00056-6 Abstract >>
N/A
|
1411. |
Mendes AC,
Rodrigues C,
Pires J,
Amorim J,
Ramos MH,
Novais ?,
Peixe L,
( 2016 ) Importation of Fosfomycin Resistance fosA3 Gene to Europe. PMID : 26812028 : DOI : 10.3201/eid2202.151301 PMC : PMC4734505 Abstract >>
N/A
|
1412. |
Huja S,
Oren Y,
Trost E,
Brzuszkiewicz E,
Biran D,
Blom J,
Goesmann A,
Gottschalk G,
Hacker J,
Ron EZ,
Dobrindt U,
( 2015 ) Genomic avenue to avian colisepticemia. PMID : 25587010 : DOI : 10.1128/mBio.01681-14 PMC : PMC4313913 Abstract >>
Here we present an extensive genomic and genetic analysis of Escherichia coli strains of serotype O78 that represent the major cause of avian colisepticemia, an invasive infection caused by avian pathogenic Escherichia coli (APEC) strains. It is associated with high mortality and morbidity, resulting in significant economic consequences for the poultry industry. To understand the genetic basis of the virulence of avian septicemic E. coli, we sequenced the entire genome of a clinical isolate of serotype O78-O78:H19 ST88 isolate 789 (O78-9)-and compared it with three publicly available APEC O78 sequences and one complete genome of APEC serotype O1 strain. Although there was a large variability in genome content between the APEC strains, several genes were conserved, which are potentially critical for colisepticemia. Some of these genes are present in multiple copies per genome or code for gene products with overlapping function, signifying their importance. A systematic deletion of each of these virulence-related genes identified three systems that are conserved in all septicemic strains examined and are critical for serum survival, a prerequisite for septicemia. These are the plasmid-encoded protein, the defective ETT2 (E. coli type 3 secretion system 2) type 3 secretion system ETT2sepsis, and iron uptake systems. Strain O78-9 is the only APEC O78 strain that also carried the regulon coding for yersiniabactin, the iron binding system of the Yersinia high-pathogenicity island. Interestingly, this system is the only one that cannot be complemented by other iron uptake systems under iron limitation and in serum. Avian colisepticemia is a severe systemic disease of birds causing high morbidity and mortality and resulting in severe economic losses. The bacteria associated with avian colisepticemia are highly antibiotic resistant, making antibiotic treatment ineffective, and there is no effective vaccine due to the multitude of serotypes involved. To understand the disease and work out strategies to combat it, we performed an extensive genomic and genetic analysis of Escherichia coli strains of serotype O78, the major cause of the disease. We identified several potential virulence factors, conserved in all the colisepticemic strains examined, and determined their contribution to growth in serum, an absolute requirement for septicemia. These findings raise the possibility that specific vaccines or drugs can be developed against these critical virulence factors to help combat this economically important disease.
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1413. |
Adamczuk M,
Zaleski P,
Dziewit L,
Wolinowska R,
Nieckarz M,
Wawrzyniak P,
Kieryl P,
Plucienniczak A,
Bartosik D,
( 2015 ) Diversity and Global Distribution of IncL/M Plasmids Enabling Horizontal Dissemination of �]-Lactam Resistance Genes among the Enterobacteriaceae. PMID : 26236726 : DOI : 10.1155/2015/414681 PMC : PMC4510254 Abstract >>
Antibiotic resistance determinants are frequently associated with plasmids and other mobile genetic elements, which simplifies their horizontal transmission. Several groups of plasmids (including replicons of the IncL/M incompatibility group) were found to play an important role in the dissemination of resistance genes encoding �]-lactamases. The IncL/M plasmids are large, broad host range, and self-transmissible replicons. We have identified and characterized two novel members of this group: pARM26 (isolated from bacteria inhabiting activated sludge from a wastewater treatment plant) and pIGT15 (originating from a clinical strain of Escherichia coli). This instigated a detailed comparative analysis of all available sequences of IncL/M plasmids encoding �]-lactamases. The core genome of these plasmids is comprised of 20 genes with conserved synteny. Phylogenetic analyses of these core genes allowed clustering of the plasmids into four separate groups, which reflect their antibiotic resistance profiles. Examination of the biogeography of the IncL/M plasmids revealed that they are most frequently found in bacteria of the family Enterobacteriaceae originating from the Mediterranean region and Western Europe and that they are able to persist in various ecological niches even in the absence of direct antibiotic selection pressure.
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1414. |
Boyd AC,
Archer JA,
Sherratt DJ,
( 1989 ) Characterization of the ColE1 mobilization region and its protein products. PMID : 2671664 : DOI : 10.1007/bf02464922 Abstract >>
A third of the 6.6 kb genome of ColE1 is devoted to mobilization (mob) genes necessary to promote its specific transfer in the presence of conjugative plasmids. The mob region is genetically complex: two mob genes are entirely overlapped by a third. Oligonucleotide-directed mutagenesis was used to insert an amber codon into one of the overlapped genes and make possible a full complementation analysis of mob. Four mob genes essential for mobilization by R64drd11 were thus identified. Fragments of mob were subcloned under control of the Ptac promoter in a suitable vector, overexpressed in minicells and the mobilization proteins visualized. A comprehensive alignment of the mob region of ColE1 with those of its close relatives ColK and ColA demonstrating that the four essential mob genes are conserved is also presented.
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1415. |
Deng H,
Si HB,
Zeng SY,
Sun J,
Fang LX,
Yang RS,
Liu YH,
Liao XP,
( 2015 ) Prevalence of extended-spectrum cephalosporin-resistant Escherichia coli in a farrowing farm: ST1121 clone harboring IncHI2 plasmid contributes to the dissemination of bla CMY-2. PMID : 26579110 : DOI : 10.3389/fmicb.2015.01210 PMC : PMC4630580 Abstract >>
During a regular monitoring of antimicrobial resistance in a farrowing farm in Southern China, 117 Escherichia coli isolates were obtained from sows and piglets. Compared with the isolates from piglets, the isolates from sows exhibited higher resistance rates to the tested cephalosporins. Correspondingly, the total detection rate of the bla CMY-2/bla CTX-M genes in the sow isolates (34.2%) was also significantly higher than that of the piglet isolates (13.6%; p < 0.05). The bla CMY-2 gene had a relatively high prevalence (11.1%) in the E. coli isolates. MLST and PFGE analysis revealed the clonal spread of ST1121 E. coli in most (7/13) of the bla CMY-2-positive isolates. An indistinguishable IncHI2 plasmid harboring bla CMY-2 was also identified in each of the seven ST1121 E. coli isolates. Complete sequence analysis of this IncHI2 plasmid (pEC5207) revealed that pEC5207 may have originated through recombination of an IncHI2 plasmid with a bla CMY-2-carrying IncA/C plasmid like pCFSAN007427_01. In addition to bla CMY-2, pEC5207 also carried other resistance determinants for aminoglycosides (aacA7), sulfonamides (sul1), as well as heavy metals ions, such as Cu and Ag. The susceptibility testing showed that the pEC5207 can mediate both antibiotic and heavy metal resistance. This highlights the role of pEC5207 in co-selection of bla CMY-2-positive isolates under the selective pressure of heavy metals, cephalosporins, and other antimicrobials. In conclusion, clonal spread of an ST1121 type E. coli strain harboring an IncHI2 plasmid contributed to the dissemination of bla CMY-2 in a farrowing farm in Southern China. We also have determined the first complete sequence analysis of a bla CMY-2-carrying IncHI2 plasmid.
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1416. |
Collier DN,
Bassford PJ,
( 1989 ) Mutations that improve export of maltose-binding protein in SecB- cells of Escherichia coli. PMID : 2670890 : DOI : 10.1128/jb.171.9.4640-4647.1989 PMC : PMC210262 Abstract >>
It previously has been proposed that the Escherichia coli SecB protein promotes the export of the maltose-binding protein (MBP) from the cytoplasm by preventing the folding of the precursor MBP (preMBP) into a translocation-incompetent conformation. The export of wild-type MBP is only partially blocked in SecB- cells. In contrast, the export of MBP16-1, an MBP species with a defective signal peptide, is totally dependent on SecB; hence, SecB- cells that synthesize MBP16-1 are unable to utilize maltose as a sole carbon source. The selection of Mal+ revertants primarily yielded mutants with alterations in the MBP16-1 signal peptide that permitted SecB-independent MBP export to the periplasm to various extents. Although each of these alterations increased the overall hydrophobicity of the signal peptide, it was not possible to strictly equate changes in hydrophobicity with the degree of SecB-independent export. Somewhat unexpectedly, two mutants were obtained in which MBP export in SecB- cells was markedly superior to that of the wild-type MBP. Although wild-type MBP is not cotranslationally translocated in SecB- cells, the two mutant proteins designated MBP172 and MBP173 exhibited significant cotranslational export in the absence of SecB. Thus, the role of SecB was partially supplanted by a signal peptide that promoted more rapid movement of MBP through the export pathway. When preMBP included the MBP172 signal peptide as well as an alteration in the mature moiety that slows folding, the SecB requirement for maximal MBP export efficiency was almost totally eliminated. These results provide additional strong support for the proposed antifolding role of SecB in MBP export.
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1417. |
Anantham S,
Harmer CJ,
Hall RM,
( 2015 ) p39R861-4, A Type 2 A/C2 Plasmid Carrying a Segment from the A/C1 Plasmid RA1. PMID : 26167918 : DOI : 10.1089/mdr.2015.0133 Abstract >>
The largest plasmid in the strain 39R861, which is used as a plasmid size standard, was recovered by conjugation and sequenced to determine its exact size. Plasmid p39R861-4 transferred at high frequency. Although reported to be the A/C1 plasmid RA1, p39R861-4 is a 155794-bp Type 2 A/C2 plasmid, in which a 39-kb segment, derived from RA1 that includes a relative of the RA1 resistance island, replaces 26.5 kb of the Type 2 backbone. p39R861-4 includes a single copy of IS10 and two resistance islands with a CR2-sul2 region in each of them. The 84 kb of backbone between the resistance islands is inverted relative to other known A/C plasmids and this inversion has arisen through recombination between the CR2-sul2 regions that are inversely oriented. The two resistance islands present before this inversion occurred were one related to but longer than that found in RA1, and one that is a form of the ARI-B island and identical to ARI-B in the A/C2 plasmid R55. They contain genes conferring resistance to tetracycline (tetA(D)), sulfonamides (sul2), and florfenicol and chloramphenicol (floR). The tet(D) determinant is flanked by two IS26 in a transposon-like structure named Tntet(D). Both resistance islands contain remnants of the two ends of the integrative element GIsul2, consistent with the sul2 gene being mobilized by GIsul2 rather than by CR2.
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1418. |
Yang X,
Liu W,
Liu Y,
Wang J,
Lv L,
Chen X,
He D,
Yang T,
Hou J,
Tan Y,
Xing L,
Zeng Z,
Liu JH,
( 2014 ) F33: A-: B-, IncHI2/ST3, and IncI1/ST71 plasmids drive the dissemination of fosA3 and bla CTX-M-55/-14/-65 in Escherichia coli from chickens in China. PMID : 25566207 : DOI : 10.3389/fmicb.2014.00688 PMC : PMC4267423 Abstract >>
The purpose of this study was to examine the occurrence of fosfomycin-resistant Escherichia coli from chickens and to characterize the plasmids carrying fosA3. A total of 661 E. coli isolates of chicken origin collected from 2009 to 2011 were screened for plasmid-mediated fosfomycin resistance determinants by PCR. Plasmids were characterized using PCR-based replicon typing, plasmid multilocus sequence typing, and restriction fragment length polymorphisms. Associated addiction systems and resistance genes were identified by PCR. PCR-mapping was used for analysis of the genetic context of fosA3. Fosfomycin resistance was detected in 58 isolates that also carried the fosA3 gene. Fifty-seven, 17, and 52 FosA3-producers also harbored bla CTX-M, rmtB, and floR genes, respectively. Most of the 58 fosA3-carrying isolates were clonally unrelated, and all fosA3 genes were located on plasmids belonged to F33:A-:B- (n = 18), IncN-F33:A-:B- (n = 7), IncHI2/ST3 (n = 10), IncI1/ST71 (n = 3), IncI1/ST108 (n = 3), and others. The genetic structures, IS26-ISEcp1-bla CTX-M-55-orf477-bla TEM-1-IS26-fosA3-1758bp-IS26 and ISEcp1-bla CTX-M-65-IS903-iroN-IS26-fosA3-536bp-IS26 were located on highly similar F33:A-:B- plasmids. In addition, bla CTX-M-14-fosA3-IS26 was frequently present on similar IncHI2/ST3 plasmids. IncFII plasmids had a significantly higher frequency of addiction systems (mean 3.5) than other plasmids. Our results showed a surprisingly high prevalence of fosA3 gene in E. coli isolates recovered from chicken in China. The spread of fosA3 can be attributed to horizontal dissemination of several epidemic plasmids, especially F33:A-:B- plasmids. Since coselection by other antimicrobials is the major driving force for the diffusion of the fosA3 gene, a strict antibiotic use policy is urgently needed in China.
|
1419. |
Potron A,
Poirel L,
Dortet L,
Nordmann P,
( 2016 ) Characterisation of OXA-244, a chromosomally-encoded OXA-48-like �]-lactamase from Escherichia coli. PMID : 26655033 : DOI : 10.1016/j.ijantimicag.2015.10.015 Abstract >>
N/A
|
1420. |
Liu YY,
Wang Y,
Walsh TR,
Yi LX,
Zhang R,
Spencer J,
Doi Y,
Tian G,
Dong B,
Huang X,
Yu LF,
Gu D,
Ren H,
Chen X,
Lv L,
He D,
Zhou H,
Liang Z,
Liu JH,
Shen J,
( 2016 ) Emergence of plasmid-mediated colistin resistance mechanism MCR-1 in animals and human beings in China: a microbiological and molecular biological study. PMID : 26603172 : DOI : 10.1016/S1473-3099(15)00424-7 Abstract >>
Until now, polymyxin resistance has involved chromosomal mutations but has never been reported via horizontal gene transfer. During a routine surveillance project on antimicrobial resistance in commensal Escherichia coli from food animals in China, a major increase of colistin resistance was observed. When an E coli strain, SHP45, possessing colistin resistance that could be transferred to another strain, was isolated from a pig, we conducted further analysis of possible plasmid-mediated polymyxin resistance. Herein, we report the emergence of the first plasmid-mediated polymyxin resistance mechanism, MCR-1, in Enterobacteriaceae. The mcr-1 gene in E coli strain SHP45 was identified by whole plasmid sequencing and subcloning. MCR-1 mechanistic studies were done with sequence comparisons, homology modelling, and electrospray ionisation mass spectrometry. The prevalence of mcr-1 was investigated in E coli and Klebsiella pneumoniae strains collected from five provinces between April, 2011, and November, 2014. The ability of MCR-1 to confer polymyxin resistance in vivo was examined in a murine thigh model. Polymyxin resistance was shown to be singularly due to the plasmid-mediated mcr-1 gene. The plasmid carrying mcr-1 was mobilised to an E coli recipient at a frequency of 10(-1) to 10(-3) cells per recipient cell by conjugation, and maintained in K pneumoniae and Pseudomonas aeruginosa. In an in-vivo model, production of MCR-1 negated the efficacy of colistin. MCR-1 is a member of the phosphoethanolamine transferase enzyme family, with expression in E coli resulting in the addition of phosphoethanolamine to lipid A. We observed mcr-1 carriage in E coli isolates collected from 78 (15%) of 523 samples of raw meat and 166 (21%) of 804 animals during 2011-14, and 16 (1%) of 1322 samples from inpatients with infection. The emergence of MCR-1 heralds the breach of the last group of antibiotics, polymyxins, by plasmid-mediated resistance. Although currently confined to China, MCR-1 is likely to emulate other global resistance mechanisms such as NDM-1. Our findings emphasise the urgent need for coordinated global action in the fight against pan-drug-resistant Gram-negative bacteria. Ministry of Science and Technology of China, National Natural Science Foundation of China.
|
1421. |
Guerineau F,
Mullineaux P,
( 1989 ) Nucleotide sequence of the sulfonamide resistance gene from plasmid R46. PMID : 2662140 : DOI : 10.1093/nar/17.11.4370 PMC : PMC317944 Abstract >>
N/A
|
1422. |
Kumamoto CA,
Nault AK,
( 1989 ) Characterization of the Escherichia coli protein-export gene secB. PMID : 2656409 : DOI : 10.1016/0378-1119(89)90393-4 Abstract >>
The Escherichia coli secB gene product is required for normal export of envelope proteins out of the cell cytoplasm. In this report, we present the identification and nucleotide sequence of the secB coding sequence. The secB structural gene overlaps almost completely with a predicted open reading frame (ORF) that is encoded on the opposite strand. To establish the identity of the secB ORF, we characterized a secB mutation that caused total loss of secB function, based upon its phenotype. This mutation resulted from a nucleotide change that caused an ochre mutation in one ORF (the secB gene) and a silent (no amino acid change) codon change in the opposite ORF.
|
1423. |
Gupta YK,
Chan SH,
Xu SY,
Aggarwal AK,
( 2015 ) Structural basis of asymmetric DNA methylation and ATP-triggered long-range diffusion by EcoP15I. PMID : 26067164 : DOI : 10.1038/ncomms8363 PMC : PMC4490356 Abstract >>
Type III R-M enzymes were identified >40 years ago and yet there is no structural information on these multisubunit enzymes. Here we report the structure of a Type III R-M system, consisting of the entire EcoP15I complex (Mod2Res1) bound to DNA. The structure suggests how ATP hydrolysis is coupled to long-range diffusion of a helicase on DNA, and how a dimeric methyltransferase functions to methylate only one of the two DNA strands. We show that the EcoP15I motor domains are specifically adapted to bind double-stranded DNA and to facilitate DNA sliding via a novel 'Pin' domain. We also uncover unexpected 'division of labour', where one Mod subunit recognizes DNA, while the other Mod subunit methylates the target adenine--a mechanism that may extend to adenine N6 RNA methylation in mammalian cells. Together the structure sheds new light on the mechanisms of both helicases and methyltransferases in DNA and RNA metabolism.
|
1424. |
Fleury MA,
Mourand G,
Jouy E,
Touzain F,
Le Devendec L,
de Boisseson C,
Eono F,
Cariolet R,
Guérin A,
Le Goff O,
Blanquet-Diot S,
Alric M,
Kempf I,
( 2015 ) Impact of ceftiofur injection on gut microbiota and Escherichia coli resistance in pigs. PMID : 26077254 : DOI : 10.1128/AAC.00177-15 PMC : PMC4538500 Abstract >>
Resistance to extended-spectrum cephalosporins (ESCs) is an important health concern. Here, we studied the impact of the administration of a long-acting form of ceftiofur on the pig gut microbiota and ESC resistance in Escherichia coli. Pigs were orally inoculated with an ESC-resistant E. coli M63 strain harboring a conjugative plasmid carrying a gene conferring resistance, bla CTX-M-1. On the same day, they were given or not a unique injection of ceftiofur. Fecal microbiota were studied using quantitative PCR analysis of the main bacterial groups and quantification of short-chain fatty acids. E. coli and ESC-resistant E. coli were determined by culture methods, and the ESC-resistant E. coli isolates were characterized. The copies of the bla CTX-M-1 gene were quantified. After ceftiofur injection, the main change in gut microbiota was the significant but transitory decrease in the E. coli population. Acetate and butyrate levels were significantly lower in the treated group. In all inoculated groups, E. coli M63 persisted in most pigs, and the bla CTX-M-1 gene was transferred to other E. coli. Culture and PCR results showed that the ceftiofur-treated group shed significantly more resistant strains 1 and 3 days after ESC injection. Thereafter, on most dates, there were no differences between the groups, but notably, one pig in the nontreated group regularly excreted very high numbers of ESC-resistant E. coli, probably leading to a higher contamination level in its pen. In conclusion, the use of ESCs, and also the presence of high-shedding animals, are important features in the spread of ESC resistance.
|
1425. |
Nichols DA,
Hargis JC,
Sanishvili R,
Jaishankar P,
Defrees K,
Smith EW,
Wang KK,
Prati F,
Renslo AR,
Woodcock HL,
Chen Y,
( 2015 ) Ligand-Induced Proton Transfer and Low-Barrier Hydrogen Bond Revealed by X-ray Crystallography. PMID : 26057252 : DOI : 10.1021/jacs.5b00749 PMC : PMC4530788 Abstract >>
Ligand binding can change the pKa of protein residues and influence enzyme catalysis. Herein, we report three ultrahigh resolution X-ray crystal structures of CTX-M �]-lactamase, directly visualizing protonation state changes along the enzymatic pathway: apo protein at 0.79 ?, precovalent complex with nonelectrophilic ligand at 0.89 ?, and acylation transition state (TS) analogue at 0.84 ?. Binding of the noncovalent ligand induces a proton transfer from the catalytic Ser70 to the negatively charged Glu166, and the formation of a low-barrier hydrogen bond (LBHB) between Ser70 and Lys73, with a length of 2.53 ? and the shared hydrogen equidistant from the heteroatoms. QM/MM reaction path calculations determined the proton transfer barrier to be 1.53 kcal/mol. The LBHB is absent in the other two structures although Glu166 remains neutral in the covalent complex. Our data represents the first X-ray crystallographic example of a hydrogen engaged in an enzymatic LBHB, and demonstrates that desolvation of the active site by ligand binding can provide a protein microenvironment conducive to LBHB formation. It also suggests that LBHBs may contribute to stabilization of the TS in general acid/base catalysis together with other preorganized features of enzyme active sites. These structures reconcile previous experimental results suggesting alternatively Glu166 or Lys73 as the general base for acylation, and underline the importance of considering residue protonation state change when modeling protein-ligand interactions. Additionally, the observation of another LBHB (2.47 ?) between two conserved residues, Asp233 and Asp246, suggests that LBHBs may potentially play a special structural role in proteins.
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1426. |
Boyd D,
Taylor G,
Fuller J,
Bryce E,
Embree J,
Gravel D,
Katz K,
Kibsey P,
Kuhn M,
Langley J,
Mataseje L,
Mitchell R,
Roscoe D,
Simor A,
Thomas E,
Turgeon N,
Mulvey M,
N/A N/A,
( 2015 ) Complete Sequence of Four Multidrug-Resistant MOBQ1 Plasmids Harboring blaGES-5 Isolated from Escherichia coli and Serratia marcescens Persisting in a Hospital in Canada. PMID : 25545311 : DOI : 10.1089/mdr.2014.0205 Abstract >>
The usefulness of carbapenems for gram-negative infections is becoming compromised by organisms harboring carbapenemases, enzymes which can hydrolyze the drug. Currently KPC (class A), NDM (class B), and OXA-48 types (class D) are the most globally widespread carbapenemases. However, among the GES-type class A extended-spectrum �]-lactamases (ESBLs) there are variants that hydrolyze carbapenems, with blaGES-5 being the most common. Two Escherichia coli and two Serratia marcescens harboring blaGES-5 on plasmids were isolated by the Canadian Nosocomial Infection Surveillance Program (CNISP) from four different patients in a single hospital over a 2-year period. Complete sequencing of the blaGES-5 plasmids indicated that all four had nearly identical backbones consisting of genes for replication, partitioning, and stability, but contained variant accessory regions consisting of mobile elements and antimicrobial resistance genes. The plasmids were of a novel replicon type, but belonged to the MOBQ1 group based on relaxase sequences, and appeared to be mobilizable, but not self-transmissible. Considering the time periods of bacterial isolation, it would appear the blaGES-5 plasmid has persisted in an environmental niche for at least 2 years in the hospital. This has implications for infection control and clinical care when it is transferred to clinically relevant gram-negative organisms.
|
1427. |
Harmer CJ,
Hall RM,
( 2015 ) IS26-Mediated Precise Excision of the IS26-aphA1a Translocatable Unit. PMID : 26646012 : DOI : 10.1128/mBio.01866-15 PMC : PMC4676283 Abstract >>
We recently showed that, in the absence of RecA-dependent homologous recombination, the Tnp26 transposase catalyzes cointegrate formation via a conservative reaction between two preexisting IS26, and this is strongly preferred over replicative transposition to a new site. Here, the reverse reaction was investigated by assaying for precise excision of the central region together with a single IS26 from a compound transposon bounded by IS26. In a recA mutant strain, Tn4352, a kanamycin resistance transposon carrying the aphA1a gene, was stable. However, loss of kanamycin resistance due to precise excision of the translocatable unit (TU) from the closely related Tn4352B, leaving behind the second IS26, occurred at high frequency. Excision occurred when Tn4352B was in either a high- or low-copy-number plasmid. The excised circular segment, known as a TU, was detected by PCR. Excision required the IS26 transposase Tnp26. However, the Tnp26 of only one IS26 in Tn4352B was required, specifically the IS26 downstream of the aphA1a gene, and the excised TU included the active IS26. The frequency of Tn4352B TU loss was influenced by the context of the transposon, but the critical determinant of high-frequency excision was the presence of three G residues in Tn4352B replacing a single G in Tn4352. These G residues are located immediately adjacent to the two G residues at the left end of the IS26 that is upstream of the aphA1a gene. Transcription of tnp26 was not affected by the additional G residues, which appear to enhance Tnp26 cleavage at this end. Resistance to antibiotics limits treatment options. In Gram-negative bacteria, IS26 plays a major role in the acquisition and dissemination of antibiotic resistance. IS257 (IS431) and IS1216, which belong to the same insertion sequence (IS) family, mobilize resistance genes in staphylococci and enterococci, respectively. Many different resistance genes are found in compound transposons bounded by IS26, and multiply and extensively antibiotic-resistant Gram-negative bacteria often include regions containing several antibiotic resistance genes and multiple copies of IS26. We recently showed that in addition to replicative transposition, IS26 can use a conservative movement mechanism in which an incoming IS26 targets a preexisting one, and this reaction can create these regions. This mechanism differs from that of all the ISs examined in detail thus far. Here, we have continued to extend understanding of the reactions carried out by IS26 by examining whether the reverse precise excision reaction is also catalyzed by the IS26 transposase.
|
1428. |
Eick-Helmerich K,
Braun V,
( 1989 ) Import of biopolymers into Escherichia coli: nucleotide sequences of the exbB and exbD genes are homologous to those of the tolQ and tolR genes, respectively. PMID : 2670903 : DOI : 10.1128/jb.171.9.5117-5126.1989 PMC : PMC210325 Abstract >>
Escherichia coli with mutations in the exb region are impaired in outer membrane receptor-dependent uptake processes. They are resistant to the antibiotic albomycin and exhibit reduced sensitivity to group B colicins. A 2.2-kilobase-pair DNA fragment of the exb locus was sequenced. It contained two open reading frames, designated exbB and exbD, which encoded polypeptides of 244 and 141 amino acids, respectively. Both proteins were found in the cytoplasmic membrane. They showed strong homologies to the TolQ and TolR proteins, respectively, which are involved in uptake of group A colicins and infection by filamentous bacteriophages. exbB and exbD were required to complement exb mutations. Osmotic shock treatment rendered exb mutants sensitive to colicin M, which was taken as evidence that the ExbB and ExbD proteins are involved in transport processes across the outer membrane. It is concluded that the exb- and tol-dependent systems originate from a common uptake system for biopolymers.
|
1429. |
Zamorano L,
Miró E,
Juan C,
Gómez L,
Bou G,
González-López JJ,
Martínez-Martínez L,
Aracil B,
Conejo MC,
Oliver A,
Navarro F,
( 2015 ) Mobile genetic elements related to the diffusion of plasmid-mediated AmpC �]-lactamases or carbapenemases from Enterobacteriaceae: findings from a multicenter study in Spain. PMID : 26077249 : DOI : 10.1128/AAC.00562-15 PMC : PMC4538544 Abstract >>
We examined the genetic context of 74 acquired ampC genes and 17 carbapenemase genes from 85 of 640 Enterobacteriaceae isolates collected in 2009. Using S1 pulsed-field gel electrophoresis and Southern hybridization, 37 of 74 bla AmpC genes were located on large plasmids of different sizes belonging to six incompatibility groups. We used sequencing and PCR mapping to investigate the regions flanking the acquired ampC genes. The bla CMY-2-like genes were associated with ISEcp1; the surrounding bla DHA genes were similar to Klebsiella pneumoniae plasmid pTN60013 associated with IS26 and the psp and sap operons; and the bla ACC-1 genes were associated with IS26 elements inserted into ISEcp1. All of the carbapenemase genes (bla VIM-1, bla IMP-22, and bla IMP-28) were located in class 1 integrons. Therefore, although plasmids are the main cause of the rapid dissemination of ampC genes among Enterobacteriaceae, we need to be aware that other mobile genetic elements, such as insertion sequences, transposons, or integrons, can be involved in the mobilization of these genes of chromosomal origin. Additionally, three new integrons (In846 to In848) are described in this study.
|
1430. |
Pickett CL,
Twiddy EM,
Coker C,
Holmes RK,
( 1989 ) Cloning, nucleotide sequence, and hybridization studies of the type IIb heat-labile enterotoxin gene of Escherichia coli. PMID : 2670900 : DOI : 10.1128/jb.171.9.4945-4952.1989 PMC : PMC210301 Abstract >>
Type IIb heat-labile enterotoxin (LT-IIb) is produced by Escherichia coli 41. Restriction fragments of total cell DNA from strain 41 were cloned into a cosmid vector, and one cosmid clone that encoded LT-IIb was identified. The genes for LT-IIb were subcloned into a variety of plasmids, expressed in minicells, sequenced, and compared with the structural genes for other members of the Vibrio cholerae-E. coli enterotoxin family. The A subunits of these toxins all have similar ADP-ribosyltransferase activity. The A genes of LT-IIa and LT-IIb exhibited 71% DNA sequence homology with each other and 55 to 57% homology with the A genes of cholera toxin (CT) and the type I enterotoxins of E. coli (LTh-I and LTp-I). The A subunits of the heat-labile enterotoxins also have limited homology with other ADP-ribosylating toxins, including pertussis toxin, diphtheria toxin, and Pseudomonas aeruginosa exotoxin A. The B subunits of LT-IIa and LT-IIb differ from each other and from type I enterotoxins in their carbohydrate-binding specificities. The B genes of LT-IIa and LT-IIb were 66% homologous, but neither had significant homology with the B genes of CT, LTh-I, and LTp-I. The A subunit genes for the type I and type II enterotoxins represent distinct branches of an evolutionary tree, and the divergence between the A subunit genes of LT-IIa and LT-IIb is greater than that between CT and LT-I. In contrast, it has not yet been possible to demonstrate an evolutionary relationship between the B subunits of type I and type II heat-labile enterotoxins. Hybridization studies with DNA from independently isolated LT-II producing strains of E. coli also suggested that additional variants of LT-II exist.
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1431. |
Gigliani F,
Sporeno E,
Perri S,
Battaglia PA,
( 1989 ) The uvp1 gene of plasmid pR cooperates with mucAB genes in the DNA repair process. PMID : 2550763 : DOI : 10.1007/bf00330560 Abstract >>
We show that a DNA fragment that contains the uvp1 gene of the plasmid pR directs the synthesis in Escherichia coli minicells of a protein of apparent molecular weight 20 kDa. Inspection of the nucleotide sequence of the region reveals an open reading frame that has the capacity to encode a protein of 198 amino acids. The uvp1 gene product has been found, in two different systems, to enhance the recombinational activity of E. coli cells. We have also observed a striking similarity to resolvase and invertase proteins. The significance of this finding for the function of the uvp1 gene product requires further investigation. We conclude that the uvp1 gene encodes a 20 kDa protein which appears to be responsible for enhancement of both UV survival and recombinational activity in E. coli.
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1432. |
Sougakoff W,
Petit A,
Goussard S,
Sirot D,
Bure A,
Courvalin P,
( 1989 ) Characterization of the plasmid genes blaT-4 and blaT-5 which encode the broad-spectrum beta-lactamases TEM-4 and TEM-5 in enterobacteriaceae. PMID : 2550326 : DOI : 10.1016/0378-1119(89)90236-9 Abstract >>
We have determined the nucleotide sequence of the plasmid genes blaT-4 and blaT-5 which encode the broad-substrate-range beta-lactamases TEM-4 and TEM-5, respectively. The TEM-4 enzyme, which confers high-level resistance to cefotaxime (Ctx) and ceftazidime (Caz), differed from the TEM-1 penicillinase by four amino acid substitutions. Two of the mutations are identical to those responsible for the wide substrate range of the TEM-3 beta-lactamase which hydrolyses Ctx and Caz. The amino acid sequence of TEM-5, which confers higher levels of resistance to Caz than to other recently developed cephalosporins, differed from that of TEM-1 by three mutations distinct from those of TEM-4. Analysis of the location of the mutations in the primary and tertiary structures of class A beta-lactamases suggests that interactions between the substituted residues and beta-lactam antibiotics non-hydrolysable by TEM-1 and TEM-2 allow TEM-4 and TEM-5 to hydrolyse efficiently novel broad-spectrum cephalosporins such as Ctx and Caz.
|
1433. |
Huang YM,
Zhong LL,
Zhang XF,
Hu HT,
Li YQ,
Yang XR,
Feng LQ,
Huang X,
Tian GB,
( 2015 ) NDM-1-Producing Citrobacter freundii, Escherichia coli, and Acinetobacter baumannii Identified from a Single Patient in China. PMID : 26055374 : DOI : 10.1128/AAC.04682-14 PMC : PMC4505197 Abstract >>
We identified New Delhi metallo-�]-lactamase (NDM-1)-producing Citrobacter freundii GB032, Escherichia coli GB102, and Acinetobacter baumannii GB661 in urine and stool samples from a single patient in China. Plasmid profiling and Southern blotting indicated that blaNDM-1 from GB032 and that from GB102 were likely located on the same plasmid, while blaNDM-1 from GB661 was located on a very large (>400-kb) plasmid. This case underscores the broad host range of blaNDM-1 and its potential to spread between members of the family Enterobacteriaceae and A. baumannii.
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1434. |
Thirup SS,
Van LB,
Nielsen TK,
Knudsen CR,
( 2015 ) Structural outline of the detailed mechanism for elongation factor Ts-mediated guanine nucleotide exchange on elongation factor Tu. PMID : 26073967 : DOI : 10.1016/j.jsb.2015.06.011 Abstract >>
Translation elongation factor EF-Tu belongs to the superfamily of guanine-nucleotide binding proteins, which play key cellular roles as regulatory switches. All G-proteins require activation via exchange of GDP for GTP to carry out their respective tasks. Often, guanine-nucleotide exchange factors are essential to this process. During translation, EF-Tu:GTP transports aminoacylated tRNA to the ribosome. GTP is hydrolyzed during this process, and subsequent reactivation of EF-Tu is catalyzed by EF-Ts. The reaction path of guanine-nucleotide exchange is structurally poorly defined for EF-Tu and EF-Ts. We have determined the crystal structures of the following reaction intermediates: two structures of EF-Tu:GDP:EF-Ts (2.2 and 1.8? resolution), EF-Tu:PO4:EF-Ts (1.9? resolution), EF-Tu:GDPNP:EF-Ts (2.2? resolution) and EF-Tu:GDPNP:pulvomycin:Mg(2+):EF-Ts (3.5? resolution). These structures provide snapshots throughout the entire exchange reaction and suggest a mechanism for the release of EF-Tu in its GTP conformation. An inferred sequence of events during the exchange reaction is presented.
|
1435. |
Guja KE,
Schildbach JF,
( 2015 ) Completing the specificity swap: Single-stranded DNA recognition by F and R100 TraI relaxase domains. PMID : 25841886 : DOI : 10.1016/j.plasmid.2015.03.006 Abstract >>
During conjugative plasmid transfer, one plasmid strand is cleaved and transported to the recipient bacterium. For F and related plasmids, TraI contains the relaxase or nickase activity that cleaves the plasmid DNA strand. F TraI36, the F TraI relaxase domain, binds a single-stranded origin of transfer (oriT) DNA sequence with high affinity and sequence specificity. The TraI36 domain from plasmid R100 shares 91% amino acid sequence identity with F TraI36, but its oriT DNA binding site differs by two of eleven bases. Both proteins readily distinguish between F and R100 binding sites. In earlier work, two amino acid substitutions in the DNA binding cleft were shown to be sufficient to change the R100 TraI36 DNA-binding specificity to that of F TraI36. In contrast, three substitutions could make F TraI36 more "R100-like", but failed to completely alter the specificity. Here we identify one additional amino acid substitution that completes the specificity swap from F to R100. To our surprise, adding further substitutions from R100 to the F background were detrimental to binding instead of being neutral, indicating that their effects were influenced by their structural context. These results underscore the complex and subtle nature of DNA recognition by relaxases and have implications for the evolution of relaxase binding sites and oriT sequences.
|
1436. |
Fang LX,
Sun J,
Li L,
Deng H,
Huang T,
Yang QE,
Li X,
Chen MY,
Liao XP,
Liu YH,
( 2015 ) Dissemination of the chromosomally encoded CMY-2 cephalosporinase gene in Escherichia coli isolated from animals. PMID : 26048440 : DOI : 10.1016/j.ijantimicag.2015.04.003 Abstract >>
In this study, 619 individual Escherichia coli isolates from food-producing and companion animals were analysed to determine the prevalence of the cephalosporinase gene blaCMY-2. In total, 18 CMY-2-producers (2.9%) were detected and exhibited multidrug-resistant phenotypes. One of the CMY-2-producers was found to possess a novel blaCMY-2-like allele, blaCMY-130. The isolates belonged to distinct pulsotypes, suggesting that the blaCMY-2 gene was not disseminated by clonal expansion of blaCMY-2-positive strains. The blaCMY-2 genes were located on IncA/C-, IncHI2- or IncX-type plasmids in 9 (50%) of the 18 E. coli isolates. However, in the other nine isolates I-CeuI-PFGE and hybridisation analyses revealed that the blaCMY-2 gene was chromosomally located. A CMY gene-containing region composed of five open reading frames (ORFs) (ISEcp1-blaCMY-2-blc-sugE-�GencR) was observed in plasmids from eight strains. A CMY gene-containing region composed of ten ORFs was observed in all of the nine chromosomally encoded blaCMY-2 genes, including a putative IS66-like element inserted in this conserved CMY genetic region in three strains. This conserved CMY genetic region was also found to be inserted into the oriV�^ (putative gamma origin), part of the IncX plasmid backbone, by a complete transposition unit flanked by 5-bp DRs (direct repeat sequence) in pS62T. These results demonstrate the high prevalence of the chromosomally encoded blaCMY-2 gene in E. coli. This is the first study reporting a chromosomally encoded blaCMY-2 gene in E. coli. Chromosomally encoded blaCMY-2 might be a source of some plasmid-mediated blaCMY-2 genes and this probably facilitates the spread of cephalosporin-resistant strains.
|
1437. |
He X,
Zhou X,
Yang Z,
Xu L,
Yu Y,
Jia L,
Li G,
( 2015 ) Cloning, expression and purification of d-tagatose 3-epimerase gene from Escherichia coli JM109. PMID : 26134660 : DOI : 10.1016/j.pep.2015.06.015 Abstract >>
An unknown d-tagatose 3-epimerase (DTE) containing a IoIE domain was identified and cloned from Escherichia coli. This gene was subcloned into the prokaryotic expression vector pET-15b, and induced by IPTG in E. coli BL21 expression system. Through His-select gel column purification and fast-protein liquid chromatography, highly purified and stable DTE protein was produced. The molecular weight of the DTE protein was estimated to be 29.8kDa. The latest 83 DTE sequences from public database were selected and analyzed by molecular clustering, multi-sequence alignment. DTEs were roughly divided into five categories.
|
1438. |
Bariši? I,
Mitteregger D,
Hirschl AM,
Noehammer C,
Wiesinger-Mayr H,
( 2014 ) High diversity of beta-lactamases in the General Hospital Vienna verified by whole genome sequencing and statistical analysis. PMID : 25159028 : DOI : 10.1016/j.meegid.2014.08.014 Abstract >>
The detailed analysis of antibiotic resistance mechanisms is essential for understanding the underlying evolutionary processes, the implementation of appropriate intervention strategies and to guarantee efficient treatment options. In the present study, 110 �]-lactam-resistant, clinical isolates of Enterobacteriaceae sampled in 2011 in one of Europe's largest hospitals, the General Hospital Vienna, were screened for the presence of 31 �]-lactamase genes. Twenty of those isolates were selected for whole genome sequencing (WGS). In addition, the number of �]-lactamase genes was estimated using biostatistical models. The carbapenemase genes blaKPC-2, blaKPC-3, and blaVIM-4 were identified in carbapenem-resistant and intermediate susceptible isolates, blaOXA-72 in an extended-spectrum �]-lactamase (ESBL)-positive one. Furthermore, the observed high prevalence of the acquired blaDHA-1 and blaCMY AmpC �]-lactamase genes (70%) in phenotypically AmpC-positive isolates is alarming due to their capability to become carbapenem-resistant upon changes in membrane permeability. The statistical analyses revealed that approximately 55% of all �]-lactamase genes present in the General Hospital Vienna were detected by this study. In summary, this work gives a very detailed picture on the disseminated �]-lactamases and other resistance genes in one of Europe's largest hospitals.
|
1439. |
Zapata G,
Vann WF,
Aaronson W,
Lewis MS,
Moos M,
( 1989 ) Sequence of the cloned Escherichia coli K1 CMP-N-acetylneuraminic acid synthetase gene. PMID : 2549035 : Abstract >>
The Escherichia coli CMP-N-acetylneuraminic acid (CMP-NeuAc) synthetase gene is located on a 3.3-kilobase (kb) HindIII fragment of the plasmid pSR23 which contains the genes for K1 capsule production (Vann, W. F., Silver, R. P., Abeijon, C., Chang, K., Aaronson, W., Sutton, A., Finn, C. W., Lindner, W., and Kotsatos, M. (1987) J. Biol. Chem. 262, 17556-17562). The CMP-NeuAc synthetase gene expression was increased 10-30-fold by cloning of a 2.7-kb EcoRI-HindIII fragment onto the vector pKK223-3 containing the tac promoter. The complete nucleotide sequence of the gene encoding CMP-NeuAc synthetase was determined from progressive deletions generated by selective digestion of M13 clones containing the 2.7-kb fragment. CMP-NeuAc synthetase is located near the EcoRI site on this fragment as indicated by the detection of an open reading frame encoding a 49,000-dalton polypeptide. The amino- and carboxyl-terminal sequences of the encoded protein were confirmed by sequencing of peptides cleaved from both ends of the purified enzyme. The nucleotide deduced amino acid sequence was confirmed by sequencing several tryptic peptides of purified enzyme. The molecular weight is consistent with that determined from sodium dodecyl sulfate-gel electrophoresis. Gel filtration and ultracentrifugation experiments under nondenaturing conditions suggest that the enzyme is active as a 49,000-dalton monomer but may form aggregates.
|
1440. |
Riccobono E,
Di Pilato V,
Di Maggio T,
Revollo C,
Bartoloni A,
Pallecchi L,
Rossolini GM,
( 2015 ) Characterization of IncI1 sequence type 71 epidemic plasmid lineage responsible for the recent dissemination of CTX-M-65 extended-spectrum �]-lactamase in the Bolivian Chaco region. PMID : 26100713 : DOI : 10.1128/AAC.00589-15 PMC : PMC4538553 Abstract >>
During the last decade, a significant diffusion of CTX-M-type extended-spectrum �]-lactamases (ESBLs) was observed in commensal Escherichia coli from healthy children in the Bolivian Chaco region, with initial dissemination of CTX-M-2, which was then replaced by CTX-M-15 and CTX-M-65. In this work, we demonstrate that the widespread dissemination of CTX-M-65 observed in this context was related to the polyclonal spreading of an IncI1 sequence type 71 (ST71) epidemic plasmid lineage. The structure of the epidemic plasmid population was characterized by complete sequencing of four representatives and PCR mapping of the remainder (n = 16). Sequence analysis showed identical plasmid backbones (similar to that of the reference IncI1 plasmid, R64) and a multiresistance region (MRR), which underwent local microevolution. The MRR harbored genes responsible for resistance to �]-lactams, aminoglycosides, florfenicol, and fosfomycin (with microevolution mainly consisting of deletion events of resistance modules). The bla CTX-M-65 module harbored by the IncI1 ST71 epidemic plasmid was apparently derived from IncN-type plasmids, likely via IS26-mediated mobilization. The plasmid could be transferred by conjugation to several different enterobacterial species (Escherichia coli, Cronobacter sakazakii, Enterobacter cloacae, Klebsiella oxytoca, Klebsiella pneumoniae, and Salmonella enterica) and was stably maintained without selective pressure in these species, with the exception of K. oxytoca and S. enterica. Fitness assays performed in E. coli recipients demonstrated that the presence of the epidemic plasmid was apparently not associated with a significant biological cost.
|
1441. |
Ham LM,
Skurray R,
( 1989 ) Molecular analysis and nucleotide sequence of finQ, a transcriptional inhibitor of the F plasmid transfer genes. PMID : 2543909 : DOI : 10.1007/bf00332236 Abstract >>
We report the cloning of finQ, a gene coding for fertility inhibition of the F plasmid, from the IncI R factor R820a. The finQ gene was mapped precisely within a 1.24 kb region by ptac-transposase + min-kan mutagenesis and its product, FinQp, identified as a single polypeptide by means of SDS-polyacrylamide gel electrophoresis. Nucleotide sequencing of the finQ region allowed elucidation of the FinQp amino acid sequence and determination of its precise molecular weight as 39,895 Da. Analysis of the predicted amino acid sequence indicated that FinQp is a positively charged protein possessing a helix-turn-helix DNA binding motif. We propose a possible model for the mechanism by which FinQp terminates transcription within the F plasmid tra region. DNA-DNA hybridization established that all FinQ+ R factors examined have an homologous finQ gene.
|
1442. |
Zhao JY,
Zhu YQ,
Li YN,
Mu XD,
You LP,
Xu C,
Qin P,
Ma JL,
( 2015 ) Coexistence of SFO-1 and NDM-1 �]-lactamase genes and fosfomycin resistance gene fosA3 in an Escherichia coli clinical isolate. PMID : 25790496 : DOI : 10.1093/femsle/fnu018 Abstract >>
This study aims to characterize antimicrobial resistance and antimicrobial resistance genetic determinants of an Escherichia coli clinical isolate HD0149 from China in 2012. This strain displayed high-level resistance to cephalosporins, carbapenems, fluoroquinolones, aminoglycosides and fosfomycin. A range of antimicrobial resistance genes was detected responsible for its multiple antimicrobial resistances, involving the blaCMY-2, blaCTX-M-65, blaNDM-1, blaSFO-1, blaTEM-1, fosA3, rmtB, sul1 and sul2 genes. Four amino acid substitutions were detected in the quinolone resistance-determining regions (QRDRs) of GyrA (S83L and D87N), ParC (S80I) and ParE (S458A). Conjugation experiments revealed two multiresistance plasmids present in E. coli HD0149. The blaSFO-1 gene associated with blaNDM-1 gene was located in a 190 kb IncA/C plasmid and the blaCTX-M-65, fosA3 and rmtB genes were located in a 110 kb IncF plasmid. This is the first identification of the blaSFO-1 gene in an E. coli isolate and on a conjugative IncA/C plasmid. This may dramatically enhance the international prevalence and dissemination of blaSFO-1 among Enterobacteriaceae.
|
1443. |
Liu L,
He D,
Lv L,
Liu W,
Chen X,
Zeng Z,
Partridge SR,
Liu JH,
( 2015 ) blaCTX-M-1/9/1 Hybrid Genes May Have Been Generated from blaCTX-M-15 on an IncI2 Plasmid. PMID : 25987615 : DOI : 10.1128/AAC.00501-15 PMC : PMC4505238 Abstract >>
Three hybrid CTX-M �]-lactamases, CTX-M-64, CTX-M-123, and CTX-M-132, with N and C termini matching CTX-M-1 group enzymes and centers matching CTX-M-9 group enzymes, have been identified. The hybrid gene sequences suggested recombination between blaCTX-M-15 and blaCTX-M-14, the two most common blaCTX-M variants worldwide. However, blaCTX-M-64 and blaCTX-M-123 are found in an ISEcp1-blaCTX-M transposition unit with a 45-bp "spacer," rather than the 48 bp usually associated with blaCTX-M-15, and 112 bp of IncA/C plasmid backbone. This is closer to the context of blaCTX-M-55, which has one nucleotide difference from blaCTX-M-15, on IncI2 plasmid pHN1122-1. Here, we characterized an IncI2 plasmid carrying blaCTX-M-15 with a 45-bp spacer (pHNY2-1) by complete sequencing and also sequenced IncI2 plasmids carrying blaCTX-M-64 (pHNAH46-1) or blaCTX-M-132 (pHNLDH19) and an IncI1 plasmid carrying blaCTX-M-123 (pHNAH4-1). pHNY2-1 has the same ISEcp1-blaCTX-M-IncA/C insertion as pHN1122-1, pHNAH46-1, and pHNLDH19, and all four plasmid backbones are almost identical. pHNAH4-1 (IncI1 sequence type 108 [ST108]) carries a transposition unit that includes a 2,720-bp fragment of the IncI2 backbone, suggesting ISEcp1-mediated transfer of blaCTX-M-IncA/C-IncI2 to an IncI1 plasmid. All three hybrid blaCTX-M genes may have resulted from recombination between blaCTX-M-14 and blaCTX-M-15 with a 45-bp spacer on an IncI2 plasmid. Five additional Escherichia coli isolates of different sequence types from different provinces, farms, and/or animals had blaCTX-M-64 on a pHNAH46-1-like IncI2 plasmid and 9 had blaCTX-M-123 on a pHNAH4-1-like IncI1 ST108 plasmid. Thus, epidemic IncI plasmids may be responsible for the spread of blaCTX-M-64 and blaCTX-M-123 between different animals and different locations in China.
|
1444. |
Hall BG,
Parker LL,
Betts PW,
DuBose RF,
Sawyer SA,
Hartl DL,
( 1989 ) IS103, a new insertion element in Escherichia coli: characterization and distribution in natural populations. PMID : 2541046 : PMC : PMC1203630 Abstract >>
IS103 is a previously unknown insertion sequence found in Escherichia coli K12. We have sequenced IS103 and find that it is a 1441-bp element that consists of a 1395-bp core flanked by imperfect 23-bp inverted repeats. IS103 causes a 6-bp duplication of the target sequence into which it inserts. There is a single copy of IS103 present in wild-type E. coli K12 strain HfrC. In strain X342 and its descendents there are two additional copies, one of which is located within the bglF gene. IS103 is capable of excising from within bglF and restoring function of that gene. IS103 exhibits 44% sequence identity with IS3, suggesting that the two insertion sequences are probably derived from a common ancestor. We have examined the distribution of IS103 in the chromosomes and plasmids of the ECOR collection of natural isolates of E. coli. IS103 is found in 36 of the 71 strains examined, and it strongly tends to inhabit plasmids rather than chromosomes. Comparison of the observed distribution of IS103 with distributions predicted by nine different models for the regulation of transposition according to copy number and of the effects of copy number on fitness suggest that transposition of IS103 is strongly regulated and that it has only minor effects on fitness. The strong clustering of IS103 within one phylogenetic subgroup of the E. coli population despite its presence on plasmids suggests that plasmids tend to remain within closely related strains and that transfer to distantly related strains is inhibited.
|
1445. |
Hargreaves ML,
Shaw KM,
Dobbins G,
Snippes Vagnone PM,
Harper JE,
Boxrud D,
Lynfield R,
Aziz M,
Price LB,
Silverstein KA,
Danzeisen JL,
Youmans B,
Case K,
Sreevatsan S,
Johnson TJ,
( 2015 ) Clonal Dissemination of Enterobacter cloacae Harboring blaKPC-3 in the Upper Midwestern United States. PMID : 26438492 : DOI : 10.1128/AAC.01291-15 PMC : PMC4649179 Abstract >>
Carbapenemase-producing, carbapenem-resistant Enterobacteriaceae, or CP-CRE, are an emerging threat to human and animal health, because they are resistant to many of the last-line antimicrobials available for disease treatment. Carbapenemase-producing Enterobacter cloacae harboring blaKPC-3 recently was reported in the upper midwestern United States and implicated in a hospital outbreak in Fargo, North Dakota (L. M. Kiedrowski, D. M. Guerrero, F. Perez, R. A. Viau, L. J. Rojas, M. F. Mojica, S. D. Rudin, A. M. Hujer, S. H. Marshall, and R. A. Bonomo, Emerg Infect Dis 20:1583-1585, 2014, http://dx.doi.org/10.3201/eid2009.140344). In early 2009, the Minnesota Department of Health began collecting and screening CP-CRE from patients throughout Minnesota. Here, we analyzed a retrospective group of CP-E. cloacae isolates (n = 34) collected between 2009 and 2013. Whole-genome sequencing and analysis revealed that 32 of the strains were clonal, belonging to the ST171 clonal complex and differing collectively by 211 single-nucleotide polymorphisms, and it revealed a dynamic clone under positive selection. The phylogeography of these strains suggests that this clone existed in eastern North Dakota and western Minnesota prior to 2009 and subsequently was identified in the Minneapolis and St. Paul metropolitan area. All strains harbored identical IncFIA-like plasmids conferring a CP-CRE phenotype and an additional IncX3 plasmid. In a single patient with multiple isolates submitted over several months, we found evidence that these plasmids had transferred from the E. cloacae clone to an Escherichia coli ST131 bacterium, rendering it as a CP-CRE. The spread of this clone throughout the upper midwestern United States is unprecedented for E. cloacae and highlights the importance of continued surveillance to identify such threats to human health.
|
1446. |
Sato S,
Nakada Y,
Shiratsuchi A,
( 1989 ) IS421, a new insertion sequence in Escherichia coli. PMID : 2542093 : DOI : 10.1016/0014-5793(89)80007-9 Abstract >>
The nucleotide sequence of a new insertion sequence (IS) in Escherichia coli, IS421, was determined. It is 1340 bp long and contains inverted repeats of 22 bp at its termini. It is flanked by 13 bp direct repeats apparently generated upon insertion. There are two ORFs longer than 200 bp in IS421. One can encode a polypeptide of 371 amino acids (aa) and the other, which is on the other strand, can encode a polypeptide of 102 aa. The C-terminal part of the 371 aa polypeptide shows some homology to that of transposases encoded in some other known IS elements. The copy number of IS421 in chromosomal DNA was 4 for E. coli K-12 and B, and 5 for E. coli C, as determined by the Southern hybridization of restriction fragments.
|
1447. |
Delport TC,
Harcourt RG,
Beaumont LJ,
Webster KN,
Power ML,
( 2015 ) MOLECULAR DETECTION OF ANTIBIOTIC-RESISTANCE DETERMINANTS IN ESCHERICHIA COLI ISOLATED FROM THE ENDANGERED AUSTRALIAN SEA LION (NEOPHOCA CINEREA). PMID : 25919463 : DOI : 10.7589/2014-08-200 Abstract >>
Greater interaction between humans and wildlife populations poses significant risks of anthropogenic impact to natural ecosystems, especially in the marine environment. Understanding the spread of microorganisms at the marine interface is therefore important if we are to mitigate adverse effects on marine wildlife. We investigated the establishment of Escherichia coli in the endangered Australian sea lion (Neophoca cinerea) by comparing fecal isolation from wild and captive sea lion populations. Fecal samples were collected from wild colonies March 2009-September 2010 and from captive individuals March 2011-May 2013. Using molecular screening, we assigned a phylotype to E. coli isolates and determined the presence of integrons, mobile genetic elements that capture gene cassettes conferring resistance to antimicrobial agents common in fecal coliforms. Group B2 was the most abundant phylotype in all E. coli isolates (n = 37), with groups A, B1, and D also identified. Integrons were not observed in E. coli (n = 21) isolated from wild sea lions, but were identified in E. coli from captive animals (n = 16), from which class I integrases were detected in eight isolates. Sequencing of gene cassette arrays identified genes conferring resistance to streptomycin-spectinomycin (aadA1) and trimethoprim (dfrA17, dfrB4). Class II integrases were not detected in the E. coli isolates. The frequent detection in captive sea lions of E. coli with resistance genes commonly identified in human clinical cases suggests that conditions experienced in captivity may contribute to establishment. Identification of antibiotic resistance in the microbiota of Australian sea lions provides crucial information for disease management. Our data will inform conservation management strategies and provide a mechanism to monitor microorganism dissemination to sensitive pinniped populations.
|
1448. |
Chavda KD,
Chen L,
Jacobs MR,
Rojtman AD,
Bonomo RA,
Kreiswirth BN,
( 2015 ) Complete sequence of a bla(KPC)-harboring cointegrate plasmid isolated from Escherichia coli. PMID : 25753632 : DOI : 10.1128/AAC.00041-15 PMC : PMC4394832 Abstract >>
Horizontal transfer of bla(KPC)-harboring plasmids contributes significantly to the inter- and intraspecies spread of Klebsiella pneumoniae carbapenemase (KPC). Here we report the complete nucleotide sequence of a bla(KPC)-harboring IncFIA plasmid, pBK32533, from Escherichia coli. pBK32533 is a cointegrate plasmid comprising of a 72-kb sequence identical to that of the nonconjugative pBK30661 plasmid plus an additional 170-kb element that harbors the genes for plasmid transfer. pBK32533 demonstrates how bla(KPC) can be spread from a nonconjugative plasmid through cointegration.
|
1449. |
Sheng JF,
Doi Y,
Li JJ,
Spychala CN,
Hu F,
( 2015 ) Complete nucleotide sequences of bla(CTX-M)-harboring IncF plasmids from community-associated Escherichia coli strains in the United States. PMID : 25753630 : DOI : 10.1128/AAC.04772-14 PMC : PMC4432217 Abstract >>
Community-associated infections due to Escherichia coli producing CTX-M-type extended-spectrum �]-lactamases are increasingly recognized in the United States. The bla(CTX-M) genes are frequently carried on IncF group plasmids. In this study, bla(CTX-M-15)-harboring plasmids pCA14 (sequence type 131 [ST131]) and pCA28 (ST44) and bla(CTX-M-14)-harboring plasmid pCA08 (ST131) were sequenced and characterized. The three plasmids were closely related to other IncFII plasmids from continents outside the United States in the conserved backbone region and multiresistance regions (MRRs). Each of the bla(CTX-M-15)-carrying plasmids pCA14 and pCA28 belonged to F31:A4:B1 (FAB [FII, FIA, FIB] formula) and showed a high level of similarity (92% coverage of pCA14 and 99% to 100% nucleotide identity), suggesting a possible common origin. The blaC(TX-M-14)-carrying plasmid pCA08 belonged to F2:A2:B20 and was highly similar to pKF3-140 from China (88% coverage of pCA08 and 99% to 100% nucleotide identity). All three plasmids carried multiple antimicrobial resistance genes and modules associated with virulence and biochemical pathways, which likely confer selective advantages for their host strains. The bla(CTX-M)-carrying IncFII-IA-IB plasmids implicated in community-associated infections in the United States shared key structural features with those identified from other continents, underscoring the global nature of this plasmid epidemic.
|
1450. |
Kotsakis SD,
Miriagou V,
Vetouli EE,
Bozavoutoglou E,
Lebessi E,
Tzelepi E,
Tzouvelekis LS,
( 2015 ) Increased Hydrolysis of Oximino-�]-Lactams by CMY-107, a Tyr199Cys Mutant Form of CMY-2 Produced by Escherichia coli. PMID : 26438499 : DOI : 10.1128/AAC.01793-15 PMC : PMC4649145 Abstract >>
The cephalosporinase CMY-107, a Tyr199Cys mutant form of CMY-2 encoded by an IncI self-transferable plasmid carried by an Escherichia coli clinical strain, was characterized. The enzyme hydrolyzed oximino-cephalosporins and aztreonam more efficiently than CMY-2 did.
|
1451. |
Schulz EC,
Barabas O,
( 2014 ) Structure of an Escherichia coli Hfq:RNA complex at 0.97 ? resolution. PMID : 25372815 : DOI : 10.1107/S2053230X14020044 PMC : PMC4231850 Abstract >>
In bacteria, small RNAs (sRNAs) silence or activate target genes through base pairing with the mRNA, thereby modulating its translation. A central player in this process is the RNA chaperone Hfq, which facilitates the annealing of sRNAs with their target mRNAs. Hfq has two RNA-binding surfaces that recognize A-rich and U-rich sequences, and is believed to bind an sRNA-mRNA pair simultaneously. However, how Hfq promotes annealing remains unclear. Here, the crystal structure of Escherichia coli Hfq is presented in complex with U6-RNA bound to its proximal binding site at 0.97 ? resolution, revealing the Hfq-RNA interaction in exceptional detail.
|
1452. |
Fürste JP,
Pansegrau W,
Ziegelin G,
Kröger M,
Lanka E,
( 1989 ) Conjugative transfer of promiscuous IncP plasmids: interaction of plasmid-encoded products with the transfer origin. PMID : 2538813 : DOI : 10.1073/pnas.86.6.1771 PMC : PMC286786 Abstract >>
To characterize protein-DNA interactions involved in the initiation of conjugative transfer replication, we isolated and sequenced the transfer origins (oriT) of the promiscuous IncP plasmids RP4 and R751. The central initiating event at the transfer origin of a conjugative plasmid is the cleavage at a unique site (nic) of the strand to be transferred to a recipient cell. This process can be triggered after the assembly of "relaxosomes" (plasmid DNA-protein relaxation complexes), requiring plasmid-encoded gene products. We analyzed the nicking reaction for plasmid RP4 and demonstrated that one of the plasmid strands is specifically cleaved within oriT. The fully functional oriT of RP4 represents an intergenic DNA region of approximately 350 base pairs. Dissection of oriT revealed that a portion carrying nic and symmetric sequence repeats determines oriT specificity. This part of oriT is contiguous to a region that is essential for efficient mobilization of oriT plasmids. In addition, oriT contains potential promoter sites allowing divergent transcription of two operons flanking oriT. We over-produced gene products and, from analyzing the products of defined deletion mutants, deduced the gene arrangements. Formation of RP4 relaxosomes is likely to depend on the presence of at least two plasmid-encoded components, which act in trans. Corresponding genes map on one side of oriT. Purification of the traJ product revealed it to be an 11-kDa polypeptide that binds to oriT DNA in vitro. The protein recognizes the part of oriT that is responsible for oriT specificity.
|
1453. |
Cowan GM,
Gann AA,
Murray NE,
( 1989 ) Conservation of complex DNA recognition domains between families of restriction enzymes. PMID : 2642743 : DOI : 10.1016/0092-8674(89)90988-4 Abstract >>
One polypeptide, designated S, confers sequence-specificity to the multisubunit type I restriction enzymes. Two families of such enzymes, K and A, include members that recognize diverse, bipartite, target sequences. The S polypeptides of the K family, while having areas of near identity, also contain two extensive regions of variable sequence. We now show that one of these, comprising the N-terminal 150 amino acids, specifies recognition of one component of the bipartite target sequence. We have determined the sequence recognized by EcoE, a member of the A family. This sequence, 5'GAG(N7)ATGC, has the trinucleotide GAG in common with EcoA and with StySB of the K family. We determined the nucleotide sequences of the S genes of EcoA and EcoE, and compared their predicted amino acid sequences with each other and with those of the five members of the K family. There is no general sequence similarity between families, but the domain of the S polypeptide of StySB, which specifies GAG, shows nearly 50 per cent identity with the amino variable region of the S polypeptides of EcoA and EcoE. A complex domain that recognizes and directs methylation of GAG is therefore common to enzymes of generally dissimilar amino acid sequence.
|
1454. |
Jalajakumari MB,
Thomas CJ,
Halter R,
Manning PA,
( 1989 ) Genes for biosynthesis and assembly of CS3 pili of CFA/II enterotoxigenic Escherichia coli: novel regulation of pilus production by bypassing an amber codon. PMID : 2576094 : DOI : 10.1111/j.1365-2958.1989.tb00154.x Abstract >>
The complete nucleotide sequence of the 4746bp HindIII fragment encoding the genes for the biosynthesis and assembly of CS3 pili has been determined. By site-directed mutagenesis in conjunction with analysis of the plasmid-encoded proteins in minicells, the actual reading frames for the various products have been determined. This demonstrated that the genes for four of the proteins (63 kD, 48 kD, 33 kD, and 20 kD in size) are encoded entirely within the same open reading frame as a fifth protein (104 kD). However, for synthesis of this latter protein, suppression or readthrough of an internal amber codon is required. Termination at this codon is also necessary for synthesis of the former proteins. Two further proteins are also encoded within the HindIII fragment: a 27 kD precursor of a periplasmic protein and the 17.5kD precursor of the major CS3 fimbrial subunit.
|
1455. |
Hall RH,
Maneval DR,
Collins JH,
Theibert JL,
Levine MM,
( 1989 ) Purification and analysis of colonization factor antigen I, coli surface antigen 1, and coli surface antigen 3 fimbriae from enterotoxigenic Escherichia coli. PMID : 2572583 : DOI : 10.1128/jb.171.11.6372-6374.1989 PMC : PMC210515 Abstract >>
Enterotoxigenic Escherichia coli fimbriae are immunogenic and play a key role in intestinal colonization. Native colonization factor antigen I, coli surface antigen 1, and coli surface antigen 3 fimbriae were purified by a common method involving shearing, differential centrifugation, gel filtration, and density gradient ultracentrifugation. The compositions and N-terminal sequences were determined. Coli surface antigen 3 possesses two N-terminal isoforms, one of which matches the published DNA sequence, except for the previously proposed signal sequence cleavage point.
|
1456. |
Lindberg F,
Tennent JM,
Hultgren SJ,
Lund B,
Normark S,
( 1989 ) PapD, a periplasmic transport protein in P-pilus biogenesis. PMID : 2572580 : DOI : 10.1128/jb.171.11.6052-6058.1989 PMC : PMC210471 Abstract >>
The product of the papD gene of uropathogenic Escherichia coli is required for the biogenesis of digalactoside-binding P pili. Mutations within papD result in complete degradation of the major pilus subunit, PapA, and of the pilinlike proteins PapE and PapF and also cause partial breakdown of the PapG adhesin. The papD gene was sequenced, and the gene product was purified from the periplasm. The deduced amino acid sequence and the N-terminal sequence obtained from the purified protein revealed that PapD is a basic and hydrophilic peripheral protein. A periplasmic complex between PapD and PapE was purified from cells that overproduced and accumulated these proteins in the periplasm. Antibodies raised against this complex reacted with purified wild-type P pili but not with pili purified from a papE mutant. In contrast, anti-PapD serum did not react with purified pili or with the culture fluid of piliated cells. However, this serum was able to specifically precipitate the PapE protein from periplasmic extracts, confirming that PapD and PapE were associated as a complex. It is suggested that PapD functions in P-pilus biogenesis as a periplasmic transport protein. Probably PapD forms complexes with pilus subunits at the outer surface of the inner membrane and transports them in a stable configuration across the periplasmic space before delivering them to the site(s) of pilus polymerization.
|
1457. |
Kotsakis SD,
Tzouvelekis LS,
Lebessi E,
Doudoulakakis A,
Bouli T,
Tzelepi E,
Miriagou V,
( 2015 ) Characterization of a mobilizable IncQ plasmid encoding cephalosporinase CMY-4 in Escherichia coli. PMID : 25691650 : DOI : 10.1128/AAC.05017-14 PMC : PMC4394814 Abstract >>
N/A
|
1458. |
Zhang WJ,
Wang XM,
Dai L,
Hua X,
Dong Z,
Schwarz S,
Liu S,
( 2015 ) Novel conjugative plasmid from Escherichia coli of swine origin that coharbors the multiresistance gene cfr and the extended-spectrum-�]-lactamase gene blaCTX-M-14b. PMID : 25421479 : DOI : 10.1128/AAC.04631-14 PMC : PMC4335840 Abstract >>
Two porcine Escherichia coli isolates harbored the cfr gene on conjugative plasmids of 38,405 bp (pGXEC6) and 41,646 bp (pGXEC3). In these two plasmids, the cfr gene was located within a 4,612-bp region containing a tnpA-IS26-cfr-IS26-�Ghyp element. Plasmid pGXEC3 was almost identical to pGXEC6 except for a 3,235-bp ISEcp1-blaCTX-M-14b insertion. The colocation of the multiresistance cfr gene with an extended-spectrum-�]-lactamase gene on a conjugative plasmid may support the dissemination of these genes by coselection.
|
1459. |
Bilge SS,
Clausen CR,
Lau W,
Moseley SL,
( 1989 ) Molecular characterization of a fimbrial adhesin, F1845, mediating diffuse adherence of diarrhea-associated Escherichia coli to HEp-2 cells. PMID : 2568985 : DOI : 10.1128/jb.171.8.4281-4289.1989 PMC : PMC210202 Abstract >>
A fimbrial adhesin, designated F1845, was found to be responsible for the diffuse HEp-2 cell adherence of a diarrheal Escherichia coli isolate. The genetic determinant of F1845 was cloned, and the order of the genes necessary for production of F1845 was determined by maxicell analysis. Five polypeptides with apparent sizes of 10, 95, 27, 15.5, and 14.3 kilodaltons (kDa) were found to be encoded in that order by the F1845 determinant. The nucleotide sequence of the 14.3-kDa subunit gene was determined and found to share extensive homology in its signal sequence with the gene encoding the structural subunit of the AFA-1 hemagglutinin of a uropathogenic E. coli strain (A. Labigne-Roussel, M.A. Schmidt, W. Walz, and S. Falkow, J. Bacteriol. 162:1285-1292, 1985) but not in the region encoding the mature protein. Southern blot hybridizations indicated that the F1845 determinants are of chromosomal origin. Hybridization studies using a probe from the region encoding the 95-kDa polypeptide indicated that related sequences may be plasmid associated in some strains and chromosomal in others. Additional hybridization studies of E. coli isolates possessing sequence homology to the F1845 determinant suggest that the sequences in the 5' region of the F1845 structural subunit gene are more highly conserved than sequences in the 3' region.
|
1460. |
Tseng SP,
Wang SF,
Kuo CY,
Huang JW,
Hung WC,
Ke GM,
Lu PL,
( 2015 ) Characterization of Fosfomycin Resistant Extended-Spectrum �]-Lactamase-Producing Escherichia coli Isolates from Human and Pig in Taiwan. PMID : 26280832 : DOI : 10.1371/journal.pone.0135864 PMC : PMC4539220 Abstract >>
To investigate the efficacy of fosfomycin against extended-spectrum �]-lactamases (ESBL) producing Escherichia coli in Taiwan and the resistance mechanisms and characterization of human and pig isolates, we analyzed 145 ESBL-producing isolates collected from two hospitals (n = 123) and five farms (n = 22) in Taiwan from February to May, 2013. Antimicrobial susceptibilities were determined. Clonal relatedness was determined by PFGE and multi-locus sequence typing. ESBLs, ampC, and fosfomycin resistant genes were detected by PCR, and their flanking regions were determined by PCR mapping and sequencing. The fosfomycin resistant mechanisms, including modification of the antibiotic target (MurA), functionless transporters (GlpT and UhpT) and their regulating genes such as uhpA, cyaA, and ptsI, and antibiotic inactivation by enzymes (FosA and FosC), were examined. The size and replicon type of plasmids carrying fosfomycin resistant genes were analyzed. Our results revealed the susceptibility rates of fosfomycin were 94% for human ESBL-producing E. coli isolates and 77% for pig isolates. The PFGE analysis revealed 79 pulsotypes. No pulsotype was found existing in both human and pig isolates. Three pulsotypes were distributed among isolates from two hospitals. ISEcp1 carrying blaCTX-M-group 9 was the predominant transposable elements of the ESBL genes. Among the thirteen fosfomycin resistant isolates, functionless transporters were identified in 9 isolates. Three isolates contained novel amino acid substitutions (Asn67Ile, Phe151Ser and Trp164Ser, Val146Ala and His159Tyr, respectively) in MurA (the target of fosfomycin). Four isolates had fosfomycin modified enzyme (fosA3) in their plasmids. The fosA3 gene was harboured in an IncN-type plasmid (101 kbp) in the three pig isolates and an IncB/O-type plasmid (113 kbp) in the human isolate. In conclusion, we identified that 6% and 23% of the ESBL-producing E. coli from human and pigs were resistant to fosfomycin, respectively, in Taiwan. No clonal spread was found between human and pig isolates. Functionless transporters were the major cause of fosfomycin resistance, and the fosA3-transferring plasmid between isolates warrants further monitoring.
|
1461. |
Chiyo PI,
Grieneisen LE,
Wittemyer G,
Moss CJ,
Lee PC,
Douglas-Hamilton I,
Archie EA,
( 2014 ) The influence of social structure, habitat, and host traits on the transmission of Escherichia coli in wild elephants. PMID : 24705319 : DOI : 10.1371/journal.pone.0093408 PMC : PMC3976290 Abstract >>
Social structure is proposed to influence the transmission of both directly and environmentally transmitted infectious agents. However in natural populations, many other factors also influence transmission, including variation in individual susceptibility and aspects of the environment that promote or inhibit exposure to infection. We used a population genetic approach to investigate the effects of social structure, environment, and host traits on the transmission of Escherichia coli infecting two populations of wild elephants: one in Amboseli National Park and another in Samburu National Reserve, Kenya. If E. coli transmission is strongly influenced by elephant social structure, E. coli infecting elephants from the same social group should be genetically more similar than E. coli sampled from members of different social groups. However, we found no support for this prediction. Instead, E. coli was panmictic across social groups, and transmission patterns were largely dominated by habitat and host traits. For instance, habitat overlap between elephant social groups predicted E. coli genetic similarity, but only in the relatively drier habitat of Samburu, and not in Amboseli, where the habitat contains large, permanent swamps. In terms of host traits, adult males were infected with more diverse haplotypes, and males were slightly more likely to harbor strains with higher pathogenic potential, as compared to adult females. In addition, elephants from similar birth cohorts were infected with genetically more similar E. coli than elephants more disparate in age. This age-structured transmission may be driven by temporal shifts in genetic structure of E. coli in the environment and the effects of age on bacterial colonization. Together, our results support the idea that, in elephants, social structure often will not exhibit strong effects on the transmission of generalist, fecal-oral transmitted bacteria. We discuss our results in the context of social, environmental, and host-related factors that influence transmission patterns.
|
1462. |
Zong Z,
Ginn AN,
Dobiasova H,
Iredell JR,
Partridge SR,
( 2015 ) Different IncI1 plasmids from Escherichia coli carry ISEcp1-blaCTX-M-15 associated with different Tn2-derived elements. PMID : 25929173 : DOI : 10.1016/j.plasmid.2015.04.007 Abstract >>
The bla(CTX-M-15) gene, encoding the globally dominant CTX-M-15 extended-spectrum �]-lactamase, has generally been found in a 2.971-kb ISEcp1-bla(CTX-M-15)-orf477�G transposition unit, with ISEcp1 providing a promoter. In available IncF plasmid sequences from Escherichia coli, this transposition unit interrupts a truncated copy of transposon Tn2 that lies within larger multiresistance regions. In E. coli, bla(CTX-M-15) is also commonly associated with IncI1 plasmids and here three such plasmids from E. coli clinical isolates from western Sydney 2006-2007 have been sequenced. The plasmid backbones are organised similarly to those of other IncI1 plasmids, but have insertions and/or deletions and sequence differences. Each plasmid also has a different insertion carrying bla(CTX-M-15). pJIE113 (IncI1 sequence type ST31) is almost identical to plasmids isolated from the 2011 E. coli O104:H4 outbreak in Europe, where the typical bla(CTX-M-15) transposition unit interrupts a complete Tn2 inserted directly in the plasmid backbone. In the novel plasmid pJIE139 (ST88), ISEcp1-blaC(TX-M-15)-orf477�G lies within a Tn2/3 hybrid transposon. Homologous recombination could explain movement of ISEcp1-bla(CTX-M-15)-orf477�G between copies of Tn2 on IncF and IncI1 plasmids and generation of the Tn2/3 hybrid. pJIE174 (ST37) is almost identical to pESBL-12 from the Netherlands and in these plasmids bla(CTX-M-15) is flanked by two copies of IS26 that truncate the transposition unit within a larger region bounded by the ends of Tn2. bla(CTX-M-15) and the associated ISEcp1-derived promoter may be able to move from this structure by the actions of IS26, independently of both ISEcp1 and Tn2.
|
1463. |
Hamers AM,
Pel HJ,
Willshaw GA,
Kusters JG,
van der Zeijst BA,
Gaastra W,
( 1989 ) The nucleotide sequence of the first two genes of the CFA/I fimbrial operon of human enterotoxigenic Escherichia coli. PMID : 2569152 : Abstract >>
An oligonucleotide probe, derived from the N-terminal amino acid sequence of the CFA/I fimbrial subunit protein, was used to identify the gene encoding this protein within a cloned DNA fragment encoding CFA/I fimbriae. The gene (cfa b) was found and sequenced. Flanking it upstream was a gene (cfa a) encoding a protein of 206 amino acids and downstream a gene (cfa c) probably encoding an 85 kDa protein was found. This genetic organisation of the CFA/I operon differs from that of other fimbrial operons in Escherichia coli. All three proteins have signal peptides. The nucleotide sequence was analysed for homology with other sequences, secondary structure, ribosomal binding sites and possible promoter sequences. A region of dyad symmetry probably involved in the regulation of translation of the cfa c gene was found at the 5' end of this gene. A region of dyad symmetry was also observed within the cfa b gene. In front of the CFA/I operon part of insertion sequence IS2 was found. This IS2 sequence was found in a number of CFA/I plasmids, obtained from strains isolated from various geographic locations. The insertion of the IS2 element in the CFA/I operon therefore probably happened rather early during evolution of CFA/I producing Escherichia coli strains.
|
1464. |
Khan Z,
Nisar MA,
Hussain SZ,
Arshad MN,
Rehman A,
( 2015 ) Cadmium resistance mechanism in Escherichia coli P4 and its potential use to bioremediate environmental cadmium. PMID : 26278537 : DOI : 10.1007/s00253-015-6901-x Abstract >>
A cadmium-resistant bacterium was isolated from industrial wastewater and identified as Escherichia coli (dubbed as P4) on the basis of morphological, biochemical tests and 16S rRNA ribotyping. It showed optimum growth at 30 �XC and pH 7. E. coli P4 found to resist Cd(+2) (10.6 mM) as well as Zn(+2) (4.4 mM), Pb(+2) (17 mM), Cu(+2) (3.5 mM), Cr(+6) (4.4 mM), As(+2) (10.6 mM), and Hg(+2) (0.53 mM). It could remove 18.8, 37, and 56 % Cd(+2) from aqueous medium after 48, 96, and 144 h, respectively. Fourier transform infrared spectroscopy (FTIR), scanning electron microscope (SEM), and Energy-dispersive X-ray (EDX) analysis also confirmed the biosorption of Cd(+2) by E. coli P4. However, temperature and pH were found to be the most critical factors in biosorption of Cd(+2) by E. coli P4. Cd(+2) stress altered E. coli P4 cell physiology analyzed by measuring glutathione (GSH) and non-protein thiol (cysteine) levels which were increased up to 130 and 48 %, respectively. Quantitative real-time polymerase chain reaction (qRT-PCR) showed alteration in the expression levels of ftsZ, mutS, clpB, ef-tu, and dnaK genes in the presence of Cd(+2). Total protein profiles of E. coli P4 in the absence and presence of Cd(+2) were compared by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), which showed remarkable difference in the banding pattern. czcB gene, a component of czcCBA operon, was amplified from genomic DNA which suggested the chromosomal-borne Cd(+2) resistance in E. coli P4. Furthermore, it harbors smtAB gene which plays a significant role in Cd(+2) resistance.
|
1465. |
Ewers C,
Dematheis F,
Singamaneni HD,
Nandanwar N,
Fruth A,
Diehl I,
Semmler T,
Wieler LH,
( 2014 ) Correlation between the genomic o454-nlpD region polymorphisms, virulence gene equipment and phylogenetic group of extraintestinal Escherichia coli (ExPEC) enables pathotyping irrespective of host, disease and source of isolation. PMID : 25349632 : DOI : 10.1186/s13099-014-0037-x PMC : PMC4209514 Abstract >>
The mutS-rpoS intergenic region in E. coli displays a mosaic structure which revealed pathotype specific patterns. To assess the importance of this region as a surrogate marker for the identification of highly virulent extraintestinal pathogenic E. coli (ExPEC) strains we aimed to: (i) characterize the genetic diversity of the mutS gene and the o454-nlpD genomic region among 510 E. coli strains from animals and humans; (ii) delineate associations between the polymorphism of this region and features such as phylogenetic background of E. coli, pathotype, host species, clinical condition, serogroup and virulence associated genes (VAG)s; and (iii) identify the most important VAGs for classification of the o454-nlpD region. Size variation in the o454-nlpD region was investigated by PCR amplification and sequencing. Phylogenetic relationships were assessed by Ecor- and Multilocus sequence- typing (MLST), and a comparative analysis between mutS gene phylogenetic tree obtained with RAxML and the MLST grouping method was performed. Correlation between o454-nlpD patterns and the features described above were analysed. In addition, the importance of 47 PCR-amplified ExPEC-related VAGs for classification of o454-nlpD patterns was investigated by means of Random Forest algorithm. Four main structures (patterns I-IV) of the o454-nlpD region among ExPEC and commensal E. coli strains were identified. Statistical analysis showed a positive and exclusive association between pattern III and the ExPEC strains. A strong association between pattern III and either the Ecor group B2 or the sequence type complexes known to represent the phylogenetic background of highly virulent ExPEC strains (such as STC95, STC73 and STC131) was found as well. RF analyses determined five genes (csgA, malX, chuA, sit, and vat) to be suitable to predict pattern III strains. The significant association between pattern III and group B2 strains suggested the o454-nlpD region to be of great value in identifying highly virulent strains among the mixed population of E. coli promising to be the basis of a future typing tool for ExPEC and their gut reservoir. Furthermore, top-ranked VAGs for classification and prediction of pattern III were identified. These data are most valuable for defining ExPEC pathotype in future in vivo assays.
|
1466. |
Heuzenroeder MW,
Neal BL,
Thomas CJ,
Halter R,
Manning PA,
( 1989 ) Characterization and molecular cloning of the PCF8775 CS5 antigen from an enterotoxigenic Escherichia coli 0115:H40 isolated in Central Australia. PMID : 2568574 : DOI : 10.1111/j.1365-2958.1989.tb00175.x Abstract >>
The genes determining the biosynthesis of the colonization factor CS5 have been cloned from Escherichia coli 0115:H40:PCF8775 isolated during an outbreak of diarrhoea among aboriginal children in Central Australia. Electron microscopy has shown purified CS5 to be of semi-rigid fimbrial type. NH2-terminal analysis has shown the CS5 determinant to be distinct from other fimbriae, although there is some conservation of certain residues. Expression in minicells of the cloned fimbrial genes encoded on pPM1312 has shown that proteins of 70 and 46.5 kD which co-purity with the 23 kD major fimbrial subunit protein are also co-expressed along with proteins of 45, 31, 17 and 14 kD. The major CS5 subunit is synthesized in precursor form (approximately 26 kD). A synthetic oligonucleotide to the NH2-terminal amino acid coding sequence of the purified protein has been used in Southern hybridization analyses to define the region on pPM1312 encoding the structural gene for the major pilin subunit.
|
1467. |
Wang L,
Wang W,
Li F,
Zhang J,
Wu J,
Gong Q,
Shi Y,
( 2015 ) Structural insights into the recognition of the internal A-rich linker from OxyS sRNA by Escherichia coli Hfq. PMID : 25670676 : DOI : 10.1093/nar/gkv072 PMC : PMC4344510 Abstract >>
Small RNA OxyS is induced during oxidative stress in Escherichia coli and it is an Hfq-dependent negative regulator of mRNA translation. OxyS represses the translation of fhlA and rpoS mRNA, which encode the transcriptional activator and �m(s) subunit of RNA polymerase, respectively. However, little is known regarding how Hfq, an RNA chaperone, interacts with OxyS at the atomic level. Here, using fluorescence polarization and tryptophan fluorescence quenching assays, we verified that the A-rich linker region of OxyS sRNA binds Hfq at its distal side. We also report two crystal structures of Hfq in complex with A-rich RNA fragments from this linker region. Both of these RNA fragments bind to the distal side of Hfq and adopt a different conformation compared with those previously reported for the (A-R-N)n tripartite recognition motif. Furthermore, using fluorescence polarization, electrophoresis mobility shift assays and in vivo translation assays, we found that an Hfq mutant, N48A, increases the binding affinity of OxyS for Hfq in vitro but is defective in the negative regulation of fhlA translation in vivo, suggesting that the normal function of OxyS depends on the details of the interaction with Hfq that may be related to the rapid recycling of Hfq in the cell.
|
1468. |
Elkins MF,
Earhart CF,
( 1989 ) Nucleotide sequence and regulation of the Escherichia coli gene for ferrienterobactin transport protein FepB. PMID : 2529253 : DOI : 10.1128/jb.171.10.5443-5451.1989 PMC : PMC210382 Abstract >>
The Escherichia coli fepB gene encodes a periplasmic protein required for ferrienterobactin transport; four fepB-related polypeptides are resolved by standard sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In vitro DNA-directed protein-synthesizing systems and experiments with the inhibitors dinitrophenol, carbonyl cyanide m-chlorophenylhydrazone, and ethanol demonstrated that the initial fepB translation product is processed. The nucleotide sequence of fepB and neighboring regions was determined. The predicted proFepB has a molecular weight of 34,255, consists of 318 amino acids, and is devoid of cysteine residues. A leader peptide is present, as are three possible leader peptidase cleavage sites after positions 22, 23, and 26. The upstream regulatory region included a Fur box, indicating that fepB is iron regulated, which was verified by RNA dot blot experiments. The regulatory region included a 68-amino-acid open reading frame (ORF) that encompassed a sequence capable of forming a large stem-and-loop structure. Indirect evidence indicated that this ORF must be translated for fepB transcription to occur. Six copies of the nonomer CCCTC(A/T)CCC or its invert were present in the stem-and-loop region. An ORF of unknown significance was found downstream from fepB; its product would have a molecular weight of 18,036 and be rich in proline and alanine. Processing of proFepB remains unclear, but the appearance of the three smaller members of the FepB family required the action of leader peptidase and the presence of the entire fepB gene.
|
1469. |
Sumrall ET,
Gallo EB,
Aboderin AO,
Lamikanra A,
Okeke IN,
( 2014 ) Dissemination of the transmissible quinolone-resistance gene qnrS1 by IncX plasmids in Nigeria. PMID : 25340787 : DOI : 10.1371/journal.pone.0110279 PMC : PMC4207749 Abstract >>
The plasmid-encoded quinolone resistance gene qnrS1 was recently found to be commonly associated with ciprofloxacin resistance in Nigeria. We mapped the qnrS1 gene from an Escherichia coli isolate obtained in Nigeria to a 43.5 Kb IncX2 plasmid. The plasmid, pEBG1, was sufficient to confer ciprofloxacin non-susceptibility, as well as tetracycline and trimethoprim resistance, on E. coli K-12. Deletion analysis confirmed that qnrS1 accounted for all the ciprofloxacin non-suceptibility conferred by pEBG1 and tetracycline and trimethoprim resistance could be attributed to tetAR and dfrA14 genes respectively. While it contained a complete IncX conjugation system, pEBG1 was not self-transmissible likely due to an IS3 element inserted between the pilX5 and pilX6 genes. The plasmid was however efficiently mobilizable. pEBG1 was most similar to another qnrS1-bearing IncX2 plasmid from Nigeria, but both plasmids acquired qnrS1 independently and differ in their content of other resistance genes. Screening qnrS1-positive isolates from other individuals in Nigeria revealed that they carried neither pEBG1 nor pNGX2-QnrS1 but that IncX plasmids were prevalent. This study demonstrates that the IncX backbone is a flexible platform that has contributed to qnrS1 dissemination in Nigeria.
|
1470. |
Vingopoulou EI,
Siarkou VI,
Batzias G,
Kaltsogianni F,
Sianou E,
Tzavaras I,
Koutinas A,
Saridomichelakis MN,
Sofianou D,
Tzelepi E,
Miriagou V,
( 2014 ) Emergence and maintenance of multidrug-resistant Escherichia coli of canine origin harbouring a blaCMY-2-IncI1/ST65 plasmid and topoisomerase mutations. PMID : 24722836 : DOI : 10.1093/jac/dku090 Abstract >>
To characterize the mechanisms implicated in fluoroquinolone (FQ) and expanded-spectrum cephalosporin (ESC) resistance in three clinical and seven faecal multidrug-resistant (MDR; resistant to at least three antimicrobial classes) Escherichia coli isolates from a dog with atopic dermatitis, also suffering from recurrent otitis, that had already been exposed to prolonged antimicrobial treatment and colonized for a long period. MICs of FQs, ESCs and other antimicrobials were determined by the broth microdilution method. Phenotypic tests (efflux pump inhibition and combination disc tests) and isoelectric focusing were combined with genotypic analyses [PCRs, sequencing, conjugation, S1 nuclease PFGE, PCR-based replicon typing, plasmid multilocus sequence typing (pMLST) and PCR mapping] to characterize the molecular basis of FQ and ESC resistance. Isolates were further characterized by MLST and PFGE. Three otitis and five faecal isolates with enrofloxacin MICs of 32 to >128 mg/L displayed the GyrA:S83L+D87N/ParC:E62K/ParE:G545D pattern harbouring novel ParC and ParE substitutions, whereas the two remaining faecal isolates were susceptible or borderline resistant single-step mutants (GyrA:S83L pattern) and carried qnrS1. Efflux pump overexpression also contributed to FQ resistance and the MDR phenotype. The three otitis and five faecal isolates also exhibited cefoxitin/ceftazidime MICs of 32-64 mg/L and harboured blaCMY-2, adjusted to ISEcp1, on an IncI1/ST65 conjugative plasmid, previously described in Salmonella Heidelberg from poultry. Interestingly, all isolates shared an identical MLST type (ST212), with the otitis isolates showing indistinguishable patterns with the high-level resistant faecal E. coli isolates. The long-term maintenance of FQ- and ESC-resistant clones harbouring topoisomerase mutations and a blaCMY-2-IncI1/ST65 plasmid in canine commensal flora after prolonged antimicrobial use may contribute to the dissemination of multidrug resistance.
|
1471. |
Thiry G,
Clippe A,
Scarcez T,
Petre J,
( 1989 ) Cloning of DNA sequences encoding foreign peptides and their expression in the K88 pili. PMID : 2471451 : PMC : PMC184235 Abstract >>
A genetic system that allows the cloning of a peptide-coding sequence in the Escherichia coli K88ac and K88ad pilin genes and their expression as recombinant pili has been constructed. Two insertion vectors were created by subcloning the pilin genes in a pBR322 plasmid and replacing the coding sequence of two nonconserved clusters by a linker. The K88ac helper genes were subcloned in the compatible pACYC184 plasmid, and expression of pili by bacteria carrying both plasmids occurred by complementation. Two peptide-coding sequences of the influenza hemagglutinin were cloned in both insertion vectors, and recombinant pilins were shown to be assembled in pili. One recombinant pilus was shown to elicit antibodies against the synthetic peptide in immunized rats. The somatostatin-coding sequence was cloned in both vectors and led in one case to detectable pilus production. The fused somatostatin was shown to be recognized by specific monoclonal and polyclonal antibodies.
|
1472. |
Sánchez S,
Llorente MT,
Echeita MA,
Herrera-León S,
( 2015 ) Development of three multiplex PCR assays targeting the 21 most clinically relevant serogroups associated with Shiga toxin-producing E. coli infection in humans. PMID : 25629697 : DOI : 10.1371/journal.pone.0117660 PMC : PMC4309606 Abstract >>
Escherichia coli serogroups O5, O15, O26, O45, O55, O76, O91, O103, O104, O111, O113, O118, O121, O123, O128, O145, O146, O157, O165, O172, and O177 are the O-antigen forms of the most clinically relevant Shiga toxin-producing E. coli (STEC) serotypes. In this study, three multiplex PCR assays able to specifically detect these 21 serogroups were developed and validated. For this purpose, the O-antigen gene clusters of E. coli O5 and O76 were fully sequenced, their associated genes were identified on the basis of homology, and serogroup-specific primers were designed. After preliminary evaluation, these two primer pairs were proven to be highly specific and suitable for the development of PCR assays for O5 and O76 serogroup identification. Specific primers were also designed for serogroups O15, O45, O55, O91, O104, O113, O118, O123, O128, O146, O157, O165, O172, and O177 based on previously published sequences, and previously published specific primers for serogroups O26, O103, O111, O121, and O145 were also included. These 21 primer pairs were shown to be specific for their target serogroup when tested against E. coli type strains representing 169 known O-antigen forms of E. coli and Shigella and therefore suitable for being used in PCR assays for serogroup identification. In order to validate the three multiplex PCR assays, 22 E. coli strains belonging to the 21 covered serogroups and 18 E. coli strains belonging to other serogroups were screened in a double-blind test and their sensitivity was determined as 1 ng chromosomal DNA. The PCR assays developed in this study could be a faster, simpler, and less expensive strategy for serotyping of the most clinically relevant STEC strains in both clinical microbiology and public health laboratories, and so their development could benefit for clinical diagnosis, epidemiological investigations, surveillance, and control of STEC infections.
|
1473. |
Kim H,
Choi J,
Kim D,
Kim KK,
( 2015 ) Crystal structure analysis of c4763, a uropathogenic Escherichia coli-specific protein. PMID : 26249697 : DOI : 10.1107/S2053230X15013035 PMC : PMC4528939 Abstract >>
Urinary-tract infections (UTIs), which are some of the most common infectious diseases in humans, can cause sepsis and death without proper treatment. Therefore, it is necessary to understand their pathogenicity for proper diagnosis and therapeutics. Uropathogenic Escherichia coli, the major causative agents of UTIs, contain several genes that are absent in nonpathogenic strains and are therefore considered to be relevant to UTI pathogenicity. c4763 is one of the uropathogenic E. coli-specific proteins, but its function is unknown. To investigate the function of c4763 and its possible role in UTI pathogenicity, its crystal structure was determined at a resolution of 1.45 ? by a multiple-wavelength anomalous diffraction method. c4763 is a homodimer with 129 residues in one subunit that contains a GGCT-like domain with five �\-helices and seven �]-strands. c4763 shows structural similarity to the C-terminal domain of allophanate hydrolase from Kluyveromyces lactis, which is involved in the degradation of urea. These results suggest that c4763 might be involved in the utilization of urea, which is necessary for bacterial survival in the urinary tract. Further biochemical and physiological investigation will elucidate its functional relevance in UTIs.
|
1474. |
Forsman K,
Göransson M,
Uhlin BE,
( 1989 ) Autoregulation and multiple DNA interactions by a transcriptional regulatory protein in E. coli pili biogenesis. PMID : 2568258 : PMC : PMC400944 Abstract >>
An operon mediating biogenesis of digalactoside-binding pilus-adhesin of serotype F13 in uropathogenic Escherichia coli includes the regulatory gene papB. The papB gene product was found to act as transcriptional activator of an operon which includes the papB gene and several pap cistrons encoding the proteins of the pilus polymer. Studies of how pap gene expression was affected by increasing amounts of PapB protein in the cells showed that high levels did not stimulate transcription but caused repression. Results from in vitro studies demonstrated that the PapB protein was a sequence-specific DNA-binding protein. Binding studies using gel mobility shift assays and DNase I protection (footprinting) showed that PapB protein binds to three separate sites. A sequence greater than 200 bp upstream of the promoter, and directly adjacent to a binding site for the cAMP receptor protein-cAMP complex, appeared as a preferential PapB binding site. A second site was localized to sequences overlapping the -10 region of the promoter and a third binding site was found within the coding sequence of the papB gene itself. The data suggest that the PapB protein has a dual function as activator/repressor of pilus-adhesin transcription and that its autoregulatory mode of action involves differential binding to separate sites.
|
1475. |
Guan Q,
Wang X,
Wang X,
Teng D,
Mao R,
Zhang Y,
Wang J,
( 2015 ) Recombinant outer membrane protein A induces a protective immune response against Escherichia coli infection in mice. PMID : 25567514 : DOI : 10.1007/s00253-014-6339-6 Abstract >>
Pathogenic Escherichia coli (E. coli) is an important infectious Gram-negative bacterium causing millions of death every year. Outer membrane protein A (OmpA) has been suggested as a potential vaccine candidate for conferring protection against bacterial infection. In this study, a universal vaccine candidate for E. coli infection was developed and evaluated. Bioinformatics analysis revealed the OmpA protein from E. coli shares 96~100%, 90~94%, and 45% identity with Shigella, Salmonella, and Pseudomonas strains, respectively. The ompA gene was cloned from the genomic DNA of E. coli, and then the OmpA protein was expressed in BL21 (DE3) using the auto-induction method. The recombinant OmpA (rOmpA) protein had an average molecular weight of 36 kDa with the purity of 93.5%. Immunological analysis indicated that the titers of anti-rOmpA sera against rOmpA and whole cells were 1:642,000 and 1:140,000, respectively. Moreover, rOmpA not only conferred a high level of immunogenicity to protect mice against the challenge of E. coli, but also generated cross-protection against Shigella and Salmonella. The anti-rOmpA sera could enhance the phagocytic activity of neutrophils against E. coli. The survive ratios of mice immunized with rOmpA and PBS were 50% and 20% after 48 h post-challenge, indicating mice were protected from E. coli infection after immunization with rOmpA. All these results clearly indicate that rOmpA may be a promising candidate for the development of a subunit vaccine to prevent E. coli infection.
|
1476. |
Fidock DA,
McNicholas PA,
Lehrbach PR,
( 1989 ) Nucleotide sequence of the F41 fimbriae subunit gene in Escherichia coli B41. PMID : 2566150 : DOI : 10.1093/nar/17.7.2849 PMC : PMC317661 Abstract >>
N/A
|
1477. |
Kim SR,
Komano T,
( 1989 ) Cloning and nucleotide sequence of the ColIb shufflon. PMID : 2623084 : Abstract >>
The R64 shufflon is a novel type of DNA rearrangement in which four DNA segments invert independently or in groups. The related plasmid ColIb carries a variant shufflon. The present sequence analysis shows that the ColIb shufflon consists of three DNA segments that are highly homologous to the A, B, and C segments of the R64 shufflon. The 329-bp D segment of R64 is not present in the ColIb shufflon. As in the case of R64, the ColIb shufflon may act as a biological switch to select one of the six open reading frames in which the N-terminal region is constant while the C-terminal region is variable.
|
1478. |
Conlan S,
Thomas PJ,
Deming C,
Park M,
Lau AF,
Dekker JP,
Snitkin ES,
Clark TA,
Luong K,
Song Y,
Tsai YC,
Boitano M,
Dayal J,
Brooks SY,
Schmidt B,
Young AC,
Thomas JW,
Bouffard GG,
Blakesley RW,
N/A N/A,
Mullikin JC,
Korlach J,
Henderson DK,
Frank KM,
Palmore TN,
Segre JA,
( 2014 ) Single-molecule sequencing to track plasmid diversity of hospital-associated carbapenemase-producing Enterobacteriaceae. PMID : 25232178 : DOI : 10.1126/scitranslmed.3009845 PMC : PMC4203314 Abstract >>
Public health officials have raised concerns that plasmid transfer between Enterobacteriaceae species may spread resistance to carbapenems, an antibiotic class of last resort, thereby rendering common health care-associated infections nearly impossible to treat. To determine the diversity of carbapenemase-encoding plasmids and assess their mobility among bacterial species, we performed comprehensive surveillance and genomic sequencing of carbapenem-resistant Enterobacteriaceae in the National Institutes of Health (NIH) Clinical Center patient population and hospital environment. We isolated a repertoire of carbapenemase-encoding Enterobacteriaceae, including multiple strains of Klebsiella pneumoniae, Klebsiella oxytoca, Escherichia coli, Enterobacter cloacae, Citrobacter freundii, and Pantoea species. Long-read genome sequencing with full end-to-end assembly revealed that these organisms carry the carbapenem resistance genes on a wide array of plasmids. K. pneumoniae and E. cloacae isolated simultaneously from a single patient harbored two different carbapenemase-encoding plasmids, indicating that plasmid transfer between organisms was unlikely within this patient. We did, however, find evidence of horizontal transfer of carbapenemase-encoding plasmids between K. pneumoniae, E. cloacae, and C. freundii in the hospital environment. Our data, including full plasmid identification, challenge assumptions about horizontal gene transfer events within patients and identify possible connections between patients and the hospital environment. In addition, we identified a new carbapenemase-encoding plasmid of potentially high clinical impact carried by K. pneumoniae, E. coli, E. cloacae, and Pantoea species, in unrelated patients and in the hospital environment.
|
1479. |
Lim D,
Maas WK,
( 1989 ) Reverse transcriptase-dependent synthesis of a covalently linked, branched DNA-RNA compound in E. coli B. PMID : 2466573 : DOI : 10.1016/0092-8674(89)90693-4 Abstract >>
We have found a branched DNA-RNA compound in E. coli B, that is similar in its secondary structure, but not its nucleotide sequence, to the previously described branched DNA-RNA compounds in myxobacteria. This compound is not produced in E. coli K12. We have cloned a 3.5 kb chromosomal segment of E. coli B, which, when transferred into E. coli K12, leads to the production of the DNA-RNA compound. We describe the isolation of the DNA-RNA compound, the determination of its nucleotide sequence, and the nucleotide sequence of the genes required for its formation. The sequence contains the coding regions for the DNA component, the RNA component, and an open reading frame encoding a reverse transcriptase. This reverse transcriptase is shown to be required for the formation of the DNA-RNA compound in vivo and in vitro.
|
1480. |
Valat C,
Forest K,
Auvray F,
Métayer V,
Méheut T,
Polizzi C,
Gay E,
Haenni M,
Oswald E,
Madec JY,
( 2014 ) Assessment of Adhesins as an Indicator of Pathovar-Associated Virulence Factors in Bovine Escherichia coli. PMID : 25217019 : DOI : 10.1128/AEM.02365-14 PMC : PMC4249168 Abstract >>
The CS31A, F17, and F5 adhesins are usually targeted by serology-based methods to detect pathogenic Escherichia coli associated with calf enteritis. However, the virulence traits of the selected isolates are still poorly described. Here, from a set of 349 diarrheagenic E. coli isolates from cattle, we demonstrated a 70.8% concordance rate (Cohen's kappa, 0.599) between serology- and PCR-based approaches for the detection of adhesins under field conditions. A 79% to 82.4% correspondence between the two methods was found for fimbrial adhesins, whereas major discrepancies (33%) were observed for CS31A-type antigens. Various F17A variants were found, such as F17Ac (20K) (50%), F17Aa (FY) (18.9%), F17Ab (8.1%), and F17Ad (111K) (5.4%), including a high proportion (17.6%) of new F17A internal combinations (F17Aab, F17Aac, and F17Abc) or untypeable variants. In addition, the highest proportion of pathovar-associated virulence factor (VF) genes was observed among E. coli isolates that produced F5/F41 adhesins. A specific link between the heat-stable toxins related to the enterotoxigenic E. coli (ETEC) pathovar and adhesins was identified. STa was significantly linked to F5/F41 and EAST1 to CS31A adhesins (P < 0.001), respectively, whereas NTEC was associated with F17 adhesin (P = 0.001). Clustering between phylogroups according to the adhesin types was also observed. Also, few Shiga toxin-producing E. coli (STEC) or enteropathogenic E. coli (EPEC) pathovars were identified. Finally, no statistically significant difference was observed in the occurrence of extended-spectrum beta lactamase (ESBL) production according to the adhesins expressed by the isolates (P = 0.09). Altogether, this study gives new insights into the relationship between adhesins, VF, and antimicrobial resistance in calf enteritis and supports the need for further standardization of methodologies for such approaches.
|
1481. |
Bleibtreu A,
Clermont O,
Darlu P,
Glodt J,
Branger C,
Picard B,
Denamur E,
( 2014 ) The rpoS gene is predominantly inactivated during laboratory storage and undergoes source-sink evolution in Escherichia coli species. PMID : 25266386 : DOI : 10.1128/JB.01972-14 PMC : PMC4248845 Abstract >>
The rpoS gene codes for an alternative RNA polymerase sigma factor, which acts as a general regulator of the stress response. Inactivating alleles of rpoS in collections of natural Escherichia coli isolates have been observed at very variable frequencies, from less than 1% to more than 70% of strains. rpoS is easily inactivated in nutrient-deprived environments such as stab storage, which makes it difficult to determine the true frequency of rpoS inactivation in nature. We studied the evolutionary history of rpoS and compared it to the phylogenetic history of bacteria in two collections of 82 human commensal and extraintestinal E. coli strains. These strains were representative of the phylogenetic diversity of the species and differed only by their storage conditions. In both collections, the phylogenetic histories of rpoS and of the strains were congruent, indicating that horizontal gene transfer had not occurred at the rpoS locus, and rpoS was under strong purifying selection, with a ratio of the nonsynonymous mutation rate (Ka) to the synonymous substitution rate (Ks) substantially smaller than 1. Stab storage was associated with a high frequency of inactivating alleles, whereas almost no amino acid sequence variation was observed in RpoS in the collection studied directly after isolation of the strains from the host. Furthermore, the accumulation of variations in rpoS was typical of source-sink dynamics. In conclusion, rpoS is rarely inactivated in natural E. coli isolates within their mammalian hosts, probably because such strains rapidly become evolutionary dead ends. Our data should encourage bacteriologists to freeze isolates immediately and to avoid the use of stab storage.
|
1482. |
Thulin E,
Sundqvist M,
Andersson DI,
( 2015 ) Amdinocillin (Mecillinam) resistance mutations in clinical isolates and laboratory-selected mutants of Escherichia coli. PMID : 25583718 : DOI : 10.1128/AAC.04819-14 PMC : PMC4325821 Abstract >>
Amdinocillin (mecillinam) is a �]-lactam antibiotic that is used mainly for the treatment of uncomplicated urinary tract infections. The objectives of this study were to identify mutations that confer amdinocillin resistance on laboratory-isolated mutants and clinical isolates of Escherichia coli and to determine why amdinocillin resistance remains rare clinically even though resistance is easily selected in the laboratory. Under laboratory selection, frequencies of mutation to amdinocillin resistance varied from 8 �� 10(-8) to 2 �� 10(-5) per cell, depending on the concentration of amdinocillin used during selection. Several genes have been demonstrated to give amdinocillin resistance, but here eight novel genes previously unknown to be involved in amdinocillin resistance were identified. These genes encode functions involved in the respiratory chain, the ribosome, cysteine biosynthesis, tRNA synthesis, and pyrophosphate metabolism. The clinical isolates exhibited significantly greater fitness than the laboratory-isolated mutants and a different mutation spectrum. The cysB gene was mutated (inactivated) in all of the clinical isolates, in contrast to the laboratory-isolated mutants, where mainly other types of more costly mutations were found. Our results suggest that the frequency of mutation to amdinocillin resistance is high because of the large mutational target (at least 38 genes). However, the majority of these resistant mutants have a low growth rate, reducing the probability that they are stably maintained in the bladder. Inactivation of the cysB gene and a resulting loss of cysteine biosynthesis are the major mechanism of amdinocillin resistance in clinical isolates of E. coli.
|
1483. |
Zhao L,
Zhang J,
Zheng B,
Wei Z,
Shen P,
Li S,
Li L,
Xiao Y,
( 2015 ) Molecular epidemiology and genetic diversity of fluoroquinolone-resistant Escherichia coli isolates from patients with community-onset infections in 30 Chinese county hospitals. PMID : 25520451 : DOI : 10.1128/JCM.02594-14 PMC : PMC4390631 Abstract >>
The high frequency of fluoroquinolone resistance in Escherichia coli is a feature of clinical bacteriology in China, where the molecular epidemiology and genetic characteristics of this resistance in county hospitals remain unclear. A total of 590 nonduplicate E. coli isolates from 30 county hospitals located across seven Chinese regions were examined for plasmid-mediated quinolone resistance (PMQR) genes and mutations in quinolone resistance-determining regions (QRDRs). Multilocus sequence typing (MLST) and phylogenetic analysis of fluoroquinolone-resistant isolates were used to determine their genetic relatedness. The ciprofloxacin resistance rate of community-onset E. coli was 51.2%, and at least one PMQR gene was carried by 220 (37.3%) isolates. These included qnr (3.7%), aac(6')-Ib-cr (19.7%), qepA (14.4%), and oqxAB (3.8%). Two novel oqxB mutants were identified and named oqxB20 and oqxB29. From 60 sequence types (STs) isolated, 5 novel STs (ST4499 to ST4503) were identified. ST1193 (7.9%) was the second most abundant ST among fluoroquinolone-resistant isolates (ST131 was the most common, with 14.6%), and this is the first report of it in China. This is also the first report of ST2115 and ST3014 isolates from human samples. Ciprofloxacin-resistant E. coli isolates fell mainly into phylogroups B2 and D. The rates of fluoroquinolone resistance and the prevalence of PMQR genes in community-onset E. coli isolates from Chinese county hospitals were high. The wide-ranging molecular epidemiology of E. coli isolates from scattered locations across China indicates that fluoroquinolone resistance evolved from different sources.
|
1484. |
Knirel YA,
Prokhorov NS,
Shashkov AS,
Ovchinnikova OG,
Zdorovenko EL,
Liu B,
Kostryukova ES,
Larin AK,
Golomidova AK,
Letarov AV,
( 2015 ) Variations in O-antigen biosynthesis and O-acetylation associated with altered phage sensitivity in Escherichia coli 4s. PMID : 25512310 : DOI : 10.1128/JB.02398-14 PMC : PMC4325112 Abstract >>
The O polysaccharide of the lipopolysaccharide (O antigen) of Gram-negative bacteria often serves as a receptor for bacteriophages that can make the phage dependent on a given O-antigen type, thus supporting the concept of the adaptive significance of the O-antigen variability in bacteria. The O-antigen layer also modulates interactions of many bacteriophages with their hosts, limiting the access of the viruses to other cell surface receptors. Here we report variations of O-antigen synthesis and structure in an environmental Escherichia coli isolate, 4s, obtained from horse feces, and its mutants selected for resistance to bacteriophage G7C, isolated from the same fecal sample. The 4s O antigen was found to be serologically, structurally, and genetically related to the O antigen of E. coli O22, differing only in side-chain �\-D-glucosylation in the former, mediated by a gtr locus on the chromosome. Spontaneous mutations of E. coli 4s occurring with an unusually high frequency affected either O-antigen synthesis or O-acetylation due to the inactivation of the gene encoding the putative glycosyltransferase WclH or the putative acetyltransferase WclK, respectively, by the insertion of IS1-like elements. These mutations induced resistance to bacteriophage G7C and also modified interactions of E. coli 4s with several other bacteriophages conferring either resistance or sensitivity to the host. These findings suggest that O-antigen synthesis and O-acetylation can both ensure the specific recognition of the O-antigen receptor following infection by some phages and provide protection of the host cells against attack by other phages.
|
1485. |
Zhang Y,
Wang L,
Hu Y,
Jin C,
( 2014 ) Solution structure of the TatB component of the twin-arginine translocation system. PMID : 24699374 : DOI : 10.1016/j.bbamem.2014.03.015 Abstract >>
The twin-arginine protein transport (Tat) system translocates fully folded proteins across lipid membranes. In Escherichia coli, the Tat system comprises three essential components: TatA, TatB and TatC. The protein translocation process is proposed to initiate by signal peptide recognition and substrate binding to the TatBC complex. Upon formation of the TatBC-substrate protein complex, the TatA subunits are recruited and form the protein translocation pore. Experimental evidences suggest that TatB forms a tight complex with TatC at 1:1 molar ratio and the TatBC complex contains multiple copies of both proteins. Cross-linking experiments demonstrate that TatB functions in tetrameric units and interacts with both TatC and substrate proteins. However, structural information of the TatB protein is still lacking, and its functional mechanism remains elusive. Herein, we report the solution structure of TatB in DPC micelles determined by Nuclear Magnetic Resonance (NMR) spectroscopy. Overall, the structure shows an extended 'L-shape' conformation comprising four helices: a transmembrane helix (TMH) �\1, an amphipathic helix (APH) �\2, and two solvent exposed helices �\3 and �\4. The packing of TMH and APH is relatively rigid, whereas helices �\3 and �\4 display notably higher mobility. The observed floppiness of helices �\3 and �\4 allows TatB to sample a large conformational space, thus providing high structural plasticity to interact with substrate proteins of different sizes and shapes.
|
1486. |
Jønsson R,
Struve C,
Boisen N,
Mateiu RV,
Santiago AE,
Jenssen H,
Nataro JP,
Krogfelt KA,
( 2015 ) Novel aggregative adherence fimbria variant of enteroaggregative Escherichia coli. PMID : 25624357 : DOI : 10.1128/IAI.02820-14 PMC : PMC4363450 Abstract >>
Enteroaggregative Escherichia coli (EAEC) organisms belong to a diarrheagenic pathotype known to cause diarrhea and can be characterized by distinct aggregative adherence (AA) in a stacked-brick pattern to cultured epithelial cells. In this study, we investigated 118 EAEC strains isolated from the stools of Danish adults with traveler's diarrhea. We evaluated the presence of the aggregative adherence fimbriae (AAFs) by a multiplex PCR, targeting the four known major subunit variants as well as their usher-encoding genes. Almost one-half (49/118) of the clinical isolates did not possess any known AAF major fimbrial subunit, despite the presence of other AggR-related loci. Further investigation revealed the presence of an AAF-related gene encoding a yet-uncharacterized adhesin, termed agg5A. The sequence of the agg5DCBA gene cluster shared fimbrial accessory genes (usher, chaperone, and minor pilin subunit genes) with AAF/III, as well as the signal peptide present in the beginning of the agg3A gene. The complete agg5DCBA gene cluster from a clinical isolate, EAEC strain C338-14, with the typical stacked-brick binding pattern was cloned, and deletion of the cluster was performed. Transformation to a nonadherent E. coli HB101 and complementation of the nonadherent C338-14 mutant with the complete gene cluster restored the AA adhesion. Overall, we found the agg5A gene in 12% of the 118 strains isolated from Denmark, suggesting that this novel adhesin represents an important variant.
|
1487. |
Kosykh VG,
Repik AV,
Kaliman AV,
Bur'ianov IaI,
Baev AA,
( 1989 ) [Primary structure of the gene of restriction endonuclease EcoRII]. PMID : 2612358 : Abstract >>
N/A
|
1488. |
Curtis MD,
James R,
Coddington A,
( 1989 ) An evolutionary relationship between the ColE5-099 and the ColE9-J plasmids revealed by nucleotide sequencing. PMID : 2561131 : DOI : 10.1099/00221287-135-10-2783 Abstract >>
The nucleotide sequence of a 1124 bp fragment of the ColE5-099 plasmid which encodes colicin E5 immunity, a lys gene involved in colicin release from the host cell, and the 3' end of the colicin E5 structural gene has been determined. Open reading frames corresponding to the three genes have been located by analogy with similar sequences from other E colicin plasmids. The location of these open reading frames corresponds with the position of the genes as determined by subcloning and transposon mutagenesis. The amino acid sequence of the carboxy-terminal 107 amino acid residues of the colicin E5 gene shows no homology with any other E colicin, suggesting a different mode of action in killing sensitive cells. A comparison of the nucleotide sequence of this region of the ColE5-099 plasmid with that of the equivalent region of the ColE9-J plasmid suggests a close evolutionary relationship between these two plasmids.
|
1489. |
Itou H,
Yagura M,
Shirakihara Y,
Itoh T,
( 2015 ) Structural basis for replication origin unwinding by an initiator primase of plasmid ColE2-P9: duplex DNA unwinding by a single protein. PMID : 25538245 : DOI : 10.1074/jbc.M114.595645 PMC : PMC4319026 Abstract >>
Duplex DNA is generally unwound by protein oligomers prior to replication. The Rep protein of plasmid ColE2-P9 (34 kDa) is an essential initiator for plasmid DNA replication. This protein binds the replication origin (Ori) in a sequence-specific manner as a monomer and unwinds DNA. Here we present the crystal structure of the DNA-binding domain of Rep (E2Rep-DBD) in complex with Ori DNA. The structure unveils the basis for Ori-specific recognition by the E2Rep-DBD and also reveals that it unwinds DNA by the concerted actions of its three contiguous structural modules. The structure also shows that the functionally unknown PriCT domain, which forms a compact module, plays a central role in DNA unwinding. The conservation of the PriCT domain in the C termini of some archaeo-eukaryotic primases indicates that it probably plays a similar role in these proteins. Thus, this is the first report providing the structural basis for the functional importance of the conserved PriCT domain and also reveals a novel mechanism for DNA unwinding by a single protein.
|
1490. |
Heim U,
Tietze E,
Weschke W,
Tschäpe H,
Wobus U,
( 1989 ) Nucleotide sequence of a plasmid born streptothricin-acetyl-transferase gene (sat-1). PMID : 2550905 : DOI : 10.1093/nar/17.17.7103 PMC : PMC318437 Abstract >>
N/A
|
1491. |
Ogutu JO,
Zhang Q,
Huang Y,
Yan H,
Su L,
Gao B,
Zhang W,
Zhao J,
Cai W,
Li W,
Zhao H,
Chen Y,
Song W,
Chen X,
Fu Y,
Zhang F,
( 2015 ) Development of a multiplex PCR system and its application in detection of blaSHV, blaTEM, blaCTX-M-1, blaCTX-M-9 and blaOXA-1 group genes in clinical Klebsiella pneumoniae and Escherichia coli strains. PMID : 26104141 : DOI : 10.1038/ja.2015.68 Abstract >>
Resistance to �]-lactam antibiotics through �]-lactamase production by Enterobacteriaceae continues to burden the health-care sector worldwide. Traditional methods for detection of �]-lactamases are time-consuming and labor-intensive and newer methods with varying capabilities continue to be developed. The objective of this study was to develop a multiplex PCR (M-PCR) system for the detection of blaSHV, blaTEM, blaCTX-M-1, blaCTX-M-9 and blaOXA-1 group genes and to apply it in clinical Klebsiella pneumoniae and Escherichia coli strains. To do this, we used group-specific PCR primers in singleplex reactions followed by optimization into multiplex reactions. Specificity and sensitivity of the M-PCR were then evaluated using 58 reference strains before its application to detect bla group genes in 203 clinical Enterobacteriaceae strains. PCR amplicons were sequenced to determine the �]-lactamase subtypes. The M-PCR system exhibited 100% specificity and sensitivity. In all, 83.7% of K. pneumoniae and 89.8% of E. coli clinical strains harbored bla group genes with 46.9%, 40.1%, 15.0%, 21.1% and 6.1% of K. pneumoniae having blaSHV, blaTEM, blaCTX-M-1, blaCTX-M-9 and blaOXA-1 group genes, respectively, whereas 12.2%, 77.6%, 22.4%, 36.7% and 8.2% of E. coli had blaSHV, blaTEM, blaCTX-M-1, blaCTX-M-9 and blaOXA-1 group genes, respectively. BlaSHV-1, blaSHV-11, blaSHV-27, blaSHV-33, blaSHV-144, blaTEM-1, blaTEM-135, blaOXA-1, blaCTX-M-3, blaCTX-M-9, blaCTX-M-14, blaCTX-M-15, blaCTX-M-27, blaCTX-M-55, blaCTX-M-65 and blaCTX-M-104 were detected. In conclusion, the M-PCR system was efficient and versatile with an advantage of simultaneously detecting all the targeted bla group genes. Hence, it is a potential candidate for developing M-PCR kits for the screening of these genes for clinical or epidemiological purposes.
|
1492. |
Leigue L,
Warth JF,
Melo LC,
Silva KC,
Moura RA,
Barbato L,
Silva LC,
Santos AC,
Silva RM,
Lincopan N,
( 2015 ) MDR ST2179-CTX-M-15 Escherichia coli co-producing RmtD and AAC(6')-Ib-cr in a horse with extraintestinal infection, Brazil. PMID : 25538170 : DOI : 10.1093/jac/dku520 Abstract >>
N/A
|
1493. |
Moonens K,
De Kerpel M,
Coddens A,
Cox E,
Pardon E,
Remaut H,
De Greve H,
( 2014 ) Nanobody mediated inhibition of attachment of F18 Fimbriae expressing Escherichia coli. PMID : 25502211 : DOI : 10.1371/journal.pone.0114691 PMC : PMC4263667 Abstract >>
Post-weaning diarrhea and edema disease caused by F18 fimbriated E. coli are important diseases in newly weaned piglets and lead to severe production losses in farming industry. Protective treatments against these infections have thus far limited efficacy. In this study we generated nanobodies directed against the lectin domain of the F18 fimbrial adhesin FedF and showed in an in vitro adherence assay that four unique nanobodies inhibit the attachment of F18 fimbriated E. coli bacteria to piglet enterocytes. Crystallization of the FedF lectin domain with the most potent inhibitory nanobodies revealed their mechanism of action. These either competed with the binding of the blood group antigen receptor on the FedF surface or induced a conformational change in which the CDR3 region of the nanobody displaces the D?-E loop adjacent to the binding site. This D?-E loop was previously shown to be required for the interaction between F18 fimbriated bacteria and blood group antigen receptors in a membrane context. This work demonstrates the feasibility of inhibiting the attachment of fimbriated pathogens by employing nanobodies directed against the adhesin domain.
|
1494. |
Turrientes MC,
González-Alba JM,
del Campo R,
Baquero MR,
Cantón R,
Baquero F,
Galán JC,
( 2014 ) Recombination blurs phylogenetic groups routine assignment in Escherichia coli: setting the record straight. PMID : 25137251 : DOI : 10.1371/journal.pone.0105395 PMC : PMC4138120 Abstract >>
The characterization of population structures plays a main role for understanding outbreaks and the dynamics of bacterial spreading. In Escherichia coli, the widely used combination of multiplex-PCR scheme together with goeBURST has some limitations. The purpose of this study is to show that the combination of different phylogenetic approaches based on concatenated sequences of MLST genes results in a more precise assignment of E. coli phylogenetic groups, complete understanding of population structure and reconstruction of ancestral clones. A collection of 80 Escherichia coli strains of different origins was analyzed following the Clermont and Doumith's multiplex-PCR schemes. Doumith's multiplex-PCR showed only 1.7% of misassignment, whereas Clermont's-2000 protocol reached 14.0%, although the discrepancies reached 30% and 38.7% respectively when recombinant C, F and E phylogroups were considered. Therefore, correct phylogroup attribution is highly variable and depends on the clonal composition of the sample. As far as population structure of these E. coli strains, including 48 E. coli genomes from GenBank, goeBURST provides a quite dispersed population structure; whereas NeighborNet approach reveals a complex population structure. MLST-based eBURST can infer different founder genotypes, for instance ST23/ST88 could be detected as the founder genotypes for STC23; however, phylogenetic reconstructions might suggest ST410 as the ancestor clone and several evolutionary trajectories with different founders. To improve our routine understanding of E. coli molecular epidemiology, we propose a strategy based on three successive steps; first, to discriminate three main groups A/B1/C, D/F/E and B2 following Doumith's protocol; second, visualization of population structure based on MLST genes according to goeBURST, using NeighborNet to establish more complex relationships among STs; and third, to perform, a cost-free characterization of evolutionary trajectories in variants emerging along the clonal expansion using parsimony methods of phylogenetic analysis.
|
1495. |
Fischer J,
Rodríguez I,
Baumann B,
Guiral E,
Beutin L,
Schroeter A,
Kaesbohrer A,
Pfeifer Y,
Helmuth R,
Guerra B,
( 2014 ) blaCTX-M-??-carrying Escherichia coli and Salmonella isolates from livestock and food in Germany. PMID : 25074857 : DOI : 10.1093/jac/dku270 Abstract >>
The characterization of CTX-M-?? �]-lactamase-producing Escherichia coli and Salmonella isolates originating mainly from German livestock and food. E. coli (526, mainly commensals) and Salmonella (151) non-human isolates resistant to third-generation cephalosporins, originating from routine and monitoring submissions (2003-12) to the Federal Institute for Risk Assessment and different national targeted studies (2011-12), were examined for the presence of blaCTX-M-?? genes by PCR amplification/sequencing. Additional resistance and virulence genes were screened by DNA microarray and PCR amplification. E. coli isolates with blaCTX-M-?? were characterized by phylogenetic grouping, PFGE and multilocus sequence typing (MLST). The blaCTX-M-15 plasmids were analysed by replicon typing, plasmid MLST, S1 nuclease PFGE and Southern blot hybridization experiments. Twenty-one E. coli (livestock, food and a toy; 4.0%) and two Salmonella (horse and swine; 1.3%) isolates were CTX-M-?? producers. E. coli isolates were mainly ascribed to three clonal lineages of sequence types ST678 (German outbreak with enteroaggregative Shiga-toxin-producing E. coli O104:H4; salmon, cucumber and a toy), ST410 (poultry, swine and cattle farms) and ST167/617 (swine farms and turkey meat). The blaCTX-M-?? genes were located on IncI1 and multireplicon IncF plasmids or on the chromosome of E. coli ST410 isolates. The prevalence of CTX-M-??-producing isolates from non-human sources in Germany is still low. The blaCTX-M-?? gene is, however, present in multidrug-resistant E. coli clones with pathogenic potential in livestock and food. The maintenance of the blaCTX-M-?? gene due to chromosomal carriage is noteworthy. The possibility of an exchange of CTX-M-??-producing isolates or plasmids between livestock and humans (in both directions) deserves continuous surveillance.
|
1496. |
Kossykh V,
Repyk A,
Kaliman A,
Buryanov Y,
( 1989 ) Nucleotide sequence of the EcoRII restriction endonuclease gene. PMID : 2597679 : DOI : 10.1016/0167-4781(89)90117-6 Abstract >>
The nucleotide sequence of a 1394 basepair (bp) DNA fragment containing the EcoRII restriction endonuclease (R.EcoRII) gene was determined. The endonuclease gene is 1206 bp in length (predicted 402 amino acids (aa) and Mr = 45 178) and is separated by 33 bp from the EcoRII modification methylase (M.EcoRII) gene. The EcoRII restriction-modification system has a tail-to-tail organization of the two genes.
|
1497. |
Amos GC,
Hawkey PM,
Gaze WH,
Wellington EM,
( 2014 ) Waste water effluent contributes to the dissemination of CTX-M-15 in the natural environment. PMID : 24797064 : DOI : 10.1093/jac/dku079 PMC : PMC4054988 Abstract >>
Multidrug-resistant Enterobacteriaceae pose a significant threat to public health. We aimed to study the impact of sewage treatment effluent on antibiotic resistance reservoirs in a river. River sediment samples were taken from downstream and upstream of a waste water treatment plant (WWTP) in 2009 and 2011. Third-generation cephalosporin (3GC)-resistant Enterobacteriaceae were enumerated. PCR-based techniques were used to elucidate mechanisms of resistance, with a new two-step PCR-based assay developed to investigate bla(CTX-M-15) mobilization. Conjugation experiments and incompatibility replicon typing were used to investigate plasmid ecology. We report the first examples of bla(CTX-M-15) in UK river sediment; the prevalence of bla(CTX-M-15) was dramatically increased downstream of the WWTP. Ten novel genetic contexts for this gene were identified, carried in pathogens such as Escherichia coli ST131 as well as indigenous aquatic bacteria such as Aeromonas media. The bla(CTX-M-15) -gene was readily transferable to other Gram-negative bacteria. We also report the first finding of an imipenem-resistant E. coli in a UK river. The high diversity and host range of novel genetic contexts proves that evolution of novel combinations of resistance genes is occurring at high frequency and has to date been significantly underestimated. We have identified a worrying reservoir of highly resistant enteric bacteria in the environment that poses a threat to human and animal health.
|
1498. |
Holden NJ,
Wright F,
Mackenzie K,
Marshall J,
Mitchell S,
Mahajan A,
Wheatley R,
Daniell TJ,
( 2014 ) Prevalence and diversity of Escherichia coli isolated from a barley trial supplemented with bulky organic soil amendments: green compost and bovine slurry. PMID : 24151873 : DOI : 10.1111/lam.12180 Abstract >>
A barley field trial supplemented with bulky organic soil amendments, municipal compost or bovine slurry was sampled for Escherichia coli to test the hypothesis that E. coli isolated from the soil or from barley plants were derived from bovine slurry. A qualitative analysis showed that a total of 12% of the bulk soil cores and 16% of harvested grain samples yielded E. coli. The strongest association for positive detection of E. coli from soil was with time of year and for slurry-treated plots, with irrigation. However, E. coli were detected in plots from all treatment types and not exclusively associated with bovine slurry. Phylogroup, plasmid profiling and population genetics analysis (multilocus sequence typing) revealed extensive genetic diversity. Identical sequence types for slurry and soil isolates were detected, indicative of direct transfer into the soil, although not frequently. Host interaction assays with selected isolates showed a variation in the ability to colonize barley roots, but not in interactions with bovine cells. The work has implications in appropriate use of E. coli as a faecal indicator as isolates were widespread and diverse, reinforcing the view that some are a natural part of the microflora in agricultural systems. Faecal deposition is considered to be the main process that introduces Escherichia coli into soil, giving rise to their use as a faecal indication species and the potential for cycling pathogens in agricultural systems. We found that bovine slurry was not the main source of E. coli in a barley trial and a high degree of diversity was present in the collection. The findings support the hypothesis that the population structure of E. coli in secondary habitats is shaped by the environment and highlight the drawbacks of its use as a faecal indicator species.
|
1499. |
Nomura T,
Fujita N,
Ishihama A,
( 1987 ) Expression of the leuX gene in Escherichia coli. Regulation at transcription and tRNA processing steps. PMID : 2448476 : DOI : 10.1016/0022-2836(87)90472-4 Abstract >>
The leuX (supP) gene of Escherichia coli codes for a suppressor tRNA (tRNA(6Leu] that inserts leucine at the amber codon. Analysis of both in-vitro and in-vivo transcripts indicated that the gene is organized into a single gene operon, carrying its own promoter and rho-independent terminator, and its primary transcript accumulates in cells of wild-type E. coli with respect to tRNA processing. Systematic and quantitative measurements of both the unprocessed primary transcript and mature tRNA(Leu6) indicated that: (1) transcription of the leuX gene is under stringent control in vivo and is repressed in vitro by ppGpp; (2) transcription of the leuX gene is under growth rate-dependent control; but (3) the level of mature tRNA stays constant under various growth conditions. A model is proposed, which assumes that the enzyme catalyzing the first-step reaction in the leuX tRNA processing is limited, thereby keeping the level of mature tRNA(Leu6) at a constant level irrespective of changes in the level of the unprocessed primary transcript.
|
1500. |
Charfi K,
Mansour W,
Khalifa AB,
Mastouri M,
Aouni M,
Mammeri H,
( 2015 ) Emergence of OXA-204 �]-lactamase in Tunisia. PMID : 26001616 : DOI : 10.1016/j.diagmicrobio.2015.04.003 Abstract >>
A retrospective epidemiological survey was carried out to determine the prevalence of carbapenemase producers among enterobacterial clinical isolates recovered in the center of maternity and neonatology of Monastir (Tunisia). PCR screening identified 1 OXA-48 and 2 OXA-204 producers, which coexpressed the CTX-M-15 or the CMY-4 �]-lactamases. PCR mapping showed that the bla(OXA-48) gene was carried by a Tn1999.2 transposon, whereas the bla(OXA-204) gene was part of the Tn2016 transposon-like structure. The OXA-48- or OXA-204-producing Klebsiella pneumoniae clinical isolates and the OXA-204-expressing Escherichia coli clinical isolate belonged to the widespread sequence types ST11, ST101, and ST617, respectively. The OXA-204 enzyme, which is a point derivative of the OXA-48 carbapenemase, had hitherto been reported in 2013 from K. pneumoniae isolate. Our study shows for the first time the dissemination of this resistance marker in E. coli strain. The coproduction of OXA-204 with CTX-M-15 and CMY-4 enzymes may potentiate the risk of multiresistance and may enhance the risk of dissemination.
|
1501. |
Hsieh PF,
Wu MC,
Yang FL,
Chen CT,
Lou TC,
Chen YY,
Wu SH,
Sheu JC,
Wang JT,
( 2014 ) D-galactan II is an immunodominant antigen in O1 lipopolysaccharide and affects virulence in Klebsiella pneumoniae: implication in vaccine design. PMID : 25477867 : DOI : 10.3389/fmicb.2014.00608 PMC : PMC4237132 Abstract >>
In the O1 strain of Klebsiella, the lipopolysaccharide (LPS) O-antigen is composed of D-galactan I and D-galactan II. Although the composition of the O1 antigen of Klebsiella was resolved more than two decades, the genetic locus involved in the biosynthesis of D-galactan II and the role of D-galactan II in bacterial pathogenesis remain unclear. Here, we report the identification of the D-galactan II-synthesizing genes by screening a transposon mutant library of an acapsulated Klebsiella pneumoniae O1 strain with bacteriophage. K. pneumoniae strain deleted for wbbY exhibited abrogated D-galactan II production; altered serum resistance and attenuation of virulence. Serologic analysis of K. pneumoniae clinical isolates demonstrated that D-galactan II was more prevalent in community-acquired pyogenic liver abscess (PLA)-causing strains than in non-tissue-invasive strains. WbbY homologs, WbbZ homologs, and lipopolysaccharide structures based on D-galactan II also were present in several Gram-negative bacteria. Immunization of mice with the magA-mutant (K(-) 1 O1) (that is, with a LPS D-galactan II-producing strain) provided protection against infection with an O1:K2 PLA strain. Our findings indicate that both WbbY and WbbZ homologs are sufficient for the synthesis of D-galactan II. D-galactan II represents an immunodominant antigen; is conserved among multiple species of Gram-negative bacteria and could be a useful vaccine candidate.
|
1502. |
Morales M,
Attai H,
Troy K,
Bermudes D,
( 2015 ) Accumulation of single-stranded DNA in Escherichia coli carrying the colicin plasmid pColE3-CA38. PMID : 25450765 : DOI : 10.1016/j.plasmid.2014.11.001 PMC : PMC4298466 Abstract >>
We sequenced the complete 7118 bp circular plasmid pColE3-CA38 (pColE3) from Escherichia coli, located the previously identified colicin components together with two new ORFs that have homology to mobilization and transfer proteins, and found that pColE3 is highly similar to a plasmid present in enterohemorrhagic E. coli O111. We also found that unusual aspects of the plasmid include the inability to be completely digested with restriction endonucleases and asymmetric Phred DNA sequencing quality scores, with significantly lower scores in the forward direction relative to the colicin and immunity proteins consistent with plus (+) strand DNA. Comparing the A260 with picogreen double-stranded DNA (dsDNA) fluorescence and oligreen single-stranded DNA (ssDNA) fluorescence as well as metachromatic staining by acridine orange, we found that the undigested pColE3 DNA stains preferentially as ssDNA and that it coexists with dsDNA. We also identified ssDNA in pColE5 and pColE9 but not in pColE1. Colicin plasmids producing ssDNA may represent a new subclass of rolling-circle replication plasmids and add to the known similarities between colicins and filamentous phage.
|
1503. |
Lin YT,
Pan YJ,
Lin TL,
Fung CP,
Wang JT,
( 2015 ) Transfer of CMY-2 Cephalosporinase from Escherichia coli to Virulent Klebsiella pneumoniae Causing a Recurrent Liver Abscess. PMID : 25987637 : DOI : 10.1128/AAC.00492-15 PMC : PMC4505199 Abstract >>
A CMY-2-producing capsular type K2 Klebsiella pneumoniae strain (TVGHKP93) with multidrug resistance was isolated from a recurrent liver abscess in a patient who also carried a CMY-2-producing Escherichia coli strain (TVGHEC01) in the stool. TVGHKP93 retained its high virulence compared with that of the isogenic strain (TVGHKP60) with wild-type resistance from the first liver abscess. Our conjugation experiment showed the successful transfer of the blaCMY-2-carrying plasmid from TVGHEC01 into TVGHKP60. The transconjugant showed both high virulence and the multidrug-resistant phenotype, as did TVGHKP93.
|
1504. |
Lau PC,
Condie JA,
( 1989 ) Nucleotide sequences from the colicin E5, E6 and E9 operons: presence of a degenerate transposon-like structure in the ColE9-J plasmid. PMID : 2549375 : DOI : 10.1007/bf02464892 Abstract >>
The nucleotide sequences of 1288 bp of plasmid ColE5-099, 1609 bp of ColE6-CT14 and 2099 bp of ColE9-J were determined. These sequences encompass the structural genes for the C-terminal receptor-binding and nuclease domains of colicins E5, E6 and E9, their cis- or trans-acting immunity proteins and four lysis proteins including an atypical one of non-lipoprotein nature (Lys) present in the ColE9-J plasmid. The ColE6 gene organisation, in the order col-imm-E8imm-lys, is identical to that found in the previously described double-immunity gene system of ColE3-CA38 (an RNase producer). The corresponding genes in the two plasmids are 87%-94% homologous. In ColE9-J, the genes are organised as col-imm-lys-E5imm-lys. The E9 col-imm gene pair is homologous to the colicin E2-P9 type (a DNase producer). Downstream from E9imm is an E5imm (designated E5imm[E9]) which is trans-acting. Neither the predicted structures of E5Imm[E9] nor the cis-acting Imm resident in the ColE5-099 plasmid which differs by a single amino acid shows any resemblance to other immunity structures which have been sequenced. Furthermore, the E5col sequences differ from those predicted previously for other colicins except for the conserved btuB-specified receptor-binding domain. A novel 205 nucleotide long insertion sequence is found in the ColE9-J plasmid. This insertion sequence, which we named ISE9, has features reminiscent of the degenerate transposon IS101 previously found in plasmid pSC101. One effect of ISE9 is the presence of the atypical lysis gene, lys.(ABSTRACT TRUNCATED AT 250 WORDS)
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1505. |
Liu Y,
Feng Y,
Wu W,
Xie Y,
Wang X,
Zhang X,
Chen X,
Zong Z,
( 2015 ) First Report of OXA-181-Producing Escherichia coli in China and Characterization of the Isolate Using Whole-Genome Sequencing. PMID : 26014927 : DOI : 10.1128/AAC.00442-15 PMC : PMC4505247 Abstract >>
We report the first OXA-181-producing strain in China. blaOXA-181 was found in sequence type 410 (ST410) Escherichia coli strain WCHEC14828 from a Chinese patient without recent travel history. Genome sequencing and conjugation experiments were performed. blaOXA-181 was carried on a 51-kb self-transmissible IncX3 plasmid and was linked with qnrS1, a quinolone resistance gene. blaOXA-181 was introduced onto the IncX3 plasmid from a ColE2-type plasmid, and IncX3 plasmids have the potential to mediate the dissemination of blaOXA-181.
|
1506. |
Schuurmans JM,
van Hijum SA,
Piet JR,
Händel N,
Smelt J,
Brul S,
ter Kuile BH,
( 2014 ) Effect of growth rate and selection pressure on rates of transfer of an antibiotic resistance plasmid between E. coli strains. PMID : 24525238 : DOI : 10.1016/j.plasmid.2014.01.002 Abstract >>
Antibiotic resistance increases costs for health care and causes therapy failure. An important mechanism for spreading resistance is transfer of plasmids containing resistance genes and subsequent selection. Yet the factors that influence the rate of transfer are poorly known. Rates of plasmid transfer were measured in co-cultures in chemostats of a donor and a acceptor strain under various selective pressures. To document whether specific mutations in either plasmid or acceptor genome are associated with the plasmid transfer, whole genome sequencing was performed. The DM0133 TetR tetracycline resistance plasmid was transferred between Escherichia coli K-12 strains during co-culture at frequencies that seemed higher at increased growth rate. Modeling of the take-over of the culture by the transformed strain suggests that in reality more transfer events occurred at low growth rates. At moderate selection pressure due to an antibiotic concentration that still allowed growth, a maximum transfer frequency was determined of once per 10(11) cell divisions. In the absence of tetracycline or in the presence of high concentrations the frequency of transfer was sometimes zero, but otherwise reduced by at least a factor of 5. Whole genome sequencing showed that the plasmid was transferred without mutations, but two functional mutations in the genome of the recipient strain accompanied this transfer. Exposure to concentrations of antibiotics that fall within the mutant selection window stimulated transfer of the resistance plasmid most.
|
1507. |
Dong XN,
Womble DD,
Rownd RH,
( 1987 ) Transcriptional pausing in a region important for plasmid NR1 replication control. PMID : 2445727 : DOI : 10.1128/jb.169.12.5353-5363.1987 PMC : PMC213958 Abstract >>
The results of in vitro single-round transcription experiments indicated that RNA polymerase pauses during transcription of the leader region that precedes the repA1 gene of IncFII plasmid NR1. Transcription initiated at either of the two transcription promoter sites of the repA1 gene, which encodes the essential replication initiation protein of NR1, was observed to pause in this region. Pausing was specifically enhanced by addition of NusA protein, an Escherichia coli transcription accessory factor. Northern blot RNA-DNA hybridization analysis of repA1 mRNA synthesized in vivo revealed RNA species that had lengths equivalent to those of the in vitro-paused intermediates. The steady-state rate of in vivo repA1 mRNA transcription downstream from the pause sites (measured by quantitative hybridization of pulse-labeled RNA to DNA probes complementary to different segments of repA1 mRNA) was not appreciably affected, which suggests that the pause sites do not promote premature termination of transcription. The pause sites were located between the target sequence within the leader region of the mRNA that interacts with a 91-base countertranscript and the beginning of the repA1 coding sequence. Because the countertranscript is an inhibitor of translation of repA1 mRNA, transcriptional pausing in this region may be an important feature of the regulation of RepA1 synthesis, which is the mechanism by which plasmid NR1 controls its replication.
|
1508. |
Dutta S,
Pazhani GP,
Nataro JP,
Ramamurthy T,
( 2015 ) Heterogenic virulence in a diarrheagenic Escherichia coli: evidence for an EPEC expressing heat-labile toxin of ETEC. PMID : 25465159 : DOI : 10.1016/j.ijmm.2014.10.006 Abstract >>
We have encountered an Escherichia coli strain isolated from a child with acute diarrhea. This strain harbored eae and elt genes encoding for E. coli attaching and effacing property and heat-labile enterotoxin of EPEC and ETEC, respectively. Due to the presence of these distinct virulence factors, we named this uncommon strain as EPEC/ETEC hybrid. The elt gene was identified in a conjugally transferable plasmid of the EPEC/ETEC hybrid. In addition, several virulence genes in the locus of enterocyte effacement have been identified, which confirms that the EPEC/ETEC has an EPEC genetic background. The hybrid nature of this strain was further confirmed by using tissue culture assays. In the multi locus sequence typing (MLST) analysis, the EPEC/ETEC belonged to the sequence type ST328 and was belonging to ST278 Cplx. Sequence analysis of the plasmid DNA revealed presence of six large contigs with several insertion sequences. A phage integrase gene and the prophages of gp48 and gp49 have been found in the upstream of eltAB. In the downstream of elt, an urovirulence loci adhesion encoding (pap) cluster containing papG, and papC were also identified. Similar to other reports, we have identified a heterogenic virulence in a diarrheagenic E. coli but with different combination of genes.
|
1509. |
Chuba PJ,
Leon MA,
Banerjee A,
Palchaudhuri S,
( 1989 ) Cloning and DNA sequence of plasmid determinant iss, coding for increased serum survival and surface exclusion, which has homology with lambda DNA. PMID : 2546040 : DOI : 10.1007/bf00334367 Abstract >>
Escherichia coli K12 cells carrying a cloned 1.4 kb HindIII fragment from plasmid ColV2-K94, showed increased survival in guinea pig serum. The recombinant plasmid also conferred group II surface exclusion, i.e. the cells were reduced in recipient ability towards the incoming plasmid R538drd in conjugation experiments. Southern blotting suggested homology with bacteriophage lambda DNA and to the insertion element IS2. Determination of the DNA sequence of the fragment demonstrated the presence of a truncated IS2 (165 bp), separated by 250 bp from a 900 bp stretch of homology with lambda DNA, beginning within the Rz gene and continuing in the rightward direction on the lambda map. A 97 amino acid open reading frame (ORF) adjacent to Rz and on the opposite strand, remained intact in iss, with several amino acid changes. The ORF in iss is preceded by sequences resembling prokaryotic ribosome binding sites and promoters.
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1510. |
Ma Y,
Xu X,
Guo Q,
Wang P,
Wang W,
Wang M,
( 2015 ) Characterization of fosA5, a new plasmid-mediated fosfomycin resistance gene in Escherichia coli. PMID : 25441705 : DOI : 10.1111/lam.12366 Abstract >>
A clinical strain of extended-spectrum �]-lactamase-producing Escherichia coli E265, with a fosfomycin MIC of 512 �gg ml(-1), was isolated from an inpatient with hospital-acquired pneumonia. This strain was negative for known fos genes, had no mutation in the target enzyme by polymerase chain reaction amplification and had functional transport systems for fosfomycin uptake. Fosfomycin resistance could be transferred from strain E265 to E. coli J53 azide(R) by conjugation. The DNA fragment containing fosfomycin resistance determinants was cloned into E. coli TOP10. The minimal inhibitory concentrations of fosfomycin for the transconjugant and transformant were 512 and 1024 �gg ml(-1). By sequencing, a plasmid-mediated fosA subtype, designated fosA5, was found and characterized. The fosA5 gene was 420 bp in length and encoded a 139-amino-acid protein that shared 69 to 80% identity with FosA, FosA2, FosA3 and FosA4, and 31, 14 and 25% identity with FosB, FosC and FosX, respectively. The analysis of genetic environment of fosA5 suggested that a strain such as Klebsiella pneumoniae CG4 might be the origin of plasmid-mediated fosA5, with IS10 playing an important role in its mobilization. This study aimed to clone and characterize a plasmid-mediated fosA subtype gene, fosA5, in a clinical strain of ESBL-producing Escherichia coli, which confers fosfomycin resistance. Detection of the fosA5 gene clarified the mechanism of fosfomycin resistance in a strain that was negative for known fosfomycin resistance genes. Monitoring and surveillance will be important to follow the changes in fosfomycin resistance and prevent further dissemination of fos genes.
|
1511. |
Dellus-Gur E,
Elias M,
Caselli E,
Prati F,
Salverda ML,
de Visser JA,
Fraser JS,
Tawfik DS,
( 2015 ) Negative Epistasis and Evolvability in TEM-1 �]-Lactamase--The Thin Line between an Enzyme's Conformational Freedom and Disorder. PMID : 26004540 : DOI : 10.1016/j.jmb.2015.05.011 PMC : PMC4718737 Abstract >>
Epistasis is a key factor in evolution since it determines which combinations of mutations provide adaptive solutions and which mutational pathways toward these solutions are accessible by natural selection. There is growing evidence for the pervasiveness of sign epistasis--a complete reversion of mutational effects, particularly in protein evolution--yet its molecular basis remains poorly understood. We describe the structural basis of sign epistasis between G238S and R164S, two adaptive mutations in TEM-1 �]-lactamase--an enzyme that endows antibiotics resistance. Separated by 10 ?, these mutations initiate two separate trajectories toward increased hydrolysis rates and resistance toward second and third-generation cephalosporins antibiotics. Both mutations allow the enzyme's active site to adopt alternative conformations and accommodate the new antibiotics. By solving the corresponding set of crystal structures, we found that R164S causes local disorder whereas G238S induces discrete conformations. When combined, the mutations in 238 and 164 induce local disorder whereby nonproductive conformations that perturb the enzyme's catalytic preorganization dominate. Specifically, Asn170 that coordinates the deacylating water molecule is misaligned, in both the free form and the inhibitor-bound double mutant. This local disorder is not restored by stabilizing global suppressor mutations and thus leads to an evolutionary cul-de-sac. Conformational dynamism therefore underlines the reshaping potential of protein's structures and functions but also limits protein evolvability because of the fragility of the interactions networks that maintain protein structures.
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1512. |
Yamamoto T,
( 1989 ) Organization of complex transposon Tn2610 carrying two copies of tnpA and tnpR. PMID : 2546492 : DOI : 10.1128/aac.33.5.746 PMC : PMC172526 Abstract >>
Transposon Tn2610 has two elements of 3.5 kilobase pairs as inverted repeats, one set at each end. This unique terminal element contained the transposition genes tnpA and tnpR. Only the tnpA gene in the left element was functional for transposition, whereas both tnpR genes were active. Possible evolutionary relationships among class II transposable elements are proposed on the basis of the genetic and structural organization of Tn2610.
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1513. |
Tada T,
Shrestha B,
Miyoshi-Akiyama T,
Shimada K,
Ohara H,
Kirikae T,
Pokhrel BM,
( 2014 ) NDM-12, a novel New Delhi metallo-�]-lactamase variant from a carbapenem-resistant Escherichia coli clinical isolate in Nepal. PMID : 25092693 : DOI : 10.1128/AAC.03355-14 PMC : PMC4187903 Abstract >>
A novel New Delhi metallo-�]-lactamase variant, NDM-12, was identified in a carbapenem-resistant Escherichia coli clinical isolate obtained from a urine sample from a patient in Nepal. NDM-12 differed from NDM-1 by two amino acid substitutions (M154L and G222D). The enzymatic activities of NDM-12 against �]-lactams were similar to those of NDM-1, although NDM-12 showed lower kcat/Km ratios for all �]-lactams tested except doripenem. The blaNDM-12 gene was located in a plasmid of 160 kb.
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1514. |
Liu CT,
Layfield JP,
Stewart RJ,
French JB,
Hanoian P,
Asbury JB,
Hammes-Schiffer S,
Benkovic SJ,
( 2014 ) Probing the electrostatics of active site microenvironments along the catalytic cycle for Escherichia coli dihydrofolate reductase. PMID : 24977791 : DOI : 10.1021/ja5038947 PMC : PMC4183630 Abstract >>
Electrostatic interactions play an important role in enzyme catalysis by guiding ligand binding and facilitating chemical reactions. These electrostatic interactions are modulated by conformational changes occurring over the catalytic cycle. Herein, the changes in active site electrostatic microenvironments are examined for all enzyme complexes along the catalytic cycle of Escherichia coli dihydrofolate reductase (ecDHFR) by incorporation of thiocyanate probes at two site-specific locations in the active site. The electrostatics and degree of hydration of the microenvironments surrounding the probes are investigated with spectroscopic techniques and mixed quantum mechanical/molecular mechanical (QM/MM) calculations. Changes in the electrostatic microenvironments along the catalytic environment lead to different nitrile (CN) vibrational stretching frequencies and (13)C NMR chemical shifts. These environmental changes arise from protein conformational rearrangements during catalysis. The QM/MM calculations reproduce the experimentally measured vibrational frequency shifts of the thiocyanate probes across the catalyzed hydride transfer step, which spans the closed and occluded conformations of the enzyme. Analysis of the molecular dynamics trajectories provides insight into the conformational changes occurring between these two states and the resulting changes in classical electrostatics and specific hydrogen-bonding interactions. The electric fields along the CN axes of the probes are decomposed into contributions from specific residues, ligands, and solvent molecules that make up the microenvironments around the probes. Moreover, calculation of the electric field along the hydride donor-acceptor axis, along with decomposition of this field into specific contributions, indicates that the cofactor and substrate, as well as the enzyme, impose a substantial electric field that facilitates hydride transfer. Overall, experimental and theoretical data provide evidence for significant electrostatic changes in the active site microenvironments due to conformational motion occurring over the catalytic cycle of ecDHFR.
|
1515. |
Li G,
Zhang Y,
Bi D,
Shen P,
Ai F,
Liu H,
Tian Y,
Ma Y,
Wang B,
Rajakumar K,
Ou HY,
Jiang X,
( 2015 ) First report of a clinical, multidrug-resistant Enterobacteriaceae isolate coharboring fosfomycin resistance gene fosA3 and carbapenemase gene blaKPC-2 on the same transposon, Tn1721. PMID : 25367902 : DOI : 10.1128/AAC.03061-14 PMC : PMC4291370 Abstract >>
In order to understand the genetic background and dissemination mechanism of carbapenem resistance and fosfomycin resistance in Enterobacteriaceae isolates, we studied a clinical Escherichia coli strain HS102707 isolate and an Enterobacter aerogenes strain HS112625 isolate, both of which were resistant to carbapenem and fosfomycin and positive for the bla(KPC-2) and fosA3 genes. In addition, a clinical Klebsiella pneumoniae strain HS092839 isolate which was resistant to carbapenem was also studied. A 70-kb plasmid was successfully transferred to recipient E. coli J53 by a conjugation test. PCR and Southern blot analysis showed that bla(KPC-2) was located on this plasmid. The complete sequence of pHS102707 showed that this plasmid belongs to the P11 subfamily (IncP1) and has a replication gene, several plasmid-stable genes, an intact type IV secretion system gene cluster, and a composite transposon Tn1721-Tn3 that harbored bla(KPC-2). Interestingly, a composite IS26 transposon carrying fosA3 was inserted in the Tn1721-tnpA gene in pHS102707 and pHS112625, leading to the disruption of Tn1721-tnpA and the deletion of Tn1721-tnpR. However, only IS26 with a truncated Tn21-tnpR was inserted in pHS092839 at the same position. To our knowledge, this is the first report of fosA3 and bla(KPC-2) colocated in the same Tn1721-Tn3-like composite transposon on a novel IncP group plasmid.
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1516. |
Turner AK,
Grinsted J,
( 1989 ) DNA sequence of the transposase gene of the class II transposon, Tn3926. PMID : 2537961 : DOI : 10.1093/nar/17.4.1757 PMC : PMC331833 Abstract >>
N/A
|
1517. |
Mortezaei N,
Epler CR,
Shao PP,
Shirdel M,
Singh B,
McVeigh A,
Uhlin BE,
Savarino SJ,
Andersson M,
Bullitt E,
( 2015 ) Structure and function of enterotoxigenic Escherichia coli fimbriae from differing assembly pathways. PMID : 25355550 : DOI : 10.1111/mmi.12847 PMC : PMC4275653 Abstract >>
Pathogenic enterotoxigenic Escherichia coli (ETEC) are the major bacterial cause of diarrhea in young children in developing countries and in travelers, causing significant mortality in children. Adhesive fimbriae are a prime virulence factor for ETEC, initiating colonization of the small intestinal epithelium. Similar to other Gram-negative bacteria, ETEC express one or more diverse fimbriae, some assembled by the chaperone-usher pathway and others by the alternate chaperone pathway. Here, we elucidate structural and biophysical aspects and adaptations of each fimbrial type to its respective host niche. CS20 fimbriae are compared with colonization factor antigen I (CFA/I) fimbriae, which are two ETEC fimbriae assembled via different pathways, and with P-fimbriae from uropathogenic E. coli. Many fimbriae unwind from their native helical filament to an extended linear conformation under force, thereby sustaining adhesion by reducing load at the point of contact between the bacterium and the target cell. CFA/I fimbriae require the least force to unwind, followed by CS20 fimbriae and then P-fimbriae, which require the highest unwinding force. We conclude from our electron microscopy reconstructions, modeling and force spectroscopy data that the target niche plays a central role in the biophysical properties of fimbriae that are critical for bacterial pathophysiology.
|
1518. |
Strau? LM,
Dahms C,
Becker K,
Kramer A,
Kaase M,
Mellmann A,
( 2015 ) Development and evaluation of a novel universal �]-lactamase gene subtyping assay for blaSHV, blaTEM and blaCTX-M using clinical and livestock-associated Escherichia coli. PMID : 25414200 : DOI : 10.1093/jac/dku450 Abstract >>
Antibiotic resistance among Escherichia coli is globally an increasing problem in public healthcare. Understanding the spread of plasmid-mediated ESBL genes is of great importance in elucidating their molecular epidemiology. However, differentiation of subtypes and alleles is frequently hampered by the lack of comprehensive diagnostic tools. We therefore developed a novel universal blaSHV, blaTEM and blaCTX-M subtyping assay based on PCR and Sanger sequencing that results in large amplicons of >700 bp, enabling differentiation of bla alleles as precisely as possible. The assay was established using 10 reference strains with known bla genotypes that represent all examined primer groups and 101 uncharacterized ESBL-producing E. coli of clinical and livestock-associated origins from different German regions. All isolates were tested in parallel with established blaSHV, blaTEM and blaCTX-M subtyping assays for the respective �]-lactamases and their alleles. The novel assay yielded equal (n = 92) or better (n = 47) subtyping results compared with established subtyping methods and reliably detected all expected enzymes in the reference strains. Overall, the occurring enzymes could be differentiated into groups representing one (n = 9), two (n = 5) or three (n = 4) highly similar alleles. Moreover, ESBL and non-ESBL allelic variants of blaSHV and blaTEM occurring in the same isolate were distinguished reliably. We established a highly discriminatory assay for the subtyping of clinically important ESBL genes that can easily be used in epidemiological analyses.
|
1519. |
Sun J,
Deng H,
Li L,
Chen MY,
Fang LX,
Yang QE,
Liu YH,
Liao XP,
( 2015 ) Complete nucleotide sequence of cfr-carrying IncX4 plasmid pSD11 from Escherichia coli. PMID : 25403661 : DOI : 10.1128/AAC.04388-14 PMC : PMC4291432 Abstract >>
We report the complete nucleotide sequence of a plasmid carrying the multiresistance gene cfr. This plasmid was isolated from an Escherichia coli strain of swine origin in 2011. This 37,672-bp plasmid, pSD11, had an IncX4 backbone similar to those of the IncX4 plasmids obtained from the United States and Australia, in which the cfr gene was flanked by two copies of IS26 and a truncated Tn1331 was inserted.
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1520. |
Rendueles O,
Beloin C,
Latour-Lambert P,
Ghigo JM,
( 2014 ) A new biofilm-associated colicin with increased efficiency against biofilm bacteria. PMID : 24451204 : DOI : 10.1038/ismej.2013.238 PMC : PMC4030232 Abstract >>
Formation of bacterial biofilm communities leads to profound physiological modifications and increased physical and metabolic exchanges between bacteria. It was previously shown that bioactive molecules produced within the biofilm environment contribute to bacterial interactions. Here we describe new pore-forming colicin R, specifically produced in biofilms formed by the natural isolate Escherichia coli ROAR029 but that cannot be detected under planktonic culture conditions. We demonstrate that an increased SOS stress response within mature biofilms induces SOS-dependent colicin R expression. We provide evidence that colicin R displays increased activity against E. coli strains that have a reduced lipopolysaccharide length, such as the pathogenic enteroaggregative E. coli LF82 clinical isolate, therefore pointing to lipopolysaccharide size as an important determinant for resistance to colicins. We show that colicin R toxicity toward E. coli LF82 is increased under biofilm conditions compared with planktonic susceptibility and that release of colicin R confers a strong competitive advantage in mixed biofilms by rapidly outcompeting sensitive neighboring bacteria. This work identifies the first biofilm-associated colicin that preferentially targets biofilm bacteria. Furthermore, it indicates that the study of antagonistic molecules produced in biofilm and multispecies contexts could reveal unsuspected, ecologically relevant bacterial interactions influencing population dynamics in natural environments.
|
1521. |
Chen XY,
Woodward A,
Zijlstra RT,
Gänzle MG,
( 2014 ) Exopolysaccharides synthesized by Lactobacillus reuteri protect against enterotoxigenic Escherichia coli in piglets. PMID : 25015886 : DOI : 10.1128/AEM.01782-14 PMC : PMC4178603 Abstract >>
Enterotoxigenic Escherichia coli (ETEC) is a major cause of diarrhea in piglets; ETEC cells colonize the intestinal mucosa with adhesins and deliver toxins that cause fluid loss. This study determined the antiadhesive properties of bacterial exopolysaccharides (reuteran and levan) and related glycans (dextran and inulin) in a small intestinal segment perfusion (SISP) model. The SISP model used 10 jejunal segments from 5-week-old piglets. Five segments were infected with ETEC expressing K88 fimbriae (ETEC K88), while five segments were treated with saline. Every two segments (ETEC and non-ETEC infected) were infused with 65 ml of 10 g liter(-1) of glycans or saline (control) for 8 h. High-resolution melting-curve (HRM) quantitative PCR (qPCR) indicated that E. coli is the dominant bacterium in infected segments, while other bacteria were predominant in noninfected segments. Infection by ETEC K88 was also verified by qPCR; gene copy numbers of K88 fimbriae and the heat-labile toxin (LT) in mucosal scrapings and outflow fluid of infected segments were significantly higher than those in noninfected segments. Genes coding for K88 fimbriae and LT were also detected in noninfected segments. LT amplicons from infected and noninfected segments were 99% identical over 481 bp, demonstrating the presence of autochthonous ETEC K88. All glycans reduced fluid loss caused by ETEC K88 infection. Reuteran tended (P = 0.06) to decrease ETEC K88 levels in mucosal scraping sample, as judged by qPCR. Fluorescent in situ hybridization analysis demonstrated that reuteran significantly (P = 0.012) decreased levels of adherent ETEC K88. Overall, reuteran may prevent piglet diarrhea by reducing adhesion of ETEC K88.
|
1522. |
Scaletsky IC,
Nascimento HH,
Silva LE,
Souza RT,
Silva NP,
( 2014 ) Phenotypic and genotypic characteristics associated with biofilm formation in clinical isolates of atypical enteropathogenic Escherichia coli (aEPEC) strains. PMID : 25012525 : DOI : 10.1186/1471-2180-14-184 PMC : PMC4100040 Abstract >>
Biofilm formation by enteropathogenic Escherichia coli (EPEC) have been recently described in the prototype typical EPEC E2348/69 strain and in an atypical EPEC O55:H7 strain. In this study, we sought to evaluate biofilm formation in a collection of 126 atypical EPEC strains isolated from 92 diarrheic and 34 nondiarrheic children, belonging to different serotypes. The association of biofilm formation and adhesin-related genes were also investigated. Biofilm formation occurred in 37 (29%) strains of different serotypes, when the assays were performed at 26�XC and 37�XC for 24 h. Among these, four strains (A79, A87, A88, and A111) formed a stronger biofilm than did the others. The frequency of biofilm producers was higher among isolates from patients compared with isolates from controls (34.8% vs 14.7%; P = 0.029). An association was found between biofilm formation and expression of type 1 fimbriae and curli (P < 0.05). Unlike the previously described aEPEC O55:H7, one aEPEC O119:HND strain (A111) formed a strong biofilm and pellicle at the air-liquid interface, but did not express curli. Transposon mutagenesis was used to identify biofilm-deficient mutants. Transposon insertion sequences of six mutants revealed similarity with type 1 fimbriae (fimC, fimD, and fimH), diguanylate cyclase, ATP synthase F1, beta subunit (atpD), and the uncharacterized YjiC protein. All these mutants were deficient in biofilm formation ability. This study showed that the ability to adhere to abiotic surfaces and form biofilm is present in an array of aEPEC strains. Moreover, it seems that the ability to form biofilms is associated with the presence of type 1 fimbriae and diguanylate cyclase. Characterization of additional biofilm formation mutants may reveal other mechanisms involved in biofilm formation and bring new insights into aEPEC adhesion and pathogenesis.
|
1523. |
Kartsev NN,
Fursova NK,
Pachkunov DM,
Bannov VA,
Eruslanov BV,
Svetoch EA,
Dyatlov IA,
( 2015 ) Molecular Characterization of Enterotoxin-Producing Escherichia coli Collected in 2011-2012, Russia. PMID : 25923803 : DOI : 10.1371/journal.pone.0123357 PMC : PMC4414545 Abstract >>
Enterotoxin-producing Escherichia coli (ETEC) are one of the main causative agents of diarrhea in children especially in developing countries and travel diarrhoea in adults. Pathogenic properties of ETEC associated with their ability to produce a heat-stable (ST) and/or heat-labile (LT) enterotoxins, as well as adhesins providing bacterial adhesion to intestinal epithelial cells. This study presents the molecular characterization of the ETEC isolates collected from the Central and Far-Eastern regions of Russia in 2011-2012. It was shown that all ETEC under study (n=18) had the heat-labile enterotoxin-coding operon elt, and had no the genes of the heat-stable enterotoxin operon est. DNA sequencing revealed two types of nucleotide exchanges in the eltB gene coding subunit B of LT in isolates collected from Cherepovets city (Central region, Russia) and Vladivostok city (Far-East region, Russia). Only one ETEC strain carried genes cfaA, cfaB, cfaC and cfaD coding adhesion factor CFA/I. Expression of LT in four ETEC isolates in the agglutination reaction was detected using a latex test-system. The isolates were assigned to serogroups O142 (n = 6), ?6 (n = 4), ?25 (n = 5), ?26 (n = 2), and O115 (n = 1). Genotyping showed that they belonged to an earlier described sequence-type ST4 (n = 3) as well as to 11 novel sequence-types ST1043, ST1312, ST3697, ST3707, ST3708, ST3709, ST3710, ST3755, ST3756, ST3757 and ST4509. The ETEC isolates displayed different levels of antimicrobial resistance. Eight isolates were resistant to only one drug, three isolates-to two drugs, one isolate-to three drugs, two isolates-to four antibacterials, and only one isolate to each of the five, six and ten antibacterials simultaneously. Genetic determinants of the resistance to beta-lactams and other classes of antibacterials on the ETEC genomes were identified. There are blaTEM (n = 10), blaCTX-M-15 (n = 1), class 1 integron (n = 3) carrying resistance cassettes to aminoglycosides and sulphonamides dfrA17-aadA5 and dfrA12-orfF-aadA2. One isolate ETEC_Ef-6 was found to be a multidrug-resistant (MDR) pathogen that carried both the beta-lactamase gene and class 1 integron. These data suggest the circulation of ETEC in Russia. Further investigations are necessary to study the spread of the revealed ETEC sequence types (STs) and serotypes. Their role in the etiology of diarrhea should be also estimated.
|
1524. |
Spitaels F,
Wieme AD,
Janssens M,
Aerts M,
Daniel HM,
Van Landschoot A,
De Vuyst L,
Vandamme P,
( 2014 ) The microbial diversity of traditional spontaneously fermented lambic beer. PMID : 24748344 : DOI : 10.1371/journal.pone.0095384 PMC : PMC3991685 Abstract >>
Lambic sour beers are the products of a spontaneous fermentation that lasts for one to three years before bottling. The present study determined the microbiota involved in the fermentation of lambic beers by sampling two fermentation batches during two years in the most traditional lambic brewery of Belgium, using culture-dependent and culture-independent methods. From 14 samples per fermentation, over 2000 bacterial and yeast isolates were obtained and identified. Although minor variations in the microbiota between casks and batches and a considerable species diversity were found, a characteristic microbial succession was identified. This succession started with a dominance of Enterobacteriaceae in the first month, which were replaced at 2 months by Pediococcus damnosus and Saccharomyces spp., the latter being replaced by Dekkera bruxellensis at 6 months fermentation duration.
|
1525. |
Prager R,
Lang C,
Aurass P,
Fruth A,
Tietze E,
Flieger A,
( 2014 ) Two novel EHEC/EAEC hybrid strains isolated from human infections. PMID : 24752200 : DOI : 10.1371/journal.pone.0095379 PMC : PMC3994036 Abstract >>
The so far highest number of life-threatening hemolytic uremic syndrome was associated with a food-borne outbreak in 2011 in Germany which was caused by an enterohemorrhagic Escherichia coli (EHEC) of the rare serotype O104:H4. Most importantly, the outbreak strain harbored genes characteristic of both EHEC and enteroaggregative E. coli (EAEC). Such strains have been described seldom but due to the combination of virulence genes show a high pathogenicity potential. To evaluate the importance of EHEC/EAEC hybrid strains in human disease, we analyzed the EHEC strain collection of the German National Reference Centre for Salmonella and other Bacterial Enteric Pathogens (NRC). After exclusion of O104:H4 EHEC/EAEC strains, out of about 2400 EHEC strains sent to NRC between 2008 and 2012, two strains exhibited both EHEC and EAEC marker genes, specifically were stx2 and aatA positive. Like the 2011 outbreak strain, one of the novel EHEC/EAEC harbored the Shiga toxin gene type stx2a. The strain was isolated from a patient with bloody diarrhea in 2010, was serotyped as O59:H-, belonged to MLST ST1136, and exhibited genes for type IV aggregative adherence fimbriae (AAF). The second strain was isolated from a patient with diarrhea in 2012, harbored stx2b, was typed as Orough:H-, and belonged to MLST ST26. Although the strain conferred the aggregative adherence phenotype, no known AAF genes corresponding to fimbrial types I to V were detected. In summary, EHEC/EAEC hybrid strains are currently rarely isolated from human disease cases in Germany and two novel EHEC/EAEC of rare serovars/MLST sequence types were characterized.
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1526. |
Ruteshouser EC,
Richardson JP,
( 1989 ) Identification and characterization of transcription termination sites in the Escherichia coli lacZ gene. PMID : 2475637 : DOI : 10.1016/0022-2836(89)90085-5 Abstract >>
The Escherichia coli lacZ gene contains a series of latent transcriptional terminators that are responsible for the polar effects of certain mutations. We demonstrate, using gel electrophoretic size analyses and nuclease S1 mapping procedures, that RNA polymerase terminates RNA synthesis in the vicinity of five positions 180, 220, 379, 421 and 463 base-pairs downstream from the start point during transcription of lacZ DNA in vitro in the presence of rho factor. Termination at all but the 421 position depends on rho factor. In the in vitro assays with 0.05 M-KCl and excess rho (36 nM), the terminators are moderately effective, having efficiencies that range from about 8% at the 180 base-pair site to 56% at the 463 base-pair site. These termination stop points correspond to five of the 11 transcriptional pause sites between 180 and 463 base-pairs. Several stop points also correspond to 3' end points of lacZ mRNA isolated from cells containing the strongly polar lacZ-U118 mutation and from cells starved for serine, thus confirming that these latent terminators are responsible for the polar effect and demonstrating that they also function under a condition of physiological stress that prevents the transcription from being translated properly. Two other potential termination factors, NusA protein and cyclic AMP receptor protein have no effect in vitro on the efficiency of termination at the five lacZ sites.
|
1527. |
Calcuttawala F,
Hariharan C,
Pazhani GP,
Ghosh S,
Ramamurthy T,
( 2015 ) Activity spectrum of colicins produced by Shigella sonnei and genetic mechanism of colicin resistance in conspecific S. sonnei strains and Escherichia coli. PMID : 25331695 : DOI : 10.1128/AAC.04122-14 PMC : PMC4291344 Abstract >>
Colicin-mediated killing is an example of allelopathy, which has been found among several bacteria. Screening of 42 strains of Shigella sonnei isolated from diarrheal patients revealed that 39 (93%) S. sonnei strains were positive for colicin production against Escherichia coli DH5�\. In the PCR-based detection of the colicin types, 36 (92.3%) were identified as E3, 2 (5.1%) as E3 and E8, and 1 (2.6%) as E3 and E2. Representative S. sonnei strains producing heterologous colicins exhibited antagonism against diarrheagenic Escherichia coli (DEC) groups. Although it is known that mutation in the colicin receptor renders the host resistant to colicin, there is a dearth of information on the genetic characterization of such mutants. In the fluctuation test, colicin-resistant E. coli mutants were found to occur spontaneously at the rates of 2.51 �� 10(-8) and 5.52 �� 10(-8) per generation when exposed to colicins E3 and E8 and colicins E3 and E2, respectively. Genotypic characterization of colicin-resistant E. coli (EC(Cr)) and S. sonnei (SS(Cr)) strains displayed mutations in the btuB gene, which encodes the receptor for vitamin B12 uptake. This gene was interrupted by various insertion sequences, such as IS1, IS2, and IS911. Complementation of EC(Cr) and SS(Cr) with plasmid-borne btuB (pbtuB) accomplished restoration of the colicin-susceptible phenotype. The vitamin B12 uptake assay gave an insight into the physiological relevance of the btuB mutation. Our studies provide insights into the latent influence of S. sonnei colicins in governing the existence of some of the shigellae and all of the DEC and the genetic mechanism underlying the emergence of resistance.
|
1528. |
Freire Martín I,
AbuOun M,
Reichel R,
La Ragione RM,
Woodward MJ,
( 2014 ) Sequence analysis of a CTX-M-1 IncI1 plasmid found in Salmonella 4,5,12:i:-, Escherichia coli and Klebsiella pneumoniae on a UK pig farm. PMID : 24729584 : DOI : 10.1093/jac/dku098 Abstract >>
In 2009, CTX-M Enterobacteriaceae and Salmonella isolates were recovered from a UK pig farm, prompting studies into the dissemination of the resistance and to establish any relationships between the isolates. PFGE was used to elucidate clonal relationships between isolates whilst plasmid profiling, restriction analysis, sequencing and PCR were used to characterize the CTX-M-harbouring plasmids. Escherichia coli, Klebsiella pneumoniae and Salmonella 4,5,12:i:- and Bovismorbificans resistant to cefotaxime (n = 65) were recovered and 63 were shown by PCR to harbour a group 1 CTX-M gene. The harbouring hosts were diverse, but the group 1 CTX-M plasmids were common. Three sequenced CTX-M plasmids from E. coli, K. pneumoniae and Salmonella enterica serotype 4,5,12:i:- were identical except for seven mutations and highly similar to IncI1 plasmid ColIb-P9. Two antimicrobial resistance regions were identified: one inserted upstream of yacABC harbouring ISCR2 transposases, sul2 and floR; and the other inserted within shfB of the pilV shufflon harbouring the ISEcp1 transposase followed by blaCTX-M-1. These data suggest that an ST108 IncI1 plasmid encoding a blaCTX-M-1 gene had disseminated across multiple genera on this farm, an example of horizontal gene transfer of the blaCTX-M-1 gene.
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1529. |
Vanwetswinkel S,
Volkov AN,
Sterckx YG,
Garcia-Pino A,
Buts L,
Vranken WF,
Bouckaert J,
Roy R,
Wyns L,
van Nuland NA,
( 2014 ) Study of the structural and dynamic effects in the FimH adhesin upon �\-d-heptyl mannose binding. PMID : 24476493 : DOI : 10.1021/jm401666c Abstract >>
Uropathogenic Escherichia coli cause urinary tract infections by adhering to mannosylated receptors on the human urothelium via the carbohydrate-binding domain of the FimH adhesin (FimHL). Numerous �\-d-mannopyranosides, including �\-d-heptyl mannose (HM), inhibit this process by interacting with FimHL. To establish the molecular basis of the high-affinity HM binding, we solved the solution structure of the apo form and the crystal structure of the FimHL-HM complex. NMR relaxation analysis revealed that protein dynamics were not affected by the sugar binding, yet HM addition promoted protein dimerization, which was further confirmed by small-angle X-ray scattering. Finally, to address the role of Y48, part of the "tyrosine gate" believed to govern the affinity and specificity of mannoside binding, we characterized the FimHL Y48A mutant, whose conformational, dynamical, and HM binding properties were found to be very similar to those of the wild-type protein.
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1530. |
Tucker SD,
Murgola EJ,
Pagel FT,
( 1989 ) Missense and nonsense suppressors can correct frameshift mutations. PMID : 2502189 : DOI : 10.1016/0300-9084(89)90089-8 Abstract >>
Missense and nonsense suppressor tRNAs, selected for their ability to read a new triplet codon, were observed to suppress one or more frameshift mutations in trpA of Escherichia coli. Two of the suppressible frameshift mutants, trpA8 and trpA46AspPR3, were cloned, sequenced, and found to be of the +1 type, resulting from the insertion of four nucleotides and one nucleotide, respectively. Twenty-two suppressor tRNAs were examined, 20 derived from one of the 3 glycine isoacceptor species, one from lysT, and one from trpT. The sequences of all but four of the mutant tRNAs are known, and two of those four were converted to suppressor tRNAs that were subsequently sequenced. Consideration of the coding specificities and anticodon sequences of the suppressor tRNAs does not suggest a unitary mechanism of frameshift suppression. Rather, the results indicate that different suppressors may shift frame according to different mechanisms. Examination of the suppression windows of the suppressible frameshift mutations indicates that some of the suppressors may work at cognate codons, either in the 0 frame or in the +1 frame, and others may act at noncognate codons (in either frame) by some as-yet-unspecified mechanism. Whatever the mechanisms, it is clear that some +1 frameshifting can occur at non-monotonous sequences. A striking example of a frameshifting missense suppressor is a mutant lysine tRNA that differs from wild-type lysine tRNA by only a single base in the amino acid acceptor stem, a C to U70 transition that results in a G.U base pair. It is suggested that when this mutant lysine tRNA reads its cognate codon, AAA, the presence of the G.U base pair sometimes leads either to a conformational change in the tRNA or to an altered interaction with some component of the translation machinery involved in translocation, resulting in a shift of reading frame. In general, the results indicate that translocation is not simply a function of anticodon loop size, that different frameshifting mechanisms may operate with different tRNAs, and that conformational features, some far removed from the anticodon region, are involved in maintaining fidelity in translocation.
|
1531. |
Turner PE,
Williams ES,
Okeke C,
Cooper VS,
Duffy S,
Wertz JE,
( 2014 ) Antibiotic resistance correlates with transmission in plasmid evolution. PMID : 25351426 : DOI : 10.1111/evo.12537 Abstract >>
Conjugative (horizontally transmissible) plasmids are autonomous replicators, whose "self-interests" do not necessarily overlap with those of their hosts. This situation causes plasmids and bacteria to sometimes experience differing selection pressures. Escherichia coli plasmid pB15 contains genes for resistance to several antibiotics, including tetracycline. When plasmid-bearing cells were experimentally evolved in the laboratory, changes in resistance level in the unselected tetracycline marker coincided with changes in plasmid rates of vertical versus horizontal transmission. Here, we used minimum inhibitory assays that measure resistance levels as quantitative traits to determine phenotypic correlations among plasmid characters and to estimate divergence among plasmid lineages. Results suggested that plasmid-level evolution led to formation of two phenotypically dissimilar groups: virulent (highly infectious) and avirulent (weakly infectious) plasmids. In contrast, measures of carbon-source utilization, and fitness assays relative to a common competitor revealed that bacterial hosts generally converged in phenotypic performance, despite divergence among their associated plasmids. Preliminary sequence analyses suggested that divergence in plasmid conjugation was due to altered configurations of a shufflon region (a site-specific recombination system), where genetic rearrangements affect conjugative ability. Furthermore, we proposed that correlated resistance and transmission in pB15 derivatives were caused by a tetracycline-resistance transposon inserted into a transfer operon, allowing transcription from its promoter to simultaneously affect both plasmid resistance and transmission.
|
1532. |
Okello E,
Moonens K,
Erume J,
De Greve H,
( 2015 ) Enterotoxigenic Escherichia coli strains are highly prevalent in Ugandan piggeries but disease outbreaks are masked by antibiotic prophylaxis. PMID : 25311441 : DOI : 10.1007/s11250-014-0694-2 Abstract >>
Post-weaning diarrhea (PWD) caused by enterotoxigenic Escherichia coli (ETEC) is an important disease of newly weaned piglets. ETEC strains commonly express F4 and/or F18 fimbriae that attach to carbohydrate receptors present on the intestinal epithelium during colonization. The disease status in the Ugandan piggeries had previously not been studied. In this cross-sectional sero-survey and clinical outbreak monitoring, we found very high sero-prevalence levels of both anti-F4 (70.5%) and anti-F18 (73.7%) antibodies, despite limited cases of clinical outbreaks. Strains isolated from these cases were typically F18(+) ETEC. High antibiotic resistance and multi-drug resistance were characteristics of the isolates, with highest resistance level of over 95% to commonly used antibiotics such as penicillin and tetracycline. We conclude that ETEC infections are widely spread on farms in Central Uganda but clinical disease outbreaks were masked by the management practices on these farms, like the use of extensive antibiotic prophylaxis.
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1533. |
Altmeyer M,
Amtmann E,
Heyl C,
Marschner A,
Scheidig AJ,
Klein CD,
( 2014 ) Beta-aminoketones as prodrugs for selective irreversible inhibitors of type-1 methionine aminopeptidases. PMID : 25293447 : DOI : 10.1016/j.bmcl.2014.09.047 Abstract >>
We identified and characterized �]-aminoketones as prodrugs for irreversible MetAP inhibitors that are selective for the MetAP-1 subtype. �]-Aminoketones with certain structural features form �\,�]-unsaturated ketones under physiological conditions, which bind covalently and selectively to cysteines in the S1 pocket of MetAP-1. The binding mode was confirmed by X-ray crystallography and assays with the MetAPs from Escherichia coli, Staphylococcus aureus and both human isoforms. The initially identified tetralone derivatives showed complete selectivity for E. coli MetAP versus human MetAP-1 and MetAP-2. Rational design of indanone analogs yielded compounds with selectivity for the human type-1 versus the human type-2 MetAP.
|
1534. |
Zurfluh K,
Wang J,
Klumpp J,
Nüesch-Inderbinen M,
Fanning S,
Stephan R,
( 2014 ) Vertical transmission of highly similar bla CTX-M-1-harboring IncI1 plasmids in Escherichia coli with different MLST types in the poultry production pyramid. PMID : 25324838 : DOI : 10.3389/fmicb.2014.00519 PMC : PMC4179741 Abstract >>
The purpose of this study was to characterize sets of extended-spectrum �]-lactamases (ESBL)-producing Enterobacteriaceae collected longitudinally from different flocks of broiler breeders, meconium of 1-day-old broilers from theses breeder flocks, as well as from these broiler flocks before slaughter. Five sets of ESBL-producing Escherichia coli were studied by multi-locus sequence typing (MLST), phylogenetic grouping, PCR-based replicon typing and resistance profiling. The bla CTX-M-1-harboring plasmids of one set (pHV295.1, pHV114.1, and pHV292.1) were fully sequenced and subjected to comparative analysis. Eleven different MLST sequence types (ST) were identified with ST1056 the predominant one, isolated in all five sets either on the broiler breeder or meconium level. Plasmid sequencing revealed that bla CTX-M-1 was carried by highly similar IncI1/ST3 plasmids that were 105 076 bp, 110 997 bp, and 117 269 bp in size, respectively. The fact that genetically similar IncI1/ST3 plasmids were found in ESBL-producing E. coli of different MLST types isolated at the different levels in the broiler production pyramid provides strong evidence for a vertical transmission of these plasmids from a common source (nucleus poultry flocks).
|
1535. |
Lina TT,
Khajanchi BK,
Azmi IJ,
Islam MA,
Mahmood B,
Akter M,
Banik A,
Alim R,
Navarro A,
Perez G,
Cravioto A,
Talukder KA,
( 2014 ) Phenotypic and molecular characterization of extended-spectrum beta-lactamase-producing Escherichia coli in Bangladesh. PMID : 25302491 : DOI : 10.1371/journal.pone.0108735 PMC : PMC4193765 Abstract >>
Resistance to cephalosporins in Enterobacteriaceae is mainly due to the production of extended-spectrum beta-lactamase (ESBL). Little is known about ESBL-producing bacteria in Bangladesh. Therefore, the study presents results of phenotypic and molecular characterization of ESBL-producing Escherichia coli from hospitals in Bangladesh. A total of 339 E. coli isolated from patients with urinary tract and wound infections attending three different medical hospitals in urban and rural areas of Bangladesh between 2003-2007 were screened for ESBL-production by the double disk diffusion test. Isolates with ESBL-phenotype were further characterized by antibiotic susceptibility testing, PCR and sequencing of different �]-lactamase and virulence genes, serotyping, and XbaI-macrorestriction followed by pulsed-field gel electrophoresis (PFGE). We identified 40 E. coli with ESBL phenotype. These isolates were resistant to ceftriaxone, ceftazidime, cefotaxime, aztreonam, cefepime, and nalidixic acid but remained susceptible to imipenem. All but one isolate were additionally resistant to ciprofloxacin, and 3 isolates were resistant to cefoxitin. ESBL genes of blaCTX-M-1-group were detected in all isolates; blaTEM-type and blaOXA-1-type genes were detected in 33 (82.5%) and 19 (47.5%) isolates, respectively. Virulence genes that are present in diarrhoeagenic E. coli were not found. Class-1 integron was present in 20 (50%) isolates. All the ESBL-producing E. coli isolates harbored plasmids ranging between 1.1 and 120 MDa. PFGE-typing revealed 26 different pulsotypes, but identical pulsotype showed 6 isolates of serotype O25:H4. The prevalence of multidrug-resistant ESBL-producing E. coli isolates appears to be high and the majority of the isolates were positive for blaCTX-M. Although there was genetic heterogeneity among isolates, presence of a cluster of isolates belonging to serotype O25:H4 indicates dissemination of the pandemic uropathogenic E. coli clone in Bangladesh.
|
1536. |
Pitart C,
Solé M,
Roca I,
Román A,
Moreno A,
Vila J,
Marco F,
( 2015 ) Molecular characterization of blaNDM-5 carried on an IncFII plasmid in an Escherichia coli isolate from a nontraveler patient in Spain. PMID : 25313215 : DOI : 10.1128/AAC.04040-14 PMC : PMC4291412 Abstract >>
A carbapenem-resistant Escherichia coli isolate (sequence type 448 [ST448]) was recovered from a urine culture of a female patient with no recent record of traveling. PCR screening identified the presence of bla(NDM-5), bla(TEM-1), bla(OXA-1), bla(CMY-42), and rmtB. bla(NDM-5) was carried in a conjugative IncFII-type plasmid (90 kb) together with bla(TEM-1) and rmtB. The genetic environment of bla(NDM-5) showed a structure similar to those of pMC-NDM and pGUE-NDM, identified in Poland and France in E. coli of African and Indian origin, respectively.
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1537. |
Hazen TH,
Zhao L,
Boutin MA,
Stancil A,
Robinson G,
Harris AD,
Rasko DA,
Johnson JK,
( 2014 ) Comparative genomics of an IncA/C multidrug resistance plasmid from Escherichia coli and Klebsiella isolates from intensive care unit patients and the utility of whole-genome sequencing in health care settings. PMID : 24914121 : DOI : 10.1128/AAC.02573-14 PMC : PMC4135983 Abstract >>
The IncA/C plasmids have been implicated for their role in the dissemination of �]-lactamases, including gene variants that confer resistance to expanded-spectrum cephalosporins, which are often the treatment of last resort against multidrug-resistant, hospital-associated pathogens. A bla(FOX-5) gene was detected in 14 Escherichia coli and 16 Klebsiella isolates that were cultured from perianal swabs of patients admitted to an intensive care unit (ICU) of the University of Maryland Medical Center (UMMC) in Baltimore, MD, over a span of 3 years. Four of the FOX-encoding isolates were obtained from subsequent samples of patients that were initially negative for an AmpC �]-lactamase upon admission to the ICU, suggesting that the AmpC �]-lactamase-encoding plasmid was acquired while the patient was in the ICU. The genomes of five E. coli isolates and six Klebsiella isolates containing bla(FOX-5) were selected for sequencing based on their plasmid profiles. An ? 167-kb IncA/C plasmid encoding the FOX-5 �]-lactamase, a CARB-2 �]-lactamase, additional antimicrobial resistance genes, and heavy metal resistance genes was identified. Another FOX-5-encoding IncA/C plasmid that was nearly identical except for a variable region associated with the resistance genes was also identified. To our knowledge, these plasmids represent the first FOX-5-encoding plasmids sequenced. We used comparative genomics to describe the genetic diversity of a plasmid encoding a FOX-5 �]-lactamase relative to the whole-genome diversity of 11 E. coli and Klebsiella isolates that carry this plasmid. Our findings demonstrate the utility of whole-genome sequencing for tracking of plasmid and antibiotic resistance gene distribution in health care settings.
|
1538. |
Karczmarczyk M,
Wang J,
Leonard N,
Fanning S,
( 2014 ) Complete nucleotide sequence of a conjugative IncF plasmid from an Escherichia coli isolate of equine origin containing blaCMY-2 within a novel genetic context. PMID : 24386888 : DOI : 10.1111/1574-6968.12364 Abstract >>
A blaCMY-2 -containing conjugative IncF plasmid denoted as pEQ011, previously identified in a multidrug-resistant Escherichia coli isolate of equine origin, was characterized. The plasmid consisted of 85 507 bp, with 118 predicted open reading frames. This is the first known report demonstrating the association of a blaCMY-2 gene with an IncF incompatibility-type plasmid backbone. A novel genetic arrangement was identified wherein the blaCMY-2 resistance gene was proximally flanked by IS1294 along with a partial blc gene located distally and within a yacABC operon.
|
1539. |
Al Bayssari C,
Olaitan AO,
Dabboussi F,
Hamze M,
Rolain JM,
( 2015 ) Emergence of OXA-48-producing Escherichia coli clone ST38 in fowl. PMID : 25348536 : DOI : 10.1128/AAC.03552-14 PMC : PMC4291434 Abstract >>
N/A
|
1540. |
Kim K,
Meyer RJ,
( 1986 ) Copy-number of broad host-range plasmid R1162 is regulated by a small RNA. PMID : 2430262 : DOI : 10.1093/nar/14.20.8027 PMC : PMC311832 Abstract >>
We have shown previously [Kim, K. and Meyer, R.J. (1985) J. Mol. Biol. 185,755-767] that copy-number of the broad host-range plasmid R1162 is controlled by the amounts of two proteins, encoded by cotranscribed genes comprising a region of the plasmid called RepI. We have now demonstrated that expression of RepI is negatively regulated by a 75 base RNA that is complementary to a segment of the RepI message. Increased intracellular amounts of RNA molecules that include this segment relieve the inhibition of RepI gene expression, suggesting that the target for regulation is the mRNA itself. A mutation decreasing the amount of the 75 base RNA results in elevated plasmid copy-number. Thus, consistent with our previous observations, regulation of the expression of the RepI genes is a factor in controlling plasmid copy-number.
|
1541. |
Stokes JM,
Davis JH,
Mangat CS,
Williamson JR,
Brown ED,
( 2014 ) Discovery of a small molecule that inhibits bacterial ribosome biogenesis. PMID : 25233066 : DOI : 10.7554/eLife.03574 PMC : PMC4371806 Abstract >>
While small molecule inhibitors of the bacterial ribosome have been instrumental in understanding protein translation, no such probes exist to study ribosome biogenesis. We screened a diverse chemical collection that included previously approved drugs for compounds that induced cold sensitive growth inhibition in the model bacterium Escherichia coli. Among the most cold sensitive was lamotrigine, an anticonvulsant drug. Lamotrigine treatment resulted in the rapid accumulation of immature 30S and 50S ribosomal subunits at 15 �XC. Importantly, this was not the result of translation inhibition, as lamotrigine was incapable of perturbing protein synthesis in vivo or in vitro. Spontaneous suppressor mutations blocking lamotrigine activity mapped solely to the poorly characterized domain II of translation initiation factor IF2 and prevented the binding of lamotrigine to IF2 in vitro. This work establishes lamotrigine as a widely available chemical probe of bacterial ribosome biogenesis and suggests a role for E. coli IF2 in ribosome assembly.
|
1542. |
Okubo T,
Sato T,
Yokota S,
Usui M,
Tamura Y,
( 2014 ) Comparison of broad-spectrum cephalosporin-resistant Escherichia coli isolated from dogs and humans in Hokkaido, Japan. PMID : 24709044 : DOI : 10.1016/j.jiac.2013.12.003 Abstract >>
Resistance to broad-spectrum cephalosporins (BSCs) in Enterobacteriaceae in companion animals has become a great concern for public health. To estimate the dissemination of BSC-resistant bacteria between dog and human, we examined the BSC-resistance determinants of and genetic similarities between 69 BSC-resistant Escherichia coli isolates derived from canine rectal swabs (n = 28) and human clinical samples (n = 41). Some E. coli isolates possessed blaTEM-1b (14 canine and 16 human isolates), blaCTx-M-2 (6 human isolates), blaCTx-M-14 (3 canine and 14 human isolates), blaCTx-M-27 (1 canine and 15 human isolates), and blaCMY-2 (11 canine and 3 human isolates). The possession of CTX-M-type �]-lactamases was significantly more frequent in human isolates, whereas CMY-2 was more common in canine isolates. Bacterial typing methods (phylogenetic typing, O-antigen serotyping, and pulsed-field gel electrophoresis) showed little clonal relationship between canine isolates and human isolates. Plasmid analysis and Southern blotting indicated that the plasmids encoding CMY-2 were similar among canine and human isolates. Based on the differences in the major �]-lactamase and the divergence of bacterial types between canine and human isolates, it seems that clonal dissemination of BSC-resistant E. coli between canines and humans is limited. The similarity of the CMY-2-encoding plasmid suggests that plasmid-mediated �]-lactamase gene transmission plays a role in interspecies diffusion of BSC-resistant E. coli between dog and human.
|
1543. |
Berry AA,
Yang Y,
Pakharukova N,
Garnett JA,
Lee WC,
Cota E,
Marchant J,
Roy S,
Tuittila M,
Liu B,
Inman KG,
Ruiz-Perez F,
Mandomando I,
Nataro JP,
Zavialov AV,
Matthews S,
( 2014 ) Structural insight into host recognition by aggregative adherence fimbriae of enteroaggregative Escherichia coli. PMID : 25232738 : DOI : 10.1371/journal.ppat.1004404 PMC : PMC4169507 DOI : 10.1371/journal.ppat.1004404 PMC : PMC4169507 Abstract >>
Enteroaggregative Escherichia coli (EAEC) is a leading cause of acute and persistent diarrhea worldwide. A recently emerged Shiga-toxin-producing strain of EAEC resulted in significant mortality and morbidity due to progressive development of hemolytic-uremic syndrome. The attachment of EAEC to the human intestinal mucosa is mediated by aggregative adherence fimbria (AAF). Using X-ray crystallography and NMR structures, we present new atomic resolution insight into the structure of AAF variant I from the strain that caused the deadly outbreak in Germany in 2011, and AAF variant II from archetype strain 042, and propose a mechanism for AAF-mediated adhesion and biofilm formation. Our work shows that major subunits of AAF assemble into linear polymers by donor strand complementation where a single minor subunit is inserted at the tip of the polymer by accepting the donor strand from the terminal major subunit. Whereas the minor subunits of AAF have a distinct conserved structure, AAF major subunits display large structural differences, affecting the overall pilus architecture. These structures suggest a mechanism for AAF-mediated adhesion and biofilm formation. Binding experiments using wild type and mutant subunits (NMR and SPR) and bacteria (ELISA) revealed that despite the structural differences AAF recognize a common receptor, fibronectin, by employing clusters of basic residues at the junction between subunits in the pilus. We show that AAF-fibronectin attachment is based primarily on electrostatic interactions, a mechanism not reported previously for bacterial adhesion to biotic surfaces.
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1544. |
Wang J,
Stephan R,
Power K,
Yan Q,
Hächler H,
Fanning S,
( 2014 ) Nucleotide sequences of 16 transmissible plasmids identified in nine multidrug-resistant Escherichia coli isolates expressing an ESBL phenotype isolated from food-producing animals and healthy humans. PMID : 24920651 : DOI : 10.1093/jac/dku206 Abstract >>
Nine extended-spectrum �]-lactamase (ESBL)-producing Escherichia coli isolated from healthy humans and food-producing animals were found to transfer their cefotaxime resistance marker at high frequency in laboratory conjugation experiments. The objective of this study was to completely characterize 16 transmissible plasmids that were detected in these bacterial isolates. The nucleotide sequences of all 16 plasmids were determined from transconjugants using next-generation sequencing technology. Open reading frames were assigned using Rapid Annotation using Subsystem Technology and analysed by BLASTn and BLASTp. The standard method was used for plasmid multilocus sequence typing (pMLST) analysis. Plasmid structures were subsequently confirmed by PCR amplification of selected regions. The complete circularized nucleotide sequence of 14 plasmids was determined, along with that of a further two plasmids that could not be confirmed as closed. These ranged in size from 1.8 to 166.6 kb. Incompatibility groups and pMLSTs identified included IncI1/ST3, IncI1/ST36, IncN/ST1, IncF and IncB/O, and those of the same Inc types presented a similar backbone structure despite being isolated from different sources. Eight plasmids contained bla(CTX-M-1) genes that were associated with either ISEcp1 or IS26 insertion sequence elements. Six plasmids isolated from humans and chickens were identical or closely related to the IncI1 reference plasmid, R64. These data, based on comparative sequence analysis, highlight the successful spread of blaESBL-harbouring plasmids of different Inc types among isolates of human and food-producing animal origin and provide further evidence for potential dissemination routes.
|
1545. |
Chen X,
He L,
Li Y,
Zeng Z,
Deng Y,
Liu Y,
Liu JH,
( 2014 ) Complete sequence of a F2:A-:B- plasmid pHN3A11 carrying rmtB and qepA, and its dissemination in China. PMID : 25236985 : DOI : 10.1016/j.vetmic.2014.08.023 Abstract >>
Previous studies have confirmed that the spread of rmtB and qepA was mainly mediated by similar F2:A-:B- plasmids. In this study, a representative rmtB and qepA-harbouring F2:A-:B- plasmid, pHN3A11, originating from an Escherichia coli strain of feline origin, was fully sequenced and compared with other IncFII plasmids. pHN3A11 is 76,626bp long with a backbone similar to that of the IncFII plasmids obtained from China (pHK23a, pFOS-HK151325, pXZ) and Canada (pC15-1a). It contains genes encoding addiction (pemI/pemK, hok/mok/sok) and partitioning (parM, parB, and stbB) systems that promote plasmid maintenance during vertical transmission. rmtB, qepA, blaTEM-1, and dfr were found in previously observed contexts, interspersed with different complete or truncated insertion sequences and transposons (�GIS1, �GTn2, �GintI1, ISCR3, 3 IS26, Tn21). Further analyses confirmed that pHN3A11-like plasmids have disseminated in E. coli isolates from pets, food animals and farm environments in China. The successful dissemination of F2:A-:B- type multidrug resistant plasmid among animals may represent a public health risk, and may further worsen the clinical impact.
|
1546. |
Fiett J,
Baraniak A,
Izdebski R,
Sitkiewicz I,
Żabicka D,
Meler A,
Filczak K,
Hryniewicz W,
Gniadkowski M,
( 2014 ) The first NDM metallo-�]-lactamase-producing Enterobacteriaceae isolate in Poland: evolution of IncFII-type plasmids carrying the bla(NDM-1) gene. PMID : 24247128 : DOI : 10.1128/AAC.01197-13 PMC : PMC3910837 Abstract >>
Poland's first Enterobacteriaceae isolate producing the New Delhi metallo-�]-lactamase (NDM) was identified in August 2011. Escherichia coli sequence type ST410 NDM-1 was cultured from a critically ill patient who had been transferred directly from the Congo. The blaNDM-1 gene was carried by conjugative IncFII-type plasmid pMC-NDM (87,619 bp), which showed structural similarity to plasmid pGUE-NDM, which was identified earlier in France in an E. coli ST131 isolate of Indian origin.
|
1547. |
San Francisco MJ,
Tisa LS,
Rosen BP,
( 1989 ) Identification of the membrane component of the anion pump encoded by the arsenical resistance operon of R-factor R773. PMID : 2523997 : DOI : 10.1111/j.1365-2958.1989.tb00098.x Abstract >>
The arsenical resistance (ars) operon of the conjugative R-factor R773 encodes an ATP-driven anion extrusion pump, producing bacterial resistance to arsenicals. There are three structural genes, of which the product of the middle gene, arsB, has not previously been identified. From nucleotide sequence data, the ArsB protein is predicted to be a 45577 Dalton hydrophobic protein. A mini-Mu transposition procedure was used to construct an arsB-lacZ gene fusion, producing a hybrid ArsB-beta-galactosidase protein which was localized in the inner membrane. The operon was cloned into a T7 RNA polymerase expression vector. In addition to the previously identified ArsA and ArsC proteins, the cells synthesized an inner membrane protein with an apparent mass of 36 kD identified as the ArsB protein.
|
1548. |
Lampson BC,
Sun J,
Hsu MY,
Vallejo-Ramirez J,
Inouye S,
Inouye M,
( 1989 ) Reverse transcriptase in a clinical strain of Escherichia coli: production of branched RNA-linked msDNA. PMID : 2466332 : DOI : 10.1126/science.2466332 Abstract >>
Branched RNA-linked multicopy single-stranded DNA (msDNA) originally detected in myxobacteria has now been found in a clinical isolate of Escherichia coli. Although lacking homology in the primary structure, the E. coli msDNA is similar in secondary structure to the myxobacterial msDNA's, including the 2',5'-phosphodiester linkage between RNA and DNA. A chromosomal DNA fragment responsible for the production of msDNA was cloned in an E. coli K12 strain; its DNA sequence revealed an open reading frame (ORF) of 586 amino acid residues. The ORF shows sequence similarity with retroviral reverse transcriptases and ribonuclease H. Disruption of the ORF blocked msDNA production, indicating that this gene is essential for msDNA synthesis.
|
1549. |
Wang D,
Liang H,
Chen J,
Mou Y,
Qi Y,
( 2014 ) Structural and environmental features of novel mdfA variant and mdfA genes in recombinant regions of Escherichia coli. PMID : 24684286 : DOI : 10.1089/mdr.2013.0201 Abstract >>
Novel mdfA gene variants were identified simultaneously from 3 of 13 positive isolates of PCR amplification in Escherichia coli from patients. These 13 positive isolates showed resistance to chloramphenicol, tetracycline, and erythromycin. The 3 mdfA gene variants were of the same genotype and all the 13 positive isolates were investigated by conjugation experiment, EcoRI restriction, and gene mapping. Conjugation experiments demonstrated that the novel mdfA variant and mdfA genes were located on plasmids that were restricted by EcoRI for ?8.2 kb-length, which was also validated by gene mapping. Further study indicated three types of genetic structures (A, B, and C) in the recombinant plasmids harboring mdfA and surrounding genes, and structure B was first reported in the article. Structure A comprises two partial-length and six full-length genes, including the mdfA gene variant in the recombinant plasmid; structure B comprises four full-length genes, the mdfA, ybjG, dacC, and ybjI; structure C comprises two full-length genes, the mdfA and dacC. These results suggested that the mdfA gene can function as transporter responsible for multidrug resistance and also mediated the synergistic function with its surrounding genes in conjugative plasmids.
|
1550. |
Futai K,
( 2014 ) Attenuated colicin-based screening to discover and create novel resistance genes. PMID : 24657850 : DOI : 10.1016/j.mimet.2014.03.003 Abstract >>
I formulated a systematic approach to screen for colicin resistance-associated genes by using an attenuated colicin mutant and demonstrated its utility in a screen of genes related to colicin E5 resistance. Screening of an Escherichia coli genome library revealed rstA as a partial resistance gene to colicin E5. Transcript expression of BtuB and OmpF, proteins responsible for translocation of nuclease E colicins, was clearly inhibited in an rstA-overexpressing strain. In addition, the tatA::recN fusion gene provides resistance to an attenuated colicin E5 mutant. Improving tatA::recN by directed coevolution, I created a novel gene with enhanced resistance to colicin E5 and concluded that attenuated colicin-based screening is useful for the discovery and creation of novel colicin resistance-associated genes.
|
1551. |
Roos G,
Wellens A,
Touaibia M,
Yamakawa N,
Geerlings P,
Roy R,
Wyns L,
Bouckaert J,
( 2013 ) Validation of Reactivity Descriptors to Assess the Aromatic Stacking within the Tyrosine Gate of FimH. PMID : 24900609 : DOI : 10.1021/ml400269v PMC : PMC4027451 Abstract >>
Antagonists of the FimH adhesin, a protein almost universally present at the extremity of type-1 fimbriae expressed by Escherichia coli, have been abundantly in the spotlight as alternative treatments of urinary tract infections. The antagonists function as bacterial antiadhesives through highly specific �\-d-mannose binding in a charged and polar pocket at the tip of the FimH lectin domain and by the stacking of alkyl or aromatic moieties substituted on the mannose with two tyrosine residues (Tyr48 and Tyr137) at the entrance of the mannose-binding pocket. Using high-resolution crystal data, interaction energies are calculated for the different observed aromatic stacking modes between the tyrosines and the antagonist. The dispersion component of the interaction energy correlates with the observed electron density. The quantum chemical reactivity descriptors local hardness and polarizability were successfully validated as prediction tools for ligand affinity in the tyrosine gate of FimH and therefore have potential for rapid drug screening.
|
1552. |
Alves MS,
Pereira A,
Araújo SM,
Castro BB,
Correia AC,
Henriques I,
( 2014 ) Seawater is a reservoir of multi-resistant Escherichia coli, including strains hosting plasmid-mediated quinolones resistance and extended-spectrum beta-lactamases genes. PMID : 25191308 : DOI : 10.3389/fmicb.2014.00426 PMC : PMC4138442 Abstract >>
The aim of this study was to examine antibiotic resistance (AR) dissemination in coastal water, considering the contribution of different sources of fecal contamination. Samples were collected in Berlenga, an uninhabited island classified as Natural Reserve and visited by tourists for aquatic recreational activities. To achieve our aim, AR in Escherichia coli isolates from coastal water was compared to AR in isolates from two sources of fecal contamination: human-derived sewage and seagull feces. Isolation of E. coli was done on Chromocult agar. Based on genetic typing 414 strains were established. Distribution of E. coli phylogenetic groups was similar among isolates of all sources. Resistances to streptomycin, tetracycline, cephalothin, and amoxicillin were the most frequent. Higher rates of AR were found among seawater and feces isolates, except for last-line antibiotics used in human medicine. Multi-resistance rates in isolates from sewage and seagull feces (29 and 32%) were lower than in isolates from seawater (39%). Seawater AR profiles were similar to those from seagull feces and differed significantly from sewage AR profiles. Nucleotide sequences matching resistance genes bla TEM, sul1, sul2, tet(A), and tet(B), were present in isolates of all sources. Genes conferring resistance to 3rd generation cephalosporins were detected in seawater (bla CTX-M-1 and bla SHV-12) and seagull feces (bla CMY-2). Plasmid-mediated determinants of resistance to quinolones were found: qnrS1 in all sources and qnrB19 in seawater and seagull feces. Our results show that seawater is a relevant reservoir of AR and that seagulls are an efficient vehicle to spread human-associated bacteria and resistance genes. The E. coli resistome recaptured from Berlenga coastal water was mainly modulated by seagulls-derived fecal pollution. The repertoire of resistance genes covers antibiotics critically important for humans, a potential risk for human health.
|
1553. |
Brouwer MS,
Bossers A,
Harders F,
van Essen-Zandbergen A,
Mevius DJ,
Smith HE,
( 2014 ) Complete Genome Sequences of IncI1 Plasmids Carrying Extended-Spectrum �]-Lactamase Genes. PMID : 25169863 : DOI : 10.1128/genomeA.00859-14 PMC : PMC4148731 Abstract >>
Extended spectrum beta-lactamases (ESBLs) confer resistance to clinically relevant antibiotics. Often, the resistance genes are carried by conjugative plasmids which are responsible for dissemination. Five IncI1 plasmids carrying ESBLs from commensal and clinical Escherichia coli isolates were completely sequenced and annotated along with a non-ESBL carrying IncI1 plasmid.
|
1554. |
Tschauner K,
Hörnschemeyer P,
Müller VS,
Hunke S,
( 2014 ) Dynamic interaction between the CpxA sensor kinase and the periplasmic accessory protein CpxP mediates signal recognition in E. coli. PMID : 25207645 : DOI : 10.1371/journal.pone.0107383 PMC : PMC4160245 Abstract >>
Two-component systems, consisting of an inner membrane sensor kinase and a cytosolic response regulator, allow bacteria to respond to changes in the environment. Some two-component systems are additionally orchestrated by an accessory protein that integrates additional signals. It is assumed that spatial and temporal interaction between an accessory protein and a sensor kinase modifies the activity of a two-component system. However, for most accessory proteins located in the bacterial envelope the mechanistic details remain unclear. Here, we analyzed the interaction between the periplasmic accessory protein CpxP and the sensor kinase CpxA in Escherichia coli in dependency of three specific stimuli. The Cpx two-component system responds to envelope stress and plays a pivotal role for the quality control of multisubunit envelope structures, including type three secretion systems and pili of different pathogens. In unstressed cells, CpxP shuts off the Cpx response by a yet unknown mechanism. We show for the first time the physical interaction between CpxP and CpxA in unstressed cells using bacterial two-hybrid system and membrane-Strep-tagged protein interaction experiments. In addition, we demonstrate that a high salt concentration and the misfolded pilus subunit PapE displace CpxP from the sensor kinase CpxA in vivo. Overall, this study provides clear evidence that CpxP modulates the activity of the Cpx system by dynamic interaction with CpxA in response to specific stresses.
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1555. |
Sakikawa T,
Akimoto S,
Ohnishi Y,
( 1989 ) The pnd gene in E. coli plasmid R16: nucleotide sequence and gene expression leading to cell Mg2+ release and stable RNA degradation. PMID : 2465777 : DOI : 10.1016/0167-4781(89)90034-1 Abstract >>
The pnd gene promotes the degradation of stable RNA in the presence of rifampicin at 42 degrees C, but is repressed during normal growth (Ohnishi, Y. and Akimoto, S. (1980) J. Bacteriol. 144, 833-835). We have determined the sequence of a third srnB-pnd-type gene, and have analyzed the effects of its expression from an inducible promoter. The nucleotide sequence of the pnd gene of the R16 plasmid exhibits an open reading frame for a polypeptide with 50 amino-acid residues, with high sequence homology to the pnd gene of a plasmid (R483) of a different incompatibility group. A possible base-paired stem and loop structure, which may participate in the regulation of gene expression, was detected between the promoter and the initiation codon, analogous to that in two comparable genes, srnB in the F and pnd in the R483 plasmid. When bacterial cells containing a lac-pnd fusion plasmid were incubated with a lac inducer at 30 degrees C, magnesium was released from the cells in bulk, and spheroplasts of the cells lysed even in hypertonic solution. Furthermore, when Mg2+ efflux was inhibited in the medium containing 5 mM Mg2+ or in Tris-HCl buffer, the degradation of stable RNA at 42 degrees C was inhibited. These results suggest that expression of the pnd gene effects a release of cellular magnesium by a membrane alterations, resulting in the stable RNA degradation at a higher temperature.
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1556. |
Moura A,
Araújo S,
Alves MS,
Henriques I,
Pereira A,
Correia AC,
( 2014 ) The contribution of Escherichia coli from human and animal sources to the integron gene pool in coastal waters. PMID : 25161650 : DOI : 10.3389/fmicb.2014.00419 PMC : PMC4129628 Abstract >>
To understand the contribution of animal- and human-derived fecal pollution sources in shaping integron prevalence and diversity in beach waters, 414 Escherichia coli strains were collected from beach waters (BW, n = 166), seagull feces (SF, n = 179), and wastewaters (WW, n = 69), on the World Biosphere Reserve of the Berlenga Island, Portugal. Statistical differences were found between the prevalence of integrons in BW (21%) and WW (10%), but not between BW and SF (19%). The majority of integrase-positive (intI (+))-strains affiliated to commensal phylogroups B1 (37%), A0 (24%), and A1 (20%). Eighteen different gene cassette arrays were detected, most of them coding for resistances to aminoglycosides, trimethoprim, chloramphenicol, and quaternary ammonia compounds. Common arrays were found among strains from different sources. Multi-resistance to three or more different classes of antibiotics was observed in 89, 82, and 57% of intI (+)-strains from BW, SF and WW, respectively. Plasmids were detected in 79% of strains (60/76) revealing a high diversity of replicons in all sources, mostly belonging to IncF (Frep, FIA, and FIB subgroups), IncI1, IncN, IncY, and IncK incompatibility groups. In 20% (15/76) of strains, integrons were successfully mobilized through conjugation to E. coli CV601. Results obtained support the existence of a diverse integron pool in the E. coli strains from this coastal environment, associated with different resistance traits and plasmid incompatibility groups, mainly shaped by animal fecal pollution inputs. These findings underscore the role of wild life in dissemination of integrons and antibiotic resistance traits in natural environments.
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1557. |
Chen L,
Chavda KD,
Melano RG,
Jacobs MR,
Koll B,
Hong T,
Rojtman AD,
Levi MH,
Bonomo RA,
Kreiswirth BN,
( 2014 ) Comparative genomic analysis of KPC-encoding pKpQIL-like plasmids and their distribution in New Jersey and New York Hospitals. PMID : 24614371 : DOI : 10.1128/AAC.00120-14 PMC : PMC3993205 Abstract >>
The global spread of Klebsiella pneumoniae carbapenemase (KPC) is predominately associated with K. pneumoniae strains genotyped as sequence type 258 (ST258). The first ST258-associated plasmid, pKpQIL, was described in Israel in 2006, but its history in the northeastern United States remains unknown. Six pKpQIL-like plasmids from four K. pneumoniae isolates (three ST258 and one ST234), one Escherichia coli isolate, and one Enterobacter aerogenes isolate, collected from 2003 to 2010 in New York (NY) and New Jersey (NJ) hospitals, were completely sequenced. The sequences and overall sizes of the six plasmids are highly similar to those of pKpQIL; the major difference is that five of six NJ/NY strains harbor blaKPC-2, while pKpQIL contains blaKPC-3. Moreover, a 26.7-kb fragment was inverted in pKpQIL-234 (from ST234 K. pneumoniae), while a 14.5-kb region was deleted in pKpQIL-Ec (from ST131 E. coli). PCR screening of 284 other clinical K. pneumoniae isolates identified 101 (35.6%) harboring pKpQIL-like plasmids from 9 of 10 surveyed hospitals, demonstrating the wide dissemination of pKpQIL in this region of endemicity. Among the positive isolates, 87.1% were typed as ST258 and 88.1% carried blaKPC-2. The finding of pKpQIL-like plasmid in this study from strains that predate the initial report of KPC in Israel provides evidence that pKpQIL may have originated in the United States. Our findings demonstrate that pKpQIL plasmids are both spreading clonally in ST258 strains and spreading horizontally to different sequence types and species, further highlighting the clinical and public health concerns associated with carbapenem resistance.
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1558. |
Tian GB,
Huang YM,
Fang ZL,
Qing Y,
Zhang XF,
Huang X,
( 2014 ) CTX-M-137, a hybrid of CTX-M-14-like and CTX-M-15-like �]-lactamases identified in an Escherichia coli clinical isolate. PMID : 24777903 : DOI : 10.1093/jac/dku126 Abstract >>
To characterize a novel CTX-M chimera, CTX-M-137, from Escherichia coli clinical isolates in China. Isolates were collected from five hospitals between 22 February 2009 and 20 December 2011. Resistance genes were investigated by PCR. blaCTX-M-137 was cloned and purified for kinetic measurements. Conjugation experiments, S1-PFGE and Southern blotting were performed to study the plasmid harbouring blaCTX-M-137. The genetic environment of blaCTX-M-137 was determined by genomic cloning and sequencing. A total of 247 cephalosporin-resistant E. coli were identified. blaCTX-M group genes were the most prevalent extended-spectrum �]-lactamase (ESBL) genes, with 71 isolates harbouring blaCTX-M-1 group genes and 137 isolates harbouring blaCTX-M-9 group genes. A novel chimera of CTX-M-14-like and CTX-M-15-like ESBLs, designated CTX-M-137, was identified from a 60-year-old man with a urinary tract infection. The N-terminus of CTX-M-137 matched CTX-M-14 and the C-terminus matched CTX-M-15. CTX-M-137 conferred resistance to ceftazidime, cefotaxime and aztreonam. Purified CTX-M-137 showed good hydrolytic activity against ceftazidime and cefotaxime, and was inhibited by clavulanic acid. The blaCTX-M-137 was carried on an ?83 kb IncI1 plasmid. blaCTX-M-137 was carried on a complete transposition unit ISEcp1-blaCTX-M-137-�Gorf477 inserted into yagA, which is part of the IncI1 plasmid backbone. We identified a novel CTX-M chimera, CTX-M-137, with a CTX-M-14-like N-terminus and a CTX-M-15-like C-terminus. Our findings suggest an ongoing diversification of CTX-M-type ESBLs through recombination events.
|
1559. |
Chen CJ,
Wu TL,
Lu PL,
Chen YT,
Fung CP,
Chuang YC,
Lin JC,
Siu LK,
( 2014 ) Closely related NDM-1-encoding plasmids from Escherichia coli and Klebsiella pneumoniae in Taiwan. PMID : 25144712 : DOI : 10.1371/journal.pone.0104899 PMC : PMC4140731 Abstract >>
Two plasmids carrying blaNDM-1 isolated from carbapenem-resistant Klebsiella pneumoniae (CR-KP) and carbapenem-resistant Escherichia coli (CR-EC) were sequenced. CR-KP and CR-EC were isolated from two Taiwanese patients without travel histories. Complete sequencing of the plasmids (pLK75 and pLK78) was conducted using a shotgun approach. Annotation of the contigs was performed using the RAST Server, followed by manual inspection and correction. These similar plasmids were obtained from two patients with overlapping stays at the same hospital. The pLK75 and pLK78 plasmids were 56,489-bp and 56,072-bp in length, respectively. Plasmid annotation revealed a common backbone similar to the IncN plasmid pR46. The regions flanking the blaNDM-1 genes in these plasmids were very similar to plasmid pNDM-HU01 in Japan, which contains a complex class 1 integron located next to an ISCR1 element. The ISCR1 element has been suggested to provide a powerful mechanism for mobilising antibiotic resistance genes. Two indigenous NDM-1-producing Enterobacteriaceae cases were identified for the first time in Taiwan, highlighting the alarming introduction of NDM-1-producing Enterobacteriaceae in this region.
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1560. |
Kotlarska E,
?uczkiewicz A,
Pisowacka M,
Burzy?ski A,
( 2015 ) Antibiotic resistance and prevalence of class 1 and 2 integrons in Escherichia coli isolated from two wastewater treatment plants, and their receiving waters (Gulf of Gdansk, Baltic Sea, Poland). PMID : 25167818 : DOI : 10.1007/s11356-014-3474-7 PMC : PMC4308648 Abstract >>
In this study, antimicrobial-resistance patterns were analyzed in Escherichia coli isolates from raw (RW) and treated wastewater (TW) of two wastewater treatment plants (WWTPs), their marine outfalls (MOut), and mouth of the Vistula River (VR). Susceptibility of E. coli was tested against different classes of antibiotics. Isolates resistant to at least one antimicrobial agent were PCR tested for the presence of integrons. Ampicillin-resistant E. coli were the most frequent, followed by amoxicillin/clavulanate (up to 32 %), trimethoprim/sulfamethoxazole (up to 20 %), and fluoroquinolone (up to 15 %)-resistant isolates. Presence of class 1 and 2 integrons was detected among tested E. coli isolates with rate of 32.06 % (n = 84) and 3.05 % (n = 8), respectively. The presence of integrons was associated with increased frequency of resistance to fluoroquinolones, trimethoprim/sulfamethoxazole, amoxicillin/clavulanate, piperacillin/tazobactam, and presence of multidrug-resistance phenotype. Variable regions were detected in 48 class 1 and 5 class 2 integron-positive isolates. Nine different gene cassette arrays were confirmed among sequenced variable regions, with predominance of dfrA1-aadA1, dfrA17-aadA5, and aadA1 arrays. These findings illustrate the importance of WWTPs in spreading of resistance genes in the environment and the need for inclusion of at least monitoring efforts in the regular WWTP processes.
|
1561. |
Ghasriani H,
Kwok JK,
Sherratt AR,
Foo AC,
Qureshi T,
Goto NK,
( 2014 ) Micelle-catalyzed domain swapping in the GlpG rhomboid protease cytoplasmic domain. PMID : 25162988 : DOI : 10.1021/bi500919v Abstract >>
Three-dimensional domain swapping is a mode of self-interaction that can give rise to altered functional states and has been identified as the trigger event in some protein deposition diseases, yet rates of interconversion between oligomeric states are usually slow, with the requirement for transient disruption of an extensive network of interactions giving rise to a large kinetic barrier. Here we demonstrate that the cytoplasmic domain of the Escherichia coli GlpG rhomboid protease undergoes slow dimerization via domain swapping and that micromolar concentrations of micelles can be used to enhance monomer-dimer exchange rates by more than 1000-fold. Detergents bearing a phosphocholine headgroup are shown to be true catalysts, with hexadecylphosphocholine reducing the 26 kcal/mol free energy barrier by >11 kcal/mol while preserving the 5 kcal/mol difference between monomer and dimer states. Catalysis involves the formation of a micelle-bound intermediate with a partially unfolded structure that is primed for domain swapping. Taken together, these results are the first to demonstrate true catalysis for domain swapping, by using micelles that work in a chaperonin-like fashion to unfold a kinetically trapped state and allow access to the domain-swapped form.
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1562. |
Hansen KH,
Bortolaia V,
Damborg P,
Guardabassi L,
( 2014 ) Strain diversity of CTX-M-producing Enterobacteriaceae in individual pigs: insights into the dynamics of shedding during the production cycle. PMID : 25128344 : DOI : 10.1128/AEM.01730-14 PMC : PMC4249051 Abstract >>
The aim of this study was to evaluate the population dynamics of CTX-M-producing Enterobacteriaceae in individual pigs on a farm positive for CTX-M-14-producing Escherichia coli. Fecal samples were collected once around the farrowing time from five sows and four times along the production cycle from two of their respective offspring. Multiple colonies per sample were isolated on cefotaxime-supplemented MacConkey agar with or without prior enrichment, resulting in 98 isolates identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry and tested for blaCTX-M. CTX-M-positive isolates (n = 86) were typed by pulsed-field gel electrophoresis (PFGE). Plasmids harboring blaCTX-M were characterized in 22 representative isolates by replicon typing and restriction fragment length polymorphism. Based on the PFGE results, all individuals shed unrelated CTX-M-14-producing E. coli strains during the course of life. Concomitant shedding of CTX-M-2/97-producing Proteus mirabilis or Providencia rettgeri was observed in two sows and two offspring. At least two genetically unrelated CTX-M-producing E. coli strains were isolated from approximately one-fourth of the samples, with remarkable differences between isolates obtained by enrichment and direct plating. A clear decrease in strain diversity was observed after weaning. Dissemination of blaCTX-M-14 within the farm was attributed to horizontal transfer of an IncK plasmid that did not carry additional resistance genes and persisted in the absence of antimicrobial selective pressure. Assessment of strain diversity was shown to be influenced by the production stage from which samples were collected, as well as by the isolation method, providing useful information for the design and interpretation of future epidemiological studies of CTX-M-producing Enterobacteriaceae in pig farms.
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1563. |
Carballeira JD,
González-Pérez B,
Moncalián G,
de la Cruz F,
( 2014 ) A high security double lock and key mechanism in HUH relaxases controls oriT-processing for plasmid conjugation. PMID : 25123661 : DOI : 10.1093/nar/gku741 PMC : PMC4176350 Abstract >>
Relaxases act as DNA selection sieves in conjugative plasmid transfer. Most plasmid relaxases belong to the HUH endonuclease family. TrwC, the relaxase of plasmid R388, is the prototype of the HUH relaxase family, which also includes TraI of plasmid F. In this article we demonstrate that TrwC processes its target nic-site by means of a highly secure double lock and key mechanism. It is controlled both by TrwC-DNA intermolecular interactions and by intramolecular DNA interactions between several nic nucleotides. The sequence specificity map of the interaction between TrwC and DNA was determined by systematic mutagenesis using degenerate oligonucleotide libraries. The specificity map reveals the minimal nic sequence requirements for R388-based conjugation. Some nic-site sequence variants were still able to form the U-turn shape at the nic-site necessary for TrwC processing, as observed by X-ray crystallography. Moreover, purified TrwC relaxase effectively cleaved ssDNA as well as dsDNA substrates containing these mutant sequences. Since TrwC is able to catalyze DNA integration in a nic-site-containing DNA molecule, characterization of nic-site functionally active sequence variants should improve the search quality of potential target sequences for relaxase-mediated integration in any target genome.
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1564. |
Søborg DA,
Hendriksen NB,
Kroer N,
( 2014 ) Occurrence and expression of bacterial human virulence gene homologues in natural soil bacteria. PMID : 25118010 : DOI : 10.1111/1574-6941.12413 Abstract >>
The presence and in vitro expression of homologues to 22 bacterial human virulence determinants amongst culturable soil bacteria were investigated. About 25% of the bacterial isolates contained virulence gene homologues representing toxin (hblA, cytK2), adhesin (fimH), regulator (phoQ) and resistance (yfbI) determinants in pathogenic bacteria. The homologues of the toxin genes were found in Actinobacteria and Firmicutes (hblA), and in Firmicutes and Alpha- and Gammaproteobacteria (cytK2). The homologues to the type 1 fimbrial adhesin gene, fimH, and the L-Ara4N transferase gene, yfbI, were observed in Actinobacteria, Firmicutes and Gammaproteobacteria. The regulator gene, phoQ, was only found in Gammaproteobacteria. The presence of cytK2 in Alpha- and Gammaproteobacteria, fimH in Actinobacteria and Firmicutes, and hblA in Actinobacteria has not previously been described. A close sequence similarity (84-100%) was observed between the genes of environmental and clinical isolates, and expression assays suggested that the genes in some cases were expressed in vitro. The presence of functional virulence gene homologues underpins their importance for the survival of environmental bacteria. Furthermore, the high degree of sequence conservation to clinical sequences indicates that natural environments may be 'evolutionary cribs' of emerging pathogens.
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1565. |
Maloj?i? G,
Owen RL,
Glockshuber R,
( 2014 ) Structural and mechanistic insights into the PAPS-independent sulfotransfer catalyzed by bacterial aryl sulfotransferase and the role of the DsbL/Dsbl system in its folding. PMID : 24601529 : DOI : 10.1021/bi401725j Abstract >>
Bacterial aryl sulfotransferases (ASSTs) catalyze sulfotransfer from a phenolic sulfate to a phenol. These enzymes are frequently found in pathogens and upregulated during infection. Their mechanistic understanding is very limited, and their natural substrates are unknown. Here, the crystal structures of Escherichia coli CFT073 ASST trapped in its presulfurylation state with model donor substrates bound in the active site are reported, which reveal the molecular interactions governing substrate recognition. Furthermore, spectroscopic titrations with donor substrates and sulfurylation kinetics of ASST illustrate that this enzyme binds substrates in a 1:1 stoichiometry and that the active sites of the ASST homooligomer act independently. Mass spectrometry and crystallographic experiments of ASST incubated with human urine demonstrate that urine contains a sulfuryl donor substrate. In addition, we examined the capability of the two paralogous dithiol oxidases present in uropathogenic E. coli CFT073, DsbA, and the ASST-specific enzyme DsbL, to introduce the single, conserved disulfide bond into ASST. We show that DsbA and DsbL introduce the disulfide bond into unfolded ASST at similar rates. Hence, a chaperone effect of DsbL, not present in DsbA, appears to be responsible for the dependence of efficient ASST folding on DsbL in vivo. The conservation of paralogous dithiol oxidases with different substrate specificities in certain bacterial strains may therefore be a consequence of the complex folding pathways of their substrate proteins.
|
1566. |
Porres-Osante N,
Azcona-Gutiérrez JM,
Rojo-Bezares B,
Undabeitia E,
Torres C,
Sáenz Y,
( 2014 ) Emergence of a multiresistant KPC-3 and VIM-1 carbapenemase-producing Escherichia coli strain in Spain. PMID : 24583362 : DOI : 10.1093/jac/dku055 Abstract >>
To characterize the mechanisms involved in carbapenem resistance, as well as the genetic elements supporting their mobilization, in a multidrug-resistant Escherichia coli isolate. The E. coli isolate was obtained from a patient with fatal urinary sepsis. Antimicrobial susceptibility testing was performed by the disc diffusion and agar dilution methods. The E. coli molecular type and phylogroup were determined using multilocus sequence typing and the triple PCR technique, respectively. PCR and sequencing were used for virulence and resistance genotype characterization. Plasmid content and gene location were analysed by S1-PFGE, I-Ceu1-PFGE and hybridization experiments. Transformation assays were performed. The E. coli strain, typed as ST448 and phylogroup B1, was resistant to all tested antibiotics except fosfomycin, tigecycline and tetracycline. The following resistance and virulence genetic structures were obtained: ISKpn7 + bla(KPC-3) + ISKpn6 linked to Tn4401; tnpR + aac(6')-Ib'-9 + aadA1 + bla(OXA-9) + tnpR + bla(TEM-1a) + tnpB + strB + strA + sul2; intI1 + bla(VIM-1) + aac(6')-Ib' + aphA15 + aadA1 + catB2 + qacE�G1-sul1 + orf5; ISEcp1 + bla(CMY-2); IS26 + bla(SHV-12); aph(3')-I; aac(3)-IV; floR; catA; and fimA. Mutations in the ampC promoter (-18, -1 and +58) and substitutions in the GyrA (Ser-83��Leu and Asp-87��Asn) and ParC (Ser-80��Ile) proteins were observed. IncFII (ST2), IncA/C and ColE(TP) plasmids of 145.5, 87 and <2 kb, respectively, were found. The bla(VIM-1) gene was located in a non-typeable plasmid of >300 kb, and the bla(KPC-3) gene in the 145.5 kb IncFII plasmid. Transformant strains carried the IncFII and ColE(TP) plasmids, and the bla(KPC-3), bla(TEM-1a), bla(OXA-9), aadA1, aac(6')-Ib'-9, aac(3)-IV and floR genes. This is the first report of the co-production of KPC-3, VIM-1, SHV-12, OXA-9 and CMY-2 in a unique clinical multiresistant E. coli isolate. The dissemination of these genes on mobile genetic elements is alarming and complicates antimicrobial therapies.
|
1567. |
Santos JA,
Alonso-García N,
Macedo-Ribeiro S,
Pereira PJ,
( 2014 ) The unique regulation of iron-sulfur cluster biogenesis in a Gram-positive bacterium. PMID : 24847070 : DOI : 10.1073/pnas.1322728111 PMC : PMC4050560 Abstract >>
Iron-sulfur clusters function as cofactors of a wide range of proteins, with diverse molecular roles in both prokaryotic and eukaryotic cells. Dedicated machineries assemble the clusters and deliver them to the final acceptor molecules in a tightly regulated process. In the prototypical Gram-negative bacterium Escherichia coli, the two existing iron-sulfur cluster assembly systems, iron-sulfur cluster (ISC) and sulfur assimilation (SUF) pathways, are closely interconnected. The ISC pathway regulator, IscR, is a transcription factor of the helix-turn-helix type that can coordinate a [2Fe-2S] cluster. Redox conditions and iron or sulfur availability modulate the ligation status of the labile IscR cluster, which in turn determines a switch in DNA sequence specificity of the regulator: cluster-containing IscR can bind to a family of gene promoters (type-1) whereas the clusterless form recognizes only a second group of sequences (type-2). However, iron-sulfur cluster biogenesis in Gram-positive bacteria is not so well characterized, and most organisms of this group display only one of the iron-sulfur cluster assembly systems. A notable exception is the unique Gram-positive dissimilatory metal reducing bacterium Thermincola potens, where genes from both systems could be identified, albeit with a diverging organization from that of Gram-negative bacteria. We demonstrated that one of these genes encodes a functional IscR homolog and is likely involved in the regulation of iron-sulfur cluster biogenesis in T. potens. Structural and biochemical characterization of T. potens and E. coli IscR revealed a strikingly similar architecture and unveiled an unforeseen conservation of the unique mechanism of sequence discrimination characteristic of this distinctive group of transcription regulators.
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1568. |
Dhara L,
Tripathi A,
Pal A,
( 2013 ) Molecular characterization and in silico analysis of naturally occurring TEM beta-lactamase variants among pathogenic Enterobacteriaceae infecting Indian patients. PMID : 24286084 : DOI : 10.1155/2013/783540 PMC : PMC3826465 Abstract >>
Cephalosporin resistance, particularly due to bla(TEM) encoded �]-lactamases, among Enterobacteriaceae is, though, an increasing public health problem in India; their circulating genetic variants remain unknown. The present study deals with determination of bla(TEM) variants among 134 pathogenic Enterobacteriaceae of Indian origin. Their resistance profile against 3rd generation cephalosporins was determined. The presence of bla(TEM) variants among the bacterial plasmids was characterized by PCR followed by sequencing. Intergenic relations among the variants was determined by phylogenetic analysis. bla(TEM) protein were modeled by Modeller9v5 and verified. The catalytic pockets were characterized, and their interaction with cephalosporins was analyzed using AutoDock tools. More than 87% of isolates showed cephalosporin resistance with ESBL production among 57.8% of Escherichia coli and 50.6% of klebsiella pneumoniae. bla(TEM-1) (84.21%), bla(TEM-1) like (3.94%), bla(TEM-33) (3.94%), bla(TEM-116) (3.94%), bla(TEM-169) (3.94%), and bla(TEM-190) (7.89%) were detected in 76 isolates. Four variants, namely, bla(TEM-1) like, bla(TEM-33), bla(TEM-169), and bla(TEM-190), coexisted in 3 isolates. The largest catalytic pocket of bla(TEM-33) explained its expanded activity towards �]-lactam-�]-lactamase inhibitor combinations. Molecular docking indicated differential resistance pattern of bla(TEM) variants.
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1569. |
Holmgren A,
Bränden CI,
( 1989 ) Crystal structure of chaperone protein PapD reveals an immunoglobulin fold. PMID : 2478891 : DOI : 10.1038/342248a0 Abstract >>
The chaperone protein PapD mediates assembly of pili in Escherichia coli. Its polypeptide chain folds into two immunoglobulin-type domains that are homologous in sequence to the human lymphocyte differentiation antigen Leu-1/CD5.
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1570. |
Woegerbauer M,
Zeinzinger J,
Springer B,
Hufnagl P,
Indra A,
Korschineck I,
Hofrichter J,
Kopacka I,
Fuchs R,
Steinwider J,
Fuchs K,
Nielsen KM,
Allerberger F,
( 2014 ) Prevalence of the aminoglycoside phosphotransferase genes aph(3')-IIIa and aph(3')-IIa in Escherichia coli, Enterococcus faecalis, Enterococcus faecium, Pseudomonas aeruginosa, Salmonella enterica subsp. enterica and Staphylococcus aureus isolates in Austria. PMID : 24194558 : DOI : 10.1099/jmm.0.065789-0 Abstract >>
The aminoglycoside phosphotransferase aph(3')-IIa primarily inactivates kanamycin and neomycin, whilst aph(3')-IIIa also inactivates amikacin. The aim of this study was to determine the frequency of both resistance genes in major human pathogens to obtain their baseline prevalence in the gene pool of these bacterial populations in Austria. In total, 10 541 Escherichia coli, Enterococcus faecalis, Enterococcus faecium, Pseudomonas aeruginosa, Salmonella enterica subsp. enterica and Staphylococcus aureus isolates were collected representatively without selection bias between 2008 and 2011. Isolates were analysed by aph(3')-IIIa/nptIII- and aph(3')-IIa/nptII-specific TaqMan real-time PCR. For positive strains, MICs using Etests were performed and resistance gene sequences were determined. The overall prevalence of aph(3')-IIIa/nptIII was 1.62 % (95 % confidence interval: 1.38-1.88 %). In Escherichia coli, enterococci, Staphylococcus aureus, P. aeruginosa and Salmonella spp., the aph(3')-IIIa/nptIII prevalence was 0.47 % (0-1.47 %), 37.53 % (32.84-42.40 %), 2.90 % (1.51-5.02 %), 0 % (0-0.32 %) and 0 % (0-0.037 %), respectively. Eleven of a total of 169 carriers showed single-nucleotide polymorphisms in the resistance allele. The overall prevalence of aph(3')-IIa/nptII was 0.0096 % (0-0.046 %). Escherichia coli (0-0.70 %), enterococci (0-0.75 %), Staphylococcus aureus (0-0.73 %) and P. aeruginosa (0-0.32 %) did not carry aph(3')-IIa. A single Salmonella isolate was positive, resulting in an aph(3')-IIa prevalence of 0.013 % (0-0.058 %). aph(3')-IIIa/nptIII carriers were moderately prevalent in the strains tested except for in enterococci, which appeared to be an important reservoir for aph(3')-IIIa. aph(3')-IIa/nptII genes were detected at clinically irrelevant frequencies and played no significant role in the aminoglycoside resistance gene pool during the observation period.
|
1571. |
Ruhe ZC,
Nguyen JY,
Beck CM,
Low DA,
Hayes CS,
( 2014 ) The proton-motive force is required for translocation of CDI toxins across the inner membrane of target bacteria. PMID : 25174572 : DOI : 10.1111/mmi.12779 PMC : PMC4191985 Abstract >>
Contact-dependent growth inhibition (CDI) is a mode of bacterial competition orchestrated by the CdiB/CdiA family of two-partner secretion proteins. The CdiA effector extends from the surface of CDI(+) inhibitor cells, binds to receptors on neighbouring bacteria and delivers a toxin domain derived from its C-terminal region (CdiA-CT). Here, we show that CdiA-CT toxin translocation requires the proton-motive force (pmf) within target bacteria. The pmf is also critical for the translocation of colicin toxins, which exploit the energized Ton and Tol systems to cross the outer membrane. However, CdiA-CT translocation is clearly distinct from known colicin-import pathways because �GtolA �GtonB target cells are fully sensitive to CDI. Moreover, we provide evidence that CdiA-CT toxins can be transferred into the periplasm of de-energized target bacteria, indicating that transport across the outer membrane is independent of the pmf. Remarkably, CDI toxins transferred under de-energized conditions remain competent to enter the target-cell cytoplasm once the pmf is restored. Collectively, these results indicate that outer- and inner-membrane translocation steps can be uncoupled, and that the pmf is required for CDI toxin transport from the periplasm to the target-cell cytoplasm.
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1572. |
Kokotek W,
Lotz W,
( 1989 ) Construction of a lacZ-kanamycin-resistance cassette, useful for site-directed mutagenesis and as a promoter probe. PMID : 2515118 : DOI : 10.1016/0378-1119(89)90522-2 Abstract >>
A lacZ gene without a promoter, but containing its ribosome-binding site, was cloned next to the kanamycin-resistance (KmR) gene of plasmid pUC4K, yielding a lacZ-KmR cassette. From the resulting plasmid, pKOK5, the lacZ-KmR cassette was recloned by means of BamHI into plasmid pKOK4, a mobilizable derivative of pBR322 which mediates ampicillin, chloramphenicol and tetracycline resistance. The lacZ-KmR cassette can be excised from pKOK5 or pKOK6 by digestion with BamHI, SalI or PstI. It can be used for insertion mutagenesis by ligation of the cassette to target DNA that has been linearized by one of these enzymes. Insertions can be selected by the KmR phenotype and mapped by digestion, e.g., with PstI and SalI. The orientation of the inserted cassette can be determined by digestion, e.g., with EcoRI or HindIII. Within the lacZ-KmR cassette, the transcription of the lacZ and the KmR genes are directed towards each other, and the two genes are separated by the bidirectionally active terminator from phage fd. In Escherichia coli, no transcription emanating from the cassette was detected. Transcription within DNA mutagenized by the cassette can be monitored by the promoterless lacZ gene. The lacZ-KmR cassette is currently used by us for the site-directed mutagenesis of hydrogen uptake gene-specific DNA from Rhizobium leguminosarum B10.
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1573. |
Fagerquist CK,
Zaragoza WJ,
Sultan O,
Woo N,
Quiñones B,
Cooley MB,
Mandrell RE,
( 2014 ) Top-down proteomic identification of Shiga toxin 2 subtypes from Shiga toxin-producing Escherichia coli by matrix-assisted laser desorption ionization-tandem time of flight mass spectrometry. PMID : 24584253 : DOI : 10.1128/AEM.04058-13 PMC : PMC3993300 Abstract >>
We have analyzed 26 Shiga toxin-producing Escherichia coli (STEC) strains for Shiga toxin 2 (Stx2) production using matrix-assisted laser desorption ionization (MALDI)-tandem time of flight (TOF-TOF) tandem mass spectrometry (MS/MS) and top-down proteomic analysis. STEC strains were induced to overexpress Stx2 by overnight culturing on solid agar supplemented with either ciprofloxacin or mitomycin C. Harvested cells were lysed by bead beating, and unfractionated bacterial cell lysates were ionized by MALDI. The A2 fragment of the A subunit and the mature B subunit of Stx2 were analyzed by MS/MS. Sequence-specific fragment ions were used to identify amino acid subtypes of Stx2 using top-down proteomic analysis using software developed in-house at the U.S. Department of Agriculture (USDA). Stx2 subtypes (a, c, d, f, and g) were identified on the basis of the mass of the A2 fragment and the B subunit as well as from their sequence-specific fragment ions by MS/MS (postsource decay). Top-down proteomic identification was in agreement with DNA sequencing of the full Stx2 operon (stx2) for all strains. Top-down results were also compared to a bioassay using a Vero-d2EGFP cell line. Our results suggest that top-down proteomic identification is a rapid, highly specific technique for distinguishing Stx2 subtypes.
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1574. |
Netikul T,
Sidjabat HE,
Paterson DL,
Kamolvit W,
Tantisiriwat W,
Steen JA,
Kiratisin P,
( 2014 ) Characterization of an IncN2-type blaNDM-?-carrying plasmid in Escherichia coli ST131 and Klebsiella pneumoniae ST11 and ST15 isolates in Thailand. PMID : 25096073 : DOI : 10.1093/jac/dku275 Abstract >>
N/A
|
1575. |
Fei X,
Ye X,
LaRonde NA,
Lorimer GH,
( 2014 ) Formation and structures of GroEL:GroES2 chaperonin footballs, the protein-folding functional form. PMID : 25136110 : DOI : 10.1073/pnas.1412922111 PMC : PMC4156775 Abstract >>
The GroE chaperonins assist substrate protein (SP) folding by cycling through several conformational states. With each cycle the SP is, in turn, captured, unfolded, briefly encapsulated (t1/2 ? 1 s), and released by the chaperonin complex. The protein-folding functional form is the US-football-shaped GroEL:GroES2 complex. We report structures of two such "football" complexes to ? 3.7-? resolution; one is empty whereas the other contains encapsulated SP in both chambers. Although encapsulated SP is not visible on the electron density map, using calibrated FRET and order-of-addition experiments we show that owing to SP-catalyzed ADP/ATP exchange both chambers of the football complex encapsulate SP efficiently only if the binding of SP precedes that of ATP. The two rings of GroEL thus behave as a parallel processing machine, rather than functioning alternately. Compared with the bullet-shaped GroEL:GroES1 complex, the GroEL:GroES2 football complex differs conformationally at the GroEL-GroES interface and also at the interface between the two GroEL rings. We propose that the electrostatic interactions between the �`-NH(3+) of K105 of helix D in one ring with the negatively charged carboxyl oxygen of A109 at the carboxyl end of helix D of the other ring provide the structural basis for negative inter-ring cooperativity.
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1576. |
Li J,
Lan R,
Xiong Y,
Ye C,
Yuan M,
Liu X,
Chen X,
Yu D,
Liu B,
Lin W,
Bai X,
Wang Y,
Sun Q,
Wang Y,
Zhao H,
Meng Q,
Chen Q,
Zhao A,
Xu J,
( 2014 ) Sequential isolation in a patient of Raoultella planticola and Escherichia coli bearing a novel ISCR1 element carrying blaNDM-1. PMID : 24594606 : DOI : 10.1371/journal.pone.0089893 PMC : PMC3940617 Abstract >>
The gene for New Delhi metallo-�]-lactamase 1 (NDM-1) has been reported to be transmitted via plasmids which are easily transferable and capable of wide distribution. We report the isolation of two NDM-1 producing strains and possible in vivo transfer of blaNDM-1 in a patient. Clinical samples were collected for bacterial culture and antibiotic susceptibility testing from a patient during a 34-day hospitalization. The presence of blaNDM-1 was detected by PCR and sequencing. Plasmids of interest were sequenced. Medical records were reviewed for evidence of association between the administration of antibiotics and the acquisition of the NDM-1 resistance. A NDM-1 positive Raoultella planticola was isolated from blood on the ninth day of hospitalization without administration of any carbapenem antibiotics and a NDM-1 positive Escherichia coli was isolated from feces on the 29th day of hospitalization and eight days after imipenem administration. The blaNDM-1 was carried by a 280 kb plasmid pRpNDM1-1 in R. planticola and a 58 kb plasmid pEcNDM1-4 in E. coli. The two plasmids shared a 4812 bp NDM-1-ISCR1 element which was found to be excisable from the plasmid as a free form and transferrable in vitro to a NDM-1 negative plasmid from E. coli. blaNDM-1 was embedded in an ISCR1 complex class 1 integron as a novel 4812 bp NDM-1-ISCR1 element. The element was found to be able to self excise to become a free form, which may provide a new vehicle for NDM-1 dissemination. This mechanism could greatly accelerate the spread of NDM-1 mediated broad spectrum �]-lactam resistance.
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1577. |
Maluta RP,
Fairbrother JM,
Stella AE,
Rigobelo EC,
Martinez R,
de ?vila FA,
( 2014 ) Potentially pathogenic Escherichia coli in healthy, pasture-raised sheep on farms and at the abattoir in Brazil. PMID : 24438985 : DOI : 10.1016/j.vetmic.2013.12.013 Abstract >>
Sheep harbor pathogenic Escherichia coli, which may cause severe disease in humans. In this study, the prevalence of Shiga toxin-producing E. coli (STEC) and enteropathogenic E. coli (EPEC) was examined in sheep feces and carcasses on three farms and at an abattoir in Brazil. The isolates were further characterized for the presence of markers recently associated with disease in humans, to investigate their possible origin and role as food-borne pathogens. At the abattoir, 99 carcass samples yielded two STEC and 10 EPEC isolates while 101 fecal samples yielded five EPEC and eight STEC isolates. On the other hand, on the farms, 202 samples yielded 44 STEC and eight EPEC isolates. The 77 isolates were typed by PFGE. Isolates with the same PFGE pattern and also those that were not restricted with XbaI were termed as "clones" (n=49). The isolates of any one clone mostly originated from the same sampling site. In addition, seven isolates encoded for novel Stx2 variants and five for Stx2e, the subtype related to porcine edema disease, which was for the first time isolated from sheep feces and carcasses. Also, three stx2-only isolates harbored genes of predicted Stx2 variants that were formed by A and B subunits of different types including Stx2a and Stx2d. The EPEC isolates were heterogeneous, 21 (91.3%) of them possessing efa1, ehxA, lpfAO113 or paa genes associated with diarrhea in humans. Thus, using markers recently associated with disease, we have demonstrated that E. coli similar to those pathogenic for humans are present in the sheep intestinal microflora, particularly at the abattoir, underlining the potential for food-borne transmission.
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1578. |
Delannoy S,
Fach P,
Beutin L,
Miko A,
Rivas M,
Bentancor A,
( 2014 ) Emerging types of Shiga toxin-producing E. coli (STEC) O178 present in cattle, deer, and humans from Argentina and Germany. PMID : 24987616 : DOI : 10.3389/fcimb.2014.00078 PMC : PMC4060028 Abstract >>
More than 400 serotypes of Shiga toxin-producing Escherichia coli (STEC) have been implicated in outbreaks and sporadic human diseases. In recent years STEC strains belonging to serogroup O178 have been commonly isolated from cattle and food of bovine origin in South America and Europe. In order to explore the significance of these STEC strains as potential human pathogens, 74 German and Argentinean E. coli O178 strains from animals, food and humans were characterized phenotypically and investigated for their serotypes, stx-genotypes and 43 virulence-associated markers by a real-time PCR-microarray. The majority (n = 66) of the O178 strains belonged to serotype O178:H19. The remaining strains divided into O178:H7 (n = 6), O178:H10 (n = 1), and O178:H16 (n = 1). STEC O178:H19 strains were mainly isolated from cattle and food of bovine origin, but one strain was from a patient with hemolytic uremic syndrome (HUS). Genotyping of the STEC O178:H19 strains by pulsed-field gel electrophoresis revealed two major clusters of genetically highly related strains which differ in their stx-genotypes and non-Stx putative virulence traits, including adhesins, toxins, and serine-proteases. Cluster A-strains including the HUS-strain (n = 35) carried genes associated with severe disease in humans (stx2a, stx2d, ehxA, saa, subAB1, lpfAO113 , terE combined with stx1a, espP, iha). Cluster B-strains (n = 26) showed a limited repertoire of virulence genes (stx2c, pagC, lpfAO113 , espP, iha). Among O178:H7 strains isolated from deer meat and patients with uncomplicated disease a new STEC variant was detected that is associated with the genotype stx1c/stx2b/ehxA/subAB2/espI/[terE]/espP/iha. None of the STEC O178 strains was positive for locus of enterocyte effacement (LEE)- and nle-genes. Results indicate that STEC O178:H19 strains belong to the growing group of LEE-negative STEC that should be considered with respect to their potential to cause diseases in humans.
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1579. |
Zhang W,
Nadirk J,
Kossow A,
Bielaszewska M,
Leopold SR,
Witten A,
Fruth A,
Karch H,
Ammon A,
Mellmann A,
( 2014 ) Phylogeny and phenotypes of clinical and environmental Shiga toxin-producing Escherichia coli O174. PMID : 24034719 : DOI : 10.1111/1462-2920.12234 Abstract >>
Shiga toxin (Stx)-producing Escherichia coli (STEC) of serogroup O174 are human pathogenic intimin gene (eae)-negative STEC. To facilitate diagnosis and subtyping, we genotypically and phenotypically characterized 25 STEC O174 isolates from humans with different clinical outcomes and from animals and the environment. fliC genotyping resulted in four different genotypes (fliCH2 : n = 5; fliCH8 : n = 8; fliCH21 : n = 11; fliCH46 : n = 1). Twenty-three strains were motile expressing the corresponding H antigen; two non-motile isolates possessed fliCH8 . The stx genotypes and non-stx virulence loci, including toxins, serine-proteases and adhesins correlated well with serotypes but showed no differences with respect to the isolates' origins. Multilocus sequence typing identified seven sequence types that correlated with serotypes. Core gene typing further specified the four serotypes, including a previously unknown O174:H46 combination, and revealed distant relationships of the different serotypes within serogroup O174 and in relation to other haemolytic uremic syndrome (HUS)-associated STEC. Only serotype O174:H21 was associated with HUS. Differences in virulence factors and in the adherence capacity of STEC O174 corroborated this separation into four distinct groups. Our study provides a basis for O174 subtyping, unravels considerable genotypic and phenotypic heterogeneity and sheds light to potential environmental and animal reservoirs.
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1580. |
Girón JA,
Curtiss R,
Mellata M,
Stacy AK,
Mitchell NM,
Maddux JT,
De la Cruz MA,
Durán L,
( 2014 ) Evaluation of the prevalence and production of Escherichia coli common pilus among avian pathogenic E. coli and its role in virulence. PMID : 24466152 : DOI : 10.1371/journal.pone.0086565 PMC : PMC3900561 Abstract >>
Avian pathogenic Escherichia coli (APEC) strains cause systemic and localized infections in poultry, jointly termed colibacillosis. Avian colibacillosis is responsible for significant economic losses to the poultry industry due to disease treatment, decrease in growth rate and egg production, and mortality. APEC are also considered a potential zoonotic risk for humans. Fully elucidating the virulence and zoonotic potential of APEC is key for designing successful strategies against their infections and their transmission. Herein, we investigated the prevalence of a newly discovered E. coli common pilus (ECP) for the subunit protein of the ECP pilus (ecpA) and ECP expression amongst APEC strains as well as the role of ECP in virulence. A PCR-based ecpA survey of a collection of 167 APEC strains has shown that 76% (127/167) were ecpA+. An immunofluorescence assay using anti-EcpA antibodies, revealed that among the ecpA+ strains, 37.8% (48/127) expressed ECP when grown in DMEM +0.5% Mannose in contact with HeLa cells at 37�XC and/or in biofilm at 28�XC; 35.4% (17/48) expressed ECP in both conditions and 64.6% (31/48) expressed ECP in biofilm only. We determined that the ecp operon in the APEC strain �q7122 (ecpA+, ECP-) was not truncated; the failure to detect ECP in some strains possessing non-truncated ecp genes might be attributed to differential regulatory mechanisms between strains that respond to specific environmental signals. To evaluate the role of ECP in the virulence of APEC, we generated ecpA and/or ecpD-deficient mutants from the strain �q7503 (ecpA+, ECP+). Deletion of ecpA and/or ecpD abolished ECP synthesis and expression, and reduced biofilm formation and motility in vitro and virulence in vivo. All together our data show that ecpA is highly prevalent among APEC isolates and its expression could be differentially regulated in these strains, and that ECP plays a role in the virulence of APEC.
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1581. |
Murakami K,
Etoh Y,
Ichihara S,
Maeda E,
Takenaka S,
Horikawa K,
Narimatsu H,
Kawano K,
Kawamura Y,
Ito K,
( 2014 ) Isolation and characteristics of Shiga toxin 2f-producing Escherichia coli among pigeons in Kyushu, Japan. PMID : 24465879 : DOI : 10.1371/journal.pone.0086076 PMC : PMC3900449 Abstract >>
An increasing number of Shiga toxin 2f-producing Escherichia coli (STEC2f) infections in humans are being reported in Europe, and pigeons have been suggested as a reservoir for the pathogen. In Japan, there is very little information regarding carriage of STEC2f by pigeons, prompting the need for further investigation. We collected 549 samples of pigeon droppings from 14 locations in Kyushu, Japan, to isolate STEC2f and to investigate characteristics of the isolates. Shiga toxin stx 2f gene fragments were detected by PCR in 16 (2.9%) of the 549 dropping samples across four of the 14 locations. We obtained 23 STEC2f-isolates from seven of the original samples and from three pigeon dropping samples collected in an additional sampling experiment (from a total of seven locations across both sampling periods). Genotypic and phenotypic characteristics were then examined for selected isolates from each of 10 samples with pulsed-field gel electrophoresis profiles. Eight of the stx 2f gene fragments sequenced in this study were homologous to others that were identified in Europe. Some isolates also contained virulence-related genes, including lpfA O26, irp 2, and fyuA, and all of the 10 selected isolates maintained the eae, astA, and cdt genes. Moreover, five of the 10 selected isolates contained sfpA, a gene that is restricted to Shiga toxin-producing E. coli O165:H2 and sorbitol-fermenting Shiga toxin-producing E. coli O157:NM. We document serotypes O152:HNM, O128:HNM, and O145:H34 as STEC2f, which agrees with previous studies on pigeons and humans. Interestingly, O119:H21 was newly described as STEC2f. O145:H34, with sequence type 722, was described in a German study in humans and was also isolated in the current study. These results revealed that Japanese zoonotic STEC2f strains harboring several virulence-related factors may be of the same clonal complexes as some European strains. These findings provide useful information for public health-related disease management strategies in Japan.
|
1582. |
Jurado-Rabadán S,
de la Fuente R,
Ruiz-Santa-Quiteria JA,
Orden JA,
de Vries LE,
Agersø Y,
( 2014 ) Detection and linkage to mobile genetic elements of tetracycline resistance gene tet(M) in Escherichia coli isolates from pigs. PMID : 25015125 : DOI : 10.1186/1746-6148-10-155 PMC : PMC4105395 Abstract >>
In Escherichia coli the genes involved in the acquisition of tetracycline resistance are mainly tet(A) and tet(B). In addition, tet(M) is the most common tetracycline resistance determinant in enterococci and it is associated with conjugative transposons and plasmids. Although tet(M) has been identified in E. coli, to our knowledge, there are no previous reports studying the linkage of the tet(M) gene in E. coli to different mobile genetic elements. The aim of this study was to determine the occurrence of tet(A), tet(B), and tet(M) genes in doxycycline-resistant E. coli isolates from pigs, as well as the detection of mobile genetic elements linked to tet(M) in E. coli and its possible transfer from enterococci. tet(A) was the most frequently detected gene (87.9%) in doxycycline-resistant isolates. tet(M) was found in 13.1% E. coli isolates. The tet(M) gene was detected in relation with conjugative transposons in 10 out of 36 enterococci isolates analyzed but not in any of E. coli isolates positive for tet(M). Southern blot showed that in E. coli and in most of the enterococci isolates the tet(M) gene was carried on a plasmid. According to the phylogenetic analysis, E. coli contained a new tet(M) allele grouping separately. Mating experiments revealed that tet(M) was carried on a mobile element successfully transferred between enterococci and between enterococci and E. coli. The detection of tet(M) in E. coli isolates from pigs was higher than expected. In our study, tet(M) detected in E. coli seems not to have been transferred from enterococci, although it can not be ruled out that the horizontal transfer of this gene occurred from other intestinal tract bacteria.
|
1583. |
Ma J,
Bao Y,
Sun M,
Dong W,
Pan Z,
Zhang W,
Lu C,
Yao H,
( 2014 ) Two functional type VI secretion systems in avian pathogenic Escherichia coli are involved in different pathogenic pathways. PMID : 24980972 : DOI : 10.1128/IAI.01769-14 PMC : PMC4187841 Abstract >>
Type VI secretion systems (T6SSs) are involved in the pathogenicity of several Gram-negative bacteria. The VgrG protein, a core component and effector of T6SS, has been demonstrated to perform diverse functions. The N-terminal domain of VgrG protein is a homologue of tail fiber protein gp27 of phage T4, which performs a receptor binding function and determines the host specificity. Based on sequence analysis, we found that two putative T6SS loci exist in the genome of the avian pathogenic Escherichia coli (APEC) strain TW-XM. To assess the contribution of these two T6SSs to TW-XM pathogenesis, the crucial clpV clusters of these two T6SS loci and their vgrG genes were deleted to generate a series of mutants. Consequently, T6SS1-associated mutants presented diminished adherence to and invasion of several host cell lines cultured in vitro, decreased pathogenicity in duck and mouse infection models in vivo, and decreased biofilm formation and bacterial competitive advantage. In contrast, T6SS2-associated mutants presented a significant decrease only in the adherence to and invasion of mouse brain microvascular endothelial cell (BMEC) line bEnd.3 and brain tissue of the duck infection model. These results suggested that T6SS1 was involved in the proliferation of APEC in systemic infection, whereas VgrG-T6SS2 was responsible only for cerebral infection. Further study demonstrated that VgrG-T6SS2 was able to bind to the surface of bEnd.3 cells, whereas it did not bind to DF-1 (chicken embryo fibroblast) cells, which further proved the interaction of VgrG-T6SS2 with the surface of BMECs.
|
1584. |
Rao L,
Lv L,
Zeng Z,
Chen S,
He D,
Chen X,
Wu C,
Wang Y,
Yang T,
Wu P,
Liu Y,
Liu JH,
( 2014 ) Increasing prevalence of extended-spectrum cephalosporin-resistant Escherichia coli in food animals and the diversity of CTX-M genotypes during 2003-2012. PMID : 24999233 : DOI : 10.1016/j.vetmic.2014.06.013 Abstract >>
The aim of this study was to investigate the trends and the diversity of CTX-M types of extended-spectrum �]-lactamase (ESBL) in Escherichia coli isolated from food animals in China over a ten-year period. From 2003 to 2012, 2815 E. coli isolates collected from diseased animals (chickens, pigs, and waterfowl) were screened for the prevalence of CTX-M genes. CTX-M-positive isolates were tested for their susceptibilities to 10 antimicrobial agents and the clonal relationship of CTX-M-producing E. coli isolates was also assessed. Overall, 677 (20.1%) of the 2815 E. coli isolates carried CTX-M genes. Eighteen different types of CTX-M ESBLs were identified, with CTX-M-14, CTX-M-55, and CTX-M-65 being the most dominant genotypes. The occurrence of CTX-M-producing E. coli increased significantly from 5.7% in 2003-2005 to 35.3% in 2009-2012 (p<0.0001). High genetic heterogeneities were observed in the CTX-M-producing E. coli isolates. Most CTX-M-producing strains were also resistant to other classes of antimicrobials. Compared to isolates carrying CTX-M-9 subgroup of ESBLs, isolates carrying CTX-M-1 subgroup ESBLs showed significantly higher resistance rates to ceftazidime, amikacin, and fosfomycin (p<0.01). The study reported the dramatic increase of CTX-M ESBLs in E. coli isolated from animals overtime in China. The increasing incidence of CTX-M-55 with high hydrolytic activity against ceftazidime and the widely spread co-resistance in CTX-M-producing isolates alarm the serious antimicrobial resistance situation in China and highlight the need for urgent control strategies to limit the dissemination of those resistant genes in China.
|
1585. |
Wolf MK,
Andrews GP,
Tall BD,
McConnell MM,
Levine MM,
Boedeker EC,
( 1989 ) Characterization of CS4 and CS6 antigenic components of PCF8775, a putative colonization factor complex from enterotoxigenic Escherichia coli E8775. PMID : 2491834 : PMC : PMC313061 Abstract >>
PCF8775 is a putative colonization factor complex present on the surface of 10 to 20% of enterotoxigenic Escherichia coli strains and has been reported to be composed of antigen CS6 (morphology undefined) expressed alone or together with either of the rigid fimbrial antigens CS4 and CS5. To better define the individual components of this complex and the determinants of their expression, we prepared antiserum to the PCF8775 complex as it was expressed on prototype strain E8775 and then used the antiserum to identify the subunit structure of the antigens, to study their morphology, and to detect expression of individual components of the complex after transfer of plasmids into laboratory strain HB101. CS4 was purified from strain E8775, confirmed to be fimbrial by electron microscopy, and found to be composed of a 22-kilodalton protein subunit whose N-terminal amino acid sequence (1 to 20) was similar to that of colonization factor antigen I. Transconjugants that express CS6 but not CS4 were obtained by mating prototype strain E8775 with HB101. CS6 expression was mediated by a 61-megadalton plasmid. Expression of CS6 in the transconjugants correlates with expression of a 16-kilodalton cell surface protein. The CS6 antigen was confirmed to be present on the cell surface by immunogold labeling, but its morphology was beyond the limits of resolution by electron microscopy.
|
1586. |
Sassi A,
Loucif L,
Gupta SK,
Dekhil M,
Chettibi H,
Rolain JM,
( 2014 ) NDM-5 carbapenemase-encoding gene in multidrug-resistant clinical isolates of Escherichia coli from Algeria. PMID : 24982080 : DOI : 10.1128/AAC.02818-13 PMC : PMC4135848 Abstract >>
Here, we report the first autochthonous cases of infections caused by blaNDM-5 New Delhi metallo-�]-lactamase-producing Escherichia coli strains recovered from urine and blood specimens of three patients from Algeria between January 2012 and February 2013. The three isolates belong to sequence type 2659 and they coexpress blaCTX-M-15 with the blaTEM-1 and blaaadA2 genes.
|
1587. |
Chan J,
Lo WU,
Lai EL,
Cheung YY,
Lau TC,
Chow KH,
Ho PL,
( 2013 ) Prevalence and molecular epidemiology of plasmid-mediated fosfomycin resistance genes among blood and urinary Escherichia coli isolates. PMID : 23988630 : DOI : 10.1099/jmm.0.062653-0 Abstract >>
A total of 1878 non-duplicate clinical Escherichia coli isolates (comprising 1711 urinary isolates and 167 blood-culture isolates), which were collected from multiple centres in Hong Kong during 1996-2008, were used to investigate the prevalence and molecular epidemiology of plasmid-mediated fosfomycin (fos) resistance genes. Eighteen of the 1878 clinical E. coli isolates were fosfomycin resistant, of which six were fosA3 positive and two were positive for another fosA variant (designated fosKP96). No isolates had the fosC2 gene. The clones of the eight isolates were diverse: sequence type (ST) 95 (n = 2), ST118 (n = 1), ST131 (n = 1), ST617 (n = 1), ST648 (n = 1), ST1488 (n = 1) and ST2847 (n = 1). In the isolates, fosA3 and blaCTX-M genes were co-harboured on conjugative plasmids with F2:A-:B- (n = 2), N (n = 1), F-:A-:B1 and N (n = 1) and untypable (n = 2) replicons. Both fosKP96-carrying plasmids belonged to replicon N. RFLP analysis showed that the two F2:A-:B- plasmids carrying fosA3 and blaCTX-M-3 genes shared the same pattern. Complete sequencing of one of the two F2:A-:B- plasmids, pFOS-HK151325 (69 768 bp) demonstrated it to be >99 % identical to the previously sequenced plasmid pHK23a originating from a pig E. coli isolate in the same region. This study demonstrated the dissemination of fosA3 genes in diverse E. coli clones on multiple blaCTX-M-carrying plasmid types, of which F2:A-:B- plasmids closely related to pHK23a were shared by isolates from human and animal sources.
|
1588. |
Chen L,
Hu H,
Chavda KD,
Zhao S,
Liu R,
Liang H,
Zhang W,
Wang X,
Jacobs MR,
Bonomo RA,
Kreiswirth BN,
( 2014 ) Complete sequence of a KPC-producing IncN multidrug-resistant plasmid from an epidemic Escherichia coli sequence type 131 strain in China. PMID : 24395232 : DOI : 10.1128/AAC.02587-13 PMC : PMC4023777 Abstract >>
We report here the nucleotide sequence of a novel blaKPC-2-harboring incompatibility group N (IncN) plasmid, pECN580, from a multidrug-resistant Escherichia coli sequence type 131 (ST131) isolate recovered from Beijing, China. pECN580 harbors �]-lactam resistance genes blaKPC-2, blaCTX-M-3, and blaTEM-1; aminoglycoside acetyltransferase gene aac(6')-Ib-cr; quinolone resistance gene qnrS1; rifampin resistance gene arr-3; and trimethoprim resistance gene dfrA14. The emergence of a blaKPC-2-harboring multidrug-resistant plasmid in an epidemic E. coli ST131 clone poses a significant potential threat in community and hospital settings.
|
1589. |
Le Nours J,
Paton AW,
Byres E,
Troy S,
Herdman BP,
Johnson MD,
Paton JC,
Rossjohn J,
Beddoe T,
( 2013 ) Structural basis of subtilase cytotoxin SubAB assembly. PMID : 23921389 : DOI : 10.1074/jbc.M113.462622 PMC : PMC3779744 Abstract >>
Pathogenic strains of Escherichia coli produce a number of toxins that belong to the AB5 toxin family, which comprise a catalytic A-subunit that induces cellular dysfunction and a B-pentamer that recognizes host glycans. Although the molecular actions of many of the individual subunits of AB5 toxins are well understood, how they self-associate and the effect of this association on cytotoxicity are poorly understood. Here we have solved the structure of the holo-SubAB toxin that, in contrast to other AB5 toxins whose molecular targets are located in the cytosol, cleaves the endoplasmic reticulum chaperone BiP. SubA interacts with SubB in a similar manner to other AB5 toxins via the A2 helix and a conserved disulfide bond that joins the A1 domain with the A2 helix. The structure revealed that the active site of SubA is not occluded by the B-pentamer, and the B-pentamer does not enhance or inhibit the activity of SubA. Structure-based sequence comparisons with other AB5 toxin family members, combined with extensive mutagenesis studies on SubB, show how the hydrophobic patch on top of the B-pentamer plays a dominant role in binding the A-subunit. The structure of SubAB and the accompanying functional characterization of various mutants of SubAB provide a framework for understanding the important role of the B-pentamer in the assembly and the intracellular trafficking of this AB5 toxin.
|
1590. |
Wang D,
Hu E,
Chen J,
Tao X,
Gutierrez K,
Qi Y,
( 2013 ) Characterization of novel ybjG and dacC variants in Escherichia coli. PMID : 23912810 : DOI : 10.1099/jmm.0.062893-0 Abstract >>
A total of 69 strains of Escherichia coli from patients in the Taizhou Municipal Hospital, China, were isolated, and 11 strains were identified that were resistant to bacitracin, chloramphenicol, tetracycline and erythromycin. These strains were PCR positive for at least two out of three genes, ybjG, dacC and mdfA, by gene mapping with conventional PCR detection. Conjugation experiments demonstrated that these genes existed in plasmids that conferred resistance. Novel ybjG and dacC variants were isolated from E. coli strains EC2163 and EC2347, which were obtained from the sputum of intensive care unit patients. Genetic mapping showed that the genes were located on 8200 kb plasmid regions flanked by EcoRI restriction sites. Three distinct genetic structures were identified among the 11 PCR-positive strains of E. coli, and two contained the novel ybjG and dacC variants. The putative amino acid differences in the ybjG and dacC gene variants were characterized. These results provide evidence for novel variants of ybjG and dacC, and suggest that multiple drug resistance in hospital strains of E. coli depends on the synergistic function of ybjG, dacC and mdfA within three distinct genetic structures in conjugative plasmids.
|
1591. |
Krebs MP,
Reznikoff WS,
( 1986 ) Transcriptional and translational initiation sites of IS50. Control of transposase and inhibitor expression. PMID : 2438419 : DOI : 10.1016/0022-2836(86)90028-8 Abstract >>
We have examined transcriptional start sites responsible for expression of the transposase and transposition inhibitor proteins encoded by IS50R, and determined the likely translational start site of transposase. Amino-terminal analysis of a transposase-beta-galactosidase fusion protein gave the sequence Met-Ile-Thr-Ser-Ala, which corresponds to the predicted amino acid sequence starting at position 93 of IS50. S1 nuclease mapping of IS50 RNA produced in vivo indicated that three transcripts, T1, T2 and T3, start near this position. Only T1 starts upstream from the transposase amino terminus. T2 corresponds to an in-vitro transcript described previously. Analysis of the transcripts and proteins produced from deletion derivatives of an IS50-lacZ construct suggested that the three transcripts initiate at independent but overlapping promoters clustered near the end of IS50. This analysis confirmed that only T1 can encode transposase, and that T2 is largely responsible for expression of the inhibitor protein. The coding capacity of T3 was not determined. Finally, transcripts that originate outside of IS50 are prevented from expressing transposase because of a secondary structure that is present in these transcripts only.
|
1592. |
Seok SH,
Im H,
Won HS,
Seo MD,
Lee YS,
Yoon HJ,
Cha MJ,
Park JY,
Lee BJ,
( 2014 ) Structures of inactive CRP species reveal the atomic details of the allosteric transition that discriminates cyclic nucleotide second messengers. PMID : 24914983 : DOI : 10.1107/S139900471400724X Abstract >>
The prokaryotic global transcription factor CRP has been considered to be an ideal model for in-depth study of both the allostery of the protein and the differential utilization of the homologous cyclic nucleotide second messengers cAMP and cGMP. Here, atomic details from the crystal structures of two inactive CRP species, an apo form and a cGMP-bound form, in comparison with a known active conformation, the cAMP-CRP complex, provide macroscopic and microscopic insights into CRP allostery, which is coupled to specific discrimination between the two effectors. The cAMP-induced conformational transition, including dynamic fluctuations, can be driven by the fundamental folding forces that cause water-soluble globular proteins to construct an optimized hydrophobic core, including secondary-structure formation. The observed conformational asymmetries underlie a negative cooperativity in the sequential binding of cyclic nucleotides and a stepwise manner of binding with discrimination between the effector molecules. Additionally, the finding that cGMP, which is specifically recognized in a syn conformation, induces an inhibitory conformational change, rather than a null effect, on CRP supports the intriguing possibility that cGMP signalling could be widely utilized in prokaryotes, including in aggressive inhibition of CRP-like proteins.
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1593. |
Aizawa J,
Neuwirt N,
Barbato L,
Neves PR,
Leigue L,
Padilha J,
Pestana de Castro AF,
Gregory L,
Lincopan N,
( 2014 ) Identification of fluoroquinolone-resistant extended-spectrum �]-lactamase (CTX-M-8)-producing Escherichia coli ST224, ST2179 and ST2308 in buffalo (Bubalus bubalis). PMID : 24928853 : DOI : 10.1093/jac/dku218 Abstract >>
N/A
|
1594. |
Baty D,
Knibiehler M,
Verheij H,
Pattus F,
Shire D,
Bernadac A,
Lazdunski C,
( 1987 ) Site-directed mutagenesis of the COOH-terminal region of colicin A: effect on secretion and voltage-dependent channel activity. PMID : 2434951 : DOI : 10.1073/pnas.84.5.1152 PMC : PMC304384 Abstract >>
A large number of mutants introducing point mutations and deletions into the COOH-terminal domain of colicin A have been constructed by using site-directed mutagenesis. The COOH-terminal domain carries the channel activity. The effects of the alterations in the polypeptide chain on the secretion of colicin A by colicinogenic cells have been investigated. All deletions and some mutations were found to lead to protein aggregation in the cytoplasm, thereby preventing release into the medium. The mutated colicin A proteins have been purified, and their activity in vivo (on sensitive cells) and in vitro (in planar lipid bilayers) has been assayed. Deletions in the region containing putative helices 4, 5, and 6 (predicted to be involved in pore formation) and the transitions (Ala----Asp-492, Phe----Pro-493) in helix 4 abolished the activity. No correlation was observed between mutations leading to protein aggregation and those leading to loss of channel activity. Some mutations were found to alter characteristic properties of the single channels, such as stability, current-relaxation kinetics, voltage dependence, and pore conductance. Site-directed mutagenesis provides a powerful tool for studying structure-function relationships of voltage-sensitive ionic channels.
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1595. |
Chandran SP,
Diwan V,
Tamhankar AJ,
Joseph BV,
Rosales-Klintz S,
Mundayoor S,
Lundborg CS,
Macaden R,
( 2014 ) Detection of carbapenem resistance genes and cephalosporin, and quinolone resistance genes along with oqxAB gene in Escherichia coli in hospital wastewater: a matter of concern. PMID : 24975198 : DOI : 10.1111/jam.12591 Abstract >>
This study was performed to detect the presence of Escherichia coli resistant to cephalosporins, carbapenems and quinolones in hospital wastewater. Wastewaters from a rural (H1) and an urban (H2) hospital were tested for E. coli resistant to cephalosporins, carbapenem and quinolones. Genes coding for chromosomal and plasmid-mediated resistance and phylogenetic grouping was detected by multiplex polymerase chain reaction (PCR) and for genetic relatedness by rep-PCR. Of 190 (H1 = 94; H2 = 96) E. coli examined, 44% were resistant to both cephalosporins and quinolones and 3% to imipenem. ESBLs were detected phenotypically in 96% of the isolates, the gene blaCTX-M coding for 87% and blaTEM for 63%. Quinolone resistance was due to mutations in gyrA and parC genes in 97% and plasmid-coded aac-(6')-Ib-cr in 89% of isolates. Only in one carbapenem-resistant E. coli, NDM-1 was detected. Nearly 67% of the isolates belonged to phylogenetic group B2. There was no genetic relatedness among the isolates. Hospital wastewater contains genetically diverse multidrug-resistant E. coli. This study stresses the need for efficient water treatment plants in healthcare settings as a public health measure to minimize spread of multidrug-resistant bacteria into the environment.
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1596. |
Pérez-Llarena FJ,
Kerff F,
Zamorano L,
Fernández MC,
Nuñez ML,
Miró E,
Oliver A,
Navarro F,
Bou G,
( 2013 ) Characterization of the new AmpC �]-lactamase FOX-8 reveals a single mutation, Phe313Leu, located in the R2 loop that affects ceftazidime hydrolysis. PMID : 23877692 : DOI : 10.1128/AAC.00818-13 PMC : PMC3811397 Abstract >>
A novel class C �]-lactamase (FOX-8) was isolated from a clinical strain of Escherichia coli. The FOX-8 enzyme possessed a unique substitution (Phe313Leu) compared to FOX-3. Isogenic E. coli strains carrying FOX-8 showed an 8-fold reduction in resistance to ceftazidime relative to FOX-3. In a kinetic analysis, FOX-8 displayed a 33-fold reduction in kcat/Km for ceftazidime compared to FOX-3. In the FOX family of �]-lactamases, the Phe313 residue located in the R2 loop affects ceftazidime hydrolysis and alters the phenotype of E. coli strains carrying this variant.
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1597. |
Nakamura G,
Wachino J,
Sato N,
Kimura K,
Yamada K,
Jin W,
Shibayama K,
Yagi T,
Kawamura K,
Arakawa Y,
( 2014 ) Practical agar-based disk potentiation test for detection of fosfomycin-nonsusceptible Escherichia coli clinical isolates producing glutathione S-transferases. PMID : 24951800 : DOI : 10.1128/JCM.01094-14 PMC : PMC4313133 Abstract >>
The number of reports concerning Escherichia coli clinical isolates that produce glutathione S-transferases responsible for fosfomycin resistance (FR-GSTs) has been increasing. We have developed a disk-based potentiation test in which FR-GST producers expand the growth inhibition zone around a Kirby-Bauer disk containing fosfomycin in combination with sodium phosphonoformate (PPF). PPF, an analog of fosfomycin, is a transition-state inhibitor of FosA(PA), a type of FR-GST from Pseudomonas aeruginosa. Considering its mechanism of action, PPF was expected to inhibit a variety of FR-GSTs. In the presence of PPF, zone enlargement around the disk containing fosfomycin was observed for FosA3-, FosA4-, and FosC2-producing E. coli clinical isolates. Moreover, the growth inhibition zone was remarkably enlarged when the Mueller-Hinton (MH) agar plate contained 25 �gg/ml glucose-6-phosphate (G6P). When we retrospectively tested 12 fosfomycin-resistant (MIC, ?256 �gg/ml) E. coli clinical isolates from our hospital with the potentiation test, 6 FR-GST producers were positive phenotypically by potentiation disk and were positive for FR-GST genes: 5 harbored fosA3 and 1 harbored fosA4. To identify the production of FR-GSTs, we set the provisional cutoff value, 5-mm enlargement, by adding PPF to a fosfomycin disk on the MH agar plates containing G6P. Our disk-based potentiation test reliably identifies FR-GST producers and can be performed easily; therefore, it will be advantageous in epidemiological surveys and infection control of fosfomycin-resistant bacteria in clinical settings.
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1598. |
Kusumoto M,
Fukamizu D,
Ogura Y,
Yoshida E,
Yamamoto F,
Iwata T,
Ooka T,
Akiba M,
Hayashi T,
( 2014 ) Lineage-specific distribution of insertion sequence excision enhancer in enterotoxigenic Escherichia coli isolated from swine. PMID : 24334665 : DOI : 10.1128/AEM.03696-13 PMC : PMC3911043 Abstract >>
Insertion sequences (ISs) are the simplest transposable elements and are widely distributed in bacteria; however, they also play important roles in genome evolution. We recently identified a protein called IS excision enhancer (IEE) in enterohemorrhagic Escherichia coli (EHEC) O157. IEE promotes the excision of IS elements belonging to the IS3 family, such as IS629, as well as several other families. IEE-mediated IS excision generates various genomic deletions that lead to the diversification of the bacterial genome. IEE has been found in a broad range of bacterial species; however, among sequenced E. coli strains, IEE is primarily found in EHEC isolates. In this study, we investigated non-EHEC pathogenic E. coli strains isolated from domestic animals and found that IEE is distributed in specific lineages of enterotoxigenic E. coli (ETEC) strains of serotypes O139 or O149 isolated from swine. The iee gene is located within integrative elements that are similar to SpLE1 of EHEC O157. All iee-positive ETEC lineages also contained multiple copies of IS629, a preferred substrate of IEE, and their genomic locations varied significantly between strains, as observed in O157. These data suggest that IEE may have been transferred among EHEC and ETEC in swine via SpLE1 or SpLE1-like integrative elements. In addition, IS629 is actively moving in the ETEC O139 and O149 genomes and, as in EHEC O157, is promoting the diversification of these genomes in combination with IEE.
|
1599. |
Wendel AF,
Brodner AH,
Wydra S,
Ressina S,
Henrich B,
Pfeffer K,
Toleman MA,
Mackenzie CR,
( 2013 ) Genetic characterization and emergence of the metallo-�]-lactamase GIM-1 in Pseudomonas spp. and Enterobacteriaceae during a long-term outbreak. PMID : 23877696 : DOI : 10.1128/AAC.00118-13 PMC : PMC3811479 Abstract >>
Since the first isolation in 2002, the metallo-�]-lactamase GIM-1 has not been detected outside Germany. The data presented here, for 50 clinical blaGIM-1-positive isolates, including Pseudomonas spp. and Enterobacteriaceae (Enterobacter cloacae, Klebsiella oxytoca, Serratia marcescens, Escherichia coli, and Citrobacter freundii), collected between 2007 and 2012 at the original site in an ongoing outbreak, demonstrate a diverse genetic background and dissemination of the gene conferring resistance to enteric bacteria.
|
1600. |
Liu BT,
Li L,
Fang LX,
Sun J,
Liao XP,
Yang QE,
Huang T,
Liu YH,
( 2014 ) Characterization of plasmids carrying oqxAB in bla(CTX-M)-negative Escherichia coli isolates from food-producing animals. PMID : 24927154 : DOI : 10.1089/mdr.2014.0022 Abstract >>
To study the characteristics of plasmids harboring oqxAB among bla(CTX-M)-negative Escherichia coli isolates and search for oqxAB-harboring plasmids similar to plasmids carrying oqxAB-bla(CTX-M) reported previously, conjugation experiment was performed for 115 randomly selected oqxAB-positive but bla(CTX-M)-negative E. coli isolates from diseased animals in Guangdong, China. S1 nuclease pulsed-field gel electrophoresis (PFGE) and southern blotting experiments were performed to investigate the location of oqxAB and other resistance genes. The EcoRI digestion profiles of the plasmids with oqxAB were also analyzed. The clonal relatedness of donor isolates was investigated by PFGE. In this study, 32 oqxAB transconjugants were successfully obtained and most transconjugants showed multidrug resistances. Eleven replicon combination types were found in these transconjugants. floR and oqxAB were found on the same plasmids in all nine transconjugants resistant to florfenicol. The sequences between floR and oqxAB were identical in most transconjugants and the two genes were both linked with tnp in insertion sequences. Nine F18:A-:B1 plasmids with only oqxAB shared identical EcoRI digestion profiles and the profiles were also identical with that of a plasmid carrying oqxAB-bla(CTX-M) found previously. Co-transfer of plasmids carrying oqxAB and fosA3, respectively, was also observed in one isolate. This study demonstrates the dissemination of oqxAB among bla(CTX-M)-negative E. coli isolates was mainly mediated by identical F18:A-:B1 plasmids. A novel arrangement of regions between floR and oqxAB might play an important role in the dissemination of floR-oqxAB. This is the first description of the genetic environment of the relationship between oqxAB and floR in E. coli.
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1601. |
Smet A,
Vaes R,
Praud K,
Doublet B,
Daminet S,
Cloeckaert A,
Haesebrouck F,
( 2014 ) New broad-spectrum �]-lactamases emerging among Enterobacteriaceae from healthy cats and dogs: a public health concern? PMID : 24916079 : DOI : 10.1016/j.ijantimicag.2014.03.006 Abstract >>
N/A
|
1602. |
Schramm E,
Mende J,
Braun V,
Kamp RM,
( 1987 ) Nucleotide sequence of the colicin B activity gene cba: consensus pentapeptide among TonB-dependent colicins and receptors. PMID : 2439491 : DOI : 10.1128/jb.169.7.3350-3357.1987 PMC : PMC212389 Abstract >>
Colicin B formed by Escherichia coli kills sensitive bacteria by dissipating the membrane potential through channel formation. The nucleotide sequence of the structural gene (cba) which encodes colicin B and of the upstream region was determined. A polypeptide consisting of 511 amino acids was deduced from the open reading frame. The active colicin had a molecular weight of 54,742. The carboxy-terminal amino acid sequence showed striking homology to the corresponding channel-forming region of colicin A. Of 216 amino acids, 57% were identical and an additional 19% were homologous. In this part 66% of the nucleotides were identical in the colicin A and B genes. This region contained a sequence of 48 hydrophobic amino acids. Sequence homology to the other channel-forming colicins, E1 and I, was less pronounced. A homologous pentapeptide was detected in colicins B, M, and I whose uptake required TonB protein function. The same consensus sequence was found in all outer membrane proteins involved in the TonB-dependent uptake of iron siderophores and of vitamin B12. Upstream of cba a sequence comprising 294 nucleotides was identical to the sequence upstream of the structural gene of colicin E1, with the exception of 43 single-nucleotide replacements, additions, or deletions. Apparently, the region upstream of colicins B and E1 and the channel-forming sequences of colicins A and B have a common origin.
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1603. |
Stokes MO,
Abuoun M,
Umur S,
Wu G,
Partridge SR,
Mevius DJ,
Coldham NG,
Fielder MD,
( 2013 ) Complete sequence of pSAM7, an IncX4 plasmid carrying a novel blaCTX-M-14b transposition unit isolated from Escherichia coli and Enterobacter cloacae from cattle. PMID : 23836183 : DOI : 10.1128/AAC.01157-13 PMC : PMC3754312 Abstract >>
The same plasmid carrying blaCTX-M-14b was identified from an Escherichia coli isolate and an Enterobacter cloacae isolate collected from cattle in the United Kingdom by complete plasmid sequencing. This 35,341-bp plasmid, pSAM7, had an IncX4 backbone that is 99% identical to that of pJIE143 from a human isolate in Australia. PCR screening identified pSAM7-like plasmids in three other E. coli isolates of different multilocus sequence types isolated from cattle on different farms in the United Kingdom.
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1604. |
Beck CM,
Diner EJ,
Kim JJ,
Low DA,
Hayes CS,
( 2014 ) The F pilus mediates a novel pathway of CDI toxin import. PMID : 24889811 : DOI : 10.1111/mmi.12658 PMC : PMC4107189 Abstract >>
Contact-dependent growth inhibition (CDI) is a widespread form of inter-bacterial competition that requires direct cell-to-cell contact. CDI(+) inhibitor cells express CdiA effector proteins on their surface. CdiA binds to specific receptors on susceptible target bacteria and delivers a toxin derived from its C-terminal region (CdiA-CT). Here, we show that purified CdiA-CT(536) toxin from uropathogenic Escherichia coli 536 translocates into bacteria, thereby by-passing the requirement for cell-to-cell contact during toxin delivery. Genetic analyses demonstrate that the N-terminal domain of CdiA-CT(536) is necessary and sufficient for toxin import. The CdiA receptor plays no role in this import pathway; nor do the Tol and Ton systems, which are exploited to internalize colicin toxins. Instead, CdiA-CT(536) import requires conjugative F pili. We provide evidence that the N-terminal domain of CdiA-CT(536) interacts with F pilin, and that pilus retraction is critical for toxin import. This pathway is reminiscent of the strategy used by small RNA leviviruses to infect F(+) cells. We propose that CdiA-CT(536) mimics the pilin-binding maturation proteins of leviviruses, allowing the toxin to bind F pili and become internalized during pilus retraction.
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1605. |
Dolejska M,
Villa L,
Minoia M,
Guardabassi L,
Carattoli A,
( 2014 ) Complete sequences of IncHI1 plasmids carrying blaCTX-M-1 and qnrS1 in equine Escherichia coli provide new insights into plasmid evolution. PMID : 24862095 : DOI : 10.1093/jac/dku172 Abstract >>
To determine the structure of two multidrug-resistant IncHI1 plasmids carrying blaCTX-M-1 in Escherichia coli isolates disseminated in an equine clinic in the Czech Republic. A complete nucleotide sequencing of 239 kb IncHI1 (pEQ1) and 287 kb IncHI1/X1 (pEQ2) plasmids was performed using the 454-Genome Sequencer FLX system. The sequences were compared using bioinformatic tools with other sequenced IncHI1 plasmids. A comparative analysis of pEQ1 and pEQ2 identified high nucleotide identity with the IncHI1 type 2 plasmids. A novel 24 kb module containing an operon involved in short-chain fructooligosaccharide uptake and metabolism was found in the pEQ backbones. The role of the pEQ plasmids in the metabolism of short-chain fructooligosaccharides was demonstrated by studying the growth of E. coli cells in the presence of these sugars. The module containing the blaCTX-M-1 gene was formed by a truncated macrolide resistance cluster and flanked by IS26 as previously observed in IncI1 and IncN plasmids. The IncHI1 plasmid changed size and gained the quinolone resistance gene qnrS1 as a result of IS26-mediated fusion with an IncX1 plasmid. Our data highlight the structure and evolution of IncHI1 from equine E. coli. A plasmid-mediated sugar metabolic element could play a key role in strain fitness, contributing to the successful dissemination and maintenance of these plasmids in the intestinal microflora of horses.
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1606. |
Tagg KA,
Iredell JR,
Partridge SR,
( 2014 ) Complete sequencing of IncI1 sequence type 2 plasmid pJIE512b indicates mobilization of blaCMY-2 from an IncA/C plasmid. PMID : 24890591 : DOI : 10.1128/AAC.02773-14 PMC : PMC4135994 Abstract >>
Sequencing of pJIE512b, a 92.3-kb IncI1 sequence type 2 (ST2) plasmid carrying bla(CMY-2), revealed a bla(CMY-2) context that appeared to have been mobilized from an IncA/C plasmid by the insertion sequence IS1294. A comparison with published plasmids suggests that bla(CMY-2) has been mobilized from IncA/C to IncI1 plasmids more than once by IS1294-like elements. Alignment of pJIE512b with the only other available IncI1 ST2 plasmid revealed differences across the backbones, indicating variability within this sequence type.
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1607. |
Danzeisen JL,
Wannemuehler Y,
Nolan LK,
Johnson TJ,
( 2013 ) Comparison of multilocus sequence analysis and virulence genotyping of Escherichia coli from live birds, retail poultry meat, and human extraintestinal infection. PMID : 23678737 : DOI : 10.1637/10218-042812-ResNote.1 Abstract >>
To examine the correlations between virulence genotyping and multilocus sequence analysis of Escherichia coli from poultry and humans, 88 isolates were examined. The isolates were selected from a population of over 1000 based on their assignment to nine different virulence genotyping clusters. Clustering based on multilocus sequence analysis mostly correlated with virulence genotyping, although multilocus sequence analysis demonstrated higher discriminatory ability and greater reliability related to inferred phylogenetic relationships. No distinct patterns in host source were observed using inferred phylogeny through multilocus sequence analysis, indicating that human, avian, and retail meat isolates are diverse, and some belong to multiple shared clonal complexes. Clonal complexes with host source overlap included ST95 and ST23 and additional novel groups, underscoring the diversity of avian pathogenic E. coli and the potential importance of these novel groups as avian and zoonotic pathogens.
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1608. |
Timofte D,
Maciuca IE,
Evans NJ,
Williams H,
Wattret A,
Fick JC,
Williams NJ,
( 2014 ) Detection and molecular characterization of Escherichia coli CTX-M-15 and Klebsiella pneumoniae SHV-12 �]-lactamases from bovine mastitis isolates in the United Kingdom. PMID : 24247146 : DOI : 10.1128/AAC.00752-13 PMC : PMC3910873 Abstract >>
Recent reports raised concerns about the role that farm stock may play in the dissemination of extended-spectrum �]-lactamase (ESBL)-producing bacteria. This study characterized the ESBLs in two Escherichia coli and three Klebsiella pneumoniae subsp. pneumoniae isolates from cases of clinical bovine mastitis in the United Kingdom. Bacterial culture and sensitivity testing of bovine mastitic milk samples identified Gram-negative cefpodoxime-resistant isolates, which were assessed for their ESBL phenotypes. Conjugation experiments and PCR-based replicon typing (PBRT) were used for characterization of transferable plasmids. E. coli isolates belonged to sequence type 88 (ST88; determined by multilocus sequence typing) and carried blaCTX-M-15 and blaTEM-1, while K. pneumoniae subsp. pneumoniae isolates carried blaSHV-12 and blaTEM-1. Conjugation experiments demonstrated that blaCTX-M-15 and blaTEM-1 were carried on a conjugative plasmid in E. coli, and PBRT identified this to be an IncI1 plasmid. The resistance genes were nontransferable in K. pneumoniae subsp. pneumoniae isolates. Moreover, in the E. coli isolates, an association of ISEcp1 and IS26 with blaCTX-M-15 was found where the IS26 element was inserted upstream of both ISEcp1 and the blaCTX-M promoter, a genetic arrangement highly similar to that described in some United Kingdom human isolates. We report the first cases in Europe of bovine mastitis due to E. coli CTX-M-15 and also of bovine mastitis due to K. pneumoniae subsp. pneumoniae SHV-12 �]-lactamases in the United Kingdom. We also describe the genetic environment of blaCTX-M-15 and highlight the role that IncI1 plasmids may play in the spread and dissemination of ESBL genes, which have been described in both human and cattle isolates.
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1609. |
Dhara L,
Tripathi A,
( 2014 ) Genetic and structural insights into plasmid-mediated extended-spectrum �]-lactamase activity of CTX-M and SHV variants among pathogenic Enterobacteriaceae infecting Indian patients. PMID : 24794736 : DOI : 10.1016/j.ijantimicag.2014.03.002 Abstract >>
Resistance to third-generation cephalosporins (3GCs) mediated by extended-spectrum �]-lactamases (ESBLs) in pathogenic Enterobacteriaceae is considered a major public health threat in India. This study deals with the detection of plasmid-mediated blaCTX-M, blaSHV and blaOXA genes, understanding their contribution to the ESBL phenotype, and their molecular interaction with 3GCs. More than 87% of isolates showed 3GC resistance, with ESBL production in 60.0% of Escherichia coli and 47.7% of Klebsiella pneumoniae. Molecular characterisation revealed the presence of blaCTX-M-15 (29.8%), blaCTX-M-truncated (1.3%), blaCTX-M-27 (0.7%), blaSHV-1 (20.5%), blaSHV-11 (2.0%), blaSHV-42 (0.7%) and blaOXA-1 (9.9%), among which blaCTX-M variants and blaSHV-42 were ESBLs. Phylogenetic analysis predicted strong selection pressure on all blaCTX-M variants, blaSHV-11 and blaSHV-42. The instability index and Gibbs free folding energy change (�G�GG) predicted decreased stability of SHV-11 and SHV-42. Mutations of CTX-M-truncated, SHV-11 and SHV-42 located in the core region of the enzymes were found to be functional/pathogenic in nature. The catalytic pockets of CTX-M-15 and SHV-42 had the greatest molecular surface area, which might explain their expanded substrate spectrum towards oxyimino-cephalosporins. Molecular dynamics analysis indicated different structural flexibility of CTX-M-truncated compared with the other enzymes. Amino acid alterations resulted in a change of orientation of catalytic residues of class A �]-lactamases that might affect their catalytic processes. Molecular interactions revealed higher catalytic efficiency (�GG and Km) of CTX-M-15, CTX-M-truncated, CTX-M-27, SHV-11 and SHV-42 compared with their respective wild-types. This study provides useful insights into ESBL production of pathogenic Enterobacteriaceae in India that might help in the development of new antibiotics.
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1610. |
Nardi RM,
Camargo IL,
Lima-Bittencourt CI,
Paiva MC,
Nascimento AM,
( 2012 ) The first report of the qnrB19, qnrS1 and aac(6?)-Ib-cr genes in urinary isolates of ciprofloxacin-resistant Escherichia coli in Brazil. PMID : 22850962 : DOI : 10.1590/s0074-02762012000500018 Abstract >>
In this study, we investigated the presence of plasmid-mediated quinolone resistance (PMQR) genes among 101 ciprofloxacin-resistant urinary Escherichia coli isolates and searched for mutations in the quinolone-resistance-determining regions (QRDRs) of the DNA gyrase and topoisomerase IV genes in PMQR-carrying isolates. Eight isolates harboured the qnr and aac(6')-Ib-cr genes (3 qnrS1, 1 qnrB19 and 4 aac(6')-Ib-cr). A mutational analysis of the QRDRs in qnr and aac(6')-Ib-cr-positive isolates revealed mutations in gyrA, parC and parE that might be associated with high levels of resistance to quinolones. No mutation was detected in gyrB. Rare gyrA, parC and parE mutations were detected outside of the QRDRs. This is the first report of qnrB19, qnrS1 and aac(6')-Ib-cr -carrying E. coli isolates in Brazil.
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1611. |
Zou L,
Meng J,
McDermott PF,
Wang F,
Yang Q,
Cao G,
Hoffmann M,
Zhao S,
( 2014 ) Presence of disinfectant resistance genes in Escherichia coli isolated from retail meats in the USA. PMID : 24908046 : DOI : 10.1093/jac/dku197 Abstract >>
To examine the distribution of all genes known to be responsible for resistance to quaternary ammonium compounds (QACs), and their association with resistance to QACs and other antimicrobials, in Escherichia coli recovered from retail meats. A total of 570 strains of E. coli isolated from US retail meats in 2006 were screened for the presence of 10 QAC resistance genes [qacE, qacE�G1, qacF, qacG, emrE, sugE(c), sugE(p), mdfA and ydgE/ydgF]. The MICs of six common disinfectants were determined using an agar dilution method. Possible associations between the presence of the gene and bacterial resistance to QACs and antimicrobials were investigated. emrE, sugE(c), mdfA and ydgE/ydgF were commonly present (77.2%-100%) in the E. coli isolates, but qac and sugE(p) were less prevalent (0.4%-22.3%). emrE-mdfA-sugE(c)-ydgE/F was the most common QAC resistance gene profile. A significant association was found between antimicrobial resistance and the presence of sugE(p) and qacE�G1 (P < 0.05). Antimicrobial-resistant E. coli isolates tended to contain more diverse combinations of disinfectant resistance genes than susceptible ones. All isolates showed reduced susceptibility to five of six disinfectants compared with the control strains. Higher MICs were generally associated with the presence of qac and sugE(p) genes. The QAC resistance genes were commonly present among E. coli isolated from retail meats, and the qac and sugE(p) genes were highly associated with multidrug resistance phenotypes. Using QACs in the food industry may not be as effective as expected and could provide selection pressure for strains with acquired resistance to other antimicrobials.
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1612. |
Lonardi E,
Moonens K,
Buts L,
de Boer AR,
Olsson JD,
Weiss MS,
Fabre E,
Guérardel Y,
Deelder AM,
Oscarson S,
Wuhrer M,
Bouckaert J,
( 2013 ) Structural Sampling of Glycan Interaction Profiles Reveals Mucosal Receptors for Fimbrial Adhesins of Enterotoxigenic Escherichia coli. PMID : 24833052 : DOI : 10.3390/biology2030894 PMC : PMC3960879 DOI : 10.3390/biology2030894 PMC : PMC3960879 Abstract >>
Fimbriae are long, proteinaceous adhesion organelles expressed on the bacterial envelope, evolutionarily adapted by Escherichia coli strains for the colonization of epithelial linings. Using glycan arrays of the Consortium for Functional Glycomics (CFG), the lectin domains were screened of the fimbrial adhesins F17G and FedF from enterotoxigenic E. coli (ETEC) and of the FimH adhesin from uropathogenic E. coli. This has led to the discovery of a more specific receptor for F17G, GlcNAcb1,3Gal. No significant differences emerged from the glycan binding profiles of the F17G lectin domains from five different E. coli strains. However, strain-dependent amino acid variations, predominantly towards the positively charged arginine, were indicated by sulfate binding in FedF and F17G crystal structures. For FedF, no significant binders could be observed on the CFG glycan array. Hence, a shotgun array was generated from microvilli scrapings of the distal jejunum of a 3-week old piglet about to be weaned. On this array, the blood group A type 1 hexasaccharide emerged as a receptor for the FedF lectin domain and remarkably also for F18-fimbriated E. coli. F17G was found to selectively recognize glycan species with a terminal GlcNAc, typifying intestinal mucins. In conclusion, F17G and FedF recognize long glycan sequences that could only be identified using the shotgun approach. Interestingly, ETEC strains display a large capacity to adapt their fimbrial adhesins to ecological niches via charge-driven interactions, congruent with binding to thick mucosal surfaces displaying an acidic gradient along the intestinal tract.
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1613. |
Weber PC,
Palchaudhuri S,
( 1986 ) Incompatibility repressor in a RepA-like replicon of the IncFI plasmid ColV2-K94. PMID : 2423502 : DOI : 10.1128/jb.166.3.1106-1112.1986 PMC : PMC215238 Abstract >>
The replication region Rep1 of the IncFI plasmid ColV2-K94 was cloned on self-replicating restriction fragments. Rep1 was structurally and functionally homologous to the RepA replicon of IncFII R plasmids. Despite this close relationship, these two replication systems were compatible with each other. The nucleotide sequence of the copA incompatibility-replication control gene of Rep1 was determined and compared with the copA sequence of RepA. Six base changes were found in a 24-base-pair span of the copA gene; these may result in the formation of a new, more stable, 49-base stem-loop structure in the potential CopA RNA repressor molecule. We postulate that these alterations weaken the interaction between RNA transcripts of the Rep1 and RepA replicons.
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1614. |
Martinez-Jéhanne V,
Pichon C,
du Merle L,
Poupel O,
Cayet N,
Bouchier C,
Le Bouguénec C,
( 2012 ) Role of the vpe carbohydrate permease in Escherichia coli urovirulence and fitness in vivo. PMID : 22615242 : DOI : 10.1128/IAI.00457-12 PMC : PMC3434581 Abstract >>
Uropathogenic Escherichia coli (UPEC) strains are a leading cause of infections in humans, but the mechanisms governing host colonization by this bacterium remain poorly understood. Previous studies have identified numerous gene clusters encoding proteins involved in sugar transport, in pathogen-specific islands. We investigated the role in fitness and virulence of the vpe operon encoding an EII complex of the phosphotransferase (PTS) system, which is found more frequently in human strains from infected urine and blood (45%) than in E. coli isolated from healthy humans (15%). We studied the role of this locus in vivo, using the UPEC E. coli strain AL511, mutants, and complemented derivatives in two experimental mouse models of infection. Mutant strains displayed attenuated virulence in a mouse model of sepsis. A role in kidney colonization was also demonstrated by coinfection experiments in a mouse model of pyelonephritis. Electron microscopy examinations showed that the vpeBC mutant produced much smaller amounts of a capsule-like surface material than the wild type, particularly when growing in human urine. Complementation of the vpeBC mutation led to an increase in the amount of exopolysaccharide, resistance to serum killing, and virulence. It was therefore clear that the loss of vpe genes was responsible for all the observed phenotypes. We also demonstrated the involvement of the vpe locus in gut colonization in the streptomycin-treated mouse model of intestinal colonization. These findings confirm that carbohydrate transport and metabolism underlie the ability of UPEC strains to colonize the host intestine and to infect various host sites.
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1615. |
Lowary TL,
Whitfield C,
Richards MR,
( 2012 ) Biosynthesis of the polymannose lipopolysaccharide O-antigens from Escherichia coli serotypes O8 and O9a requires a unique combination of single- and multiple-active site mannosyltransferases. PMID : 22875852 : DOI : 10.1074/jbc.M112.401000 PMC : PMC3471746 Abstract >>
The Escherichia coli O9a and O8 O-antigen serotypes represent model systems for the ABC transporter-dependent synthesis of bacterial polysaccharides. The O9a and O8 antigens are linear mannose homopolymers containing conserved reducing termini (the primer-adaptor), a serotype-specific repeat unit domain, and a terminator. Synthesis of these glycans occurs on the polyisoprenoid lipid-linked primer, undecaprenol pyrophosphoryl-GlcpNAc, by two conserved mannosyltransferases, WbdC and WbdB, and a serotype-specific mannosyltransferase, WbdA. The glycan structure and pattern of conservation in the O9a and O8 mannosyltransferases are not consistent with the existing model of O9a biosynthesis. Here we establish a revised pathway using a combination of in vivo (mutant complementation) experiments and in vitro strategies with purified enzymes and synthetic acceptors. WbdC and WbdB synthesize the adaptor region, where they transfer one and two �\-(1��3)-linked mannose residues, respectively. The WbdA enzymes are solely responsible for forming the repeat unit domains of these O-antigens. WbdA(O9a) has two predicted active sites and polymerizes a tetrasaccharide repeat unit containing two �\-(1��3)- and two �\-(1��2)-linked mannopyranose residues. In contrast, WbdA(O8) polymerizes trisaccharide repeat units containing single �\-(1��3)-, �\-(1��2)-, and �]-(1��2)-mannopyranoses. These studies illustrate assembly systems exploiting several mannosyltransferases with flexible active sites, arranged in single- and multiple-domain formats.
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1616. |
Venturini C,
Hassan KA,
Roy Chowdhury P,
Paulsen IT,
Walker MJ,
Djordjevic SP,
( 2013 ) Sequences of two related multiple antibiotic resistance virulence plasmids sharing a unique IS26-related molecular signature isolated from different Escherichia coli pathotypes from different hosts. PMID : 24223859 : DOI : 10.1371/journal.pone.0078862 PMC : PMC3817090 Abstract >>
Enterohemorrhagic Escherichia coli (EHEC) and atypical enteropathogenic E. coli (aEPEC) are important zoonotic pathogens that increasingly are becoming resistant to multiple antibiotics. Here we describe two plasmids, pO26-CRL125 (125 kb) from a human O26:H- EHEC, and pO111-CRL115 (115kb) from a bovine O111 aEPEC, that impart resistance to ampicillin, kanamycin, neomycin, streptomycin, sulfathiazole, trimethoprim and tetracycline and both contain atypical class 1 integrons with an identical IS26-mediated deletion in their 3?-conserved segment. Complete sequence analysis showed that pO26-CRL125 and pO111-CRL115 are essentially identical except for a 9.7 kb fragment, present in the backbone of pO26-CRL125 but absent in pO111-CRL115, and several indels. The 9.7 kb fragment encodes IncI-associated genes involved in plasmid stability during conjugation, a putative transposase gene and three imperfect repeats. Contiguous sequence identical to regions within these pO26-CRL125 imperfect repeats was identified in pO111-CRL115 precisely where the 9.7 kb fragment is missing, suggesting it may be mobile. Sequences shared between the plasmids include a complete IncZ replicon, a unique toxin/antitoxin system, IncI stability and maintenance genes, a novel putative serine protease autotransporter, and an IncI1 transfer system including a unique shufflon. Both plasmids carry a derivate Tn21 transposon with an atypical class 1 integron comprising a dfrA5 gene cassette encoding resistance to trimethoprim, and 24 bp of the 3?-conserved segment followed by Tn6026, which encodes resistance to ampicillin, kanymycin, neomycin, streptomycin and sulfathiazole. The Tn21-derivative transposon is linked to a truncated Tn1721, encoding resistance to tetracycline, via a region containing the IncP-1�\ oriV. Absence of the 5 bp direct repeats flanking Tn3-family transposons, indicates that homologous recombination events played a key role in the formation of this complex antibiotic resistance gene locus. Comparative sequence analysis of these closely related plasmids reveals aspects of plasmid evolution in pathogenic E. coli from different hosts.
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1617. |
Burbelo PD,
Ching KH,
Morse CG,
Alevizos I,
Bayat A,
Cohen JI,
Ali MA,
Kapoor A,
Browne SK,
Holland SM,
Kovacs JA,
Iadarola MJ,
( 2013 ) Altered antibody profiles against common infectious agents in chronic disease. PMID : 24312567 : DOI : 10.1371/journal.pone.0081635 PMC : PMC3847058 Abstract >>
Despite the important diagnostic value of evaluating antibody responses to individual human pathogens, antibody profiles against multiple infectious agents have not been used to explore health and disease mainly for technical reasons. We hypothesized that the interplay between infection and chronic disease might be revealed by profiling antibodies against multiple agents. Here, the levels of antibodies against a panel of 13 common infectious agents were evaluated with the quantitative Luciferase Immunoprecipitation Systems (LIPS) in patients from three disease cohorts including those with pathogenic anti-interferon-�^ autoantibodies (IFN-�^ AAB), HIV and Sj?gren's syndrome (SjS) to determine if their antibody profiles differed from control subjects. The IFN-�^ AAB patients compared to controls demonstrated statistically higher levels of antibodies against VZV (p=0.0003), EBV (p=0.002), CMV (p=0.003), and C. albicans (p=0.03), but lower antibody levels against poliovirus (p=0.04). Comparison of HIV patients with blood donor controls revealed that the patients had higher levels of antibodies against CMV (p=0.0008), HSV-2 (p=0.0008), EBV (p=0.001), and C. albicans (p=0.01), but showed decreased levels of antibodies against coxsackievirus B4 (p=0.0008), poliovirus (p=0.0005), and HHV-6B (p=0.002). Lastly, SjS patients had higher levels of anti-EBV antibodies (p=0.03), but lower antibody levels against several enteroviruses including a newly identified picornavirus, HCoSV-A (p=0.004), coxsackievirus B4 (p=0.04), and poliovirus (p=0.02). For the IFN-�^ AAB and HIV cohorts, principal component analysis revealed unique antibody clusters that showed the potential to discriminate patients from controls. The results suggest that antibody profiles against these and likely other common infectious agents may yield insight into the interplay between exposure to infectious agents, dysbiosis, adaptive immunity and disease activity.
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1618. |
Tondi D,
Venturelli A,
Bonnet R,
Pozzi C,
Shoichet BK,
Costi MP,
( 2014 ) Targeting class A and C serine �]-lactamases with a broad-spectrum boronic acid derivative. PMID : 24882105 : DOI : 10.1021/jm5006572 PMC : PMC4079326 Abstract >>
Production of �]-lactamases (BLs) is the most widespread resistance mechanism adopted by bacteria to fight �]-lactam antibiotics. The substrate spectrum of BLs has become increasingly broad, posing a serious health problem. Thus, there is an urgent need for novel BL inhibitors. Boronic acid transition-state analogues are able to reverse the resistance conferred by class A and C BLs. We describe a boronic acid analogue possessing interesting and potent broad-spectrum activity vs class A and C serine-based BLs. Starting from benzo(b)thiophene-2-boronic acid (BZBTH2B), a nanomolar non-�]-lactam inhibitor of AmpC that can potentiate the activity of a third-generation cephalosporin against AmpC-producing resistant bacteria, we designed a novel broad-spectrum nanomolar inhibitor of class A and C BLs. Structure-based drug design (SBDD), synthesis, enzymology data, and X-ray crystallography results are discussed. We clarified the inhibitor binding geometry responsible for broad-spectrum activity vs serine-active BLs using double mutant thermodynamic cycle studies.
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1619. |
Rahman M,
Shukla SK,
Prasad KN,
Ovejero CM,
Pati BK,
Tripathi A,
Singh A,
Srivastava AK,
Gonzalez-Zorn B,
( 2014 ) Prevalence and molecular characterisation of New Delhi metallo-�]-lactamases NDM-1, NDM-5, NDM-6 and NDM-7 in multidrug-resistant Enterobacteriaceae from India. PMID : 24831713 : DOI : 10.1016/j.ijantimicag.2014.03.003 Abstract >>
The growing prevalence of carbapenem resistance in Enterobacteriaceae worldwide is a major concern. New Delhi metallo-�]-lactamase (NDM)-mediated carbapenem resistance has been identified in Enterobacteriaceae from numerous countries including those of the Indian subcontinent. Currently, seven NDM �]-lactamase variants (NDM-1 to -7) have been identified. This study evaluated the detection and molecular characterisation of NDM variants in Enterobacteriaceae at a tertiary care hospital in India. A total of 464 isolates were tested; 57 (12.3%) were resistant or showed reduced susceptibility to imipenem and meropenem. All carbapenem-resistant isolates were blaNDM-positive by PCR, but 13 isolates bore variants that differed in sequence from blaNDM-1. NDM-5, NDM-6 and NDM-7 were identified in two, eight and three isolates, respectively. blaNDM variants were located on plasmids of >100kb with IncF, IncA/C and untypeable replicon types. Genes encoding the 16S rRNA methyltransferases RmtB, RmtC and ArmA as well as those for AmpC �]-lactamases were also located on the same plasmids as blaNDM in different combinations. The prevalence of NDM-5 to -7 variants was significantly higher in Escherichia coli (P=0.015) and they were more frequently isolated from the urology ward (P=0.037) than NDM-1. The mortality rate was comparable between patients infected with isolates positive for blaNDM-1 and blaNDM variants [25% (11/44) vs. 23% (3/13)]. Expression of blaNDM variants in E. coli using the same promoter showed that NDM-7 conferred higher resistance to imipenem. The diverse genotypic features of blaNDM indicate rapid evolution of NDM resulting from their wide spread in the Indian subcontinent.
|
1620. |
Lo AW,
Moonens K,
De Kerpel M,
Brys L,
Pardon E,
Remaut H,
De Greve H,
( 2014 ) The molecular mechanism of Shiga toxin Stx2e neutralization by a single-domain antibody targeting the cell receptor-binding domain. PMID : 25053417 : DOI : 10.1074/jbc.M114.566257 PMC : PMC4155698 Abstract >>
Shiga toxin Stx2e is the major known agent that causes edema disease in newly weaned pigs. This severe disease is characterized by neurological disorders, hemorrhagic lesions, and frequent fatal outcomes. Stx2e consists of an enzymatically active A subunit and five B subunits that bind to a specific glycolipid receptor on host cells. It is evident that antibodies binding to the A subunit or the B subunits of Shiga toxin variants may have the capability to inhibit their cytotoxicity. Here, we report the discovery and characterization of a VHH single domain antibody (nanobody) isolated from a llama phage display library that confers potent neutralizing capacity against Stx2e toxin. We further present the crystal structure of the complex formed between the nanobody (NbStx2e1) and the Stx2e toxoid, determined at 2.8 ? resolution. Structural analysis revealed that for each B subunit of Stx2e, one NbStx2e1 is interacting in a head-to-head orientation and directly competing with the glycolipid receptor binding site on the surface of the B subunit. The neutralizing NbStx2e1 can in the future be used to prevent or treat edema disease.
|
1621. |
Jacobson JM,
Yin J,
Kitov PI,
Mulvey G,
Griener TP,
James MN,
Armstrong G,
Bundle DR,
( 2014 ) The crystal structure of shiga toxin type 2 with bound disaccharide guides the design of a heterobifunctional toxin inhibitor. PMID : 24225957 : DOI : 10.1074/jbc.M113.518886 PMC : PMC3887212 Abstract >>
Shiga toxin type 2 (Stx2a) is clinically most closely associated with enterohemorrhagic E. coli O157:H7-mediated hemorrhagic colitis that sometimes progresses to hemolytic-uremic syndrome. The ability to express the toxin has been acquired by other Escherichia coli strains, and outbreaks of food poisoning have caused significant mortality rates as, for example, in the 2011 outbreak in northern Germany. Stx2a, an AB5 toxin, gains entry into human cells via the glycosphingolipid receptor Gb3. We have determined the first crystal structure of a disaccharide analog of Gb3 bound to the B5 pentamer of Stx2a holotoxin. In this Gb3 analog,-GalNAc replaces the terminal-Gal residue. This co-crystal structure confirms previous inferences that two of the primary binding sites identified in theB5 pentamer of Stx1 are also functional in Stx2a. This knowledge provides a rationale for the synthesis and evaluation of heterobifunctional antagonists for E. coli toxins that target Stx2a. Incorporation of GalNAc Gb3 trisaccharide in a heterobifunctional ligand with an attached pyruvate acetal, a ligand for human amyloid P component, and conjugation to poly[acrylamide-co-(3-azidopropylmethacrylamide)] produced a polymer that neutralized Stx2a in a mouse model of Shigatoxemia.
|
1622. |
Kaur B,
Chakraborty D,
Kumar B,
( 2014 ) Metabolic engineering of Pediococcus acidilactici BD16 for production of vanillin through ferulic acid catabolic pathway and process optimization using response surface methodology. PMID : 25077778 : DOI : 10.1007/s00253-014-5950-x Abstract >>
Occurrence of feruloyl-CoA synthetase (fcs) and enoyl-CoA hydratase (ech) genes responsible for the bioconversion of ferulic acid to vanillin have been reported and characterized from Amycolatopsis sp., Streptomyces sp., and Pseudomonas sp. Attempts have been made to express these genes in Escherichia coli DH5�\, E. coli JM109, and Pseudomonas fluorescens. However, none of the lactic acid bacteria strain having GRAS status was previously proposed for heterologous expression of fcs and ech genes for production of vanillin through biotechnological process. Present study reports heterologous expression of vanillin synthetic gene cassette bearing fcs and ech genes in a dairy isolate Pediococcus acidilactici BD16. After metabolic engineering, statistical optimization of process parameters that influence ferulic acid to vanillin biotransformation in the recombinant strain was carried out using central composite design of response surface methodology. After scale-up of the process, 3.14 mM vanillin was recovered from 1.08 mM ferulic acid per milligram of recombinant cell biomass within 20 min of biotransformation. From LCMS-ESI spectral analysis, a metabolic pathway of phenolic biotransformations was predicted in the recombinant P. acidilactici BD16 (fcs (+)/ech (+)).
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1623. |
Zdorovenko EL,
Varbanets LD,
Liu B,
Valueva OA,
Wang Q,
Shashkov AS,
Garkavaya EG,
Brovarskaya OS,
Wang L,
Knirel YA,
( 2014 ) Structure and gene cluster of the O antigen of Escherichia coli L-19, a candidate for a new O-serogroup. PMID : 25061042 : DOI : 10.1099/mic.0.080804-0 Abstract >>
Escherichia coli L-19 isolated from a healthy individual did not agglutinate with any of 21 polyvalent antisera that cover 174 E. coli O-serogroups. The strain was studied in respect to the O-antigen (O-specific polysaccharide, OPS) structure and genetics. The LPS was isolated by phenol-water extraction of bacterial cells and cleaved by mild acid hydrolysis to yield the OPS. The OPS was studied by sugar and methylation analyses, along with 1D and 2D (1)H and (13)C NMR spectroscopy. The established structure of the linear tetrasaccharide repeating unit was found to be unique among known bacterial polysaccharide structures. A peculiar component of the L-19 OPS was an amide of glucuronic acid with 2-amino-1,3-propanediol (2-amino-2-deoxyglycerol) (GroN). The O-antigen gene cluster of L-19 between the conserved genes galF and gnd was sequenced, and gene functions were tentatively assigned by a comparison with sequences in the available databases and found to be in agreement with the OPS structure. Except for putative genes for synthesis and transfer of GroN, the sequences in the L-19 O-antigen gene cluster were little related to those of reference strains of the 174 known E. coli O-serogroups. The data obtained suggest that L-19 can be considered as a candidate for a new E. coli O-serogroup.
|
1624. |
Bouché F,
Bouché JP,
( 1989 ) Genetic evidence that DicF, a second division inhibitor encoded by the Escherichia coli dicB operon, is probably RNA. PMID : 2477663 : DOI : 10.1111/j.1365-2958.1989.tb00249.x Abstract >>
The dicB operon of Escherichia coli, which was previously shown to code for a small protein inhibiting cell division, expresses a second inhibitor, DicF. Inhibition by DicF requires the transcription of a short (at the most 65 nucleotides long) stretch of DNA, acts in trans, and does not require the expression of other components of the dicABCF locus. The characteristics of the DNA sequence strongly suggest that division inhibition does not involve the translation of dicF mRNA into protein.
|
1625. |
La MV,
Jureen R,
Lin RT,
Teo JW,
( 2014 ) Unusual detection of an Acinetobacter class D carbapenemase gene, blaOXA-23, in a clinical Escherichia coli isolate. PMID : 25031438 : DOI : 10.1128/JCM.01566-14 PMC : PMC4187746 Abstract >>
N/A
|
1626. |
Dykes CW,
Halliday IJ,
Read MJ,
Hobden AN,
Harford S,
( 1985 ) Nucleotide sequences of four variants of the K88 gene of porcine enterotoxigenic Escherichia coli. PMID : 2412961 : PMC : PMC262168 Abstract >>
The nucleotide sequences of four variants of the Escherichia coli K88 antigen gene, K88ab1, K88ab2, K88ac, and K88ad, have been determined. The K88ab2 and K88ac sequences have not been reported previously. The K88ab1 sequence is very similar to that determined by other workers, but the K88ad sequence differs considerably from that described in a previous report. Comparison of the amino acid sequences inferred from the gene sequences revealed certain clusters of amino acid substitutions which have been correlated with areas of potential antigenicity in the mature proteins.
|
1627. |
Campagne S,
Marsh ME,
Capitani G,
Vorholt JA,
Allain FH,
( 2014 ) Structural basis for -10 promoter element melting by environmentally induced sigma factors. PMID : 24531660 : DOI : 10.1038/nsmb.2777 Abstract >>
Bacterial transcription is controlled by sigma factors, the RNA polymerase subunits that act as initiation factors. Although a single housekeeping sigma factor enables transcription from thousands of promoters, environmentally induced sigma factors redirect gene expression toward small regulons to carry out focused responses. Using structural and functional analyses, we determined the molecular basis of -10 promoter element recognition by Escherichia coli �m(E), which revealed an unprecedented way to achieve promoter melting. Group IV sigma factors induced strand separation at the -10 element by flipping out a single nucleotide from the nontemplate-strand DNA base stack. Unambiguous selection of this critical base was driven by a dynamic protein loop, which can be substituted to modify specificity of promoter recognition. This mechanism of promoter melting explains the increased promoter-selection stringency of environmentally induced sigma factors.
|
1628. |
Bortolaia V,
Hansen KH,
Nielsen CA,
Fritsche TR,
Guardabassi L,
( 2014 ) High diversity of plasmids harbouring blaCMY-2 among clinical Escherichia coli isolates from humans and companion animals in the upper Midwestern USA. PMID : 24500191 : DOI : 10.1093/jac/dku011 Abstract >>
To determine the population structure and genetic relatedness of plasmids encoding CMY-2 �]-lactamase in clinical Escherichia coli from humans and companion animals within a defined geographical area. In total, 42 human and 73 companion animal isolates displaying an AmpC phenotype were isolated at a regional diagnostic reference laboratory in the upper Midwestern USA during 2009-11. Following PCR screening for transferable AmpC genes and plasmid transformation, blaCMY-2-positive plasmids were characterized by S1 nuclease PFGE, PCR-based replicon typing, antimicrobial susceptibility testing of transformants, conjugation experiments, plasmid multilocus sequence typing and restriction fragment length polymorphism. blaCMY-2 occurred in 6 (14%), 56 (86%) and 6 (75%) isolates from humans, dogs and cats, respectively. Usually plasmids carrying blaCMY-2 were conjugative (78%) and did not contain additional resistance genes (82%). The replicon types were IncI1 (52%), IncA/C (13%), IncFII (10%), IncI2 (5%), IncL/M (3%), IncB/O (2%) or non-typeable (15%). Related IncI1/ST12 plasmids were detected in one human and five canine isolates, while the remaining plasmids did not show similarity across host species. A novel epidemiological linkage of blaCMY-2 with IncL/M plasmids and a new CMY gene variant (blaCMY-108) were found in human isolates. This study is one of the first One Health attempts to compare plasmids encoding CMY-2 �]-lactamase among clinical isolates from humans and companion animals in the same region. The results indicate an unforeseen heterogeneity of plasmid backgrounds and suggest limited exchange between the two populations, in which blaCMY-2 occurred at very different frequencies and was harboured by distinct plasmid types.
|
1629. |
Deng H,
Sun J,
Ma J,
Li L,
Fang LX,
Zhang Q,
Liu YH,
Liao XP,
( 2014 ) Identification of the multi-resistance gene cfr in Escherichia coli isolates of animal origin. PMID : 25036029 : DOI : 10.1371/journal.pone.0102378 PMC : PMC4103833 Abstract >>
Previous study indicated that the multi-resistance gene cfr was mainly found in gram-positive bacteria, such as Staphylococcus and Enterococcus, and was sporadically detected in Escherichia coli. Little is known about the prevalence and transmission mechanism of cfr in E. coli. In this study, the presence of cfr in E. coli isolates collected during 2010-2012 from food-producing animals in Guangdong Province of China was investigated, and the cfr-positive E. coli isolates were characterized by PFGE, plasmid profiling, and genetic environment analysis. Of the 839 E. coli isolates, 10 isolates from pig were cfr positive. All the cfr-positive isolates presented a multi-resistance phenotype and were genetically divergent as determined by PFGE. In 8 out of the 10 strains, the cfr gene was located on plasmids of ?30 kb. Restriction digestion of the plasmids with EcoRI and sequence hybridization with a cfr-specific probe revealed that the cfr-harboring fragments ranged from 6 to 23 kb and a ?18 kb cfr-carrying fragment was common for the plasmids that were ?30 kb. Four different genetic environments of cfr were detected, in which cfr is flanked by two identical copies of IS26, which may loop out the intervening sequence through homologous recombination. Among the 8 plasmids of ?30 kb, 7 plasmids shared the same genetic environment. These results demonstrate plasmid-carried cfr in E. coli and suggest that transposition and homologous recombination mediated by IS26 might have played a rule in the transfer of the cfr gene in E. coli.
|
1630. |
Lemaître C,
Mahjoub-Messai F,
Dupont D,
Caro V,
Diancourt L,
Bingen E,
Bidet P,
Bonacorsi S,
( 2013 ) A conserved virulence plasmidic region contributes to the virulence of the multiresistant Escherichia coli meningitis strain S286 belonging to phylogenetic group C. PMID : 24086343 : DOI : 10.1371/journal.pone.0074423 PMC : PMC3784414 Abstract >>
Recent isolation of the non-K1 Escherichia coli neonatal meningitis strain S286, belonging to phylogroup C, which is closely related to major group B1, and producing an extended-spectrum beta-lactamase, encouraged us to seek the genetic determinants responsible for its virulence. We show that S286 belongs to the sequence O type ST23O78 and harbors 4 large plasmids. The largest one, pS286colV (~120 kb), not related to resistance, contains genes characteristic of a Conserved Virulence Plasmidic (CVP) region initially identified in B2 extra-intestinal avian pathogenic E. coli (APEC) strains and in the B2 neonatal meningitis E. coli strain S88. The sequence of this CVP region has a strong homology (98%) with that of the recently sequenced plasmid pChi7122-1 of the O78 APEC strain Chi7122. A CVP plasmid-cured variant of S286 was less virulent than the wild type strain in a neonatal rat sepsis model with a significant lower level of bacteremia at 24 h (4.1 �� 1.41 versus 2.60 �� 0.16 log CFU/ml, p = 0.001) and mortality. However, the mortality in the model of adult mice was comparable between wild type and variant indicating that pS286colV is not sufficient by itself to fully explain the virulence of S286. Gene expression analysis of pS286colV in iron depleted environment was very close to that of pS88, suggesting that genes of CVP region may be expressed similarly in two very different genetic backgrounds (group C versus group B2). Screening a collection of 178 human A/B1 extraintestinal pathogenic E. coli (ExPEC) strains revealed that the CVP region is highly prevalent (23%) and MLST analysis indicated that these CVP positive strains belong to several clusters and mostly to phylogroup C. The virulence of S286 is explained in part by the presence of CVP region and this region has spread in different clusters of human A/B1 ExPEC, especially in group C.
|
1631. |
Tang D,
Liu YH,
Hawkey PM,
Xu L,
( 2012 ) Prevalence and characteristics of �]-lactamase and plasmid-mediated quinolone resistance genes in Escherichia coli isolated from farmed fish in China. PMID : 22809702 : DOI : 10.1093/jac/dks250 Abstract >>
To determine the molecular epidemiology of extended-spectrum �]-lactamases (ESBLs) and plasmid-mediated quinolone resistance (PMQR) in Escherichia coli isolated from farmed fish in China. E. coli was isolated from fish gut samples from fish farmed throughout Guangdong province and tested for the presence of the �]-lactamase genes and PMQR-encoding genes using PCR and DNA sequence analysis. Co-transfer of plasmids encoding for ESBLs as well as PMQR determinants was explored by conjugation into E. coli. A total of 218 non-duplicate E. coli were recovered from fish gut samples. �]-Lactamase genes were identified in 19 (17%) of 112 strains with reduced susceptibility to ampicillin, and PMQR genes were identified in 59 (73.8%) of 80 strains with reduced susceptibility to ciprofloxacin. Only three ESBL genes were identified in three isolates: bla(CTX-M-14), bla(CTX-M-79) and bla(SHV-27). PMQR gene screening identified qnr genes (n = 59) as the most common, including qnrB (n = 33), qnrS (n = 21) and qnrD (n = 5), with aac(6')-Ib-cr (n = 6) being rarely found. The co-carriage of two or three PMQR genes in one strain was found in 7 (11.9%) isolates. The ESBL gene bla(CTX-M-79) was found to be co-carried with qnrS. Co-transfer of qnrS was observed with bla(CTX-M-79). Our study is the first to demonstrate the existence of high levels of mobile genes conferring reduced susceptibility to fluoroquinolones as well as the presence of ESBL genes in fish produced in China, and identifies a significant reservoir of antibiotic resistance genes relevant to human medicine.
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1632. |
Fischer J,
Rodríguez I,
Schmoger S,
Friese A,
Roesler U,
Helmuth R,
Guerra B,
( 2012 ) Escherichia coli producing VIM-1 carbapenemase isolated on a pig farm. PMID : 22454489 : DOI : 10.1093/jac/dks108 Abstract >>
N/A
|
1633. |
Sato N,
Kawamura K,
Nakane K,
Wachino J,
Arakawa Y,
( 2013 ) First detection of fosfomycin resistance gene fosA3 in CTX-M-producing Escherichia coli isolates from healthy individuals in Japan. PMID : 23909549 : DOI : 10.1089/mdr.2013.0061 Abstract >>
We examined the prevalence and mechanism of fosfomycin resistance in CTX-M-producing Escherichia coli isolates from healthy Japanese individuals. One hundred thirty-eight CTX-M-producing E. coli isolates were subjected to fosfomycin susceptibility testing. The presence of acquired fosfomycin resistance genes such as fosA, fosA3, and fosC2 was explored, and the transmissibility of fosfomycin resistance, replicon type of plasmid, and genetic environment of fosA3 were investigated. Eight isolates (5.8%) showed resistance to fosfomycin, five of which harbored fosA3, which was in genetic linkage with blaCTX-M. The replicon types of the five transferred fosA3-carrying plasmids were as follows: IncI1 (n=2), IncN (n=1), and IncFII (n=2). Each fosA3 gene was located close to the blaCTX-M gene and was flanked by IS26 elements. These genetic environments of fosA3 in E. coli from healthy individuals were quite similar to those observed in the clinical and veterinary settings. Our results indicate that fosA3 genes possibly inserted by small mobile genetic elements flanked by two IS26 elements have already spread throughout the plasmids along with the blaCTX-M genes of commensal E. coli colonizing in healthy Japanese people.
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1634. |
Blanco J,
Mora A,
Mamani R,
López C,
Blanco M,
Dahbi G,
Herrera A,
Marzoa J,
Fernández V,
de la Cruz F,
Martínez-Martínez L,
Alonso MP,
Nicolas-Chanoine MH,
Johnson JR,
Johnston B,
López-Cerero L,
Pascual A,
Rodríguez-Baño J,
N/A N/A,
( 2013 ) Four main virotypes among extended-spectrum-�]-lactamase-producing isolates of Escherichia coli O25b:H4-B2-ST131: bacterial, epidemiological, and clinical characteristics. PMID : 23926164 : DOI : 10.1128/JCM.01555-13 PMC : PMC3811668 Abstract >>
A total of 1,021 extended-spectrum-�]-lactamase-producing Escherichia coli (ESBLEC) isolates obtained in 2006 during a Spanish national survey conducted in 44 hospitals were analyzed for the presence of the O25b:H4-B2-ST131 (sequence type 131) clonal group. Overall, 195 (19%) O25b-ST131 isolates were detected, with prevalence rates ranging from 0% to 52% per hospital. Molecular characterization of 130 representative O25b-ST131 isolates showed that 96 (74%) were positive for CTX-M-15, 15 (12%) for CTX-M-14, 9 (7%) for SHV-12, 6 (5%) for CTX-M-9, 5 (4%) for CTX-M-32, and 1 (0.7%) each for CTX-M-3 and the new ESBL enzyme CTX-M-103. The 130 O25b-ST131 isolates exhibited relatively high virulence scores (mean, 14.4 virulence genes). Although the virulence profiles of the O25b-ST131 isolates were fairly homogeneous, they could be classified into four main virotypes based on the presence or absence of four distinctive virulence genes: virotypes A (22%) (afa FM955459 positive, iroN negative, ibeA negative, sat positive or negative), B (31%) (afa FM955459 negative, iroN positive, ibeA negative, sat positive or negative), C (32%) (afa FM955459 negative, iroN negative, ibeA negative, sat positive), and D (13%) (afa FM955459 negative, iroN positive or negative, ibeA positive, sat positive or negative). The four virotypes were also identified in other countries, with virotype C being overrepresented internationally. Correspondingly, an analysis of XbaI macrorestriction profiles revealed four major clusters, which were largely virotype specific. Certain epidemiological and clinical features corresponded with the virotype. Statistically significant virotype-specific associations included, for virotype B, older age and a lower frequency of infection (versus colonization), for virotype C, a higher frequency of infection, and for virotype D, younger age and community-acquired infections. In isolates of the O25b:H4-B2-ST131 clonal group, these findings uniquely define four main virotypes, which are internationally distributed, correspond with pulsed-field gel electrophoresis (PFGE) profiles, and exhibit distinctive clinical-epidemiological associations.
|
1635. |
Kim YC,
Grable JC,
Love R,
Greene PJ,
Rosenberg JM,
( 1990 ) Refinement of Eco RI endonuclease crystal structure: a revised protein chain tracing. PMID : 2399465 : DOI : 10.1126/science.2399465 Abstract >>
N/A
|
1636. |
Zhang WJ,
Xu XR,
Schwarz S,
Wang XM,
Dai L,
Zheng HJ,
Liu S,
( 2014 ) Characterization of the IncA/C plasmid pSCEC2 from Escherichia coli of swine origin that harbours the multiresistance gene cfr. PMID : 24013193 : DOI : 10.1093/jac/dkt355 PMC : PMC3937595 Abstract >>
To determine the complete nucleotide sequence of the multidrug resistance plasmid pSCEC2, isolated from a porcine Escherichia coli strain, and to analyse it with particular reference to the cfr gene region. Plasmid pSCEC2 was purified from its E. coli J53 transconjugant and then sequenced using the 454 GS-FLX System. After draft assembly, predicted gaps were closed by PCR with subsequent sequencing of the amplicons. Plasmid pSCEC2 is 135 615 bp in size and contains 200 open reading frames for proteins of ?100 amino acids. Analysis of the sequence of pSCEC2 revealed two resistance gene segments. The 4.4 kb cfr-containing segment is flanked by two IS256 elements in the same orientation, which are believed to be involved in the dissemination of the rRNA methylase gene cfr. The other segment harbours the resistance genes floR, tet(A)-tetR, strA/strB and sul2, which have previously been found on other IncA/C plasmids. Except for these two resistance gene regions, the pSCEC2 backbone displayed >99% nucleotide sequence identity to that of other IncA/C family plasmids isolated in France, Chile and the USA. The cfr gene was identified on an IncA/C plasmid, which is well known for its broad host range and transfer and maintenance properties. The location on such a plasmid will further accelerate the dissemination of cfr and co-located resistance genes among different Gram-negative bacteria. The genetic context of cfr on plasmid pSCEC2 underlines the complexity of cfr transfer events and confirms the role that insertion sequences play in the spread of cfr.
|
1637. |
Beutin L,
Hammerl JA,
Reetz J,
Strauch E,
( 2013 ) Shiga toxin-producing Escherichia coli strains from cattle as a source of the Stx2a bacteriophages present in enteroaggregative Escherichia coli O104:H4 strains. PMID : 24012149 : DOI : 10.1016/j.ijmm.2013.08.001 Abstract >>
Enteroaggregative, Shiga toxin-producing E. coli (EAEC-STEC) O104:H4 strains are emerging pathogens causing life threatening diseases in humans. EAEC-STEC O104:H4 strains isolated between 2001 and 2011 were found to harbor a distinct type of Shiga toxin 2a- (Stx2a) encoding prophage. This phage type shows only <65% genetic similarity to so far described viable Stx phages due to differences in the modules for DNA replication, metabolism, regulation and host specificity. Stx production in EAEC is rarely observed and the source of the Stx2a phage in the EAEC-STEC O104:H4 strains is not known. We identified two DNA segments derived from orf15 and the cI gene of the O104:H4 Stx2a phage P13374 that are characteristic for Stx2a prophages present in EAEC-STEC O104:H4 strains. By PCR, these sequences were detected in 14 (5.8%) of 241 Stx2-positive STEC from animals and food. Infectious Stx2a phages could be isolated from four bovine STEC strains. These were found highly similar to P13374 for orf15, cI and stx2a sequences, the chromosomal integration site (wrbA), for phage DNA restriction profiles, virion morphology and superinfection immunity. Stx2a phages of the four bovine STEC strains formed lysogens on the E. coli K-12 strain C600. Phage P13374 from an EAEC-STEC O104:H4 outbreak strain and one of the bovine STEC phages (P13803) lysogenized the Stx-negative EAEC O104:H4 strain CB14647 by integrating in the wrbA gene of CB14647 and converted it into a Stx2a producer. Our findings provide experimental evidence that EAEC-STEC O104:H4 strains have evolved by uptake of Stx2a phages from the bovine reservoir.
|
1638. |
Brolund A,
Franzén O,
Melefors O,
Tegmark-Wisell K,
Sandegren L,
( 2013 ) Plasmidome-analysis of ESBL-producing escherichia coli using conventional typing and high-throughput sequencing. PMID : 23785449 : DOI : 10.1371/journal.pone.0065793 PMC : PMC3681856 Abstract >>
Infections caused by Extended spectrum �]-lactamase (ESBL)-producing E. coli are an emerging global problem, threatening the effectiveness of the extensively used �]-lactam antibiotics. ESBL dissemination is facilitated by plasmids, transposons, and other mobile elements. We have characterized the plasmid content of ESBL-producing E. coli from human urinary tract infections. Ten diverse isolates were selected; they had unrelated pulsed-field gel electrophoresis (PFGE) types (<90% similarity), were from geographically dispersed locations and had diverging antibiotic resistance profiles. Three isolates belonged to the globally disseminated sequence type ST131. ESBL-genes of the CTX-M-1 and CTX-M-9 phylogroups were identified in all ten isolates. The plasmid content (plasmidome) of each strain was analyzed using a combination of molecular methods and high-throughput sequencing. Hidden Markov Model-based analysis of unassembled sequencing reads was used to analyze the genetic diversity of the plasmid samples and to detect resistance genes. Each isolate contained between two and eight distinct plasmids, and at least 22 large plasmids were identified overall. The plasmids were variants of pUTI89, pKF3-70, pEK499, pKF3-140, pKF3-70, p1ESCUM, pEK204, pHK17a, p083CORR, R64, pLF82, pSFO157, and R721. In addition, small cryptic high copy-number plasmids were frequent, containing one to seven open reading frames per plasmid. Three clustered groups of such small cryptic plasmids could be distinguished based on sequence similarity. Extrachromosomal prophages were found in three isolates. Two of them resembled the E. coli P1 phage and one was previously unknown. The present study confirms plasmid multiplicity in multi-resistant E. coli. We conclude that high-throughput sequencing successfully provides information on the extrachromosomal gene content and can be used to generate a genetic fingerprint of possible use in epidemiology. This could be a valuable tool for tracing plasmids in outbreaks.
|
1639. |
Roth AL,
Lister PD,
Hanson ND,
( 2013 ) Effect of drug treatment options on the mobility and expression of blaKPC. PMID : 23861308 : DOI : 10.1093/jac/dkt280 Abstract >>
Both transposition and increases in gene expression have been implicated in the success of KPC-producing pathogens, but the stimulus required for these phenomena are unknown. It is possible that exposure to antimicrobials during patient treatment increases bla(KPC) expression or induces Tn4401 transposition. The purpose of this study was to determine if exposure to carbapenems or other antimicrobial drug classes could stimulate expression of bla(KPC) or the in vitro transposition of Tn4401. Five KPC-producing clinical isolates were evaluated in this study. Gene expression of RNA from each isolate exposed to subinhibitory, MIC or suprainhibitory levels of antibiotics was evaluated using real-time RT-PCR. Southern blots were performed on plasmids from isolates exposed to subinhibitory levels of antibiotics. There were subtle changes in bla(KPC) RNA expression following antibiotic exposure that were both strain and drug dependent. Multiple plasmids ranging from ~8 to >200 kb were observed for the Enterobacteriaceae isolates, whereas the Pseudomonas aeruginosa isolate had one ~55 kb plasmid. No changes in hybridization patterns or binding intensity for the bla(KPC) probe were observed after antibiotic exposure. While the changes in bla(KPC) RNA expression are subtle, the different responses observed suggest both strain- and genera-specific variations in response to different antibiotic treatments.
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1640. |
Nichols DA,
Jaishankar P,
Larson W,
Smith E,
Liu G,
Beyrouthy R,
Bonnet R,
Renslo AR,
Chen Y,
( 2012 ) Structure-based design of potent and ligand-efficient inhibitors of CTX-M class A �]-lactamase. PMID : 22296601 : DOI : 10.1021/jm2014138 Abstract >>
The emergence of CTX-M class A extended-spectrum �]-lactamases poses a serious health threat to the public. We have applied structure-based design to improve the potency of a novel noncovalent tetrazole-containing CTX-M inhibitor (K(i) = 21 �gM) more than 200-fold via structural modifications targeting two binding hot spots, a hydrophobic shelf formed by Pro167 and a polar site anchored by Asp240. Functional groups contacting each binding hot spot independently in initial designs were later combined to produce analogues with submicromolar potencies, including 6-trifluoromethyl-3H-benzoimidazole-4-carboxylic acid [3-(1H-tetrazol-5-yl)-phenyl]-amide, which had a K(i) value of 89 nM and reduced the MIC of cefotaxime by 64-fold in CTX-M-9 expressing Escherichia coli . The in vitro potency gains were accompanied by improvements in ligand efficiency (from 0.30 to 0.39) and LipE (from 1.37 to 3.86). These new analogues represent the first nM-affinity noncovalent inhibitors of a class A �]-lactamase. Their complex crystal structures provide valuable information about ligand binding for future inhibitor design.
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1641. |
Liu Z,
Li W,
Wang J,
Pan J,
Sun S,
Yu Y,
Zhao B,
Ma Y,
Zhang T,
Qi J,
Liu G,
Lu F,
( 2013 ) Identification and characterization of the first Escherichia coli strain carrying NDM-1 gene in China. PMID : 23762496 : DOI : 10.1371/journal.pone.0066666 PMC : PMC3677923 Abstract >>
New Delhi metallo-�]-lactamase-1 (NDM-1), an acquired class B carbapenemase, is a significant clinical threat due to its extended hydrolysis of �]-lactams including carbapenems. In this study, we identified the first confirmed clinical isolate of Escherichia coli BJ01 harboring bla(NDM-1) in China. The isolate is highly resistant to all tested antimicrobials except polymyxin. bla(NDM-1), bla(CTX-M-57), and bla TEM-1 were identified in the isolate. bla(NDM-1) was transferable to E. coli EC600 and DH5�\ in both plasmid conjugation experiments and plasmid transformation tests. BJ01 was identified as a new sequence type, ST224, by multilocus sequence typing. Analysis of genetic environment shows complex transposon-like structures surrounding the bla NDM-1 gene. Genetic analysis revealed that the region flanking bla(NDM-1) was very similar to previously identified NDM-positive Acinetobacter spp. isolated in China. The findings of this study raise attention to the emergence and spread of NDM-1-carrying Enterobacteriaceae in China.
|
1642. |
Ruhe ZC,
Wallace AB,
Low DA,
Hayes CS,
( 2013 ) Receptor polymorphism restricts contact-dependent growth inhibition to members of the same species. PMID : 23882017 : DOI : 10.1128/mBio.00480-13 PMC : PMC3735181 Abstract >>
Bacteria that express contact-dependent growth inhibition (CDI) systems outcompete siblings that lack immunity, suggesting that CDI mediates intercellular competition. To further explore the role of CDI in competition, we determined the target cell range of the CDIEC93 system from Escherichia coli EC93. The CdiAEC93 effector protein recognizes the widely conserved BamA protein as a receptor, yet E. coli EC93 does not inhibit other enterobacterial species. The predicted membrane topology of BamA indicates that three of its extracellular loops vary considerably between species, suggesting that loop heterogeneity may control CDI specificity. Consistent with this hypothesis, other enterobacteria are sensitized to CDIEC93 upon the expression of E. coli bamA and E. coli cells become CDIEC93 resistant when bamA is replaced with alleles from other species. Our data indicate that BamA loops 6 and 7 form the CdiAEC93-binding epitope and their variation between species restricts CDIEC93 target cell selection. Although BamA loops 6 and 7 vary dramatically between species, these regions are identical in hundreds of E. coli strains, suggesting that BamAEcoli and CdiAEC93 play a role in self-nonself discrimination. Contact-dependent growth inhibition (CDI) systems are widespread among Gram-negative bacteria, enabling them to bind to neighboring bacterial cells and deliver protein toxins that inhibit cell growth. In this study, we tested the role of CDI in interspecies competition using intestinal isolate Escherichia coli EC93 as an inhibitor cell model. Although E. coli EC93 inhibits different E. coli strains, other bacterial species from the intestine are completely resistant to CDI. We show that resistance is due to small variations in the CDI receptor that prevent other species from being recognized as target cells. CDI receptor interactions thus provide a mechanism by which bacteria can distinguish siblings and other close relatives (self) from more distant relatives or other species of bacteria (nonself). Our results provide a possible means by which antimicrobials could be directed to one or only a few related bacterial pathogens by using a specific receptor zip code.""
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1643. |
Ghatak S,
Singha A,
Sen A,
Guha C,
Ahuja A,
Bhattacharjee U,
Das S,
Pradhan NR,
Puro K,
Jana C,
Dey TK,
Prashantkumar KL,
Das A,
Shakuntala I,
Biswas U,
Jana PS,
( 2013 ) Detection of New Delhi metallo-beta-lactamase and extended-spectrum beta-lactamase genes in Escherichia coli isolated from mastitic milk samples. PMID : 23870003 : DOI : 10.1111/tbed.12119 Abstract >>
In this study, eight Escherichia coli isolates were obtained from milk samples of dairy cattle suffering from clinical/subclinical mastitis. Isolates were characterized for antimicrobial resistance traits and virulence genes. Results revealed that one isolate was harbouring New Delhi metallo-beta-lactamase gene (blaNDM). Cloning and sequencing of the PCR amplicon confirmed the identity of the gene (GenBank accession no. KC769583) having 100% homology with blaNDM-5 (GenBank accession no. JN104597.1), and this isolate was susceptible to colistin, chloramphenicol and tetracycline only. Moreover, another isolate carried extended-spectrum beta-lactamase (ESBL) gene - blaCTX-M , and all isolates possessed blaTEM gene. Of the eight isolates, only one isolate was positive for shiga toxin gene (stx2), and none were harbouring stx1 gene. Occurrence of New Delhi metallo-beta-lactamase (blaNDM) in one E. coli isolate and ESBL genes in other isolates poses a potential threat to human health following possible entry and spread through food chain.
|
1644. |
Fei X,
Yang D,
LaRonde-LeBlanc N,
Lorimer GH,
( 2013 ) Crystal structure of a GroEL-ADP complex in the relaxed allosteric state at 2.7 ? resolution. PMID : 23861496 : DOI : 10.1073/pnas.1311996110 PMC : PMC3740897 Abstract >>
The chaperonin proteins GroEL and GroES are cellular nanomachines driven by the hydrolysis of ATP that facilitate the folding of structurally diverse substrate proteins. In response to ligand binding, the subunits of a ring cycle in a concerted manner through a series of allosteric states (T, R, and R?), enabling work to be performed on the substrate protein. Removing two salt bridges that ordinarily break during the allosteric transitions of the WT permitted the structure of GroEL-ADP in the R state to be solved to 2.7 ? resolution. Whereas the equatorial domain displays almost perfect sevenfold symmetry, the apical domains, to which substrate proteins bind, and to a lesser extent, the intermediate domains display a remarkable asymmetry. Freed of intersubunit contacts, the apical domain of each subunit adopts a different conformation, suggesting a flexibility that permits interaction with diverse substrate proteins. This result contrasts with a previous cryo-EM study of a related allosteric ATP-bound state at lower resolution. After artificially imposing sevenfold symmetry it was concluded that a GroEL ring in the R-ATP state existed in six homogeneous but slightly different states. By imposing sevenfold symmetry on each of the subunits of the crystal structure of GroEL-ADP, we showed that the synthetic rings of (X-ray) GroEL-ADP and (cryo-EM) GroEL-ATP are structurally closely related. A deterministic model, the click stop mechanism, that implied temporal transitions between these states was proposed. Here, however, these conformational states are shown to exist as a structurally heterogeneous ensemble within a single ring.
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1645. |
Schink AK,
Kadlec K,
Schwarz S,
( 2012 ) Detection of qnr genes among Escherichia coli isolates of animal origin and complete sequence of the conjugative qnrB19-carrying plasmid pQNR2078. PMID : 22334601 : DOI : 10.1093/jac/dks024 Abstract >>
The aims of this study were to identify qnr genes among quinolone-resistant Escherichia coli isolates from defined disease conditions of companion and farm animals obtained in the BfT-GermVet study, and to gain insight into their localization and the organization of the qnr gene regions. The qnr genes were detected by PCR and confirmed by sequencing. qnr-positive isolates were checked for mutations in DNA gyrase and topoisomerase IV genes by PCR and sequencing of the quinolone resistance-determining regions. Multilocus sequence typing (MLST) was performed for the qnr-positive E. coli isolates. Plasmids harbouring qnr genes were transferred by conjugation into E. coli recipients, subjected to PCR-based replicon typing and plasmid-MLST, and one qnrB19-carrying plasmid was sequenced completely. Only 2 of 417 E. coli isolates investigated carried qnr genes. Both isolates originated from horses and showed MLST type ST86. They harboured conjugative qnrB19-carrying plasmids, which proved to be indistinguishable by restriction analysis, belonged to incompatibility group IncN, showed plasmid-MLST type ST8 and did not carry other resistance genes. The qnrB19 gene was flanked by copies of the insertion sequence IS26. One of these plasmids, pQNR2078, was sequenced completely and had a size of 42 379 bp. Except for the resistance gene region, plasmid pQNR2078 closely resembled the bla(CTX-M-65)-carrying plasmid pKC396 from E. coli. qnr genes were rarely detected among E. coli from animals in the BfT-GermVet study. The qnrB19 gene was detected on conjugative plasmids, with IS26 being likely involved in the mobility of qnrB19.
|
1646. |
Poirel L,
Potron A,
De La Cuesta C,
Cleary T,
Nordmann P,
Munoz-Price LS,
( 2012 ) Wild coastline birds as reservoirs of broad-spectrum-�]-lactamase-producing Enterobacteriaceae in Miami Beach, Florida. PMID : 22314536 : DOI : 10.1128/AAC.05982-11 PMC : PMC3346599 Abstract >>
A high rate of broad-spectrum-�]-lactamase-producing Escherichia coli isolates was identified from seagull and pelican feces collected in the Miami Beach, Florida, area. The most commonly identified resistance determinants were CMY-2 and CTX-M-15. Those wild birds might be therefore considered vehicles for wide dissemination of multidrug-resistant Enterobacteriaceae in the United States.
|
1647. |
Kim J,
Xiao H,
Bonanno JB,
Kalyanaraman C,
Brown S,
Tang X,
Al-Obaidi NF,
Patskovsky Y,
Babbitt PC,
Jacobson MP,
Lee YS,
Almo SC,
( 2013 ) Structure-guided discovery of the metabolite carboxy-SAM that modulates tRNA function. PMID : 23676670 : DOI : 10.1038/nature12180 PMC : PMC3895326 Abstract >>
The identification of novel metabolites and the characterization of their biological functions are major challenges in biology. X-ray crystallography can reveal unanticipated ligands that persist through purification and crystallization. These adventitious protein-ligand complexes provide insights into new activities, pathways and regulatory mechanisms. We describe a new metabolite, carboxy-S-adenosyl-l-methionine (Cx-SAM), its biosynthetic pathway and its role in transfer RNA modification. The structure of CmoA, a member of the SAM-dependent methyltransferase superfamily, revealed a ligand consistent with Cx-SAM in the catalytic site. Mechanistic analyses showed an unprecedented role for prephenate as the carboxyl donor and the involvement of a unique ylide intermediate as the carboxyl acceptor in the CmoA-mediated conversion of SAM to Cx-SAM. A second member of the SAM-dependent methyltransferase superfamily, CmoB, recognizes Cx-SAM and acts as a carboxymethyltransferase to convert 5-hydroxyuridine into 5-oxyacetyl uridine at the wobble position of multiple tRNAs in Gram-negative bacteria, resulting in expanded codon-recognition properties. CmoA and CmoB represent the first documented synthase and transferase for Cx-SAM. These findings reveal new functional diversity in the SAM-dependent methyltransferase superfamily and expand the metabolic and biological contributions of SAM-based biochemistry. These discoveries highlight the value of structural genomics approaches in identifying ligands within the context of their physiologically relevant macromolecular binding partners, and in revealing their functions.
|
1648. |
Giani T,
Conte V,
Di Pilato V,
Aschbacher R,
Weber C,
Larcher C,
Rossolini GM,
( 2012 ) Escherichia coli from Italy producing OXA-48 carbapenemase encoded by a novel Tn1999 transposon derivative. PMID : 22290939 : DOI : 10.1128/AAC.00035-12 PMC : PMC3318333 Abstract >>
N/A
|
1649. |
Zheng H,
Zeng Z,
Chen S,
Liu Y,
Yao Q,
Deng Y,
Chen X,
Lv L,
Zhuo C,
Chen Z,
Liu JH,
( 2012 ) Prevalence and characterisation of CTX-M �]-lactamases amongst Escherichia coli isolates from healthy food animals in China. PMID : 22325120 : DOI : 10.1016/j.ijantimicag.2011.12.001 Abstract >>
The impact of extended-spectrum �]-lactamase (ESBL)-producing Enterobacteriaceae of food animal origins on human health has caught considerable attention worldwide. Intestinal Escherichia coli obtained from healthy food animals (pigs, cattle and poultry) in China were tested for the presence of ESBL genes. CTX-M-producing isolates were further characterised by pulsed-field gel electrophoresis (PFGE), phylogenetic grouping, genetic environment analysis, conjugation and plasmid replicon typing. A total of 127 of the 896 E. coli isolates showed reduced susceptibility to cefotaxime (minimal inhibitory concentration?2 �gg/mL). bla(CTX-M) genes were detected in 111 of the 127 isolates. The most common CTX-M types were CTX-M-14 (n=40), CTX-M-55 (n=29) and CTX-M-65 (n=22), followed by CTX-M-27, -15, -98, -24, -3, -102 and -104. CMY-2 was detected in two isolates. High clonal diversity was found amongst CTX-M-producing isolates. Insertion sequence ISEcp1 was observed 42 bp upstream of the start codon of all CTX-M-9 group genes, whereas the spacer region between the right inverted repeats and CTX-M-1 group genes varied from 45 bp to 127 bp. Most bla(CTX-M) genes were transferable by conjugation. IncFII, IncI1, IncFIB, IncN and IncA/C replicons were detected in 28, 21, 7, 5 and 1 of the 70 transconjugants carrying bla(CTX-M), respectively. This study demonstrates that commensal E. coli from healthy food animals can be important reservoirs of bla(CTX-M) genes and may contribute to the dissemination and transfer of these �]-lactamase genes throughout China.
|
1650. |
Roper DI,
Cooper RA,
( 1990 ) Subcloning and nucleotide sequence of the 3,4-dihydroxyphenylacetate (homoprotocatechuate) 2,3-dioxygenase gene from Escherichia coli C. PMID : 2261999 : DOI : 10.1016/0014-5793(90)81437-s Abstract >>
A cloned gene encoding the Escherichia coli C homoprotocatechuate (HPC) dioxygenase, an aromatic ring cleavage enzyme, was used to produce large amounts of the protein. Preparations of E. coli C HPC dioxygenase, whether expressed from the cloned gene or produced by the bacterium, lost activity very rapidly. The pure protein showed one type of subunit of Mr 33,000. The first 21 N-terminal amino acids were sequenced and the data used to confirm that the open reading frame of 831 bp, identified from the nucleotide sequence, encoded HPC dioxygenase. Comparison of the derived amino acid sequence with those of other extradiol and intradiol dioxygenases showed no obvious similarity to any of them.
|
1651. |
Rasheed JK,
Kitchel B,
Zhu W,
Anderson KF,
Clark NC,
Ferraro MJ,
Savard P,
Humphries RM,
Kallen AJ,
Limbago BM,
( 2013 ) New Delhi metallo-�]-lactamase-producing Enterobacteriaceae, United States. PMID : 23731823 : DOI : 10.3201/eid1906.121515 PMC : PMC3713825 Abstract >>
We characterized 9 New Delhi metallo-�]-lactamase-producing Enterobacteriaceae (5 Klebsiella pneumoniae, 2 Escherichia coli, 1 Enterobacter cloacae, 1 Salmonella enterica serovar Senftenberg) isolates identified in the United States and cultured from 8 patients in 5 states during April 2009-March 2011. Isolates were resistant to �]-lactams, fluoroquinolones, and aminoglycosides, demonstrated MICs ?1 ?g/mL of colistin and polymyxin, and yielded positive metallo-�]-lactamase screening results. Eight isolates had blaNDM-1, and 1 isolate had a novel allele (blaNDM-6). All 8 patients had recently been in India or Pakistan, where 6 received inpatient health care. Plasmids carrying blaNDM frequently carried AmpC or extended spectrum �]-lactamase genes. Two K. pneumoniae isolates and a K. pneumoniae isolate from Sweden shared incompatibility group A/C plasmids with indistinguishable restriction patterns and a common blaNDM fragment; all 3 were multilocus sequence type 14. Restriction profiles of the remaining New Delhi metallo-�]-lactamase plasmids, including 2 from the same patient, were diverse.
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1652. |
Ho WS,
Tan LK,
Ooi PT,
Yeo CC,
Thong KL,
( 2013 ) Prevalence and characterization of verotoxigenic-Escherichia coli isolates from pigs in Malaysia. PMID : 23731465 : DOI : 10.1186/1746-6148-9-109 PMC : PMC3681573 Abstract >>
Postweaning diarrhea caused by pathogenic Escherichia coli, in particular verotoxigenic E. coli (VTEC), has caused significant economic losses in the pig farming industry worldwide. However, there is limited information on VTEC in Malaysia. The objective of this study was to characterize pathogenic E. coli isolated from post-weaning piglets and growers with respect to their antibiograms, carriage of extended-spectrum beta-lactamases, pathotypes, production of hemolysins and fimbrial adhesins, serotypes, and genotypes. PCR detection of virulence factors associated with different E. coli pathotypes (ETEC, EPEC, EHEC, and VTEC) revealed that VTEC was the only pathotype identified from six swine farms located at north-western Peninsular Malaysia. A low prevalence rate of VTEC was found among the swine samples (n = 7/345) and all 7 VTEC isolates were multidrug resistant. Five of these isolates from different hosts raised in the same pen were likely to be of the same clone as they shared identical sero-pathotypes (O139:H1, VT2e/�\-hly/F18), resistance profiles and DNA fingerprinting profiles. Two other serotypes, O130: H26 (n = 1) and O168: H21 (n = 1) carrying virulence factors were also identified. O168: H21 is possibly a new serotype as this has not been previously reported. The occurrence of VTEC with infrequently encountered serotypes that are multidrug resistant and harbouring virulence factors may be of public health concern. The detection of possible clones in this study also showed that the combination of different typing tools including phenotyping and genotyping methods is useful for molecular epidemiologic surveillance and studies.
|
1653. |
Redzej A,
Ilangovan A,
Lang S,
Gruber CJ,
Topf M,
Zangger K,
Zechner EL,
Waksman G,
( 2013 ) Structure of a translocation signal domain mediating conjugative transfer by type IV secretion systems. PMID : 23710762 : DOI : 10.1111/mmi.12275 PMC : PMC3912908 Abstract >>
Relaxases are proteins responsible for the transfer of plasmid and chromosomal DNA from one bacterium to another during conjugation. They covalently react with a specific phosphodiester bond within DNA origin of transfer sequences, forming a nucleo-protein complex which is subsequently recruited for transport by a plasmid-encoded type IV secretion system. In previous work we identified the targeting translocation signals presented by the conjugative relaxase TraI of plasmid R1. Here we report the structure of TraI translocation signal TSA. In contrast to known translocation signals we show that TSA is an independent folding unit and thus forms a bona fide structural domain. This domain can be further divided into three subdomains with striking structural homology with helicase subdomains of the SF1B family. We also show that TSA is part of a larger vestigial helicase domain which has lost its helicase activity but not its single-stranded DNA binding capability. Finally, we further delineate the binding site responsible for translocation activity of TSA by targeting single residues for mutations. Overall, this study provides the first evidence that translocation signals can be part of larger structural scaffolds, overlapping with translocation-independent activities.
|
1654. |
Tuntufye HN,
Lebeer S,
Gwakisa PS,
Goddeeris BM,
( 2012 ) Identification of Avian pathogenic Escherichia coli genes that are induced in vivo during infection in chickens. PMID : 22344666 : DOI : 10.1128/AEM.07677-11 PMC : PMC3346453 Abstract >>
Avian pathogenic Escherichia coli (APEC) is associated with extraintestinal infections in poultry causing a variety of diseases collectively known as colibacillosis. The host and bacterial factors influencing and/or responsible for carriage and systemic translocation of APEC inside the host are poorly understood. Identification of such factors could help in the understanding of its pathogenesis and in the subsequent development of control strategies. Recombination-based in vivo expression technology (RIVET) was used to identify APEC genes specifically expressed during infection in chickens. A total of 21 clones with in vivo-induced promoters were isolated from chicken livers and spleens, indicative of systemic infection. DNA sequencing of the cloned fragments revealed that 12 of the genes were conserved E. coli genes (metH, lysA, pntA, purL, serS, ybjE, ycdK [rutC], wcaJ, gspL, sdsR, ylbE, and yjiY), 6 of the genes were phage related/associated, and 3 genes were pathogen specific (tkt1, irp2, and eitD). These genes are involved in various cellular functions, such as metabolism, cell envelope and integrity, transport systems, and virulence. Others were phage related or have yet-unknown functions.
|
1655. |
Okeke IN,
Newman MJ,
Lamikanra A,
Opintan JA,
Bishar RA,
Aboderin AO,
( 2012 ) Regional dissemination of a trimethoprim-resistance gene cassette via a successful transposable element. PMID : 22666464 : DOI : 10.1371/journal.pone.0038142 PMC : PMC3364232 Abstract >>
Antimicrobial resistance is a growing international problem. We observed a 50% increase in the prevalence of trimethoprim resistance among fecal Escherichia coli from healthy Nigerian students between 1998 and 2005, a trend to increase that continued in 2009. A PCR-based screen revealed that 131 (43.1%) of isolates obtained in Nigeria in 2005 and 2009 carried integron-borne dfrA cassettes. In the case of 67 (51.1%) of these isolates, the cassette was a class 1-integron-borne dfrA7 gene, which has been reported at high prevalence from E. coli isolates from other parts of Africa. Complete sequencing of a 27 Kb dfrA7-bearing plasmid from one isolate located the dfrA7 gene within a Tn21-type transposon. The transposon also contained an IS26-derived bla/sul/str element, encoding resistance to �]-lactams, sulphonamides and streptomycin, and mercury resistance genes. Although the plasmid backbone was only found in 12 (5.8%) of trimethoprim-resistant isolates, dfrA7 and other transposon-borne genes were detected in 14 (16.3%) and 32 (26.3%) of trimethoprim resistant isolates collected in Nigeria in 2005 and 2009, respectively. Additionally, 37 (19.3%) of trimethoprim-resistant E. coli isolates collected between 2006 and 2008 from Ghana were positive for the dfrA7 and a transposon marker, but only 4 (2.1%) harbored the plasmid backbone. Our data point to transposition as a principal mechanism for disseminating dfrA7 among E. coli from Nigeria and Ghana. On-going intensive use of the affordable broad-spectrum antibacterials is likely to promote selective success of a highly prevalent transposable element in West Africa.
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1656. |
Sato T,
Yokota S,
Uchida I,
Okubo T,
Usui M,
Kusumoto M,
Akiba M,
Fujii N,
Tamura Y,
( 2013 ) Fluoroquinolone resistance mechanisms in an Escherichia coli isolate, HUE1, without quinolone resistance-determining region mutations. PMID : 23745120 : DOI : 10.3389/fmicb.2013.00125 PMC : PMC3662882 Abstract >>
Fluoroquinolone resistance can cause major clinical problems. Here, we investigated fluoroquinolone resistance mechanisms in a clinical Escherichia coli isolate, HUE1, which had no mutations quinolone resistance-determining regions (QRDRs) of DNA gyrase and topoisomerase IV. HUE1 demonstrated MICs that exceeded the breakpoints for ciprofloxacin, levofloxacin, and norfloxacin. HUE1 harbored oqxAB and qnrS1 on distinct plasmids. In addition, it exhibited lower intracellular ciprofloxacin concentrations and higher mRNA expression levels of efflux pumps and their global activators than did reference strains. The genes encoding AcrR (local AcrAB repressor) and MarR (MarA repressor) were disrupted by insertion of the transposon IS3-IS629 and a frameshift mutation, respectively. A series of mutants derived from HUE1 were obtained by plasmid curing and gene knockout using homologous recombination. Compared to the MICs of the parent strain HUE1, the fluoroquinolone MICs of these mutants indicated that qnrS1, oqxAB, acrAB, acrF, acrD, mdtK, mdfA, and tolC contributed to the reduced susceptibility to fluoroquinolone in HUE1. Therefore, fluoroquinolone resistance in HUE1 is caused by concomitant acquisition of QnrS1 and OqxAB and overexpression of AcrAB-TolC and other chromosome-encoded efflux pumps. Thus, we have demonstrated that QRDR mutations are not absolutely necessary for acquiring fluoroquinolone resistance in E. coli.
|
1657. |
Garza-Ramos U,
Barrios H,
Hernandez-Vargas MJ,
Rojas-Moreno T,
Reyna-Flores F,
Tinoco P,
Othon V,
Poirel L,
Nordmann P,
Cattoir V,
Ruiz-Palacios G,
Fernandez JL,
Santamaria RI,
Bustos P,
Castro N,
Silva-Sanchez J,
( 2012 ) Transfer of quinolone resistance gene qnrA1 to Escherichia coli through a 50 kb conjugative plasmid resulting from the splitting of a 300 kb plasmid. PMID : 22514263 : DOI : 10.1093/jac/dks123 Abstract >>
To analyse the in vitro transfer of the qnrA1 gene by a 50 kb (pSZ50) self-transferable plasmid that derives from a 300 kb plasmid (pSZ300) and to determine the complete nucleotide sequence of plasmid pSZ50. Extended-spectrum �]-lactamase (ESBL) and plasmid-mediated quinolone resistance (PMQR) genes of an Escherichia coli clinical isolate were analysed. Plasmid analysis included conjugation and selection on seven antibiotics examined by antimicrobial susceptibility testing, RFLP comparison, Southern hybridization, incompatibility group identification and shotgun sequencing. The E. coli 5509 isolate carries the genes encoding the ESBL CTX-M-15 and the quinolone resistance determinants qnrA1, qnrB2 and aac(6')-Ib-cr on a 300 kb plasmid. Seven transfer resistances were analysed by conjugation under two conditions (30 and 37�XC), leading to two distinct transconjugant phenotypes with different resistances. Transconjugants of phenotype A harboured a 300 kb plasmid named pSZ300 that conferred resistance to eight antibiotics and harboured the qnrA1, aac(6')-Ib-cr and bla(CTX-M-15) genes. Transconjugants of phenotype B were resistant to three antibiotics and they harboured the qnrA1 gene on an ? 50 kb plasmid named pSZ50. Both plasmids were self-transferable at a frequency of 1 �� 10(-3). Plasmid pSZ300 was typed to be both an IncF and IncN plasmid, whereas pSZ50 corresponded only to type IncN. Fingerprinting and Southern hybridization showed that plasmid pSZ50 derived from pSZ300. The complete nucleotide sequence of plasmid pSZ50 was determined (51556 bp) and 55 open reading frames were predicted. The qnrA1 gene was identified in a tandem duplicate inside a sul1-type integron structure. The plasmid pSZ300 represented a fusion of two replicons (IncF and IncN), and our observations suggest that the plasmid pSZ50 (IncN) may split and transfer antibiotic resistance determinants. This mechanism could be advantageous in the dissemination of antibiotic resistance genes.
|
1658. |
Szijártó V,
Lukasiewicz J,
Gozdziewicz TK,
Magyarics Z,
Nagy E,
Nagy G,
( 2014 ) Diagnostic potential of monoclonal antibodies specific to the unique O-antigen of multidrug-resistant epidemic Escherichia coli clone ST131-O25b:H4. PMID : 24789798 : DOI : 10.1128/CVI.00685-13 PMC : PMC4097449 Abstract >>
The Escherichia coli lineage sequence type 131 (ST131)-O25b:H4 is a globally spread multidrug-resistant clone responsible for a great proportion of extraintestinal infections. Driven by the significant medical needs associated with this successful pathogenic lineage, we generated murine monoclonal antibodies (MAbs) against its lipopolysaccharide (LPS) O25b antigen in order to develop quick diagnostic tests. Murine monoclonal antibodies were generated by immunizing mice with whole killed nonencapsulated ST131-O25b E. coli cells and screening hybridoma supernatants for binding to purified LPS molecules obtained from an E. coli ST131-O25b clinical isolate. The MAbs selected for further study bound to the surface of live E. coli O25b strains irrespective of the capsular type expressed, while they did not bind to bacteria or purified LPS from other serotypes, including the related classical O25 antigen (O25a). Using these specific MAbs, we developed a latex bead-based agglutination assay that has greater specificity and is quicker and simpler than the currently available typing methods. The high specificities of these MAbs can be explained by the novel structure of the O25b repeating unit elucidated in this article. Based on comparative analysis by nuclear magnetic resonance (NMR) and mass spectrometry, the N-acetyl-fucose in the O25a O-antigen had been replaced by O-acetyl-rhamnose in the O25b repeating unit. The genetic determinants responsible for this structural variation were identified by aligning the corresponding genetic loci and were confirmed by trans-complementation of a rough mutant by the subserotype-specific fragments of the rfb operons.
|
1659. |
Wang J,
Wing RA,
( 2014 ) Diamonds in the rough: a strong case for the inclusion of weak-intensity X-ray diffraction data. PMID : 24816117 : DOI : 10.1107/S1399004714005318 PMC : PMC4014128 Abstract >>
Overwhelming evidence exists to show that the inclusion of weak-intensity, high-resolution X-ray diffraction data helps improve the refinement of atomic models by imposing strong constraints on individual and overall temperature B factors and thus the quality of crystal structures. Some researchers consider these data to be of little value and opt to discard them during data processing, particularly at medium and low resolution, at which individual B factors of atomic models cannot be refined. Here, new evidence is provided to show that the inclusion of these data helps to improve the quality of experimental phases by imposing proper constraints on electron-density models during noncrystallographic symmetry (NCS) averaging. Using electron-density correlation coefficients as criteria, the resolution of data has successfully been extended from 3.1 to 2.5 ? resolution with redundancy-independent merging R factors from below 100% to about 310%. It is further demonstrated that phase information can be fully extracted from observed amplitudes through de novo NCS averaging. Averaging starts with uniform density inside double-shelled spherical masks and NCS matrices that are derived from bound heavy-atom clusters at the vertices of cuboctahedrally symmetric protein particles.
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1660. |
Chen YT,
Lin JC,
Fung CP,
Lu PL,
Chuang YC,
Wu TL,
Siu LK,
( 2014 ) KPC-2-encoding plasmids from Escherichia coli and Klebsiella pneumoniae in Taiwan. PMID : 24123430 : DOI : 10.1093/jac/dkt409 Abstract >>
Two plasmids carrying bla(KPC-2) isolated from carbapenem-resistant Escherichia coli (CR-EC) and carbapenem-resistant Klebsiella pneumoniae (CR-KP), respectively, were completely sequenced. The CR-KP strain was selected from an outbreak in 2012, and the CR-EC strain was the first blaKPC-2-carrying E. coli identified in the same carbapenem resistance monitoring programme in Taiwan. Antimicrobial susceptibility tests, multilocus sequence typing (MLST) and the conjugal transfer of plasmids were performed. Complete sequencing of the plasmids was performed using a shotgun approach. The CR-EC and CR-KP strains in this study were determined to be ST410 and ST11, respectively, by MLST. From CR-EC, we identified a 145 kb conjugative plasmid that carries bla(KPC-2), bla(CMY-2), bla(CTX-M-3) and bla(TEM-1). The plasmid is a chimera composed of three regions related to IncI, IncN and RepFIC replicons. From CR-KP, we identified an 86.5 kb plasmid, pKPC-LK30, which carries bla(KPC-2) and bla(SHV-11). The plasmid is very similar to two bla(KPC-2)-carrying IncFII(K) plasmids, but lacks one of the replication origins and cannot conjugate. The differences in cross-species transferability of the two plasmids can be explained by genetic differences between their backbones and could have resulted in the confined bla(KPC-2)-carrying CR-KP outbreak in Taiwan. Plasmid pKPC-LKEc is the first bla(KPC-2)-carrying plasmid identified from CR-EC in Taiwan. With relatively high transferability it should be closely monitored.
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1661. |
Swennes AG,
Buckley EM,
Parry NM,
Madden CM,
García A,
Morgan PB,
Astrofsky KM,
Fox JG,
( 2012 ) Enzootic enteropathogenic Escherichia coli infection in laboratory rabbits. PMID : 22573597 : DOI : 10.1128/JCM.00832-12 PMC : PMC3405579 Abstract >>
Enteropathogenic Escherichia coli (EPEC) is the most important cause of persistent diarrhea in children, particularly in developing countries. Animals serve as pathogenic E. coli reservoirs, and compelling evidence for cross-species EPEC transmission exists. In this report, enzootic EPEC infection associated with up to 10.5% diarrhea-associated morbidity in a large laboratory Dutch Belted rabbit colony was investigated. These rabbits were obtained from a commercial vendor and had acute diarrhea following shipment. Fecal culture of 20 rabbits yielded 48 E. coli isolates, 83% of which were eae positive. Repetitive sequence-based PCR (REP-PCR) and serologic analysis identified a single disease-associated EPEC O145:H2 strain. In sampled rabbits, EPEC-positive culture and the presence of diarrhea were significantly associated. This strain displayed a localized adherence-like HEp-2 cell adherence pattern, as seen in diarrheic human infant EPEC isolates. Treatment was instituted with the fluoroquinolone antibiotic enrofloxacin, to which all isolates were susceptible. Preshipment parenteral enrofloxacin administration reduced diarrhea-associated morbidity 22-fold and mortality 12-fold in subsequent deliveries. This report emphasizes the zoonotic potential of animal EPEC strains and the need for virulence determinant-based screening of E. coli isolates from diarrheic animals.
|
1662. |
Johnson TJ,
Bielak EM,
Fortini D,
Hansen LH,
Hasman H,
Debroy C,
Nolan LK,
Carattoli A,
( 2012 ) Expansion of the IncX plasmid family for improved identification and typing of novel plasmids in drug-resistant Enterobacteriaceae. PMID : 22470007 : DOI : 10.1016/j.plasmid.2012.03.001 Abstract >>
IncX plasmids are narrow host range plasmids of Enterobactericeae that have been isolated for over 50years. They are known to encode type IV fimbriae enabling their own conjugative transfer, and to provide accessory functions to their host bacteria such as resistance towards antimicrobial agents and biofilm formation. Previous plasmid-based replicon typing procedures have indicated that the prevalence of IncX plasmids is low among members of the Enterobacteriaceae. However, examination of a number of IncX-like plasmid sequences and their occurrence in various organisms suggests that IncX plasmid diversity and prevalence is underappreciated. To address these possible shortcomings, we generated additional plasmid sequences of IncX plasmids of interest and compared them to the genomes of all sequenced IncX-like plasmids. These comparisons revealed that IncX plasmids possess a highly syntenic plasmid backbone, but that they are quite divergent with respect to nucleotide and amino acid similarity. Based on phylogenetic comparisons of the sequenced IncX plasmids, the IncX plasmid group has been expanded to include at least four subtypes, IncX1-IncX4. A revised IncX plasmid replicon typing procedure, based upon these sequences and subtypes, was then developed. Use of this revised typing procedure revealed that IncX plasmid occurrence among bacterial populations is much more common than had previously been acknowledged. Thus, this revised procedure can be used to better discern the occurrence of IncX type plasmids among enterobacterial populations.
|
1663. |
Lienemann T,
Salo E,
Rimhanen-Finne R,
Rönnholm K,
Taimisto M,
Hirvonen JJ,
Tarkka E,
Kuusi M,
Siitonen A,
( 2012 ) Shiga toxin-producing Escherichia coli serotype O78:H(-) in family, Finland, 2009. PMID : 22469631 : DOI : 10.3201/eid1804.111310 PMC : PMC3309701 Abstract >>
Shiga toxin-producing Escherichia coli (STEC) is a pathogen that causes gastroenteritis and bloody diarrhea but can lead to severe disease, such as hemolytic uremic syndrome (HUS). STEC serotype O78:H(-) is rare among humans, and infections are often asymptomatic. We detected a sorbitol-fermenting STEC O78:H(-):stx(1c):hlyA in blood and fecal samples of a 2-week-old boy who had bacteremia and HUS and in fecal samples of his asymptomatic family members. The phenotypic and genotypic characteristics and the virulence properties of this invasive STEC were investigated. Our findings demonstrate that contrary to earlier suggestions, STEC under certain conditions can invade the human bloodstream. Moreover, this study highlights the need to implement appropriate diagnostic methods for identifying the whole spectrum of STEC strains associated with HUS.
|
1664. |
Koh TH,
Cao D,
Tee NW,
Teo JW,
( 2014 ) Escherichia coli with bla(IMP-8) in Singapore. PMID : 24145544 : DOI : 10.1128/AAC.01754-13 PMC : PMC3910795 Abstract >>
N/A
|
1665. |
Zhang H,
You C,
Ren J,
Xu D,
Han M,
Liao W,
( 2014 ) A simple one-step PCR walking method and its application of bacterial rRNA for sequencing identification. PMID : 24126601 : DOI : 10.1007/s00284-013-0462-y Abstract >>
There are many PCR walking methods applied currently, and they all have examples of successful application in organisms which are more complex than bacteria. However, to a certain extent, it will be more convenient for researchers if the complicated operation and poor specificity for bacteria can be improved. Here, we introduced an improved one-step PCR walking method of bacteria. Using a specific primer of the known sequence together with a universal semi-random primer, the unknown sequence adjacent to a known sequence can be obtained easily by just one ordinary round PCR. The products can be gel-purified and directly sequenced. Specific primers were designed according to the gene sequence of bacterial rRNA, and the variable and adjacent gene sequences were obtained by this method. The sequence analysis of the product showed that it can improve the resolution of bacterial identification to the species level.
|
1666. |
Feulner G,
Gray JA,
Kirschman JA,
Lehner AF,
Sadosky AB,
Vlazny DA,
Zhang J,
Zhao S,
Hill CW,
( 1990 ) Structure of the rhsA locus from Escherichia coli K-12 and comparison of rhsA with other members of the rhs multigene family. PMID : 2403547 : DOI : 10.1128/jb.172.1.446-456.1990 PMC : PMC208451 Abstract >>
The complete nucleotide sequence of the rhsA locus and selected portions of other members of the rhs multigene family of Escherichia coli K-12 have been determined. A definition of the limits of the rhsA and rhsC loci was established by comparing sequences from E. coli K-12 with sequences from an independent E. coli isolate whose DNA contains no homology to the rhs core. This comparison showed that rhsA comprises 8,249 base pairs (bp) in strain K-12 and that the Rhs0 strain, instead, contains an unrelated 32-bp sequence. Similarly, the K-12 rhsC locus is 9.6 kilobases in length and a 10-bp sequence resides at its location in the Rhs0 strain. The rhsA core, the highly conserved portion shared by all rhs loci, comprises a single open reading frame (ORF) 3,714 bp in length. The nucleotide sequence of the core ORF predicts an extremely hydrophilic 141-kilodalton peptide containing 28 repeats of a motif whose consensus is GxxxRYxYDxxGRL(I or T). One of the most novel aspects of the rhs family is the extension of the core ORF into the divergent adjacent region. Core extensions of rhsA, rhsB, rhsC, and rhsD add 139, 173, 159, and 177 codons to the carboxy termini of the respective core ORFs. For rhsA, the extended core protein would have a molecular mass of 156 kilodaltons. Core extensions of rhsB and rhsD are related, exhibiting 50.3% conservation of the predicted amino acid sequence. However, comparison of the core extensions of rhsA and rhsC at both the nucleotide and the predicted amino acid level reveals that each is highly divergent from the other three rhs loci. The highly divergent portion of the core extension is joined to the highly conserved core by a nine-codon segment of intermediate conservation. The rhsA and rhsC loci both contain partial repetitions of the core downstream from their primary cores. The question of whether the rhs loci should be considered accessory genetic elements is discussed but not resolved.
|
1667. |
San Francisco MJ,
Hope CL,
Owolabi JB,
Tisa LS,
Rosen BP,
( 1990 ) Identification of the metalloregulatory element of the plasmid-encoded arsenical resistance operon. PMID : 2408017 : DOI : 10.1093/nar/18.3.619 PMC : PMC333470 Abstract >>
The regulatory region of the plasmid-encoded arsenical resistance (ars) operon was cloned as a 727-bp EcoRI-HindIII fragment. When cloned into a promoter probe vector this fragment conferred arsenite inducible tetracycline resistance in Escherichia coli, indicating that the fragment carried a regulatory gene, the arsR gene. A single region corresponding to -35 and -10 promoter recognition sites was identified. The transcriptional start site of the mRNA was determined by primer extension. The sequence has an open reading frame for a potential 13,179 Da polypeptide, termed the ArsR protein. The fragment was cloned into a temperature regulated expression vector. A protein with an apparent molecular mass of about 12 kDa was induced by either temperature or arsenite. This protein was purified and used to produce antibodies specific for the ArsR protein.
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1668. |
Ma J,
Sun M,
Bao Y,
Pan Z,
Zhang W,
Lu C,
Yao H,
( 2013 ) Genetic diversity and features analysis of type VI secretion systems loci in avian pathogenic Escherichia coli by wide genomic scanning. PMID : 24120694 : DOI : 10.1016/j.meegid.2013.09.031 Abstract >>
Avian pathogenic Escherichia coli (APEC) strains frequently cause extra-intestinal infections and significant economic losses. Recent studies revealed that the type VI secretion system (T6SS) is involved in APEC pathogenesis. Here we provide the first evidence of three distinguishable and conserved T6SS loci in APEC genomes. In addition, we present the prevalence and comparative genomic analysis of these three T6SS loci in 472 APEC isolates. The prevalence of T6SS1, T6SS2 and T6SS3 loci were 14.62% (69/472), 2.33% (11/472) and 0.85% (4/472) positive in the APEC collections, respectively, and revealed that >85% of the strains contained T6SS loci which consisted of the virulent phylogenetic groups D and B2. Comprehensive analysis showed prominent characteristics of T6SS1 locus, including wildly prevalence, rich sequence diversity, versatile VgrG islands and excellent expression competence in various E. coli pathotypes. Whereas the T6SS2 locus infatuated with ECOR groups B2 and sequence conservation, of which are only expressed in meningitis E. coli. Regrettably, the T6SS3 locus was encoded in negligible APEC isolates and lacked several key genes. An in-depth analysis about VgrG proteins indicated that their COG4253 and gp27 domain were involved in the transport of putative effector islands and recognition of host cells respectively, which revealed that VgrG proteins played an important role in functions formation of T6SS.
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1669. |
Di Martino ML,
Fioravanti R,
Barbabella G,
Prosseda G,
Colonna B,
Casalino M,
( 2013 ) Molecular evolution of the nicotinic acid requirement within the Shigella/EIEC pathotype. PMID : 24120364 : DOI : 10.1016/j.ijmm.2013.09.007 Abstract >>
Nicotinamide adenine dinucleotide (NAD) is a crucial cofactor in several anabolic and catabolic reactions. NAD derives from quinolinic acid (QUIN) which in Escherichia coli is obtained through a pyridine salvage pathway or a de novo synthesis pathway. In the latter case, two enzymes, L-aspartate oxidase (NadB) and quinolinate synthase (NadA), are required for the synthesis of QUIN. In contrast to its E. coli ancestor, Shigella spp., the causative agent of bacillary dissentery, lacks the de novo pathway and strictly requires nicotinic acid for growth (Nic? phenotype). This phenotype depends on the silencing of the nadB and nadA genes and its pathoadaptive nature is suggested by the observation that QUIN attenuates the Shigella invasive process. Shigella shares the pathogenicity mechanism with enteronvasive E. coli (EIEC), a group of pathogenic E. coli. On the basis of this similarity EIEC and Shigella have been grouped into a single E. coli pathotype. However EIEC strains do not constitute a homogeneous group and do not possess the complete set of characters that define Shigella strains. In this work we have analysed thirteen EIEC strains belonging to different serotypes and originating from different geographic areas. We show that, in contrast to Shigella, only some EIEC strains require nicotinic acid for growth in minimal medium. Moreover, by studying the emergence of the Nic? phenotype in all serotypes of S. flexneri, as well as in S. sonnei and S. dysenteriae, we describe which molecular rearrangements occurred and which mutations are responsible for the inactivation of the nadA and nadB genes. Our data confirm that the genome of Shigella is extremely dynamic and support the hypothesis that EIEC might reflect an earlier stage of the pathoadaptation process undergone by Shigella.
|
1670. |
Ng NM,
Littler DR,
Paton AW,
Le Nours J,
Rossjohn J,
Paton JC,
Beddoe T,
( 2013 ) EcxAB is a founding member of a new family of metalloprotease AB5 toxins with a hybrid cholera-like B subunit. PMID : 24095060 : DOI : 10.1016/j.str.2013.08.024 Abstract >>
AB5 toxins are composed of an enzymatic A subunit that disrupts cellular function associated with a pentameric B subunit required for host cell invasion. EcxAB is an AB5 toxin isolated from clinical strains of Escherichia coli classified as part of the cholera family due to B subunit homology. Cholera-group toxins have catalytic ADP-ribosyltransferases as their A subunits, so it was surprising that EcxA did not. We confirmed that EcxAB self-associates as a functional toxin and obtained its structure. EcxAB is a prototypical member of a hybrid AB5 toxin family containing metzincin-type metalloproteases as their active A subunit paired to a cholera-like B subunit. Furthermore, EcxA is distinct from previously characterized proteases and thus founds an AB5-associated metzincin family that we term the toxilysins. EcxAB provides the first observation of conserved B subunit usage across different AB5 toxin families and provides evidence that the intersubunit interface of these toxins is far more permissive than previously supposed.
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1671. |
Vredenburg J,
Varela AR,
Hasan B,
Bertilsson S,
Olsen B,
Narciso-da-Rocha C,
Bonnedahl J,
Stedt J,
Da Costa PM,
Manaia CM,
( 2014 ) Quinolone-resistant Escherichia coli isolated from birds of prey in Portugal are genetically distinct from those isolated from water environments and gulls in Portugal, Spain and Sweden. PMID : 24034690 : DOI : 10.1111/1462-2920.12231 Abstract >>
The influence of geographic distribution and type of habitat on the molecular epidemiology of ciprofloxacin resistant Escherichia coli was investigated. Ciprofloxacin resistant E. coli from wastewater, urban water with faecal contamination and faeces of gulls, pigeons and birds of prey, from Portugal, Spain and Sweden were compared based on multi-locus sequence typing (MLST) and quinolone resistance genetic determinants. Multi-locus sequence typing allowed the differentiation of E. coli lineages associated with birds of prey from those inhabiting gulls and waters. E. coli lineages of clinical relevance, such as the complex ST131, were detected in wastewater, streams and gulls in Portugal, Spain and Sweden. Quinolone resistance was due to gyrA and parC mutations, although distinct mutations were detected in birds of prey and in wastewater, streams and gulls isolates. These differences were correlated with specific MLST lineages, suggesting resistance inheritance. Among the plasmid-mediated quinolone resistance genes, only aac(6')-ib-cr and qnrS were detected in wastewater, streams and gulls isolates, but not in birds of prey. The horizontal transfer of the gene aac(6')-ib-cr could be inferred from its occurrence in different MLST lineages.
|
1672. |
Hirai I,
Fukui N,
Taguchi M,
Yamauchi K,
Nakamura T,
Okano S,
Yamamoto Y,
( 2013 ) Detection of chromosomal blaCTX-M-15 in Escherichia coli O25b-B2-ST131 isolates from the Kinki region of Japan. PMID : 24091130 : DOI : 10.1016/j.ijantimicag.2013.08.005 Abstract >>
Escherichia coli O25b-B2-ST131 isolates harbouring bla(CTX-M-15) are distributed worldwide. The bla(CTX-M-15) transposition unit has often been found on plasmids and the genetic contexts have been examined; however, less is known about the frequency and contexts of the bla(CTX-M-15) transposition unit on the chromosome. This study was performed to determine the chromosomal location of the bla(CTX-M-15) transposition unit and to analyse the molecular structure of the region surrounding the bla(CTX-M-15) transposition unit in E. coli O25b-B2-ST131 isolates. Twenty-two E. coli O25b-B2-ST131 strains harbouring bla(CTX-M-15) that had been isolated from university hospital patients and nursing home residents in the Kinki region of Japan were examined. Inverse PCR (iPCR) targeting bla(CTX-M-15) was performed to classify the molecular structure of the region surrounding the bla(CTX-M-15) transposition unit. The isolates were classified into nine types (types A-I) considering the iPCR results; type A was the most prevalent type (13/22 isolates). Sequences of the iPCR-amplified DNA fragments showed that the bla(CTX-M-15) transposition unit consisted of ISEcp1, bla(CTX-M-15) and orf477�G. A homology search of the obtained sequences showed that the bla(CTX-M-15) transposition unit was inserted into different chromosomal regions in eight of the nine classified types. Although 21 of the 22 E. coli isolates possessed chromosomally located bla(CTX-M-15) transposition units, clonal spread was not evident on pulsed-field gel electrophoresis (PFGE) analysis. Taken together, these data indicate that certain E. coli O25b-B2-ST131 strains harbouring chromosomal bla(CTX-M-15) have emerged and spread in the Kinki region of Japan.
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1673. |
Stahlhut SG,
Chattopadhyay S,
Kisiela DI,
Hvidtfeldt K,
Clegg S,
Struve C,
Sokurenko EV,
Krogfelt KA,
( 2013 ) Structural and population characterization of MrkD, the adhesive subunit of type 3 fimbriae. PMID : 24123820 : DOI : 10.1128/JB.00753-13 PMC : PMC3889607 Abstract >>
Type 3 fimbriae are adhesive organelles found in enterobacterial pathogens. The fimbriae promote biofilm formation on biotic and abiotic surfaces; however, the exact identity of the receptor for the type 3 fimbriae adhesin, MrkD, remains elusive. We analyzed naturally occurring structural and functional variabilities of the MrkD adhesin from Klebsiella pneumoniae and Escherichia coli isolates of diverse origins. We identified a total of 33 allelic variants of mrkD among 90 K. pneumoniae isolates and 10 allelic variants among 608 E. coli isolates, encoding 11 and 9 protein variants, respectively. Based on the level of accumulated silent variability between the alleles, mrkD was acquired a relatively long time ago in K. pneumoniae but recently in E. coli. However, unlike K. pneumoniae, mrkD in E. coli is actively evolving under a strong positive selection by accumulation of mutations, often targeting the same positions in the protein. Several naturally occurring MrkD protein variants from E. coli were found to be significantly less adherent when tested in a mannan-binding assay and showed reduced biofilm-forming capacity. Functional examination of the MrkD adhesin in flow chamber experiments determined that it interacts with Saccharomyces cerevisiae cells in a shear-dependent manner, i.e., the binding is catch-bond-like and enhanced under increasing shear conditions. Homology modeling strongly suggested that MrkD has a two-domain structure, comprising a pilin domain anchoring the adhesin to the fimbrial shaft and a lectin domain containing the binding pocket; this is similar to structures found in other catch-bond-forming fimbrial adhesins in enterobacteria.
|
1674. |
Xu X,
Cui S,
Zhang F,
Luo Y,
Gu Y,
Yang B,
Li F,
Chen Q,
Zhou G,
Wang Y,
Pang L,
Lin L,
( 2014 ) Prevalence and characterization of cefotaxime and ciprofloxacin co-resistant Escherichia coli isolates in retail chicken carcasses and Ground Pork, China. PMID : 23952362 : DOI : 10.1089/mdr.2012.0224 Abstract >>
Retail meat products could serve as an important medium for the transfer of multidrug resistant isolates from food-producing animals to the community. In this study, the prevalence and characteristics of cefotaxime and ciprofloxacin co-resistant Escherichia coli isolates were investigated in retail chicken and ground pork samples from four provinces of China. The isolates were subjected to phylogenetic group typing and antimicrobial susceptibility testing. All isolates were further characterized by pulsed-field gel electrophoresis to determine the genetic relatedness. These isolates were also screened for beta-lactamase genes, quinolone resistance determinants by PCR, and followed by DNA sequence analysis. Cefotaxime and ciprofloxacin co-resistant E. coli isolates with diverse genetic origins were recovered in 31.9% (106/332) of retail meat samples. E. coli isolates of phylogenetic group A were dominant (59.4%, 63/106), and all isolates showed multidrug resistant profiles. The dominant resistant profiles were AMP-CAZ-CTX-CIP-CHL-GEN-SXT-TET (n=43) and AMP-CAZ-CTX-CIP-CHL-SXT-TET (n=43). Point mutations in quinolone resistance determination regions of topoisomerases were identified in all the isolates, and most of the isolates accumulated three (n=78) or four (n=21) point mutations. Plasmid-mediated quinolone-resistant determinants were identified in 68 isolates, including oqxAB (n=66), qnrS1 (n=7), qnrS2 (n=4), and aac(6')-Ib-cr (n=9). Eight subtypes of bla(CTX-M) were identified in 103 E. coli isolates, and blaCTX-M-55 (n=90) was dominant. This study highlights that retail meat could serve as an important reservoir of cefotaxime and ciprofloxacin co-resistant E. coli isolates. It is necessary to evaluate their contribution in the community and hospital infections.
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1675. |
Yuen AS,
Kolappan S,
Ng D,
Craig L,
( 2013 ) Structure and secretion of CofJ, a putative colonization factor of enterotoxigenic Escherichia coli. PMID : 24106767 : DOI : 10.1111/mmi.12407 Abstract >>
Enterotoxigenic Escherichia coli (ETEC) colonize the human gut, causing severe cholera-like diarrhoea. ETEC utilize a diverse array of pili and fimbriae for host colonization, including the Type IVb pilus CFA/III. The CFA/III pilus machinery is encoded on the cof operon, which is similar in gene sequence and synteny to the tcp operon that encodes another Type IVb pilus, the Vibrio cholerae toxin co-regulated pilus (TCP). Both pilus operons possess a syntenic gene encoding a protein of unknown function. In V. cholerae, this protein, TcpF, is a critical colonization factor secreted by the TCP apparatus. Here we show that the corresponding ETEC protein, CofJ, is a soluble protein secreted via the CFA/III apparatus. We present a 2.6 ? resolution crystal structure of CofJ, revealing a large �]-sandwich protein that bears no sequence or structural homology to TcpF. CofJ has a cluster of exposed hydrophobic side-chains at one end and structural homology to the pore-forming proteins perfringolysin O and �\-haemolysin. CofJ binds to lipid vesicles and epithelial cells, suggesting a role in membrane attachment during ETEC colonization.
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1676. |
Poey ME,
Albini M,
Saona G,
Laviña M,
( 2012 ) Virulence profiles in uropathogenic Escherichia coli isolated from pregnant women and children with urinary tract abnormalities. PMID : 22406645 : DOI : 10.1016/j.micpath.2012.02.006 Abstract >>
Uropathogenic Escherichia coli is the leading etiologic agent of urinary tract infections, encompassing a highly heterogeneous group of strains. Although many putative urovirulence factors have been described, none of them appear in all uropathogenic E. coli strains, a fact that suggests that this group would be composed of different pathogenic subgroups. In this work, a study was performed on two collections of E. coli isolates proceeding from urine cultures from two groups of patients with urinary tract infection: pregnant women and children with urinary tract abnormalities. The isolates were analyzed for their virulence content and for their phylogeny by means of PCR determinations and of phenotypic assays. Associations among the virulence traits analyzed were searched for and this approach led to the identification of five urovirulence profiles. From a total of 230 isolates, 123 (53%) could be assigned to one of these profiles. A few loci appeared as markers of these profiles so that their presence allowed predicting the general virulence content of the strains. It is presumed that these conserved associations among the virulence functions would be devoted to ensure the coherence of the bacterial pathogenic strategy. In addition, three profiles appeared with significantly different frequencies depending on the host of origin of the isolates, indicating the existence of a correlation between the virulence content of the strains and their host specificity.
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1677. |
Okamoto K,
Takahara M,
( 1990 ) Synthesis of Escherichia coli heat-stable enterotoxin STp as a pre-pro form and role of the pro sequence in secretion. PMID : 2203746 : DOI : 10.1128/jb.172.9.5260-5265.1990 PMC : PMC213188 Abstract >>
Escherichia coli heat-stable enterotoxin STp is presumed from its DNA sequence to be synthesized in vivo as a 72-amino-acid residue precursor that is cleaved to generate mature STp consisting of the 18 carboxy-terminal amino acid residues. There are two methionine residues in the inferred STp sequence in addition to the methionine residue at position 1. In order to confirm production of the STp 72-amino-acid residue precursor, we substituted the additional methionine residues by oligonucleotide-directed site-specific mutagenesis. Since these substitutions did not cause a significant change in STp production, it can be concluded that STp is normally synthesized as the 72-amino-acid residue precursor. The length of the STp precursor indicated the existence of a pro sequence between the signal peptide and the mature protein. In order to identify the pro sequence and determine its role in protein secretion, deletion and fusion proteins were made. A deletion mutant in which the gene fragment encoding amino acid residues 22 to 53 of STp was removed was made. STp activity was found in the culture supernatant of cells. Amino acid sequence analysis of the purified STp deletion mutant revealed that the pro sequence encompasses amino acid residues 20 to 54. A hybrid protein consisting of STp amino acids 1 to 53 fused in frame from residue 53 to nuclease A was not secreted into the culture supernatant. These results indicate that the pro sequence does not function to guide periplasmic protein into the extracellular milieu.
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1678. |
Sugihara J,
Smirnova I,
Kasho V,
Kaback HR,
( 2011 ) Sugar recognition by CscB and LacY. PMID : 22106930 : DOI : 10.1021/bi201592y PMC : PMC3249425 Abstract >>
The sucrose permease (CscB) and lactose permease (LacY) of Escherichia coli belong to the oligosaccharide/H(+) symporter subfamily of the major facilitator superfamily, and both catalyze sugar/H(+) symport across the cytoplasmic membrane. Thus far, there is no common substrate for the two permeases; CscB transports sucrose, and LacY is highly specific for galactopyranosides. Determinants for CscB sugar specificity are unclear, but the structural organization of key residues involved in sugar binding appears to be similar in CscB and LacY. In this study, several sugars containing galactopyranosyl, glucopyranosyl, or fructofuranosyl moieties were tested for transport with cells overexpressing either CscB or LacY. CscB recognizes not only sucrose but also fructose and lactulose, but glucopyranosides are not transported and do not inhibit sucrose transport. The findings indicate that CscB exhibits practically no specificity with respect to the glucopyranosyl moiety of sucrose. Inhibition of sucrose transport by CscB tested with various fructofuranosides suggests that the C(3)-OH group of the fructofuranosyl ring may be important for recognition by CscB. Lactulose is readily transported by LacY, where specificity is directed toward the galactopyranosyl ring, and the affinity of LacY for lactulose is similar to that observed for lactose. The studies demonstrate that the substrate specificity of CscB is directed toward the fructofuranosyl moiety of the substrate, while the specificity of LacY is directed toward the galactopyranosyl moiety.
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1679. |
Helinski DR,
McEachern MJ,
Filutowicz MS,
( 1990 ) N-terminal truncated forms of the bifunctional pi initiation protein express negative activity on plasmid R6K replication. PMID : 2277631 : DOI : 10.1007/bf00259447 Abstract >>
The replication initiation protein pi of the Escherichia coli plasmid R6K is a dual regulator in the control of plasmid copy number, functioning both as a specific initiator and inhibitor of replication. While the biochemical basis of these activities is not known, initiator activity requires binding of the protein to the seven 22 bp direct repeats within the gamma-origin region. By deleting C-terminal segments of the pi coding region, we have found that the N-terminal polypeptides of pi that are produced, corresponding to the first 117 and 164 amino acids, respectively, retain the negative activity of the bifunctional protein, i.e. these truncated pi proteins specifically inhibit R6K replication in vivo. These negatively acting polypeptides, however, are incapable of initiating replication in vivo and fail to bind to the gamma-origin of the R6K DNA in vitro. A correspondence between the observed negative activity of the N-terminal peptide and the negative regulatory activity of the intact pi protein is supported by the finding that point mutations introduced into the 164 amino acid N-terminal peptide that result in a decrease in its inhibitory activity also produce a plasmid high-copy phenotype when these mutations are incorporated into the full-length pi protein. These findings demonstrate that the negative domain of pi resides in the N-terminal segment of the protein. Furthermore, the data obtained suggest that inhibition of R6K replication by pi does not require direct binding to DNA.
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1680. |
Ribeiro AF,
Laroche E,
Hanin G,
Fournier M,
Quillet L,
Dupont JP,
Pawlak B,
( 2012 ) Antibiotic-resistant Escherichia coli in karstic systems: a biological indicator of the origin of fecal contamination? PMID : 22486636 : DOI : 10.1111/j.1574-6941.2012.01382.x Abstract >>
Occurrences of antibiotic-resistant Escherichia coli in two springs of a karstic system (NW France) providing drinking water were determined to study the role of aquifers in the dissemination of the resistance genes. Water samples were collected during wet and dry periods and after a heavy rainfall event to investigate E. coli density, antibiotic resistance patterns, and occurrences of class 1, 2, and 3 integrons. By observing patterns of the resistant isolates (i.e. number and type of resistances) and their occurrences, we were able to define two resistant subpopulations, introduced in the aquifer via surface water: (1) R1-2, characterized by one or two resistance(s), essentially to chloramphenicol and/or tetracycline (96.5%), was always found during the heavy rainfall event; (2) R3-10, characterized by three or more resistances, mostly resistant to tetracycline (94.1%) and beta-lactams (86%), was found transiently. Class 1 and 2 integrons were detected, mostly in the R3-10 subpopulation for class 1 integrons. The characteristics of these two subpopulations strongly suggest that the contamination originates from pasture runoff for the R1-2 subpopulation and from wastewater treatment plant effluents for the R3-10 subpopulation. These two subpopulations of E. coli could be used as biological indicators to determine the origin of groundwater contamination.
|
1681. |
Nakahigashi K,
Inokuchi H,
( 1990 ) Nucleotide sequence between the fadB gene and the rrnA operon from Escherichia coli. PMID : 2243799 : DOI : 10.1093/nar/18.21.6439 PMC : PMC332553 Abstract >>
N/A
|
1682. |
Rojo-Bezares B,
Martín C,
López M,
Torres C,
Sáenz Y,
( 2012 ) First detection of blaIMI-2 gene in a clinical Escherichia coli strain. PMID : 22106212 : DOI : 10.1128/AAC.05478-11 PMC : PMC3264261 Abstract >>
N/A
|
1683. |
Ooka T,
Seto K,
Kawano K,
Kobayashi H,
Etoh Y,
Ichihara S,
Kaneko A,
Isobe J,
Yamaguchi K,
Horikawa K,
Gomes TA,
Linden A,
Bardiau M,
Mainil JG,
Beutin L,
Ogura Y,
Hayashi T,
( 2012 ) Clinical significance of Escherichia albertii. PMID : 22377117 : DOI : 10.3201/eid1803.111401 PMC : PMC3309589 Abstract >>
Discriminating Escherichia albertii from other Enterobacteriaceae is difficult. Systematic analyses showed that E. albertii represents a substantial portion of strains currently identified as eae-positive Escherichia coli and includes Shiga toxin 2f-producing strains. Because E. albertii possesses the eae gene, many strains might have been misidentified as enterohemorrhagic or enteropathogenic E. coli.
|
1684. |
Knirel YA,
Ding P,
Shashkov AS,
Beutin L,
Xu Y,
Senchenkova SN,
( 2012 ) Structural and genetic characterization of the Escherichia coli O180 O antigen and identification of a UDP-GlcNAc 6-dehydrogenase. PMID : 22730467 : DOI : 10.1093/glycob/cws098 Abstract >>
The O antigen is an essential component of the lipopolysaccharides on the surface of Gram-negative bacteria and its variation provides a major basis for serotyping schemes. The Escherichia coli O-antigen form O180 was first designated in 2004, and O180 strains were found to contain virulence factors and cause diarrhea. Different O-antigen forms are almost entirely due to genetic variations in the O-antigen gene clusters. In this study, the chemical structure and gene cluster of E. coli O180 O antigen were investigated. A tetrasaccharide repeating unit with the following structure: ��4)-�]-D-ManpNAc3NAcA-(1 �� 2)-�\-L-Rhap(I)-(1 �� 3)-�]-L-Rhap(II)-(1 �� 4)-�\-D-GlcpNAc-(1��was identified in the E. coli O180 O antigen, including the residue D-ManpNAc3NAcA (2,3-diacetamido-2,3-dideoxy-D-mannopyranuronic acid) that had not been hitherto identified in E. coli. Genes in the O-antigen gene cluster were assigned functions based on their similarities with those from available databases, and five genes involved in the synthesis of UDP-D-ManpNAc3NAcA (the nucleotide-activated form of D-ManpNAc3NAcA) were identified. The gnaA gene, encoding the enzyme involved in the initial step of the UDP-D-ManpNAc3NAcA biosynthetic pathway, was cloned and the enzyme product was expressed, purified and assayed for its activity. GnaA was characterized using capillary electrophoresis and electrospray ionization mass spectrometry and identified as a UDP-GlcNAc 6-dehydrogenase. The kinetic and physicochemical parameters of GnaA also were determined.
|
1685. |
Iguchi A,
N/A N/A,
( 2012 ) Molecular characterization reveals three distinct clonal groups among clinical shiga toxin-producing Escherichia coli strains of serogroup O103. PMID : 22718945 : DOI : 10.1128/JCM.00789-12 PMC : PMC3421808 Abstract >>
Shiga toxin-producing Escherichia coli (STEC) is one of the most important groups of food-borne pathogens, and STEC strains belonging to the serotype O103:H2 can cause diarrhea, hemorrhagic colitis, and hemolytic-uremic syndrome in humans. STEC O103:non-H2 strains are also sometimes isolated from human patients, but their genetic characteristics and role in significant human enteric disease are not yet understood. Here, we investigated 17 STEC O103:non-H2 strains, including O103:H11, O103:H25, O103:HUT (UT [untypeable]), and O103:H- (nonmotile) isolated in Japan, and their characteristics were compared to those of STEC O103:H2 and other serotype STEC strains. Sequence analyses of fliC and eae genes revealed that strains possessed any of the following combinations: fliC-H2/eae-epsilon, fliC-H11/eae-beta1, and fliC-H25/eae-theta, where fliC-H2, -H11, and -H25 indicate fliC genes encoding H2, H11, and H25 flagella antigens, respectively, and eae-epsilon, -beta1, and -theta indicate eae genes encoding epsilon, beta1, and theta subclass intimins, respectively. Phylogenetic analysis based on the sequences of seven housekeeping genes demonstrated that the O103:H11/[fliC-H11] and O103:H25/[fliC-H25] strains formed two distinct groups, different from that of the O103:H2/[fliC-H2] strains. Interestingly, a group consisting of O103:H11 strains was closely related to STEC O26:H11, which is recognized as a most important non-O157 serotype, suggesting that the STEC O103:H11 and STEC O26:H11 clones evolved from a common ancestor. The multiplex PCR system for the rapid typing of STEC O103 strains described in the present study may aid clinical and epidemiological studies of the STEC O103:H2, O103:H11, and O103:H25 groups. In addition, our data provide further insights into the high variability of STEC stains with emerging new serotypes.
|
1686. |
Spira B,
de Almeida Toledo R,
Maharjan RP,
Ferenci T,
( 2011 ) The uncertain consequences of transferring bacterial strains between laboratories - rpoS instability as an example. PMID : 22067413 : DOI : 10.1186/1471-2180-11-248 PMC : PMC3240573 Abstract >>
Microbiological studies frequently involve exchanges of strains between laboratories and/or stock centers. The integrity of exchanged strains is vital for archival reasons and to ensure reproducible experimental results. For at least 50 years, one of the most common means of shipping bacteria was by inoculating bacterial samples in agar stabs. Long-term cultures in stabs exhibit genetic instabilities and one common instability is in rpoS. The sigma factor RpoS accumulates in response to several stresses and in the stationary phase. One consequence of RpoS accumulation is the competition with the vegetative sigma factor �m70. Under nutrient limiting conditions mutations in rpoS or in genes that regulate its expression tend to accumulate. Here, we investigate whether short-term storage and mailing of cultures in stabs results in genetic heterogeneity. We found that samples of the E. coli K-12 strain MC4100TF exchanged on three separate occasions by mail between our laboratories became heterogeneous. Reconstruction studies indicated that LB-stabs exhibited mutations previously found in GASP studies in stationary phase LB broth. At least 40% of reconstructed stocks and an equivalent proportion of actually mailed stock contained these mutations. Mutants with low RpoS levels emerged within 7 days of incubation in the stabs. Sequence analysis of ten of these segregants revealed that they harboured each of three different rpoS mutations. These mutants displayed the classical phenotypes of bacteria lacking rpoS. The genetic stability of MC4100TF was also tested in filter disks embedded in glycerol. Under these conditions, GASP mutants emerge only after a 3-week period. We also confirm that the intrinsic high RpoS level in MC4100TF is mainly due to the presence of an IS1 insertion in rssB. Given that many E. coli strains contain high RpoS levels similar to MC4100TF, the integrity of such strains during transfers and storage is questionable. Variations in important collections may be due to storage-transfer related issues. These results raise important questions on the integrity of bacterial archives and transferred strains, explain variation like in the ECOR collection between laboratories and indicate a need for the development of better methods of strain transfer.
|
1687. |
Lou H,
Chen M,
Black SS,
Bushell SR,
Ceccarelli M,
Mach T,
Beis K,
Low AS,
Bamford VA,
Booth IR,
Bayley H,
Naismith JH,
( 2011 ) Altered antibiotic transport in OmpC mutants isolated from a series of clinical strains of multi-drug resistant E. coli. PMID : 22053181 : DOI : 10.1371/journal.pone.0025825 PMC : PMC3203869 Abstract >>
Antibiotic-resistant bacteria, particularly gram negative species, present significant health care challenges. The permeation of antibiotics through the outer membrane is largely effected by the porin superfamily, changes in which contribute to antibiotic resistance. A series of antibiotic resistant E. coli isolates were obtained from a patient during serial treatment with various antibiotics. The sequence of OmpC changed at three positions during treatment giving rise to a total of four OmpC variants (denoted OmpC20, OmpC26, OmpC28 and OmpC33, in which OmpC20 was derived from the first clinical isolate). We demonstrate that expression of the OmpC K12 porin in the clinical isolates lowers the MIC, consistent with modified porin function contributing to drug resistance. By a range of assays we have established that the three mutations that occur between OmpC20 and OmpC33 modify transport of both small molecules and antibiotics across the outer membrane. This results in the modulation of resistance to antibiotics, particularly cefotaxime. Small ion unitary conductance measurements of the isolated porins do not show significant differences between isolates. Thus, resistance does not appear to arise from major changes in pore size. Crystal structures of all four OmpC clinical mutants and molecular dynamics simulations also show that the pore size is essentially unchanged. Molecular dynamics simulations suggest that perturbation of the transverse electrostatic field at the constriction zone reduces cefotaxime passage through the pore, consistent with laboratory and clinical data. This subtle modification of the transverse electric field is a very different source of resistance than occlusion of the pore or wholesale destruction of the transverse field and points to a new mechanism by which porins may modulate antibiotic passage through the outer membrane.
|
1688. |
Close TJ,
Murray IA,
Martinez-Suarez JV,
( 1990 ) Nucleotide sequences of genes encoding the type II chloramphenicol acetyltransferases of Escherichia coli and Haemophilus influenzae, which are sensitive to inhibition by thiol-reactive reagents. PMID : 2268278 : DOI : 10.1042/bj2720505 PMC : PMC1149729 Abstract >>
Sensitivity of enzymes to inhibition by thiol-reactive reagents is often presented as evidence for the possible involvement of cysteine residues in substrate binding and catalysis or to highlight possible important differences in structure and mechanism between closely related enzymes. The primary phenotypic distinction between the enterobacterial type II chloramphenicol acetyltransferase (CATII; typified by the enzyme encoded by the incW transmissible plasmid pSa) and the CATI and CATIII variants is the greatly enhanced susceptibility of CATII to inactivation by thiol-specific modifying reagents. Determination of the nucleotide sequence of the gene, catII, present on pSa and that of a related determinant, catIIH, isolated from Haemophilus influenzae indicates that sensitivity to such reagents cannot be due to the presence of additional reactive cysteine residues in CATII. Comparative analysis of the inactivation of CATII and CATIII by 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), 4,4'-dithiodipyridine (DTDP) and methyl methanethiosulphonate (MMTS) suggests that (i) inactivation occurs as a result of chemical modification of the same residue (Cys-31) in each enzyme, (ii) reagents that inactivate via a pseudo-first-order process (DTNB and DTDP) appear to bind with a greater affinity to CATII, and (iii) the intrinsic reactivity of Cys-31 in CATII greatly exceeds that of the corresponding residue in CATIII. The results lead to the conclusion that a striking difference in chemical reactivity of a unique and conserved thiol group between closely related enzyme variants may not be easily explained even when a high-resolution tertiary structure is available for one of them. Plausible explanations include more favourable access of reagents to Cys-31 in CATII or an enhanced reactivity of its thiol group imposed by the side chains of residues that are not in immediate contact with it.
|
1689. |
Chen X,
Zhang W,
Pan W,
Yin J,
Pan Z,
Gao S,
Jiao X,
( 2012 ) Prevalence of qnr, aac(6')-Ib-cr, qepA, and oqxAB in Escherichia coli isolates from humans, animals, and the environment. PMID : 22391545 : DOI : 10.1128/AAC.06191-11 PMC : PMC3370760 Abstract >>
qnr, aac(6')-Ib-cr, qepA, and oqxAB genes were detected in 5.7%, 4.9%, 2.6%, and 20.2% of 1,022 Escherichia coli isolates from humans, animals, and the environment, respectively, collected between 1993 and 2010 in China. The prevalence of oqxAB in porcine isolates (51.0%) was significantly higher than that in other isolates. This is the first report of oqxAB-positive isolates from ducks and geese and as early as 1994 from chickens.
|
1690. |
Ma J,
Liu JH,
Lv L,
Zong Z,
Sun Y,
Zheng H,
Chen Z,
Zeng ZL,
( 2012 ) Characterization of extended-spectrum �]-lactamase genes found among Escherichia coli isolates from duck and environmental samples obtained on a duck farm. PMID : 22407683 : DOI : 10.1128/AEM.07507-11 PMC : PMC3346353 Abstract >>
In this study, we focused on evaluating the occurrence of extended-spectrum �]-lactamase (ESBL)-producing Escherichia coli in fecal samples of healthy ducks and environmental samples from a duck farm in South China. Duck cloacal swabs and pond water samples were cultivated on MacConkey agar plates supplemented with ceftiofur. Individual colonies were examined for ESBL production. Bacteria identified as E. coli were screened for the presence of ESBL and plasmid-borne AmpC genes. The genetic relatedness, plasmid replicon type, and genetic background were determined. Of 245 samples analyzed, 123 had E. coli isolates with ceftiofur MICs higher than 8 �gg/ml (116 [50.4%] from 230 duck samples and 7 [46.7%] from 15 water samples). bla(CTX-M), bla(SHV-12), bla(CMY-2), and bla(DHA-1) were identified in 108, 5, 9, and 1 isolates, respectively. The most common bla(CTX-M) genes were bla(CTX-M-27) (n = 34), bla(CTX-M-55) (n = 27), bla(CTX-M-24e) (n = 22), and bla(CTX-M-105) (n = 20), followed by bla(CTX-M-14a), bla(CTX-M-14b), bla(CTX-M-24a), and bla(CTX-M-24b). Although most of the CTX-M producers had distinct pulsotypes, clonal transmission between duck and water isolates was observed. bla(CTX-M) genes were carried by transferable IncN, IncF, and untypeable plasmids. The novel CTX-M gene bla(CTX-M-105) was flanked by two hypothetical protein sequences, partial ISEcp1 upstream and truncated IS903D, iroN, orf1, and a Tn1721-like element downstream. It is suggested that the horizontal transfer of bla(CTX-M) genes mediated by mobile elements and the clonal spread of CTX-M-producing E. coli isolates contributed to the dissemination of bla(CTX-M) in the duck farm. Our findings highlight the importance of ducks for the dissemination of transferable antibiotic resistance genes into the environment.
|
1691. |
Mühlemann K,
Droz S,
Endimiani A,
Rohrer C,
Aebi S,
Kronenberg A,
Küffer M,
Stadler M,
Heiniger N,
( 2012 ) Transmission dynamics of extended-spectrum �]-lactamase-producing Enterobacteriaceae in the tertiary care hospital and the household setting. PMID : 22718774 : DOI : 10.1093/cid/cis581 PMC : PMC3436924 Abstract >>
Studies about transmission rates of extended-spectrum �]-lactamase (ESBL)-producing Enterobacteriaceae in hospitals and households are scarce. Eighty-two index patients with new carriage of ESBL-producing Escherichia coli (ESBL-Ec; n = 72) or ESBL-producing Klebsiella pneumoniae (ESBL-Kp; n = 10) and their hospital (n = 112) and household (n = 96) contacts were studied prospectively from May 2008 through September 2010. Isolates were phenotypically and molecularly characterized (sequencing of bla genes, repetitive extragenic palindromic polymerase chain reaction, pulse-field gel electrophoresis, and multilocus sequence typing). Transmission was defined as carriage of a clonally-related ESBL producer with identical bla(ESBL) gene(s) in the index patient and his or her contact(s). CTX-M-15 was the most prevalent ESBL in ESBL-Ec (58%) and ESBL-Kp (70%) in the index patients. Twenty (28%) ESBL-Ec isolates were of the hyperepidemic clone ST131. In the hospital, transmission rates were 4.5% (ESBL-Ec) and 8.3% (ESBL-Kp) and the incidences of transmissions were 5.6 (Ec) and 13.9 (Kp) per 1000 exposure days, respectively. Incidence of ESBL-Kp hospital transmission was significantly higher than that of ESBL-Ec (P < .0001), despite implementation of infection control measures in 75% of ESBL-Kp index patients but only 22% of ESBL-Ec index patients. Detection of ESBL producers not linked to an index patient was as frequent (ESBL-Ec, 5.7%; ESBL-Kp, 16.7%) as nosocomial transmission events. In households, transmission rates were 23% for ESBL-Ec and 25% for ESBL-Kp. Household outweighs nosocomial transmission of ESBL producers. The effect of hospital infection control measures may differ between different species and clones of ESBL producers.
|
1692. |
Anantham S,
Hall RM,
( 2012 ) pCERC1, a small, globally disseminated plasmid carrying the dfrA14 cassette in the strA gene of the sul2-strA-strB gene cluster. PMID : 22416992 : DOI : 10.1089/mdr.2012.0008 Abstract >>
Commensal Escherichia coli from healthy adult humans were screened for antibiotic resistance genes. Two unrelated strains contained the sul2 sulphonamide resistance gene and strAB streptomyicn resistance genes with the dfrA14 trimethoprim resistance gene cassette in the strA gene and conferred resistance to trimethoprim and sulphamethoxazole. A 6.8 kb plasmid, pCERC1, that contains these resistance genes was recovered and sequenced. Deletions were constructed, and the pCERC1 replication region was confined to a 1 kb segment carrying genes for RNAs that are closely related to the ColE1 replication initiation RNAs. Polymerase chain reaction assays, developed to detect the sul2-strA-strB gene cluster in this context, identified a streptomycin and sulphonamide resistance plasmid, pCERC2, identical to pCERC1 without the dfrA14 cassette in two further E. coli isolates. Bioinformatic analysis revealed plasmids similar to pCERC1 and two more members of this family. One, the probable progenitor, carries only the sul2 gene adjacent to the small mobile element CR2. The other has a variant resistance gene cluster that has evolved from pCERC2 via acquisition of the tet(A) tetracycline resistance determinant. pCERC1 and pCERC2 have been detected in many countries, indicating a global distribution and appear to have been circulating in Gram-negative bacteria for more than 25 years.
|
1693. |
Hayes F,
Barill? D,
Schumacher MA,
( 2012 ) Structural mechanism of ATP-induced polymerization of the partition factor ParF: implications for DNA segregation. PMID : 22674577 : DOI : 10.1074/jbc.M112.373696 PMC : PMC3406698 Abstract >>
Segregation of the bacterial multidrug resistance plasmid TP228 requires the centromere-binding protein ParG, the parH centromere, and the Walker box ATPase ParF. The cycling of ParF between ADP- and ATP-bound states drives TP228 partition; ATP binding stimulates ParF polymerization, which is essential for segregation, whereas ADP binding antagonizes polymerization and inhibits DNA partition. The molecular mechanism involved in this adenine nucleotide switch is unclear. Moreover, it is unknown how any Walker box protein polymerizes in an ATP-dependent manner. Here, we describe multiple ParF structures in ADP- and phosphomethylphosphonic acid adenylate ester (AMPPCP)-bound states. ParF-ADP is monomeric but dimerizes when complexed with AMPPCP. Strikingly, in ParF-AMPPCP structures, the dimers interact to create dimer-of-dimer "units" that generate a specific linear filament. Mutation of interface residues prevents both polymerization and DNA segregation in vivo. Thus, these data provide insight into a unique mechanism by which a Walker box protein forms polymers that involves the generation of ATP-induced dimer-of-dimer building blocks.
|
1694. |
Thomson CJ,
Young HK,
Amyes SG,
( 1990 ) N-terminal amino-acid sequence and subunit structure of the type IV trimethoprim-resistant plasmid-encoded dihydrofolate reductase. PMID : 2197414 : DOI : 10.1099/00222615-32-3-153 Abstract >>
The type IV plasmid-mediated dihydrofolate reductase (DHFR), from a clinical strain of Escherichia coli isolated in South India, was prepared from a transconjugant containing the original clinical plasmid, E. coli J62-2 (pUK1123), and from E. coli C600 (pUK1150) containing a 2.6-kb HindIII fragment of pUK1123 cloned into plasmid pBR322. Both preparations were purified by methotrexate affinity chromatography. Automatic amino-acid sequencing of the N-terminal of the purified type IV enzyme from both sources gave an identical sequence which was clearly distinct from other plasmid-mediated trimethoprim-resistant DHFRs. The type IV DHFR showed most homology with the endogenous, chromosomally-encoded E. coli enzyme. Amino-acid sequence analysis also showed that the type IV enzyme preparation from E. coli J62-2 harbouring the original clinical plasmid, pUK1123, also contained the E. coli DNA-binding protein NS1. Analysis by polyacrylamide gel electrophoresis suggested that the type IV enzyme, in its native form, consists of a DHFR of Mr 33,000 coupled to a DNA-binding protein.
|
1695. |
Loh S,
Skurray R,
Célérier J,
Bagdasarian M,
Bailone A,
Devoret R,
( 1990 ) Nucleotide sequence of the psiA (plasmid SOS inhibition) gene located on the leading region of plasmids F and R6-5. PMID : 2201950 : DOI : 10.1093/nar/18.15.4597 PMC : PMC331290 Abstract >>
N/A
|
1696. |
Yoshioka Y,
Fujita Y,
Ohtsubo E,
( 1990 ) Nucleotide sequence of the promoter-distal region of the tra operon of plasmid R100, including traI (DNA helicase I) and traD genes. PMID : 2164585 : DOI : 10.1016/0022-2836(90)90145-C Abstract >>
The nucleotide sequence of the promoter-distal region of the tra operon of R100 was determined. There are five open reading frames in the region between traT and finO, and their protein products were identified. Nucleotide sequences of plasmid F corresponding to the junction regions among the open reading frames seen in R100 were also determined. Comparison of these nucleotide sequences revealed strong homology in the regions containing traD, traI and an open reading frame (named orfD). The TraD protein (83,899 Da) contains three hydrophobic regions, of which two are located near the amino-terminal region. This protein also contains a possible ATP-binding consensus sequence at the amino-terminal region and a characteristic repeated peptide sequence (Gln-Gln-Pro)10 at the carboxy-terminal region. The TraI protein (191,679 Da) contains the sequence motif conserved in an ATP-dependent DNA helicase superfamily in its carboxy-terminal region. The protein product of orfD, which is probably a new tra gene (named traX), contains 65% hydrophobic amino acids, especially rich in alanine and leucine. There exist non-homologous regions between R100 and F that could be represented as four I-D (insertion or deletion) loops in heteroduplex molecules. Assignment of each loop to the strand of R100 or F was , however, found to be the reverse from that previously assumed. The three I-D loops that were located between traT and traD, between traD and traI, and between traI and finO had no terminal inverted repeat sequences nor had they any homology with known insertion sequences, while the fourth was IS3, located within the finO gene of F. The sequences in the I-D loops, except IS3, may also code for proteins that are, however, likely to be nonessential for transfer of plasmids.
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1697. |
López J,
Rodríguez JC,
( 1990 ) Nucleotide sequence and expression of the copy number control gene (cop) of the incFVII plasmid pSU233. PMID : 2263498 : DOI : 10.1093/nar/18.23.7177 PMC : PMC332823 Abstract >>
N/A
|
1698. |
Miwatani T,
Honda T,
Okamoto K,
Tsuji T,
Miyama A,
Inoue T,
( 1990 ) A single amino acid substitution in the A subunit of Escherichia coli enterotoxin results in a loss of its toxic activity. PMID : 2266142 : Abstract >>
A plasmid encoding a mutant gene of heat-labile enterotoxin (LT), produced by enterotoxigenic Escherichia coli, was induced by treatment of plasmid EWD 299 with hydroxylamine. A mutant strain of E. coli HB 101 carrying the mutant plasmid pTUH 6A produced a low toxic LT analogue (mutant LT), which was cross-reactive with anti-LT antibody. The mutant LT activity was less than 0.15 and 0.006% of the normal LT in the rabbit ileal loop test and in the rabbit skin permeability test, respectively. The amino acid composition of the mutant LT-B subunit was the same as that of the normal B subunit. Though the A2 fragment of the mutant LT was identical to normal LT by DNA analysis, the A1 fragment of the mutant LT differed from the normal A1 fragment in one amino acid at position 112; namely it had lysine instead of glutamic acid from the N terminus. These data suggest that glutamic acid at position 112 from the N terminus of the A1 fragment is important for the A subunit to express its biological activity.
|
1699. |
Betteridge T,
Partridge SR,
Iredell JR,
Stokes HW,
( 2011 ) Genetic context and structural diversity of class 1 integrons from human commensal bacteria in a hospital intensive care unit. PMID : 21628540 : DOI : 10.1128/AAC.01831-10 PMC : PMC3147655 Abstract >>
Most surveys for class 1 integrons are at least partly predicated on PCR screening that targets integron conserved regions. However, class 1 integrons are structurally diverse, so dependence on conserved regions may lead to missing clinically relevant examples of class 1 integrons. Here, we surveyed a commensal population of bacteria from patients in an intensive care unit to identify class 1 integrons irrespective of their structure or genetic context. We identified several examples of class 1 integrons linked to complete Tn402-like or Tn402 hybrid transposition modules and diverse insertion points with respect to the inverted repeat IRi boundary. The diversity and abundance of class 1 integrons identified are such that many novel elements seen here would not have been identified by commonly used methods, and they revealed an additional level of complexity.
|
1700. |
Hama C,
Takizawa T,
Moriwaki H,
Mizobuchi K,
( 1990 ) Role of leader peptide synthesis in repZ gene expression of the ColIb-P9 plasmid. PMID : 2191957 : Abstract >>
The frequency of replication initiation of the ColIb-P9 plasmid depends on the level of repZ expression, which has been shown to be negatively regulated by inc RNA, the approximately 70-base-long product of the inc gene. To further understand the regulatory mechanism of repZ gene expression, we isolated mutants defective in ColIb-P9 replication using a lambda:ColIb-P9 hybrid phage. Among six mutants isolated, one amber mutant, rep57, failed to synthesize the RepZ protein. The mutation occurred in the repZ leader sequence that encodes a 29-amino-acid reading frame, designated as repY. We also isolated mutants that suppressed the rep57 phenotype. These mutations were single base insertions between the repY initiation codon and the rep57 mutation site and resulted not only in a frame shift of repY but also in the formation of repY-repZ fusions without changing the amino acid sequence of RepZ. Thus, repY is not directly involved in the replication reaction but rather functions as a positive regulator for repZ expression. We propose that repZ expression is coupled with repY translation, which acts to disrupt a secondary structure sequestering the repZ translation initiation signal. The positive and negative regulations of repZ expression were discussed. The other mutants were mapped in repZ, confirming that repZ is essential for ColIb-P9 replication.
|
1701. |
Barton M,
Pang Y,
Peng H,
( 2012 ) Antimicrobial susceptibilities and resistance genes in Campylobacter strains isolated from poultry and pigs in Australia. PMID : 22672511 : DOI : 10.1111/j.1365-2672.2012.05354.x Abstract >>
To evaluate the phenotypic and genotypic profiles of Campylobacter spp. from poultry faecal samples from free range or intensively raised meat chickens and free range egg layers. In addition, a case-comparison study of antibiotic resistance genes from different groups of poultry and some pig strains previously collected was carried out. Resistance to different antibiotics was assessed using the agar dilution method. In addition, all the strains were tested for ampicillin (bla(OXA-61)), erythromycin (aph-3-1), tetracycline tet(O), streptomycin (aadE), and the energy-dependent multi-drug efflux pump (cmeB) resistance genes using multiplex polymerase chain reaction. The evaluation of phenotypic resistance revealed all of the strains from poultry were sensitive to ciprofloxacin, gentamicin, erythromycin or tylosin. But, widespread resistance to lincomycin (51-100%), extensive resistance to ampicillin (33�P3-60�P2%) and less resistance to tetracycline (5�P6-40�P7%) were observed in the different groups of chickens. Antibiotic resistance genes bla(OXA-61,) cmeB and tet(O) were found in 82�P6-92�P7%, 80�P3-89% and 22�P3-30�P9% Camp. coli isolates from pigs, whilst 59-65�P4% and 19�P2-40�P7% Camp. jejuni from chickens were found to encode bla(OXA-61) and tet(O), respectively. No significant difference between isolates from free range egg layers and meat chickens (P < 0�P05) was found. However, there were significant differences between the pig strains and all the groups of poultry strains (P < 0�P05) with regard to carriage of resistance genes. In addition, pulsed field gel electrophoresis of selected resistant isolates from the poultry and pig revealed closely related clonal groups. Our results suggest the resistant strains are persisting environmental isolates that have been acquired by the different livestock species. Furthermore, the different treatment practices in poultry and pigs have resulted in differences in resistance profiles in Campylobacter isolates.
|
1702. |
Lascols C,
Hackel M,
Hujer AM,
Marshall SH,
Bouchillon SK,
Hoban DJ,
Hawser SP,
Badal RE,
Bonomo RA,
( 2012 ) Using nucleic acid microarrays to perform molecular epidemiology and detect novel �]-lactamases: a snapshot of extended-spectrum �]-lactamases throughout the world. PMID : 22322349 : DOI : 10.1128/JCM.06115-11 PMC : PMC3347121 Abstract >>
The worldwide dissemination of extended-spectrum-�]-lactamase (ESBL)- and carbapenemase-producing Enterobacteriaceae is a major concern in both hospital and community settings. Rapid identification of these resistant pathogens and the genetic determinants they possess is needed to assist in clinical practice and epidemiological studies. A collection of Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, and Proteus mirabilis isolates, including phenotypically ESBL-positive (n = 1,093) and ESBL-negative isolates (n = 59), obtained in 2008-2009 from a longitudinal surveillance study (SMART) was examined using an in vitro nucleic acid-based microarray. This approach was used to detect and identify bla(ESBL) (bla(SHV), bla(TEM), and bla(CTX-M) genes of groups 1, 2, 9, and 8/25) and bla(KPC) genes and was combined with selective PCR amplification and DNA sequencing for complete characterization of the bla(ESBL) and bla(KPC) genes. Of the 1,093 phenotypically ESBL-positive isolates, 1,041 were identified as possessing at least one bla(ESBL) gene (95.2% concordance), and 59 phenotypically ESBL-negative isolates, used as negative controls, were negative. Several ESBL variants of bla(TEM) (n = 5), bla(SHV) (n = 11), bla(CTX-M) (n = 19), and bla(KPC) (n = 3) were detected. A new bla(SHV) variant, bla(SHV-129), and a new bla(KPC) variant, bla(KPC-11), were also identified. The most common bla genes found in this study were bla(CTX-M-15), bla(CTX-M-14), and bla(SHV-12). Using nucleic acid microarrays, we obtained a "molecular snapshot" of bla(ESBL) genes in a current global population; we report that CTX-M-15 is still the dominant ESBL and provide the first report of the new �]-lactamase variants bla(SHV-129) and bla(KPC-11).
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1703. |
Tran T,
Andres P,
Petroni A,
Soler-Bistué A,
Albornoz E,
Zorreguieta A,
Reyes-Lamothe R,
Sherratt DJ,
Corso A,
Tolmasky ME,
( 2012 ) Small plasmids harboring qnrB19: a model for plasmid evolution mediated by site-specific recombination at oriT and Xer sites. PMID : 22290975 : DOI : 10.1128/AAC.06036-11 PMC : PMC3318318 Abstract >>
Plasmids pPAB19-1, pPAB19-2, pPAB19-3, and pPAB19-4, isolated from Salmonella and Escherichia coli clinical strains from hospitals in Argentina, were completely sequenced. These plasmids include the qnrB19 gene and are 2,699, 3,082, 2,989, and 2,702 nucleotides long, respectively, and they share extensive homology among themselves and with other previously described small qnrB19-harboring plasmids. The genetic environment of qnrB19 in all four plasmids is identical to that in these other plasmids and in transposons such as Tn2012, Tn5387, and Tn5387-like. Nucleotide sequence comparisons among these and previously described plasmids showed a variable region characterized by being flanked by an oriT locus and a Xer recombination site. We propose that this arrangement could play a role in the evolution of plasmids and present a model for DNA swapping between plasmid molecules mediated by site-specific recombination events at oriT and a Xer target site.
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1704. |
Côté JP,
Berthiaume F,
Houle S,
Fairbrother JM,
Dozois CM,
Mourez M,
( 2012 ) Identification and mechanism of evolution of new alleles coding for the AIDA-I autotransporter of porcine pathogenic Escherichia coli. PMID : 22522689 : DOI : 10.1128/AEM.00906-12 PMC : PMC3370479 Abstract >>
Autotransporters are a large family of virulence factors of Gram-negative bacterial pathogens. The autotransporter adhesin involved in diffuse adherence (AIDA-I) is an outer membrane protein of Escherichia coli, which allows binding to epithelial cells as well as the autoaggregation of bacteria. AIDA-I is glycosylated by a specific heptosyltransferase encoded by the aah gene that forms an operon with the aidA gene. aidA is highly prevalent in strains that cause disease in pigs. Nevertheless, there are only two published whole-length sequences for this gene. In this study, we sequenced the aah and aidA genes of 24 aidA-positive porcine strains harboring distinct virulence factor profiles. We compared the obtained sequences and performed phylogenetic and pulsed-field electrophoresis analyses. Our results suggest that there are at least 3 different alleles for aidA, which are associated with distinct virulence factor profiles. The genes are found on high-molecular-weight plasmids and seem to evolve via shuffling mechanisms, with one of the sequences showing evidence of genetic recombination. Our work suggests that genetic plasticity allows the evolution of aah-aidA alleles that are selected during pathogenesis.
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1705. |
Shahid M,
Sobia F,
Singh A,
Khan HM,
( 2012 ) Concurrent occurrence of blaampC families and blaCTX-M genogroups and association with mobile genetic elements ISEcp1, IS26, ISCR1, and sul1-type class 1 integrons in Escherichia coli and Klebsiella pneumoniae isolates originating from India. PMID : 22337978 : DOI : 10.1128/JCM.06661-11 PMC : PMC3347111 Abstract >>
Cefoxitin-resistant Escherichia coli (n = 109) and Klebsiella pneumoniae (n = 16) isolates collected from patients in India in 2009 to 2010 were screened for bla(ampC) families and mobilizing elements (ISEcp1, IS26, ISCR1, and sul-1-type class 1 integrons) and their association with bla(ampC) and for the occurrence of class A beta-lactamases (BLs) (CTX-M, TEM, and SHV). The concurrent occurrences of two distinct AmpC families (bla(CIT) and bla(EBC)) and of class A with class C beta-lactamase were observed. All but one of the isolates harboring CTX-M extended-spectrum BLs (ESBLs) were carrying bla(CTX-M) genogroup 1; the remaining isolate carried bla(CTX-M) genogroup 9. The mobilizing elements occurred in different combinations in the study isolates.
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1706. |
Torres AG,
Pop M,
Popov V,
Ruiz-Perez F,
Stine OC,
Levine MM,
Del Canto F,
( 2012 ) Identification of Coli Surface Antigen 23, a novel adhesin of enterotoxigenic Escherichia coli. PMID : 22645287 : DOI : 10.1128/IAI.00263-12 PMC : PMC3434557 Abstract >>
Enterotoxigenic Escherichia coli (ETEC) is an important cause of diarrhea, mainly in developing countries. Although there are 25 different ETEC adhesins described in strains affecting humans, between 15% and 50% of the clinical isolates from different geographical regions are negative for these adhesins, suggesting that additional unidentified adhesion determinants might be present. Here, we report the discovery of Coli Surface Antigen 23 (CS23), a novel adhesin expressed by an ETEC serogroup O4 strain (ETEC 1766a), which was negative for the previously known ETEC adhesins, albeit it has the ability to adhere to Caco-2 cells. CS23 is encoded by an 8.8-kb locus which contains 9 open reading frames (ORFs), 7 of them sharing significant identity with genes required for assembly of K88-related fimbriae. This gene locus, named aal (adhesion-associated locus), is required for the adhesion ability of ETEC 1766a and was able to confer this adhesive phenotype to a nonadherent E. coli HB101 strain. The CS23 major structural subunit, AalE, shares limited identity with known pilin proteins, and it is more closely related to the CS13 pilin protein CshE, carried by human ETEC strains. Our data indicate that CS23 is a new member of the diverse adhesin repertoire used by ETEC strains.
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1707. |
Wang Y,
He T,
Schwarz S,
Zhou D,
Shen Z,
Wu C,
Wang Y,
Ma L,
Zhang Q,
Shen J,
( 2012 ) Detection of the staphylococcal multiresistance gene cfr in Escherichia coli of domestic-animal origin. PMID : 22331590 : DOI : 10.1093/jac/dks020 Abstract >>
To investigate the presence and the genetic environment of the multiresistance gene cfr in Escherichia coli found in domestic animals. A total of 1230 E. coli isolates, collected from pigs, chickens and ducks, were screened by PCR for the cfr gene. The location of the cfr gene was determined by Southern blotting, the transferability of cfr gene was tested by conjugation and transformation, and the regions flanking the cfr gene were sequenced by a modified random primer walking strategy. The location of the cfr promoter sequence was analysed by mapping the cfr transcription start site using rapid amplification of 5' cDNA ends (5' RACE). Only a single strain from the nasal swab of a pig harboured the cfr gene. Southern blotting indicated that the cfr gene was located on a ~110 kb plasmid, designated pEC-01. A cfr-carrying segment of 1545 bp with a sequence identical to that of the cfr-harbouring plasmid pSCFS1 was flanked by two IS26 elements in the same orientation. The IS26 transposition created a new hybrid promoter in which the -35 region was part of the left inverted repeat of IS26 while the -10-like sequence was part of the original cfr upstream region. To the best of our knowledge, this is the first report of the cfr gene in a naturally occurring E. coli strain. Continued surveillance of the presence of the cfr gene in Gram-negative bacteria of domestic-animal origin is warranted.
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1708. |
Luo Y,
Cui S,
Li J,
Yang J,
Lin L,
Hu C,
Jin S,
Ye L,
Zhao Q,
Ma Y,
( 2011 ) Characterization of Escherichia coli isolates from healthy food handlers in hospital. PMID : 21612511 : DOI : 10.1089/mdr.2011.0032 Abstract >>
Recent studies have reported that Escherichia coli in fecal samples of healthy humans could also serve as important reservoirs of drug-resistant bacteria. Limited data are available for E. coli-resistant profiles of healthy food handlers in hospitals who provide food service to inpatients and hospital staffs. E. coli isolates were recovered from hospital healthy food handlers, and one random selected isolate from each food handler was subjected to antimicrobial susceptibility testing, phylogenetic typing, and screening for antimicrobial-resistant mechanisms by polymerase chain reaction amplification. Ciprofloxacin-resistant isolates were further characterized by mutation analysis in the quinolone resistance determining regions (QRDRs) of GyrA and ParC. And extended-spectrum �]-lactamase (ESBL) producing isolates were screened for bla(CTX-M) by polymerase chain reaction amplification and DNA sequence analysis. In total, more than 50% (47/92) of E. coli isolates from healthy food handlers showed multidrug-resistant profiles and 50% (46/92) isolates carried intI. Resistance prevalence of the B2 phylogenetic group was significantly lower than that of the non-B2 groups for all tested antimicrobials (p < 0.05) except chloramphenicol and tetracycline. Seven isolates of phylogenetic group A (n = 3) and D (n = 4) produced ESBL, and 12 isolates of phylogenetic group A (n = 5), B2 (n = 2), and D (n = 5) were resistant to ciprofloxacin. Transferable quinolone resistance determinants were identified in four isolates. Point mutations in QRDRs of GyrA or ParC were identified among 59 out of 62 E. coli isolates showing decreased susceptibility or resistance to ciprofloxacin. Genes encoding CTX-M enzyme were identified in seven ESBL-producing isolates. The preponderance in hospital food handlers of multidrug-resistant E. coli makes it important to introduce control measures such as improved biosecurity to ensure that they do not pass through the food service and limit inpatient therapeutic options.
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1709. |
Kornacki JA,
Burlage RS,
Figurski DH,
( 1990 ) The kil-kor regulon of broad-host-range plasmid RK2: nucleotide sequence, polypeptide product, and expression of regulatory gene korC. PMID : 2160936 : DOI : 10.1128/jb.172.6.3040-3050.1990 PMC : PMC209106 Abstract >>
Broad-host-range plasmid RK2 encodes several kil operons (kilA, kilB, kilC, kilE) whose expression is potentially lethal to Escherichia coli host cells. The kil operons and the RK2 replication initiator gene (trfA) are coregulated by various combinations of kor genes (korA, korB, korC, korE). This regulatory network is called the kil-kor regulon. Presented here are studies on the structure, product, and expression of korC. Genetic mapping revealed the precise location of korC in a region near transposon Tn1. We determined the nucleotide sequence of this region and identified the korC structural gene by analysis of korC mutants. Sequence analysis predicts the korC product to be a polypeptide of 85 amino acids with a molecular mass of 9,150 daltons. The KorC polypeptide was identified in vivo by expressing wild-type and mutant korC alleles from a bacteriophage T7 RNA polymerase-dependent promoter. The predicted structure of KorC polypeptide has a net positive charge and a helix-turn-helix region similar to those of known DNA-binding proteins. These properties are consistent with the repressorlike function of KorC protein, and we discuss the evidence that KorA and KorC proteins act as corepressors in the control of the kilC and kilE operons. Finally, we show that korC is expressed from the bla promoters within the upstream transposon Tn1, suggesting that insertion of Tn1 interrupted a plasmid operon that may have originally included korC and kilC.
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1710. |
Sekizuka T,
Matsui M,
Yamane K,
Takeuchi F,
Ohnishi M,
Hishinuma A,
Arakawa Y,
Kuroda M,
( 2011 ) Complete sequencing of the bla(NDM-1)-positive IncA/C plasmid from Escherichia coli ST38 isolate suggests a possible origin from plant pathogens. PMID : 21966500 : DOI : 10.1371/journal.pone.0025334 PMC : PMC3179503 Abstract >>
The complete sequence of the plasmid pNDM-1_Dok01 carrying New Delhi metallo-�]-lactamase (NDM-1) was determined by whole genome shotgun sequencing using Escherichia coli strain NDM-1_Dok01 (multilocus sequence typing type: ST38) and the transconjugant E. coli DH10B. The plasmid is an IncA/C incompatibility type composed of 225 predicted coding sequences in 195.5 kb and partially shares a sequence with bla(CMY-2)-positive IncA/C plasmids such as E. coli AR060302 pAR060302 (166.5 kb) and Salmonella enterica serovar Newport pSN254 (176.4 kb). The bla(NDM-1) gene in pNDM-1_Dok01 is terminally flanked by two IS903 elements that are distinct from those of the other characterized NDM-1 plasmids, suggesting that the bla(NDM-1) gene has been broadly transposed, together with various mobile elements, as a cassette gene. The chaperonin groES and groEL genes were identified in the bla(NDM-1)-related composite transposon, and phylogenetic analysis and guanine-cytosine content (GC) percentage showed similarities to the homologs of plant pathogens such as Pseudoxanthomonas and Xanthomonas spp., implying that plant pathogens are the potential source of the bla(NDM-1) gene. The complete sequence of pNDM-1_Dok01 suggests that the bla(NDM-1) gene was acquired by a novel composite transposon on an extensively disseminated IncA/C plasmid and transferred to the E. coli ST38 isolate.
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1711. |
Roper DI,
Cooper RA,
( 1990 ) Purification, some properties and nucleotide sequence of 5-carboxymethyl-2-hydroxymuconate isomerase of Escherichia coli C. PMID : 2194841 : DOI : 10.1016/0014-5793(90)81507-k Abstract >>
As part of an investigation into the evolution of catabolic pathway enzymes a cloned gene encoding the Escherichia coli C 5-carboxymethyl-2-hydroxymuconate (CHM) isomerase, an enzyme of the homoprotocatechuate catabolic pathway, was used to produce large amounts of the protein. The isomerase was purified to homogeneity and some of its properties determined. The reaction occurred optimally at pH 7.6 and the specificity constant was 5.8 x 10(5) M-1.s-1 with CHM and 6.0 x 10(2) M-1.s-1 with 2-hydroxyhepta-2,4-diene-1,7-dioate, the substrate of a second isomerase in the pathway. The pure protein showed one type of subunit of Mr 14,000 whilst the molecular mass of the native enzyme was 30,000, suggesting that it was a dimer of identical subunits. The first 19 N-terminal amino acids were sequenced and the data used to confirm that the open reading frame of 378 bp, identified from the nucleotide sequence, encoded the CHM isomerase.
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1712. |
Gilson L,
Mahanty HK,
Kolter R,
( 1990 ) Genetic analysis of an MDR-like export system: the secretion of colicin V. PMID : 2249654 : PMC : PMC552155 Abstract >>
The extracellular secretion of the antibacterial toxin colicin V is mediated via a signal sequence independent process which requires the products of two linked genes: cvaA and cvaB. The nucleotide sequence of cvaB reveals that its product is a member of a subfamily of proteins, involved in the export of diverse molecules, found in both eukaryotes and prokaryotes. This group of proteins, here referred to as the 'MDR-like' subfamily, is characterized by the presence of a hydrophobic region followed by a highly conserved ATP binding fold. By constructing fusions between the structural gene for colicin V, cvaC, and a gene for alkaline phosphatase, phoA, lacking its signal sequence, it was determined that 39 codons in the N-terminus of cvaC contained the structural information to allow CvaC-PhoA fusion proteins to be efficiently translocated across the plasma membrane of Escherichia coli in a CvaA/CvaB dependent fashion. This result is consistent with the location of point mutations in the cvaC gene which yielded export deficient colicin V. The presence of the export signal at the N-terminus of CvaC contrasts with the observed C-terminal location of the export signal for hemolysin, which also utilizes an MDR-like protein for its secretion. It was also found that the CvaA component of the colicin V export system shows amino acid sequence similarities with another component involved in hemolysin export, HlyD. The role of the second component in these systems and the possibility that other members of the MDR-like subfamily will also have corresponding second components are discussed. A third component used in both colicin V and hemolysin extracellular secretion is the E. coli host outer membrane protein, TolC.
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1713. |
Kariyawasam S,
Nolan LK,
( 2011 ) papA gene of avian pathogenic Escherichia coli. PMID : 22312970 : DOI : 10.1637/9663-011911-Reg.1 Abstract >>
P fimbrial adhesins may be associated with the virulence of avian pathogenic Escherichia coli (APEC). However, most APECs are unable to express P fimbriae even when they are grown under conditions that favor P fimbrial expression. This failure can be explained by the complete absence of the pap operon or the presence of an incomplete pap operon in Pap-negative APEC strains. In the present study, we analyzed the pap operon, specifically the papA gene that encodes the major fimbrial shaft, to better understand the pap gene cluster at the genetic level. First, by PCR, we examined a collection of 500 APEC strains for the presence of 11 genes comprising the pap operon. Except for papA, all the other genes of the operon were present in 38% to 41.2% of APEC, whereas the papA was present only in 10.4% of the APEC tested. Using multiplex PCR to probe for allelic variants of papA, we sought to determine if the low prevalence of papA among APEC was related to genetic heterogeneity of the gene itself. It was determined that the papA of APEC always belongs to the F11 allelic variant. Finally, we sequenced the 'papA region' from two papA-negative strains, both of which contain all the other genes of the pap operon. Interestingly, both strains had an 11,104-bp contig interruptingpapA at the 281-bp position. This contig harbored a streptomycin resistance gene and a classic Tn10 transposon containing the genes that confer tetracycline resistance. However, we noted that the papA gene of every papA-negative APEC strain was not interrupted by an 11,104-bp contig. It is likely that transposons bearing antibiotic resistance genes have inserted within pap gene cluster of some APEC strains, and such genetic events may have been selected for by antibiotic use.
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1714. |
Hager PW,
Reich NO,
Day JP,
Coche TG,
Boyer HW,
Rosenberg JM,
Greene PJ,
( 1990 ) Probing the role of glutamic acid 144 in the EcoRI endonuclease using aspartic acid and glutamine replacements. PMID : 2254311 : Abstract >>
The x-ray structure of the EcoRI endonuclease-DNA complex (3) suggests that hydrogen bonds between amino acids, glutamic acid 144, arginine 145, and arginine 200, and major groove base moieties are the molecular determinants of specificity. We have investigated residue 144 using aspartate and glutamine substitutions introduced by site-directed mutagenesis. Substitution with glutamine results in a null phenotype (at least a 2000-fold reduction in activity). On the other hand, the aspartic acid mutant (ED144) retained in vivo activity. Substrate binding and catalytic studies were done with purified ED144 enzyme. The affinity of the ED144 enzyme for the canonical sequence 5'-GAATTC-3' is about 340-fold less than the wild-type (WT) enzyme, while its affinity for nonspecific DNA is about 50 times greater. The ED144 enzyme cleaves one strand in the EcoRI site in plasmid pBR322 with a kcat/Km similar to WT. In contrast to the WT enzyme, the ED144 enzyme dissociates after the first strand cleavage. Partitioning between cleavage and dissociation at the first and second cleavage steps for the ED144 enzyme is extremely salt-sensitive. The altered partitioning results largely from a destabilization of the enzyme-DNA complex, particularly the enzyme-nicked DNA complex, with only small changes in the respective cleavage rates. The hydrogen bonds of Glu-144 are critical, they appear to act cooperatively with other specificity contacts to stabilize the enzyme-DNA complex.
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1715. |
Williamson DA,
Sidjabat HE,
Freeman JT,
Roberts SA,
Silvey A,
Woodhouse R,
Mowat E,
Dyet K,
Paterson DL,
Blackmore T,
Burns A,
Heffernan H,
( 2012 ) Identification and molecular characterisation of New Delhi metallo-�]-lactamase-1 (NDM-1)- and NDM-6-producing Enterobacteriaceae from New Zealand hospitals. PMID : 22526013 : DOI : 10.1016/j.ijantimicag.2012.02.017 Abstract >>
The global spread of New Delhi metallo-�]-lactamase (NDM) is of significant public health concern. This study sought to determine whether bla(NDM) was present in Enterobacteriaceae isolates displaying resistance to carbapenems that were submitted to the National Antibiotic Reference Laboratory, Institute of Environmental Science and Research (Porirua, New Zealand) during 2009 and 2010. Isolates were tested for the presence of �]-lactamase genes and 16S rRNA methylase genes by polymerase chain reaction (PCR) and sequencing. Plasmid transfer studies were undertaken on isolates found to be harbouring bla(NDM). Molecular typing was performed by multilocus sequence typing (MLST). The bla(NDM-1) gene was identified in four Enterobacteriaceae isolates (two Escherichia coli, one Klebsiella pneumoniae and one Proteus mirabilis) from four patients in New Zealand hospitals in 2009 and 2010. In addition, the bla(NDM-6) gene, which differed from bla(NDM-1) by a point mutation at position 698 (C��T), was also identified in an E. coli isolate from the same patient who harboured the bla(NDM-1)-positive P. mirabilis. All four patients had recently been hospitalised or received health care in India. Four of the isolates also produced a CTX-M-15 extended-spectrum �]-lactamase and/or plasmid-mediated AmpC �]-lactamase, and all five isolates harboured the plasmid-mediated 16S rRNA methylase rmtC gene. The E. coli types were diverse by MLST, and the K. pneumoniae isolate belonged to the internationally disseminated sequence type 11 (ST11) clone. These findings further illustrate the diversity of phenotypic and genotypic features found in association with bla(NDM), in addition to documenting the international spread of this resistance mechanism, notably into a country with historically low rates of antimicrobial resistance.
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1716. |
N/A N/A,
Ichiyama S,
Takakura S,
Ito Y,
Nagao M,
Hotta G,
Matsumura Y,
Matsushima A,
( 2012 ) Emergence and spread of B2-ST131-O25b, B2-ST131-O16 and D-ST405 clonal groups among extended-spectrum-�]-lactamase-producing Escherichia coli in Japan. PMID : 22843833 : DOI : 10.1093/jac/dks278 Abstract >>
The increasing prevalence of extended-spectrum �]-lactamase (ESBL)-producing Escherichia coli has been associated with the emergence of the CTX-M-producing sequence type 131 (ST131) pandemic clonal group, a member of the O25b serogroup and the B2 phylogenetic group. To assess the clonal spread of ESBL-producing E. coli in Japan, a regional surveillance programme was conducted. A total of 581 ESBL-producing clinical specimen E. coli isolates were collected between 2001 and 2010. Clonal groups, including ST131, D-ST405, D-ST393 and D-ST69, were determined using the PCR O type, phylogenetic grouping by triplex PCR, allele-specific PCR and multilocus sequence typing (MLST). A subset of clonal groups underwent PFGE. Among clonal strains, 215 isolates (37%) were identified as belonging to the ST131 group, 185 as B2-ST131-O25b (32%), 26 as B2-ST131-O16 (4%), 3 as B1-ST131-O25b (0.5%) and 1 as B2-ST131-O-non-typeable (0.1%). Forty-one isolates (7%) were identified as belonging to the D-ST405 clonal group, seven (1%) as D-ST69 and two (0.3%) as D-ST393. The B2-ST131-O16 clonal group was characterized by CTX-M-14 and a significantly lower ciprofloxacin resistance rate than the B2-ST131-O25b clonal group. The B2-ST131-O16 and B2-ST131-O25b clonal groups each made up a single PFGE cluster, with 65% similarity. The rate of ESBL-producing E. coli increased over the years (0.2% in 2001 to 9.7% in 2010) and corresponded to increases in the numbers of the B2-ST131-O25b, B2-ST131-O16 and D-ST405 clonal groups. The B2-ST131-O25b, B2-ST131-O16 and D-ST405 clonal groups have contributed to the spread of ESBL-producing E. coli in Japan.
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1717. |
Ruiz E,
Sáenz Y,
Zarazaga M,
Rocha-Gracia R,
Martínez-Martínez L,
Arlet G,
Torres C,
( 2012 ) qnr, aac(6')-Ib-cr and qepA genes in Escherichia coli and Klebsiella spp.: genetic environments and plasmid and chromosomal location. PMID : 22223228 : DOI : 10.1093/jac/dkr548 Abstract >>
To characterize the location and genetic environments of qnr, aac(6')-Ib-cr and qepA genes related to quinolone resistance in 19 Escherichia coli, Klebsiella pneumoniae and Klebsiella oxytoca strains. Genetic environments of the indicated genes were studied by cloning, PCR mapping and sequencing. The location of these genes was analysed by S1-PFGE and PFGE-I-CeuI and hybridization with specific probes. Associated antibiotic resistance mechanisms and molecular typing of strains were also investigated. The studied strains carried the aac(6')-Ib-cr, qepA, qnrS1, qnrB6, qnrB4 and oqxAB genes, with aac(6')-Ib-cr being the most prevalent. E. coli strains belonged to sequence types (STs) ST648, ST131, ST224 and ST205, and K. pneumoniae strains to ST433, ST341, ST152, ST15 and ST431. Different genetic environments of quinolone resistance genes were observed, and some of them had not been previously detected and registered in GenBank. The aac(6')-Ib-cr gene was mainly located in class 1 integrons or associated with the Tn1721 transposon in E. coli and associated with the aac(3)-II gene in Klebsiella. All these structures contained mechanisms of gene acquisition and/or dissemination, such as IS26. The studied quinolone resistance genes were mostly detected in IncF and IncN plasmids in E. coli and in IncR plasmids in Klebsiella, but in some strains the chromosomal location of the aac(6')-Ib-cr gene was detected for the first time. The bla(CTX-M-15), bla(OXA-1), tet(A), aac(3)-II and aph(3')-Ia genes and class 1 integrons were found in most strains. The aac(6')-Ib-cr gene was detected for the first time in the chromosome, although a plasmidic location was the most frequently found, with differentiation of plasmids types in E. coli versus Klebsiella.
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1718. |
Zhou J,
Lancaster L,
Trakhanov S,
Noller HF,
( 2012 ) Crystal structure of release factor RF3 trapped in the GTP state on a rotated conformation of the ribosome. PMID : 22187675 : DOI : 10.1261/rna.031187.111 PMC : PMC3264910 Abstract >>
The class II release factor RF3 is a GTPase related to elongation factor EF-G, which catalyzes release of class I release factors RF1 and RF2 from the ribosome after termination of protein synthesis. The 3.3 ? crystal structure of the RF3�PGDPNP�Pribosome complex provides a high-resolution description of interactions and structural rearrangements that occur when binding of this translational GTPase induces large-scale rotational movements in the ribosome. RF3 induces a 7�X rotation of the body and 14�X rotation of the head of the 30S ribosomal subunit, and itself undergoes inter- and intradomain conformational rearrangements. We suggest that ordering of critical elements of switch loop I and the P loop, which help to form the GTPase catalytic site, are caused by interactions between the G domain of RF3 and the sarcin-ricin loop of 23S rRNA. The rotational movements in the ribosome induced by RF3, and its distinctly different binding orientation to the sarcin-ricin loop of 23S rRNA, raise interesting implications for the mechanism of action of EF-G in translocation.
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1719. |
Li Z,
Bouckaert J,
Deboeck F,
De Greve H,
Hernalsteens JP,
( 2012 ) Nicotinamide dependence of uropathogenic Escherichia coli UTI89 and application of nadB as a neutral insertion site. PMID : 22174382 : DOI : 10.1099/mic.0.052043-0 Abstract >>
NAD and NADP are ubiquitous in the metabolism of Escherichia coli K-12. NAD auxotrophy can be rendered by mutation in any of the three genes nadB, nadA and nadC. The nadB and nadA genes were defined as antivirulence loci in Shigella spp., as a mutation (mainly in nadB) disrupting the synthesis of quinolinate is required for virulence. Uropathogenic E. coli (UPEC) isolates from acute cystitis patients, exhibiting nicotinamide auxotrophy, were of serotype O18 : K1 : H7. E. coli UTI89, the model uropathogenic and O18 : K1 : H7 strain, requires nicotinamide or quinolinate for growth. A mutation in the nadB gene, encoding L-aspartate oxidase, was shown to be responsible for the nicotinamide requirement of UTI89. This was further confirmed by complementation of UTI89 with a recombinant plasmid harbouring the nadB gene of E. coli K-12. An Ala28Val point mutant of the recombinant plasmid failed to support the growth of UTI89 in minimal medium. This proves that the Ala28Val mutation in the NadB gene of UTI89 completely impedes de novo synthesis of nicotinamide. In spontaneous prototrophic revertants of UTI89, the nadB gene has a Val28Ala mutation. Both analyses implicate that the nicotinamide auxotrophy of UTI89 is caused by a single Ala28Val mutation in NadB. We showed that the same mutation is also present in other NAD auxotrophic E. coli O18 strains. No significant differences were observed between the virulence of isogenic NAD auxotrophic and prototrophic strains in the murine ascending urinary tract infection model. Considering these data, we applied the nadB locus as a neutral site for DNA insertions in the bacterial chromosome. We successfully restored the parental phenotype of a fimH mutant by inserting fimH, with a synthetic em7 promoter, into the nadB gene. This neutral insertion site is of significance for further research on the pathogenicity of UPEC.
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1720. |
Murase K,
Ooka T,
Iguchi A,
Ogura Y,
Nakayama K,
Asadulghani M,
Islam MR,
Hiyoshi H,
Kodama T,
Beutin L,
Hayashi T,
( 2012 ) Haemolysin E- and enterohaemolysin-derived haemolytic activity of O55/O157 strains and other Escherichia coli lineages. PMID : 22194351 : DOI : 10.1099/mic.0.054775-0 Abstract >>
Among three haemolysins identified thus far in Escherichia coli, alpha-haemolysin (HlyA) is encoded on the pathogenicity islands of extraintestinal pathogenic strains, while enterohaemolysin (EhxA) is encoded on the virulence plasmids of enterohaemorrhagic E. coli (EHEC) strains. In contrast, the gene for haemolysin E (HlyE) is located on the E. coli chromosome backbone and is therefore widely distributed among E. coli strains. However, because hlyE gene expression is repressed by the H-NS protein and because the gene has been disrupted in many strains, its haemolytic activity cannot be detected in wild-type strains by routine screening on blood agar plates. In this study, we found that the HlyE-derived haemolytic activity of enteropathogenic E. coli (EPEC) O55 : H7 can be detected after anaerobic cultivation on a washed blood agar plate (EHX plate) that is used to detect the production of EhxA. We also found that the haemolytic activity of EHEC O157 : H7 observed on EHX plates under aerobic and anaerobic growth conditions is derived from EhxA and HlyE, respectively; this differential expression of the two haemolysins occurs at the transcriptional level. Our analysis of 60 E. coli strains of various pathotypes and phylogenies for their repertoires of haemolysin genes, haemolytic phenotypes and hlyE gene sequences revealed that HlyE activity can generally be detected on EHX plates under anaerobic growth conditions if the gene is intact. Furthermore, our results indicate that hlyE gene inactivation occurred in three of the five E. coli lineages (phylogroups A, B1 and B2), which demonstrates phylogroup-specific gene disruption patterns.
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1721. |
Tacão M,
Correia A,
Henriques I,
( 2012 ) Resistance to broad-spectrum antibiotics in aquatic systems: anthropogenic activities modulate the dissemination of bla(CTX-M)-like genes. PMID : 22492443 : DOI : 10.1128/AEM.00359-12 PMC : PMC3370516 Abstract >>
We compared the resistomes within polluted and unpolluted rivers, focusing on extended-spectrum beta-lactamase (ESBL) genes, in particular bla(CTX-M). Twelve rivers from a Portuguese hydrographic basin were sampled. Physicochemical and microbiological parameters of water quality were determined, and the results showed that 9 rivers were classified as unpolluted (UP) and that 3 were classified as polluted (P). Of the 225 cefotaxime-resistant strains isolated, 39 were identified as ESBL-producing strains, with 18 carrying a bla(CTX-M) gene (15 from P and 3 from UP rivers). Analysis of CTX-M nucleotide sequences showed that 17 isolates produced CTX-M from group 1 (CTX-M-1, -3, -15, and -32) and 1 CTX-M that belonged to group 9 (CTX-M-14). A genetic environment study revealed the presence of different genetic elements previously described for clinical strains. ISEcp1 was found in the upstream regions of all isolates examined. Culture-independent bla(CTX-M)-like libraries were comprised of 16 CTX-M gene variants, with 14 types in the P library and 4 types in UP library, varying from 68% to 99% similarity between them. Besides the much lower level of diversity among CTX-M-like genes from UP sites, the majority were similar to chromosomal ESBLs such as bla(RAHN-1). The results demonstrate that the occurrence and diversity of bla(CTX-M) genes are clearly different between polluted and unpolluted lotic ecosystems; these findings favor the hypothesis that natural environments are reservoirs of resistant bacteria and resistance genes, where anthropogenic-driven selective pressures may be contributing to the persistence and dissemination of genes usually relevant in clinical environments.
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1722. |
Contreras CA,
Ochoa TJ,
Ruiz J,
Lacher DW,
Durand D,
DebRoy C,
Lanata CF,
Cleary TG,
( 2012 ) Genetic diversity of locus of enterocyte effacement genes of enteropathogenic Escherichia coli isolated from Peruvian children. PMID : 22493278 : DOI : 10.1099/jmm.0.045443-0 PMC : PMC3542133 Abstract >>
The aim of this study was to determine the frequency and allele associations of locus of enterocyte effacement encoded esp and tir genes among 181 enteropathogenic Escherichia coli (EPEC) strains (90 diarrhoea-associated and 91 controls) isolated from Peruvian children under 18 months of age. We analysed espA, espB, espD and tir alleles by PCR-RFLP. EPEC strains were isolated with higher frequency from healthy controls (91/424, 21.7%) than from diarrhoeal samples (90/936, 9.6%) (P<0.001); 28.9% of diarrhoeal and 17.6% of control samples were typical EPEC (tEPEC). The distribution of espA alleles (alpha, beta, beta2 and gamma) and espD alleles (alpha, beta, gamma and a new variant, espD-N1) between tEPEC and atypical EPEC (aEPEC) was significantly different (P<0.05). espD-alpha was more common among acute episodes (P<0.05). espB typing resulted in five alleles (alpha, beta, gamma and two new sub-alleles, espB-alpha2 and espB-alpha3), while tir-beta and tir-gamma2 were the most common intimin receptor subtypes. Seventy-two combinations of espA, espB, espD and tir alleles were found; the most prevalent combination was espA-beta, espB-beta, espD-beta, tir-beta (34/181 strains), which was more frequent among tEPEC strains (P<0.05). Our findings indicate that there is a high degree of heterogeneity among EPEC strains isolated from Peruvian children and that aEPEC and tEPEC variants cluster.
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1723. |
Humphrey B,
Thomson NR,
Thomas CM,
Brooks K,
Sanders M,
Delsol AA,
Roe JM,
Bennett PM,
Enne VI,
( 2012 ) Fitness of Escherichia coli strains carrying expressed and partially silent IncN and IncP1 plasmids. PMID : 22475035 : DOI : 10.1186/1471-2180-12-53 PMC : PMC3347995 Abstract >>
Understanding the survival of resistance plasmids in the absence of selective pressure for the antibiotic resistance genes they carry is important for assessing the value of interventions to combat resistant bacteria. Here, several poorly explored questions regarding the fitness impact of IncP1 and IncN broad host range plasmids on their bacterial hosts are examined; namely, whether related plasmids have similar fitness impacts, whether this varies according to host genetic background, and what effect antimicrobial resistance gene silencing has on fitness. For the IncP1 group pairwise in vitro growth competition demonstrated that the fitness cost of plasmid RP1 depends on the host strain. For the IncN group, plasmids R46 and N3 whose sequence is presented for the first time conferred remarkably different fitness costs despite sharing closely related backbone structures, implicating the accessory genes in fitness. Silencing of antimicrobial resistance genes was found to be beneficial for host fitness with RP1 but not for IncN plasmid pVE46. These findings suggest that the fitness impact of a given plasmid on its host cannot be inferred from results obtained with other host-plasmid combinations, even if these are closely related.
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1724. |
Kolappan S,
Roos J,
Yuen AS,
Pierce OM,
Craig L,
( 2012 ) Structural characterization of CFA/III and Longus type IVb pili from enterotoxigenic Escherichia coli. PMID : 22447901 : DOI : 10.1128/JB.00282-12 PMC : PMC3347196 Abstract >>
The type IV pili are helical filaments found on many Gram-negative pathogenic bacteria, with multiple diverse roles in pathogenesis, including microcolony formation, adhesion, and twitching motility. Many pathogenic enterotoxigenic Escherichia coli (ETEC) isolates express one of two type IV pili belonging to the type IVb subclass: CFA/III or Longus. Here we show a direct correlation between CFA/III expression and ETEC aggregation, suggesting that these pili, like the Vibrio cholerae toxin-coregulated pili (TCP), mediate microcolony formation. We report a 1.26-? resolution crystal structure of CofA, the major pilin subunit from CFA/III. CofA is very similar in structure to V. cholerae TcpA but possesses a 10-amino-acid insertion that replaces part of the �\2-helix with an irregular loop containing a 3(10)-helix. Homology modeling suggests a very similar structure for the Longus LngA pilin. A model for the CFA/III pilus filament was generated using the TCP electron microscopy reconstruction as a template. The unique 3(10)-helix insert fits perfectly within the gap between CofA globular domains. This insert, together with differences in surface-exposed residues, produces a filament that is smoother and more negatively charged than TCP. To explore the specificity of the type IV pilus assembly apparatus, CofA was expressed heterologously in V. cholerae by replacing the tcpA gene with that of cofA within the tcp operon. Although CofA was synthesized and processed by V. cholerae, no CFA/III filaments were detected, suggesting that the components of the type IVb pilus assembly system are highly specific to their pilin substrates.
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1725. |
Call DR,
Besser TE,
Sawant AA,
Brayton KA,
( 2012 ) Characterization of a novel microcin that kills enterohemorrhagic Escherichia coli O157:H7 and O26. PMID : 22773653 : DOI : 10.1128/AEM.01067-12 PMC : PMC3426703 Abstract >>
A novel phenotype was recently identified in which specific strains of Escherichia coli inhibit competing E. coli strains via a mechanism that was designated "proximity-dependent inhibition" (PDI). PDI-expressing (PDI(+)) E. coli is known to inhibit susceptible (PDI(-)) E. coli strains, including several enterohemorrhagic (EHEC) and enterotoxigenic (ETEC) E. coli strains. In this study, every strain from a genetically diverse panel of E. coli O157:H7 (n = 25) and additional strains of E. coli serovar O26 were susceptible to the PDI phenotype. LIVE/DEAD staining was consistent with inhibition by killing of susceptible cells. Comparative genome analysis identified the genetic component of PDI, which is composed of a plasmid-borne (Incl1) operon encoding a putative microcin and associated genes for transport, immunity, and microcin activation. Transfer of the plasmid to a PDI(-) strain resulted in transfer of the phenotype, and deletion of the genes within the operon resulted in loss of the inhibition phenotype. Deletion of chromosomally encoded tolC also resulted in loss of the inhibitory phenotype, and this confirmed that the putative microcin is most likely secreted via a type I secretion pathway. Deletion of an unrelated plasmid gene did not affect the PDI phenotype. Quantitative reverse transcription (RT)-PCR demonstrated that microcin expression is correlated with logarithmic-phase growth. The ability to inhibit a diversity of E. coli strains indicates that this microcin may influence gut community composition and could be useful for control of important enteric pathogens.
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1726. |
Bandyopadhyay S,
Lodh C,
Sarkar M,
Ghosh MK,
Bera AK,
Bhattacharyya D,
Mondal DK,
Baruah KK,
( 2012 ) Prevalence, molecular fingerprinting and drug resistance profile of enterovirulent Escherichia coli isolates from free-ranging yaks of Tawang district, Arunachal Pradesh, India. PMID : 22228494 : DOI : 10.1007/s11250-011-0041-9 Abstract >>
Of 273 samples (rectal swab) collected from free-ranging yaks of Tawang district, Arunachal Pradesh, 42 Shiga toxin-producing Escherichia coli (STEC), six enteropathogenic E. coli (EPEC) and 27 enterotoxigenic E. coli (ETEC) strains were isolated. All the STEC and EPEC strains were further investigated for respective stx variants (for STEC only) and additional putative virulence factors. The 27 ETEC strains were also screened for characteristic enterotoxin gene(s) and colonization factors. Occurrence of ETEC was significantly (p < 0.05) higher in the diarrheic yaks and yaks of less than 1 year of age. Majority of enterovirulent E. coli isolates were resistant to amikacin, azithromycin, chloramphenicol, colistin, doxycycline, furazolidone, nalidixic acid, nitrofurantoin, streptomycin and tetracycline. Dendrogram, constructed with molecular fingerprinting profiles obtained from RAPD (Randomly Amplified Polymorphic DNA) and ERIC (Enterobacterial Repetitive Intergenic Consensus) PCR, placed the isolates in different clusters irrespective of their serotypes, virulence gene and drug resistance pattern. Collectively, the study indicates that yaks, being a potential reservoir of multidrug resistant STEC and EPEC, may represent significant risk to public health in this region. Higher recovery of ETEC isolates from yaks with diarrhea points out that ETEC may be a major determinant for repeated occurrence of diarrhea in yaks.
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1727. |
De Greve H,
Remaut H,
De Kerpel M,
Cox E,
Panjikar S,
Tran T,
( 2012 ) Structural insight in histo-blood group binding by the F18 fimbrial adhesin FedF. PMID : 22812428 : DOI : 10.1111/j.1365-2958.2012.08174.x Abstract >>
F18-positive enterotoxigenic and Shiga toxin-producing Escherichia coli are responsible for post-weaning diarrhoea and oedema disease in pigs and lead to severe production losses in the farming industry. F18 fimbriae attach to the small intestine of young piglets by latching onto glycosphingolipids with A/H blood group determinants on type 1 core. We demonstrate the N-terminal domain of the F18 fimbrial subunit FedF to be responsible for ABH-mediated attachment and present its X-ray structure in ligand-free form and bound to A and B type 1 hexaoses. The FedF lectin domain comprises a 10-stranded immunoglobulin-like �]-sandwich. Three linear motives, Q(47) -N(50), H(88) -S(90) and R(117) -T(119), form a shallow glycan binding pocket near the tip of the domain that is selective for type 1 core glycans in extended conformation. In addition to the glycan binding pocket, a polybasic loop on the membrane proximal surface of FedF lectin domain is shown to be required for binding to piglet enterocytes. Although dispensable for ABH glycan recognition, the polybasic surface adds binding affinity in the context of the host cell membrane, a mechanism that is proposed to direct ABH-glycan binding to cell-bound glycosphingolipids and could allow bacteria to avoid clearance by secreted glycoproteins.
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1728. |
Zschüttig A,
Zimmermann K,
Blom J,
Goesmann A,
Pöhlmann C,
Gunzer F,
( 2012 ) Identification and characterization of microcin S, a new antibacterial peptide produced by probiotic Escherichia coli G3/10. PMID : 22479389 : DOI : 10.1371/journal.pone.0033351 PMC : PMC3316575 Abstract >>
Escherichia coli G3/10 is a component of the probiotic drug Symbioflor 2. In an in vitro assay with human intestinal epithelial cells, E. coli G3/10 is capable of suppressing adherence of enteropathogenic E. coli E2348/69. In this study, we demonstrate that a completely novel class II microcin, produced by probiotic E. coli G3/10, is responsible for this behavior. We named this antibacterial peptide microcin S (MccS). Microcin S is coded on a 50.6 kb megaplasmid of E. coli G3/10, which we have completely sequenced and annotated. The microcin S operon is about 4.7 kb in size and is comprised of four genes. Subcloning of the genes and gene fragments followed by gene expression experiments enabled us to functionally characterize all members of this operon, and to clearly identify the nucleotide sequences encoding the microcin itself (mcsS), its transport apparatus and the gene mcsI conferring self immunity against microcin S. Overexpression of cloned mcsI antagonizes MccS activity, thus protecting indicator strain E. coli E2348/69 in the in vitro adherence assay. Moreover, growth of E. coli transformed with a plasmid containing mcsS under control of an araC PBAD activator-promoter is inhibited upon mcsS induction. Our data provide further mechanistic insight into the probiotic behavior of E. coli G3/10.
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1729. |
Alvarado A,
Garcillán-Barcia MP,
( 2012 ) A degenerate primer MOB typing (DPMT) method to classify gamma-proteobacterial plasmids in clinical and environmental settings. PMID : 22792321 : DOI : 10.1371/journal.pone.0040438 PMC : PMC3394729 Abstract >>
Transmissible plasmids are responsible for the spread of genetic determinants, such as antibiotic resistance or virulence traits, causing a large ecological and epidemiological impact. Transmissible plasmids, either conjugative or mobilizable, have in common the presence of a relaxase gene. Relaxases were previously classified in six protein families according to their phylogeny. Degenerate primers hybridizing to coding sequences of conserved amino acid motifs were designed to amplify related relaxase genes from �^-Proteobacterial plasmids. Specificity and sensitivity of a selected set of 19 primer pairs were first tested using a collection of 33 reference relaxases, representing the diversity of �^-Proteobacterial plasmids. The validated set was then applied to the analysis of two plasmid collections obtained from clinical isolates. The relaxase screening method, which we call "Degenerate Primer MOB Typing" or DPMT, detected not only most known Inc/Rep groups, but also a plethora of plasmids not previously assigned to any Inc group or Rep-type.
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1730. |
Norman KN,
Strockbine NA,
( 2012 ) Association of nucleotide polymorphisms within the O-antigen gene cluster of Escherichia coli O26, O45, O103, O111, O121, and O145 with serogroups and genetic subtypes. PMID : 22798363 : DOI : 10.1128/AEM.01259-12 PMC : PMC3426686 Abstract >>
Shiga toxin-producing Escherichia coli (STEC) strains are important food-borne pathogens capable of causing hemolytic-uremic syndrome. STEC O157:H7 strains cause the majority of severe disease in the United States; however, there is a growing concern for the amount and severity of illness attributable to non-O157 STEC. Recently, the Food Safety and Inspection Service (FSIS) published the intent to regulate the presence of STEC belonging to serogroups O26, O45, O103, O111, O121, and O145 in nonintact beef products. To ensure the effective control of these bacteria, sensitive and specific tests for their detection will be needed. In this study, we identified single nucleotide polymorphisms (SNPs) in the O-antigen gene cluster that could be used to detect STEC strains of the above-described serogroups. Using comparative DNA sequence analysis, we identified 22 potentially informative SNPs among 164 STEC and non-STEC strains of the above-described serogroups and designed matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF) assays to test the STEC allele frequencies in an independent panel of bacterial strains. We found at least one SNP that was specific to each serogroup and also differentiated between STEC and non-STEC strains. Differences in the DNA sequence of the O-antigen gene cluster corresponded well with differences in the virulence gene profiles and provided evidence of different lineages for STEC and non-STEC strains. The SNPs discovered in this study can be used to develop tests that will not only accurately identify O26, O45, O103, O111, O121, and O145 strains but also predict whether strains detected in the above-described serogroups contain Shiga toxin-encoding genes.
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1731. |
Bandyopadhyay S,
Lodh C,
Rahaman H,
Bhattacharya D,
Bera AK,
Ahmed FA,
Mahanti A,
Samanta I,
Mondal DK,
Bandyopadhyay S,
Sarkar S,
Dutta TK,
Maity S,
Paul V,
Ghosh MK,
Sarkar M,
Baruah KK,
( 2012 ) Characterization of shiga toxin producing (STEC) and enteropathogenic Escherichia coli (EPEC) in raw yak (Poephagus grunniens) milk and milk products. PMID : 22226073 : DOI : 10.1016/j.rvsc.2011.12.011 Abstract >>
Thirty-one shiga toxin-producing (STEC) and 6 enteropathogenic Escherichia coli (EPEC) were isolated from 87 raw yak milk and 63 'churpi' samples. Of 18 stx(1) positive isolates (48.6%), 14 carried stx(1c) (77.7%). Subtyping of 28 stx(2) positive isolates (75.7%) revealed the presence of stx(2c) (9, 32.1%), stx(2d) (3, 10.7%), stx(2e) (1, 3.57%) and stx(2f) (3, 10.7%) variants. Furthermore, intimin (eaeA), enterohaemolysin (ehxA), autoagglutinating adhesin (saa), iha (adherence conferring protein), efa1 (EHEC factor for adherence), bundle forming pilli (bfpA) and toxB (type III secreted protein encoded on LEE Island, similar to toxin B of Clostridium difficile) genes were detected in 14, 16, 12, 4, 3, 2 and 2 isolates, respectively. Univariate and multivariate analysis depicted that both stx(1) and stx(2) or their variants were more likely to occur in isolates from Arunachal Pradesh (p<0.04) rather than Sikkim. Dendogram constructed on the basis of RAPD and ERIC PCR profile distributed the STEC and EPEC isolates in separate clusters irrespective of their sources and serotypes. The STEC and EPEC isolates exhibited resistance against erythromycin, amikacin, azithromycin, amoxicillin, ampicillin+cloxacillin, cephalothin, furazolidone, gentamicin, kanamycin, streptomycin and tetracycline. This is the first ever report on occurrence and characterization of STEC and EPEC isolated from yak milk and milk products.
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1732. |
Dallas WS,
( 1990 ) The heat-stable toxin I gene from Escherichia coli 18D. PMID : 2203756 : DOI : 10.1128/jb.172.9.5490-5493.1990 PMC : PMC213218 Abstract >>
The heat-stable toxin I gene in the human Escherichia coli isolate 18D is the estA1 allele. The gene is not part of a composite transposon, but inspection of the flanking DNA sequences suggests that it was at one time part of a transposon. The hypothetical transposon originated from an event other than the occurrence that formed Tn1681.
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1733. |
( 1997 ) Conformational transitions and structural deformability of EcoRV endonuclease revealed by crystallographic analysis. PMID : 9367757 : DOI : 10.1006/jmbi.1997.1315 Abstract >>
The structures of wild-type and mutant forms of the unliganded EcoRV endonuclease dimer have been determined at 2.4 A resolution in a new crystal lattice. Comparison of these structures with that of the free enzyme determined with different packing constraints shows that the conformations of the domain interfaces are not conserved between crystal forms. The unliganded enzyme and the enzyme-DNA complex delineate two distinct quaternary states separated by a 25 degrees intersubunit rotation, but considerable conformational heterogeneity, of the order of 10 degrees domain rotations, exists within each of these states. Comparison of the free enzyme structure between the two crystal forms further reveals that the C-terminal 28 amino acid residues are disordered and undergo an extensive local folding transition upon DNA binding. Introduction of the mutation T93A at the DNA-binding cleft causes large-scale effects on the protein conformation. Structural changes in the mutated unliganded enzyme propagate some 20 to 25 A to the dimerization interface and lead to a rearrangement of monomer subunits. Comparative analysis of these structures, a new structure of the enzyme cocrystallized with DNA and calcium ions, and previously determined cocrystal structures suggests important roles for a number of amino acid residues in facilitating the intersubunit motions and local folding transitions. In particular, the T93A structure reveals a pathway through the protein, by which DNA-binding may cause the domain movements required for proper alignment of catalytic groups. The key active-site residue Glu45 is located on a flexible helix inside this pathway, and this provides a direct means by which essential catalytic functions are coupled to the protein conformational change. It appears that indirect perturbation of the Glu45 conformation via an altered quaternary structure may be a contributing factor to the decreased catalytic efficiency of T93A, and this mechanism may also explain the diminished activities of other active site variants of EcoRV.
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1734. |
( 1997 ) A novel class C beta-lactamase (FOX-2) in Escherichia coli conferring resistance to cephamycins. PMID : 9303413 : PMC : PMC164064 Abstract >>
An Escherichia coli strain resistant to a broad spectrum of beta-lactams, including cephamycins, was isolated from a patient suffering from urinary tract infection. A resistance plasmid (pMVP-7) was transferred from the clinical isolate to an Escherichia coli recipient. Both strains produce a cefoxitin-hydrolyzing beta-lactamase focusing at pI 6.7. The phenotype was similar to that of a Klebsiella pneumoniae strain producing cephamycinase FOX-1, so primers were selected from the FOX-1 sequence to amplify the bla gene of the transconjugant. The PCR product obtained was sequenced. The percentage of identity of the deduced amino acid sequence with sequences of other AmpC-type beta-lactamases was 96.9% with FOX-1, 74.9% with CMY-1, and 67.7% with MOX-1. This new plasmid-mediated enzyme is most closely related to FOX-1 (11 amino acid exchanges). We therefore propose the designation FOX-2.
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1735. |
Iida S,
Sandmeier H,
Hübner P,
Hiestand-Nauer R,
Schneitz K,
Arber W,
( 1990 ) The Min DNA inversion enzyme of plasmid p15B of Escherichia coli 15T-: a new member of the Din family of site-specific recombinases. PMID : 2215218 : DOI : 10.1111/j.1365-2958.1990.tb00671.x Abstract >>
Plasmid p15B is a bacteriophage P1-related resident of Escherichia coli 15T-. Both genomes contain a segment in which DNA inversion occurs, although this part of their genomes is not identical. This DNA segment of p15B was cloned in a multicopy vector plasmid. Like its parent, the resulting plasmid, pAW800, undergoes complex multiple DNA inversions: this DNA inversion system is therefore called Min. The min gene, which codes for the p15B Min DNA invertase, can complement the P1 cin recombinase gene. The Min inversion system is thus a new member of the Din family of site-specific recombinases to which Cin belongs. The DNA sequence of the min gene revealed that Min is most closely related to the Pin recombinase of the e14 defective viral element on the E. coli K12 chromosome. Like other members of the Din family, the min gene contains a recombinational enhancer element which stimulates site-specific DNA inversion 300-fold.
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1736. |
Pedró L,
Baños RC,
Aznar S,
Madrid C,
Balsalobre C,
Juárez A,
( 2011 ) Antibiotics shaping bacterial genome: deletion of an IS91 flanked virulence determinant upon exposure to subinhibitory antibiotic concentrations. PMID : 22096603 : DOI : 10.1371/journal.pone.0027606 PMC : PMC3214074 Abstract >>
The nucleoid-associated proteins Hha and YdgT repress the expression of the toxin �\-hemolysin. An Escherichia coli mutant lacking these proteins overexpresses the toxin �\-hemolysin encoded in the multicopy recombinant plasmid pANN202-312R. Unexpectedly, we could observe that this mutant generated clones that no further produced hemolysin (Hly(-)). Generation of Hly(-) clones was dependent upon the presence in the culture medium of the antibiotic kanamycin (km), a marker of the hha allele (hha::Tn5). Detailed analysis of different Hly(-) clones evidenced that recombination between partial IS91 sequences that flank the hly operon had occurred. A fluctuation test evidenced that the presence of km in the culture medium was underlying the generation of these clones. A decrease of the km concentration from 25 mg/l to 12.5 mg/l abolished the appearance of Hly(-) derivatives. We considered as a working hypothesis that, when producing high levels of the toxin (combination of the hha ydgT mutations with the presence of the multicopy hemolytic plasmid pANN202-312R), the concentration of km of 25 mg/l resulted subinhibitory and stimulated the recombination between adjacent IS91 flanking sequences. To further test this hypothesis, we analyzed the effect of subinhibitory km concentrations in the wild type E. coli strain MG1655 harboring the parental low copy number plasmid pHly152. At a km concentration of 5 mg/l, subinhibitory for strain MG1655 (pHly152), generation of Hly(-) clones could be readily detected. Similar results were also obtained when, instead of km, ampicillin was used. IS91 is flanking several virulence determinants in different enteric bacterial pathogenic strains from E. coli and Shigella. The results presented here evidence that stress generated by exposure to subinhibitory antibiotic concentrations may result in rearrangements of the bacterial genome. Whereas some of these rearrangements may be deleterious, others may generate genotypes with increased virulence, which may resume infection.
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1737. |
De Luca F,
Benvenuti M,
Carboni F,
Pozzi C,
Rossolini GM,
Mangani S,
Docquier JD,
( 2011 ) Evolution to carbapenem-hydrolyzing activity in noncarbapenemase class D �]-lactamase OXA-10 by rational protein design. PMID : 22042844 : DOI : 10.1073/pnas.1110530108 PMC : PMC3215043 Abstract >>
Class D �]-lactamases with carbapenemase activity are emerging as carbapenem-resistance determinants in gram-negative bacterial pathogens, mostly Acinetobacter baumannii and Klebsiella pneumoniae. Carbapenemase activity is an unusual feature among class D �]-lactamases, and the structural elements responsible for this activity remain unclear. Based on structural and molecular dynamics data, we previously hypothesized a potential role of the residues located in the short-loop connecting strands �]5 and �]6 (the �]5-�]6 loop) in conferring the carbapenemase activity of the OXA-48 enzyme. In this work, the narrow-spectrum OXA-10 class D �]-lactamase, which is unable to hydrolyze carbapenems, was used as a model to investigate the possibility of evolving carbapenemase activity by replacement of the �]5-�]6 loop with those present in three different lineages of class D carbapenemases (OXA-23, OXA-24, and OXA-48). Biological assays and kinetic measurements showed that all three OXA-10-derived hybrids acquired significant carbapenemase activity. Structural analysis of the OXA-10loop24 and OXA-10loop48 hybrids revealed no significant changes in the molecular fold of the enzyme, except for the orientation of the substituted �]5-�]6 loops, which was reminiscent of that found in their parental enzymes. These results demonstrate the crucial role of the �]5-�]6 loop in the carbapenemase activity of class D �]-lactamases, and provide previously unexplored insights into the mechanism by which these enzymes can evolve carbapenemase activity.
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1738. |
Oh E,
Becker AH,
Sandikci A,
Huber D,
Chaba R,
Gloge F,
Nichols RJ,
Typas A,
Gross CA,
Kramer G,
Weissman JS,
Bukau B,
( 2011 ) Selective ribosome profiling reveals the cotranslational chaperone action of trigger factor in vivo. PMID : 22153074 : DOI : 10.1016/j.cell.2011.10.044 PMC : PMC3277850 Abstract >>
As nascent polypeptides exit ribosomes, they are engaged by a series of processing, targeting, and folding factors. Here, we present a selective ribosome profiling strategy that enables global monitoring of when these factors engage polypeptides in the complex cellular environment. Studies of the Escherichia coli chaperone trigger factor (TF) reveal that, though TF can interact with many polypeptides, �]-barrel outer-membrane proteins are the most prominent substrates. Loss of TF leads to broad outer-membrane defects and premature, cotranslational protein translocation. Whereas in vitro studies suggested that TF is prebound to ribosomes waiting for polypeptides to emerge from the exit channel, we find that in vivo TF engages ribosomes only after ~100 amino acids are translated. Moreover, excess TF interferes with cotranslational removal of the N-terminal formyl methionine. Our studies support a triaging model in which proper protein biogenesis relies on the fine-tuned, sequential engagement of processing, targeting, and folding factors.
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1739. |
( 1997 ) The chaperone-assisted membrane release and folding pathway is sensed by two signal transduction systems. PMID : 9351822 : DOI : 10.1093/emboj/16.21.6394 PMC : PMC1170246 Abstract >>
The assembly of interactive protein subunits into extracellular structures, such as pilus fibers in the Enterobacteriaceae, is dependent on the activity of PapD-like periplasmic chaperones. The ability of PapD to undergo a beta zippering interaction with the hydrophobic C-terminus of pilus subunits facilitates their folding and release from the cytoplasmic membrane into the periplasm. In the absence of the chaperone, subunits remained tethered to the membrane and were driven off-pathway via non-productive interactions. These off-pathway reactions were detrimental to cell growth; wild-type growth was restored by co-expression of PapD. Subunit misfolding in the absence of PapD was sensed by two parallel pathways: the Cpx two-component signaling system and the sigma E modulatory pathway.
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1740. |
( 1997 ) Characterization of an exported protease from Shiga toxin-producing Escherichia coli. PMID : 9379905 : DOI : 10.1046/j.1365-2958.1997.5141874.x Abstract >>
The gene for a novel, high molecular weight protein secreted by Shiga toxin-producing Escherichia coli (STEC) has been cloned, sequenced and characterized with respect to its activity. This gene, designated pssA, is localized on the large plasmid that also harbours the STEC haemolysin operon. Sequencing of a region comprising 10630nt revealed that the sequences flanking the pssA gene are composed of several remnants of different insertion elements. The PssA protein is produced as a 142kDa precursor molecule that, after N- and C-terminal processing, is released into the culture supernatant as a mature polypeptide of approximately 104kDa. The primary sequence of PssA is highly related to a family of autonomously transported putative virulence factors from different Gram-negative pathogens, which includes the Tsh protein of an avian-pathogenic E. coli strain, the SepA protein from Shigella flexneri and the EspC protein from enteropathogenic E. coli. A common motif present in all four proteins is reminiscent of the catalytic centre of certain serine proteases. PssA (protease secreted by STEC) indeed shows serine protease activity in a casein-based assay and is moreover cytotoxic for Vero cells. This activity of PssA and probably of other proteins of the Tsh family may be of functional importance during infection of the mucosal cell layer by the bacterial pathogen.
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1741. |
Chen L,
Balabanidou V,
Remeta DP,
Minetti CA,
Portaliou AG,
Economou A,
Kalodimos CG,
( 2011 ) Structural instability tuning as a regulatory mechanism in protein-protein interactions. PMID : 22152477 : DOI : 10.1016/j.molcel.2011.09.022 PMC : PMC3240846 Abstract >>
Protein-protein interactions mediate a vast number of cellular processes. Here, we present a regulatory mechanism in protein-protein interactions mediated by finely tuned structural instability and coupled with molecular mimicry. We show that a set of type III secretion (TTS) autoinhibited homodimeric chaperones adopt a molten globule-like state that transiently exposes the substrate binding site as a means to become rapidly poised for binding to their cognate protein substrates. Packing defects at the homodimeric interface stimulate binding, whereas correction of these defects results in less labile chaperones that give rise to nonfunctional biological systems. The protein substrates use structural mimicry to offset the weak spots in the chaperones and to counteract their autoinhibitory conformation. This regulatory mechanism of protein activity is evolutionarily conserved among several TSS systems and presents a lucid example of functional advantage conferred upon a biological system by finely tuned structural instability.
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1742. |
( 1997 ) Evolutionary relationships among pathogenic and nonpathogenic Escherichia coli strains inferred from multilocus enzyme electrophoresis and mdh sequence studies. PMID : 9199437 : PMC : PMC175379 Abstract >>
Within the species Escherichia coli, there are commensal strains and a variety of pathogenic strains, including enteropathogenic E. coli (EPEC), enterohemorrhagic E. coli (EHEC), enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC), and urinary tract infection (UTI) strains. The pathogenic strains are identified by serotype and by possession of specific virulence determinants (toxins and adhesions, etc.) encoded by either monocistronic genes, plasmids, or pathogenicity islands. Although there are studies on the relationships between selected pathogenic strains, the relatedness among the majority of the pathogenic forms to each other, to commensal E. coli, and to the genus Shigella (which has often been suggested to be part of E. coli) has not been determined. We used multilocus enzyme electrophoresis (MLEE) at 10 enzyme loci and the sequence of the mdh housekeeping gene to study the genetic relationships of pathogenic E. coli strains (including Shigella clones), namely, 5 EPEC strains (serotypes O111 and O55), 3 EHEC strains (serotype O157), 6 ETEC strains (serotypes O78, O159, and O148), 5 EIEC strains (serotypes O124, O28, and O112), and 13 Shigella strains representing clones Flexneri, Dysenteriae, Boydii, and Sonnei, to commensal E. coli strains. Both the MLEE and mdh sequence trees reveal that EPEC, EHEC, ETEC, EIEC, and UTI strains are distributed among the ECOR set groups, with no overall clustering of EPEC, ETEC, EIEC, or UTI strains. The genus Shigella is shown to comprise a group of closely related pathogenic E. coli strains. Six pathogenic strains, i.e., M502 (EIEC; O112ac:NM), M503 (EPEC; O111:H12), M526 (ETEC; O159:H4), M522 (EPEC; O111ac:H12), M524 (ETEC; O78:H11), and M506 (ETEC; O78:H11), were found to have mdh sequences identical to those of five ECOR group A strains (ECOR5, ECOR10, ECOR14, ECOR6, and K-12). All 11 strains are closely related by MLEE. The results indicate that pathogenic strains of E. coli do not have a single evolutionary origin within E. coli but have arisen many times. The results also suggest the possibility that any E. coli strain acquiring the appropriate virulence factors may give rise to a pathogenic form.
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1743. |
Krishnan BR,
Fobert PR,
Seitzer U,
Iyer VN,
( 1990 ) Mutations within the replicon of the IncN plasmid pCU1 that affect its Escherichia coli polA-independence but not its autonomous replication ability. PMID : 2205534 : DOI : 10.1016/0378-1119(90)90155-k Abstract >>
The minimal replicon of the incompatibility N group plasmid pCU1 is contained within a 2-kb DNA region of the plasmid. The ability of this region and of the deletion derivatives thereof, that are capable of autonomous maintenance, to direct polypeptide synthesis was examined. Two proteins of 27 and 5.5 kDa are encoded by the minimal replicon. Polypeptide chain-terminating mutations within the predicted open reading frame for the 27-kDa polypeptide abolished the synthesis of this polypeptide and also the Escherichia coli polA-independence phenotype of the pCU1 replicon. However, these mutations did not affect the autonomous replication ability of the pCU1 replicon in wild-type E. coli and the expression of incompatibility towards the parental plasmid.
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1744. |
Sizmann D,
Keilmann C,
Böck A,
( 1990 ) Primary structure requirements for the maturation in vivo of penicillin acylase from Escherichia coli ATCC 11105. PMID : 2205499 : DOI : 10.1111/j.1432-1033.1990.tb19207.x Abstract >>
The two constituent subunits of the enzyme penicillin acylase from Escherichia coli strain ATCC 11105 are derived from a single precursor polypeptide by post-translational processing. Mutant penicillin acylase precursors were constructed carrying insertions and deletions in various domains and they were analysed for their processing behaviour. It was found that an endopeptide region of appropriate size and an intact C-terminus were absolutely necessary for the maturation process. Internal deletions within the beta-subunit domain also prevented post-translational cleavage. Processing competence, therefore, was not merely determined by the amino acid sequence in the vicinity of the processing sites but relied on a correct overall conformation of the protein. The processing pathway in vivo proceeds via an intermediate comprising the alpha subunits plus endopeptide and is thus identical to the pathway which has been determined previously by in vitro analysis. The post-translational modification of the precursor is probably not carried out by a specific processing enzyme(s) as the heterologous expression of the penicillin acylase (pac) structural gene yielded processed and active enzyme in different enterobacteria and in a Pseudomonas species.
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1745. |
( 1997 ) The structure of nitric oxide synthase oxygenase domain and inhibitor complexes. PMID : 9334294 : DOI : 10.1126/science.278.5337.425 Abstract >>
The nitric oxide synthase oxygenase domain (NOSox) oxidizes arginine to synthesize the cellular signal and defensive cytotoxin nitric oxide (NO). Crystal structures determined for cytokine-inducible NOSox reveal an unusual fold and heme environment for stabilization of activated oxygen intermediates key for catalysis. A winged beta sheet engenders a curved alpha-beta domain resembling a baseball catcher's mitt with heme clasped in the palm. The location of exposed hydrophobic residues and the results of mutational analysis place the dimer interface adjacent to the heme-binding pocket. Juxtaposed hydrophobic O2- and polar L-arginine-binding sites occupied by imidazole and aminoguanidine, respectively, provide a template for designing dual-function inhibitors and imply substrate-assisted catalysis.
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1746. |
( 1997 ) TrwD, a protein encoded by the IncW plasmid R388, displays an ATP hydrolase activity essential for bacterial conjugation. PMID : 9325277 : DOI : 10.1074/jbc.272.41.25583 Abstract >>
A 1.7-kilobase pair segment from the conjugative transfer region of plasmid R388 DNA was cloned and sequenced. It contained trwD, a gene essential for plasmid R388 conjugation, for expression of the conjugative W-pilus and for sensitivity to phage PRD1. The deduced amino acid sequence of TrwD showed homology to the PulE/VirB11 superfamily of potential ATPases involved in various types of transport processes. A fusion of trwD with the glutathione S-transferase (GST) was constructed, and the resulting fusion protein was purified from overproducing bacteria. Factor Xa hydrolysis of GST-TrwD and further purification rendered TrwD protein with more than 95% purity. Antibodies raised against TrwD localized it both in the soluble fraction and in the outer membrane of Escherichia coli. TrwD is probably a peripheral outer membrane protein because it could be solubilized by increasing salt concentration to 0.5 M NaCl in the lysis buffer. Both purified GST-TrwD and TrwD could hydrolize ATP. ATPase activity increased 2-fold in the presence of detergent-phospholipid mixed micelles. To study the importance of the nucleotide-binding site, Walker box A (GXXGXGK(T/S)), present in TrwD, the conserved lysine residue was replaced by glutamine. The mutant protein, expressed and purified under the same conditions as the wild type, did not exhibit ATPase activity. TrwD(K203Q) was not able to complement the mutation in trwD of the R388 mutant plasmid, suggesting the essentiality of the ATPase activity of the protein in the conjugative process. Furthermore, the dominant character of this mutation suggested that GST-TrwD(K432Q) was still able to interact either with itself or with other component(s) of the conjugative machinery.
|
1747. |
Brun YV,
Breton R,
Lanouette P,
Lapointe J,
( 1990 ) Precise mapping and comparison of two evolutionarily related regions of the Escherichia coli K-12 chromosome. Evolution of valU and lysT from an ancestral tRNA operon. PMID : 2201776 : DOI : 10.1016/0022-2836(90)90339-N Abstract >>
Two tRNA operons have been found near the gltX gene encoding the glutamyl-tRNA synthetase of Escherichia coli K-12. The alaW operon previously undetected from genetic data and containing two identical tRNA(GGCAla) genes is 800 base-pairs downstream from the gltX terminator and is transcribed from the same strand. The valU operon containing genes for three identical tRNA(UACVal) and one tRNA(UUULys) (the wild-type allele of supN), is adjacent to gltX and is transcribed from the opposite strand. Five open reading frames were also found in this region encoding putative polypeptides of 62, 105, 130, 167 and 294 amino acid residues. ORF294 is a new member of the lysR family of bacterial transcriptional activators. The possibility that this is the xapR gene is discussed. Comparison of the physical and linkage maps of the E. coli chromosome in the 52 minute region has permitted precise mapping of most of the 18 genes in this region with the order nupC-glk- less than (alaW beta-ala W alpha)-1 kb- less than gltX-0.3 kb-(valU alpha-valU beta-valU gamma-lysV = supN) greater than xapR-xapA- less than lig-1 kb-cysK greater than -0.4 kb-ptsH greater than -0.05 kb-pstI greater than -0.05 kb-crr greater than -cysM-cysA in the clockwise order (greater than and less than indicate the direction of transcription; kb, 10(3) bases). The last two genes of valU (52 min) and lysT (16.5 min) are arranged in a similar fashion and a highly conserved region has been found in both operons. This suggests that the valU and lysT operons probably arose by a duplication of an ancestral tRNA operon. This is the first example of what may be two different tRNA operons from the same organism evolving from an ancestral tRNA gene. Comparison of the 16 and 52 minute regions of the E. coli K-12 chromosome suggests that these two regions could share a common ancestor.
|
1748. |
Sun Y,
Zeng Z,
Chen S,
Ma J,
He L,
Liu Y,
Deng Y,
Lei T,
Zhao J,
Liu JH,
( 2010 ) High prevalence of bla(CTX-M) extended-spectrum �]-lactamase genes in Escherichia coli isolates from pets and emergence of CTX-M-64 in China. PMID : 21681998 : Abstract >>
As a cause of community-acquired infections, extended-spectrum �]-lactamase (ESBL)-producing Escherichia coli constitute an emerging public-health concern. Few data on the molecular epidemiology of ESBL-producing E. coli isolates from pets are available in China. Detection and characterization of ESBL genes (bla(CTX-M), bla(SHV) and bla(TEM)) was conducted among 240 E. coli isolates recovered from healthy and sick pets in South China from 2007 to 2008. The clonal relatedness of ESBL-producing E. coli isolates was assessed by pulsed field gel electrophoresis. ESBL-encoding genes were identified in 97 (40.4%) of the 240 isolates and 96 (40.0%) of them harbored CTX-M. The most common CTX-M types were CTX-M-14 (n = 45) and CTX-M-55 (n = 24). The recently reported CTX-M-64 was identified in three isolates. Isolates producing CTX-M-27, -15, -65, -24, -3 and -9 were also identified. Ten isolates carried two or three CTX-M types, with the combination of CTX-M-14 and CTX-M-55 being the most frequent (n = 6). ISEcp1 was identified in the upstream region of 93 out of the 107 bla(CTX-M) genes (86.9%). The sequence of the spacer region (45 bp) between ISEcp1 and the start codon of all bla(CTX-M-55) genes (except four) was identical to that of bla(CTX-M-64). No major clonal relatedness was observed among these CTX-M producers. It is suggested that the horizontal transfer of bla(CTX-M) genes, mediated by mobile elements, contributes to their dissemination among E. coli isolates from pets. Our finding of high prevalence of ESBL in E. coli of companion animal origin illustrates the importance of molecular surveillance in tracking CTX-M-producing E. coli strains in pets.
|
1749. |
Muraiso K,
Mukhopadhyay G,
Chattoraj DK,
( 1990 ) Location of a P1 plasmid replication inhibitor determinant within the initiator gene. PMID : 2198259 : DOI : 10.1128/jb.172.8.4441-4447.1990 PMC : PMC213273 Abstract >>
The P1 plasmid replication initiator protein, RepA, binds to its own promoter and represses transcription efficiently. There are only about 20 RepA dimers present per repA gene. A possible reason for this highly restrained expression became evident when repA expression was increased by using foreign promoters: with fivefold overexpression, the replication rate was diminished, and with 40-fold overexpression, replication was not detectable. The inhibition was P1 specific: growth of Escherichia coli and replication of pSC101, R6K, and mini-F plasmids were not affected. The activity is apparently not from RepA itself. Excess purified RepA did not inhibit replication in vitro. Mutations of the repA translation initiation codon reduced synthesis of the initiator but not the inhibitory activity. Deletion from either the N- or C-terminal ends of repA (28 and 69 codons, respectively, out of the 286-codon open reading frame) affected the initiator but not the inhibitory activity. Further deletions affected both the activities. These results demonstrate that the integrity of the initiator is not required for inhibition, but involvement of an unstable initiator fragment or of initiator mRNA cannot be ruled out.
|
1750. |
Wormald MR,
Merrill AR,
Cramer WA,
Williams RJ,
( 1990 ) Solution NMR studies of colicin E1 C-terminal thermolytic peptide. Structural comparison with colicin A and the effects of pH changes. PMID : 2199197 : DOI : 10.1111/j.1432-1033.1990.tb19105.x Abstract >>
The aqueous solution structure of the C-terminal thermolytic peptide of colicin E1 has been investigated using both one- and two-dimensional NMR techniques. The NMR data are consistent with a fold for the peptide very similar to that reported for the colicin A C-terminal peptide in the crystalline state, although some differences have been noted. The one-dimensional NMR spectrum of the peptide has been used to follow changes in both the structure and dynamics of the peptide on changing pH. The in vitro functionally competent form of the peptide (present in solution only below pH 6) does not differ in structure significantly from the higher pH form. However, small local conformational changes are observed together with an increase in mobility in some of the more hydrophilic regions. This suggests that the effect of lower pH is to change the ease with which the major conformational changes during insertion into a membrane can occur.
|
1751. |
Li DX,
Zhang SM,
Hu GZ,
Wang Y,
Liu HB,
Wu CM,
Shang YH,
Chen YX,
Du XD,
( 2012 ) Tn3-associated rmtB together with qnrS1, aac(6')-Ib-cr and bla CTX-M-15 are co-located on an F49:A-:B- plasmid in an Escherichia coli ST10 strain in China. PMID : 22010207 : DOI : 10.1093/jac/dkr428 Abstract >>
N/A
|
1752. |
( 1997 ) A gene cluster closely related to type II secretion pathway operons of gram-negative bacteria is located on the large plasmid of enterohemorrhagic Escherichia coli O157 strains. PMID : 9084155 : DOI : 10.1111/j.1574-6968.1997.tb10299.x Abstract >>
Analysis of 14.162 kb of DNA derived from plasmid pO157 of enterohemorrhagic Escherichia coli (EHEC) O157:H7 strain EDL933, extending in the 5' direction of the recently described EHEC-hly operon, revealed 13 open reading frames (ORF) which showed great similarities to genes of members of the type II pathway secretion systems of Gram-negative bacteria. We named the ORFs etpC to etpO for EHEC type II secretion pathway. In addition, an IS911-like insertion element was found to separate the etp genes from the EHEC-hlyC gene. Hybridization experiments with a specific etp probe and various categories of enteric E. coli pathotypes revealed that the etp gene cluster occurred in all 30 EHEC strains of serogroup O157 (100%) tested and is distributed sporadically among other EHEC serogroups (60%). In addition, the etp genes were rarely detected in STEC isolated from bovine feces (10%). Moreover, it was found not to occur in enteropathogenic E. coli, enteroaggregative E. coli, enterotoxigenic E. coli and enteroinvasive E. coli. The results obtained with the etp probe were confirmed by a PCR approach to specifically detect an internal fragment of the etpD gene.
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1753. |
Clarke BR,
Richards MR,
Greenfield LK,
Hou D,
Lowary TL,
Whitfield C,
( 2011 ) In vitro reconstruction of the chain termination reaction in biosynthesis of the Escherichia coli O9a O-polysaccharide: the chain-length regulator, WbdD, catalyzes the addition of methyl phosphate to the non-reducing terminus of the growing glycan. PMID : 21990359 : DOI : 10.1074/jbc.M111.295857 PMC : PMC3308851 Abstract >>
The Escherichia coli O9a O-polysaccharide (O-PS) represents a model system for glycan biosynthesis and export by the ATP-binding cassette (ABC) transporter-dependent pathway. The polymannose O9a O-PS is synthesized using an undecaprenol-diphosphate-linked acceptor by mannosyltransferases located at the cytoplasmic membrane. An ABC-transporter subsequently exports the polymer to the periplasm where it is assembled onto lipopolysaccharide prior to translocation to the cell surface. The chain length of the O9a O-PS is regulated by the dual kinase/methyltransferase activity of the WbdD enzyme and modification of the polymer is crucial for binding and export by the ABC-transporter. Previous biochemical data provided evidence for phosphorylation/methylation at the non-reducing end of the O9a O-PS but the structure of the terminus has not been determined. Here, we describe the exploitation of a synthetic O9a O-PS repeating unit carrying a fluorescent tag as an acceptor for in vitro phosphorylation and methylation by a purified soluble form of WbdD. Phosphorylation of the acceptor was evident by both a mobility shift in thin layer chromatography and radiolabeling of the acceptor using [�^-(33)P]ATP. Methylation of the acceptor was dependent on phosphorylation and was demonstrated by radiolabeling using S-[methyl-(3)H]adenosyl-methionine as a substrate, in the presence of ATP. NMR spectroscopic and mass spectrometric methods were used to determine the precise structure of the terminal modification, leading to the conclusion that WbdD catalyzes the addition of a novel methyl phosphate group to the 3-position of the non-reducing terminal mannose of the O9a O-PS repeating unit.
|
1754. |
Yamaguchi A,
Ono N,
Akasaka T,
Noumi T,
Sawai T,
( 1990 ) Metal-tetracycline/H+ antiporter of Escherichia coli encoded by a transposon, Tn10. The role of the conserved dipeptide, Ser65-Asp66, in tetracycline transport. PMID : 2168416 : Abstract >>
The transposon Tn10-encoded tetracycline resistance protein functions as a metal-tetracycline/H+ antiporter (Yamaguchi, A., Udagawa, T., and Sawai, T. (1990) J. Biol. Chem. 265, 4809-4813). The Ser65-Asp66 dipeptide is conserved in all known tetracycline antiporter proteins and is an important target for site-directed mutagenesis. When Asp66 was replaced by Asn, the transport activity was completely lost, whereas when it was replaced by Glu, the activity was reduced to 10% of the wild-type level, indicating that a negative charge at position 66 is essential for tetracycline transport. Replacement of Ser65 by Cys or Ala, in contrast, caused only a minor change in tetracycline transport activity. However, the Cys65 mutant antiporter was sensitive to sulfhydryl reagents. Complete inactivation of the Cys65 antiporter by N-ethylmaleimide was not prevented by the substrate. A less bulky reagent, methyl methanethiosulfonate, caused partial inactivation of the Cys65 antiporter without changing its affinity to the substrate. These results indicate that a region including the dipeptide plays an important role in metal-tetracycline transport except for substrate binding. It may act as a gate which opens on the charge-charge interaction between Asp66 and the metal-tetracycline.
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1755. |
Gannon VP,
Teerling C,
Masri SA,
Gyles CL,
( 1990 ) Molecular cloning and nucleotide sequence of another variant of the Escherichia coli Shiga-like toxin II family. PMID : 2200845 : DOI : 10.1099/00221287-136-6-1125 Abstract >>
Escherichia coli strain H.I.8 (O128:B12) produces low levels of a Shiga-like toxin (SLT) which we have called SLTIIva because of its close relationship with SLTIIv. The Vero cell cytotoxicity of SLTIIva is neutralized by antisera against SLTII and SLTIIv but not by antisera against SLTI. These data indicate that the SLT of strain H.I.8 is a member of the SLTII family. Since SLTIIva shares with SLTIIv the property of having low cytotoxicity to HeLa cells compared with Vero cells, it is appropriate to consider both toxins as variants of SLTII. SLTIIva differs from SLTIIv in that it is more heat-stable. Further, SLTIIv-producing strains of E. coli have only been isolated from pigs while the SLTIIva-producing E. coli strain examined in this study was isolated from a human infant with diarrhoea. The genes for this SLT were cloned from a cosmid library of total cellular DNA by screening recombinants for Vero cell toxicity and with a DNA probe derived from SLTIIv structural genes. Nucleotide sequence analysis was performed on a 2.0 kb AvaII-HincII fragment which encodes the toxin gene. The nucleotide sequence data confirm the close relationship between SLTIIva and SLTIIv: they have 98% nucleotide sequence homology in the B subunit gene and 70.6% homology in the A subunit gene. Comparison of DNA sequences indicated that SLTIIva was most closely related to SLTIIv, closely related to SLTII and less closely related to SLTI.
|
1756. |
Pansegrau W,
Balzer D,
Kruft V,
Lurz R,
Lanka E,
( 1990 ) In vitro assembly of relaxosomes at the transfer origin of plasmid RP4. PMID : 2168553 : DOI : 10.1073/pnas.87.17.6555 PMC : PMC54575 Abstract >>
During initiation of conjugative transfer of DNA containing the transfer origin (oriT) of the promiscuous plasmid RP4, the proteins TraI, TraJ, and TraH interact and assemble a specialized nucleoprotein complex (the relaxosome) at oriT. The structure can be visualized on electron micrographs. Site- and strand-specific nicking at the transfer origin in vitro is dependent on the proteins TraI and TraJ and on Mg2+ ions. Substrate specificity is directed exclusively towards the cognate transfer origin: the RP4-specified TraJ protein cannot recognize the closely related oriT of plasmid R751. After nicking, TraI protein remains attached to the 5'-terminal 2'-deoxycytidyl residue at the nic site [Pansegrau, W., Ziegelin, G. & Lanka, E. (1990) J. Biol. Chem. 265, 10637-10644]. Nicking and relaxosome formation require supercoiled DNA. Thus, a complicated structure involving multiple plasmid-specified proteins and a defined region of DNA must be formed at the transfer origin to prepare the plasmid for generating the single strand to be transferred.
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1757. |
( 1997 ) A novel role of ImmE7 in the autoregulatory expression of the ColE7 operon and identification of possible RNase active sites in the crystal structure of dimeric ImmE7. PMID : 9135159 : DOI : 10.1093/emboj/16.6.1444 PMC : PMC1169741 Abstract >>
Site-specific cleavage of mRNA has been identified in vivo for the polycistronic colicin E7 operon (ColE7), which occurs between G and A nucleotides located at the Asp52 codon (GAT) of the immunity gene (ceiE7). In vitro, this specific cleavage occurs only in the presence of the ceiE7 gene product (ImmE7). The crystal structure of dimeric ImmE7 has been determined at 1.8 A resolution by X-ray crystallographic analysis. We found that several residues located at the interface of dimeric ImmE7 bear surprising resemblance to the active sites of some RNases. These results suggest that dimeric ImmE7 may possess a novel RNase activity that cleaves its own mRNA at a specific site and thus autoregulates translational expression of the downstream celE7 gene as well as degradation of the upstream ceaE7 mRNA.
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1758. |
Creuzburg K,
Heeren S,
Lis CM,
Kranz M,
Hensel M,
Schmidt H,
( 2011 ) Genetic background and mobility of variants of the gene nleA in attaching and effacing Escherichia coli. PMID : 22003022 : DOI : 10.1128/AEM.06492-11 PMC : PMC3233075 Abstract >>
In this study, we characterized the genetic background of various nleA variants in 106 Shiga toxin-producing Escherichia coli (STEC) and enteropathogenic Escherichia coli (EPEC) strains. The flanking regions of eight nleA variants were analyzed by DNA sequencing and compared with the corresponding regions of five previously described NleA-encoding prophages. The analyzed nleA variants were all located downstream of the DNA region responsible for phage morphogenesis. In particular, the type III effector genes avrA, ospB, nleH, and nleG and IS elements were detected in the neighborhood of nleA. The structure of the eight analyzed regions flanking nleA primarily resembled the corresponding region of the NleA????-encoding prophage BP-4795. Using PCR, the gene order flanking 13 nleA variants in strains of different serogroups was compared to the respective regions in reference strains. The analyses showed that strains which harbor prophages with conserved flanking regions of a particular nleA variant predominantly occurred, and IS elements were additionally detected in these regions. We were able to mobilize nleA by transduction in 20% of strains determined, which comprised in particular EPEC strains harboring an nleA variant, the gene encoding the protein known as "EspI-like." Plaque hybridization was used to identify phages that harbor the genes stx and nleA. However, only two strains harbored variant nleA???? in the genome of an Stx1 prophage.
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1759. |
( 1997 ) Characterization of two virulence proteins secreted by rabbit enteropathogenic Escherichia coli, EspA and EspB, whose maximal expression is sensitive to host body temperature. PMID : 9284118 : PMC : PMC175505 Abstract >>
Enteropathogenic Escherichia coli (EPEC) and rabbit EPEC (RDEC-1) cause unique histopathological features on intestinal mucosa, including attaching/effacing (A/E) lesions. Due to the human specificity of EPEC, RDEC-1 has been used as an animal model to study EPEC pathogenesis. At least two of the previously identified EPEC-secreted proteins, EspA and EspB, are required for triggering host epithelial signal transduction pathways, intimate adherence, and A/E lesions. However, the functions of these secreted proteins and their roles in pathogenesis have not been characterized. To investigate the function of EspA and EspB in RDEC-1, the espA and espB genes were cloned and their sequences were compared to that of EPEC O127. The EspA proteins showed high similarity (88.5% identity), while EspB was heterogeneous in internal regions (69.8% identity). However, RDEC-1 EspB was identical to that of enterohemorrhagic E. coli serotype O26. Mutations in RDEC-1 espA and espB revealed that the corresponding RDEC-1 gene products are essential for triggering of host signal transduction pathways and invasion into HeLa cells. Complementation with plasmids containing EPEC espA or/and espB genes into RDEC-1 mutant strains demonstrated that they were functionally interchangeable, although the EPEC proteins mediated higher levels of invasion. Furthermore, maximal expression of RDEC-1 and EPEC-secreted proteins occurred at their respective host body temperatures, which may contribute to the lack of EPEC infectivity in rabbits.
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1760. |
Kurpiel PM,
Hanson ND,
( 2011 ) Association of IS5 with divergent tandem blaCMY-2 genes in clinical isolates of Escherichia coli. PMID : 21636584 : DOI : 10.1093/jac/dkr212 Abstract >>
To characterize a unique tandem bla(CMY-2) gene arrangement found in two non-identical clinical strains of Escherichia coli. Both plasmid and chromosomal DNA were evaluated using PFGE, restriction digest analysis, plasmid profiling and Southern hybridization. bla(CMY-2) gene expression and gene copy number were evaluated by real-time PCR. Susceptibilities to selected �]-lactam antibiotics were determined by agar dilution. A tandem arrangement for bla(CMY-2) was identified in both isolates and was the only arrangement for bla(CMY-2) observed. These isolates had distinct PFGE and plasmid profiles. Each strain exhibited 2-fold higher bla(CMY-2) mRNA expression and up to 8-fold lower �]-lactam susceptibility compared with a strain with a single copy of bla(CMY-2). This is the first report of IS5 being associated with tandem bla(CMY-2). IS5 has previously been associated with antibiotic resistance through tandem gene amplification. The unique tandem arrangement provides a mechanism for increased bla(CMY-2) expression.
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1761. |
Adams MD,
Wagner LM,
Graddis TJ,
Landick R,
Antonucci TK,
Gibson AL,
Oxender DL,
( 1990 ) Nucleotide sequence and genetic characterization reveal six essential genes for the LIV-I and LS transport systems of Escherichia coli. PMID : 2195019 : Abstract >>
The nucleotide sequence of the genes encoding the high affinity, branched-chain amino acid transport systems LIV-I and LS has been determined. Seven genes are present on a 7568-base pair DNA fragment, six of which participate directly in branched-chain amino acid transport. Two periplasmic amino acid-binding proteins are encoded by the livJ (LIV-BP) and livK (LS-BP) genes. These two proteins confer specificity on the LIV-I and LS transport systems. livK is the first gene in a polycistronic message that includes four genes encoding membrane components, livHMGF. The protein products of the livHMGF genes are shared by the two systems. An analysis of the livH and livM DNA sequences suggests that they encode hydrophobic proteins capable of spanning the membrane several times. The LivG and LivF proteins are less hydrophobic, but are also tightly associated with the membrane. Both LivG and LivF contain the consensus sequence for adenine nucleotide binding observed in many other transport proteins. A deletion strain that does not express any of the liv genes was constructed. This strain was used to show that each of the membrane component genes is required for high affinity leucine transport, including two genes, livM and livF, for which no previous genetic evidence had been obtained.
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1762. |
Dassa J,
Marck C,
Boquet PL,
( 1990 ) The complete nucleotide sequence of the Escherichia coli gene appA reveals significant homology between pH 2.5 acid phosphatase and glucose-1-phosphatase. PMID : 2168385 : DOI : 10.1128/jb.172.9.5497-5500.1990 PMC : PMC213220 Abstract >>
The whole nucleotide sequence of Escherichia coli gene appA, which encodes periplasmic phosphoanhydride phosphohydrolase (optimum pH, 2.5), and its flanking regions was determined. The AppA protein is significantly homologous to the product of the nearby gene agp, acid glucose-1-phosphatase. Because identical amino acids are distributed over the whole lengths of the proteins, it is likely that appA and agp originate from the same ancestor gene.
|
1763. |
Hornsey M,
Phee L,
Wareham DW,
( 2011 ) A novel variant, NDM-5, of the New Delhi metallo-�]-lactamase in a multidrug-resistant Escherichia coli ST648 isolate recovered from a patient in the United Kingdom. PMID : 21930874 : DOI : 10.1128/AAC.05108-11 PMC : PMC3232805 Abstract >>
A new variant of the New Delhi metallo-enzyme (NDM) carbapenemase was identified in a multidrug-resistant Escherichia coli ST648 isolate recovered from the perineum and throat of a patient in the United Kingdom with a recent history of hospitalization in India. NDM-5 differed from existing enzymes due to substitutions at positions 88 (Val �� Leu) and 154 (Met �� Leu) and reduced the susceptibility of E. coli TOP10 transformants to expanded-spectrum cephalosporins and carbapenems when expressed under its native promoter.
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1764. |
Guo Y,
Wang J,
Niu G,
Shui W,
Sun Y,
Zhou H,
Zhang Y,
Yang C,
Lou Z,
Rao Z,
( 2011 ) A structural view of the antibiotic degradation enzyme NDM-1 from a superbug. PMID : 21637961 : DOI : 10.1007/s13238-011-1055-9 PMC : PMC4875342 Abstract >>
Gram-negative Enterobacteriaceae with resistance to carbapenem conferred by New Delhi metallo-�]-lactamase 1 (NDM-1) are a type of newly discovered antibioticresistant bacteria. The rapid pandemic spread of NDM-1 bacteria worldwide (spreading to India, Pakistan, Europe, America, and Chinese Taiwan) in less than 2 months characterizes these microbes as a potentially major global health problem. The drug resistance of NDM-1 bacteria is largely due to plasmids containing the blaNDM-1 gene shuttling through bacterial populations. The NDM-1 enzyme encoded by the blaNDM-1 gene hydrolyzes �]-lactam antibiotics, allowing the bacteria to escape the action of antibiotics. Although the biological functions and structural features of NDM-1 have been proposed according to results from functional and structural investigation of its homologues, the precise molecular characteristics and mechanism of action of NDM-1 have not been clarified. Here, we report the three-dimensional structure of NDM-1 with two catalytic zinc ions in its active site. Biological and mass spectroscopy results revealed that D-captopril can effectively inhibit the enzymatic activity of NDM-1 by binding to its active site with high binding affinity. The unique features concerning the primary sequence and structural conformation of the active site distinguish NDM-1 from other reported metallo-�]-lactamases (MBLs) and implicate its role in wide spectrum drug resistance. We also discuss the molecular mechanism of NDM-1 action and its essential role in the pandemic of drug-resistant NDM-1 bacteria. Our results will provide helpful information for future drug discovery targeting drug resistance caused by NDM-1 and related metallo-�]-lactamases.
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1765. |
Orden JA,
Horcajo P,
de la Fuente R,
Ruiz-Santa-Quiteria JA,
Domínguez-Bernal G,
Carrión J,
( 2011 ) Subtilase cytotoxin-coding genes in verotoxin-producing Escherichia coli strains from sheep and goats differ from those from cattle. PMID : 21965402 : DOI : 10.1128/AEM.05604-11 PMC : PMC3233036 Abstract >>
Subtilase cytotoxin (SubAB) from verotoxin (VT)-producing Escherichia coli (VTEC) strains was first described in the 98NK2 strain and has been associated with human disease. However, SubAB has recently been found in two VT-negative E. coli strains (ED 591 and ED 32). SubAB is encoded by two closely linked, cotranscribed genes (subA and subB). In this study, we investigated the presence of subAB genes in 52 VTEC strains isolated from cattle and 209 strains from small ruminants, using PCR. Most (91.9%) VTEC strains from sheep and goats and 25% of the strains from healthy cattle possessed subAB genes. The presence of subAB in a high percentage of the VTEC strains from small ruminants might increase the pathogenicity of these strains for human beings. Some differences in the results of PCRs and in the association with some virulence genes suggested the existence of different variants of subAB. We therefore sequenced the subA gene in 12 strains and showed that the subA gene in most of the subAB-positive VTEC strains from cattle was almost identical (about 99%) to that in the 98NK2 strain, while the subA gene in most of the subAB-positive VTEC strains from small ruminants was almost identical to that in the ED 591 strain. We propose the terms subAB1 to describe the SubAB-coding genes resembling that in the 98NK2 strain and subAB2 to describe those resembling that in the ED 591 strain.
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1766. |
Horcajo P,
Domínguez-Bernal G,
de la Fuente R,
Ruiz-Santa-Quiteria JA,
Blanco JE,
Blanco M,
Mora A,
Dahbi G,
López C,
Puentes B,
Alonso MP,
Blanco J,
Orden JA,
( 2012 ) Comparison of ruminant and human attaching and effacing Escherichia coli (AEEC) strains. PMID : 21958746 : DOI : 10.1016/j.vetmic.2011.08.034 Abstract >>
The presence of 12 genes associated with virulence in human attaching and effacing Escherichia coli (AEEC) was studied within a collection of 20 enterohemorrhagic E. coli (EHEC) and 206 atypical enteropathogenic E. coli (EPEC) isolated from ruminants. In addition, virulence genes and the clonal relationship of 49 atypical EPEC O26 strains isolated from humans and ruminants were compared to clarify whether ruminants serve as a reservoir of atypical EPEC for humans. A great diversity in the content of virulence gene was found. Thus, the espH, espG and map genes were detected in more than 85% of ruminant AEEC strains; the tccP2, espI, efa1/lifA, ehxA and paa genes were present in 50-70% of strains; and other genes such as tccP, espP, katP and toxB were detected in <25% of strains. EHEC strains contained more virulence genes than atypical EPEC strains. Our results suggest for the first time that the efa1/lifA gene is associated with diarrhea in newborn ruminants and that the AEEC strains with the H11 flagellar antigen are potentially more virulent than the non-H11 AEEC strains. Importantly, we identified a new intimin variant gene, eae�l, in three ruminant atypical EPEC strains. The comparison of ruminant and human EPEC O26 strains showed that some ruminant strains possess virulence gene profiles and pulse-field gel electrophoresis pulsotypes similar to those of human strains. In conclusion, our data suggest that atypical EPEC is a heterogeneous group with different pathogenic potential and that ruminants could serve as a reservoir of atypical EPEC for humans.
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1767. |
Mercier J,
Lachapelle J,
Couture F,
Lafond M,
Vézina G,
Boissinot M,
Levesque RC,
( 1990 ) Structural and functional characterization of tnpI, a recombinase locus in Tn21 and related beta-lactamase transposons. PMID : 2163386 : DOI : 10.1128/jb.172.7.3745-3757.1990 PMC : PMC213353 Abstract >>
A novel discrete mobile DNA element from Tn21 from the plasmid R100.1 is described, and its mobilization function was confirmed experimentally. In addition, the element behaves as a recombinase-active locus (tnpI) which facilitates insertions of antibiotic resistance genes as modules or cassettes at defined hot spots or integration sites. A similar tnpI sequence was detected by DNA hybridization in a series of beta-lactamase transposons and plasmids and localized on their physical maps. The genetic function of the locus cloned from Tn21 into pACYC184 was tested for conduction and integration into the plasmids R388 and pOX38Km, and the results suggested recombinase-integrase activity and recA independence. DNA sequence analysis of the tnpI locus revealed no inverted or direct terminal repeats or transposition features of class I and class II transposons. The coding capacity revealed three putative open reading frames encoding 131, 134, and 337 amino acids. Orf3 encoded a putative polypeptide product of 337 amino acids that shared highly significant identity with the carboxyl region of integrase proteins. A comparison and an alignment of the tnpI locus from Tn21 and its flanking sequences identified similar sequences in plasmids and in transposons. The alignment revealed discrete nucleotide changes in these tnpI-like loci and a conserved 3' and 5' GTTA/G hot spot as a duplicated target site. Our data confirm the remarkable ubiquity of tnpI associated with antibiotic resistance genes. We present a model of transposon modular evolution into more complex multiresistant units via tnpI and site-specific insertions, deletions, and DNA rearrangements at this locus.
|
1768. |
Metcalf WW,
Steed PM,
Wanner BL,
( 1990 ) Identification of phosphate starvation-inducible genes in Escherichia coli K-12 by DNA sequence analysis of psi::lacZ(Mu d1) transcriptional fusions. PMID : 2160940 : DOI : 10.1128/jb.172.6.3191-3200.1990 PMC : PMC209124 Abstract >>
Twenty-four independent phosphate starvation-inducible (psi) transcriptional fusions made with Mu d1(lacZbla) were analyzed by sequencing the psi::lacZ(Mu d1) chromosomal junctions by using DNAs amplified with the polymerase chain reaction or mini-Mu cloning. Our DNA sequence analysis showed that the MuR DNA in Mu d1 has an unexpected structure that is comprised of 104 bases of MuR DNA in the form of a large inverted repeat, which we denoted Mu d1-R. Also, Mu d1s in the phoA and phn (psiD) loci of the phosphate regulon showed regional specificities for the insertion sites despite the randomness of Mu d1 insertions into the genome as a whole. Gene products or open reading frames were identified for seven unknown psi::lacZ(Mu d1) transcriptional fusions by searching DNA data bases with the sequences adjacent and upstream of the Mu d1s. One psiC::lacZ(Mu d1) lies in the ugpB gene of the ugpBAEC operon, which encodes a periplasmic sn-glycerol-3-phosphate-binding protein; two psiQ::lacZ(Mu d1)s lie in the gltB gene, and one psiQ::lacZ(Mu d1) lies in the gltD gene of the gltBDF operon, encoding the large and small subunits of glutamate synthase, respectively; and the psi-51::lacZ(Mu d1) lies in the glpB gene of the glpABC operon, which codes for the anaerobically regulated glycerol-3-phosphate dehydrogenase. psiE and psiF::lacZ(Mu d1)s lie in uncharacterized open reading frames near the xylE and phoA genes, respectively. Six other psi::lacZ(Mu d1)s lie in yet unreported Escherichia coli sequences.
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1769. |
Partridge SR,
Ellem JA,
Tetu SG,
Zong Z,
Paulsen IT,
Iredell JR,
( 2011 ) Complete sequence of pJIE143, a pir-type plasmid carrying ISEcp1-blaCTX-M-15 from an Escherichia coli ST131 isolate. PMID : 21911569 : DOI : 10.1128/AAC.00639-11 PMC : PMC3232798 Abstract >>
pJIE143 (34 kb), from an Escherichia coli ST131 isolate, carries bla(CTX-M-15) but could not be typed using the standard PCR-based replicon-typing primer set. Complete sequencing revealed a backbone with similarity to IncX plasmids, including a pir-like gene encoding a �k-like replication protein and iterons related to those of other IncX plasmids. The 2.971-kb ISEcp1-bla(CTX-M-15)-orf477�G transposition unit often found within Tn2 is inserted just beyond the end of pir, flanked by 5-bp direct repeats.
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1770. |
Chiang SM,
Dong T,
Edge TA,
Schellhorn HE,
( 2011 ) Phenotypic diversity caused by differential RpoS activity among environmental Escherichia coli isolates. PMID : 21948830 : DOI : 10.1128/AEM.05274-11 PMC : PMC3209002 Abstract >>
Enteric bacteria deposited into the environment by animal hosts are subject to diverse selective pressures. These pressures may act on phenotypic differences in bacterial populations and select adaptive mutations for survival in stress. As a model to study phenotypic diversity in environmental bacteria, we examined mutations of the stress response sigma factor, RpoS, in environmental Escherichia coli isolates. A total of 2,040 isolates from urban beaches and nearby fecal pollution sources on Lake Ontario (Canada) were screened for RpoS function by examining growth on succinate and catalase activity, two RpoS-dependent phenotypes. The rpoS sequence was determined for 45 isolates, including all candidate RpoS mutants, and of these, six isolates were confirmed as mutants with the complete loss of RpoS function. Similarly to laboratory strains, the RpoS expression of these environmental isolates was stationary phase dependent. However, the expression of RpoS regulon members KatE and AppA had differing levels of expression in several environmental isolates compared to those in laboratory strains. Furthermore, after plating rpoS+ isolates on succinate, RpoS mutants could be readily selected from environmental E. coli. Naturally isolated and succinate-selected RpoS mutants had lower generation times on poor carbon sources and lower stress resistance than their rpoS+ isogenic parental strains. These results show that RpoS mutants are present in the environment (with a frequency of 0.003 among isolates) and that, similarly to laboratory and pathogenic strains, growth on poor carbon sources selects for rpoS mutations in environmental E. coli. RpoS selection may be an important determinant of phenotypic diversification and, hence, the survival of E. coli in the environment.
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1771. |
Vinué L,
Jové T,
Torres C,
Ploy MC,
( 2011 ) Diversity of class 1 integron gene cassette Pc promoter variants in clinical Escherichia coli strains and description of a new P2 promoter variant. PMID : 21917427 : DOI : 10.1016/j.ijantimicag.2011.07.007 Abstract >>
Gene cassettes of class 1 integrons may be differently expressed depending on the Pc promoter variant as well as occasionally from a second promoter located downstream of Pc, named P2. So far, the distribution of the variants has only been described in an in silico study. In this study, the prevalence of these variants in vivo was analysed in a population of 85 Escherichia coli strains from a variety of phylogenetic groups isolated from healthy subjects and clinical samples in Spain and France from 2004 to 2007. The weakest variants (PcW and PcH1) prevailed (variants associated with the integrase having the most efficient excision activity), whilst the two strongest variants, PcW(TGN-10) and PcS, were less frequent. Furthermore, a new variant of P2 associated with PcW was characterised in one integron (harbouring the gene cassette bla(OXA-1)-aadA1) from a French strain of a healthy subject. This variant was hereafter named P2m3 and shows a G��A substitution in its -10 element (TACAGT to TACAAT), a mutation that doubled the strength of P2 and approached the level of expression of the strong PcW(TGN-10) variant. When the correlation between the Pc variants and the origin of the strains was analysed, no significant difference (P<0.05) was observed in the Pc variant distribution according to the geographic origin or clinical setting.
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1772. |
Gebru E,
Damte D,
Choi MJ,
Lee SJ,
Kim YH,
Park SC,
( 2012 ) Mutant prevention concentration and phenotypic and molecular basis of fluoroquinolone resistance in clinical isolates and in vitro-selected mutants of Escherichia coli from dogs. PMID : 21893387 : DOI : 10.1016/j.vetmic.2011.07.033 Abstract >>
The antibacterial activity, selection of Escherichia coli (E. coli) mutants and mechanisms of fluoroquinolone resistance were investigated by integrating the minimum inhibitory concentration (MIC), mutant prevention concentration (MPC) and in vitro dynamic model approaches. Difloxacin and orbifloxacin, for which the above information has been scarce, were used. A range of area under curve over a 24h interval (AUC(24h))/MIC ratios and selected E. coli strains were investigated using the dynamic models. Continuous incubation for three days in the presence of difloxacin or orbifloxacin resulted in losses in E. coli susceptibility. An AUC(24h)/MIC (AUC(24h)/MPC)-dependent fluoroquinolone activity and selection of E. coli mutants was confirmed. Maximum losses in susceptibility occurred at AUC(24h)/MIC ratios of 54 (orbifloxacin) and 57.3 (difloxacin). AUC(24h)/MIC ratios of 169.8 (orbifloxacin) and 199.5 (difloxacin) were estimated to be protective against the selection of E. coli mutants, and the corresponding ratios based on AUC(24h)/MPC predictions were 34 (orbifloxacin) and 36.3 (difloxacin). When integrating our in vitro data with pharmacokinetic data in dogs, the conventional clinical doses of both drugs were found to be inadequate to attain the above protective values for 90% of the mutant subpopulation (AUC(24h)/MPC(90)). Both target mutations, esp. at codon 83 (Ser to Leu) of gyrA, and overexpression of efflux pumps contributed to resistance development, with mutants also showing decreased susceptibility to enrofloxacin and marbofloxacin. Additional studies would determine the role of mutations found outside the QRDR, at codon 24 of gyrA, and at codon 116 of parC, and establish the significance of these observations in vivo.
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1773. |
Rasheed JK,
Guzmán-Verduzco LM,
Kupersztoch YM,
( 1990 ) Two precursors of the heat-stable enterotoxin of Escherichia coli: evidence of extracellular processing. PMID : 2187146 : DOI : 10.1111/j.1365-2958.1990.tb00593.x Abstract >>
Expression of the gene of the methanol-soluble, heat-stable enterotoxin of Escherichia coli (STA) allowed the identification by SDS-PAGE of a cell-associated 7500 Dalton STA-related peptide; when similar experiments were performed with a phosphate buffer SDS-PAGE system, an additional Mr 9800 band became apparent. The 9800 Dalton form, pre-pro-STA, accumulated as an intracellular species when the experiments were performed in the presence of the proton ionophore CCCP (carbonylcyanide m-chlorophenylhydrazone); by pulse-chase experiments, it was shown that pre-pro-STA became a periplasmic Mr 7500 pro-STA and this form was chased to the culture supernatant; periplasmic and extracellular pro-STA showed the same electrophoretic mobility. A short time after the pulse, pro-STA was converted extracellularly to mature STA (Mr 4500). It is proposed that STA is synthesized as pre-pro-STA, a 72-amino-acid peptide that is subsequently cleaved between amino acids 19 and 20 as it is translocated across the inner membrane. The resulting 53-amino-acid pro-STA is first detected in the periplasm and is then secreted to the culture supernatant. Pro-STA is cleaved extracellularly to yield mature STA (Mr 4500).
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1774. |
( 1997 ) Phylogeny of mercury resistance (mer) operons of gram-negative bacteria isolated from the fecal flora of primates. PMID : 9055422 : PMC : PMC168397 Abstract >>
Nine polymorphic mer loci carried by 185 gram-negative fecal bacterial strains from humans and nonhuman primates are described. The loci were characterized with specific intragenic and intergenic PCR primers to amplify distinct regions covering approximately 80% of the typical gram-negative mer locus. These loci were grouped phylogenetically with respect to each other and with respect to seven previously sequenced mer operons from gram-negative bacteria (the latter designated loci 1, 2, 3, 6, 7, 8, and delta 8 by us here for the purpose of this analysis). Six of the mer loci recovered from primates are similar either to these previously sequenced mer loci or to another locus recently observed in environmental isolates (locus 4), and three are novel (loci 5, 9, and 10). We have observed merC, or a merC-like gene, or merF on the 5' side of merA in all of the loci except that of Tn501 (here designated mer locus 6). The merB gene was observed occasionally, always on the 3' side of merA. Unlike the initial example of a merB-containing mer locus carried by plasmid pDU1358 (locus 8), all the natural primate loci carrying merB also had large deletions of the central region of the operon (and were therefore designated locus delta 8). Four of the loci we describe (loci 2, 5, 9, and 10) have no region of homology to merB from pDU1358 and yet strains carrying them were phenylmercury resistant. Two of these loci (loci 5 and 10) also lacked merD, the putative secondary regulator of operon expression. Phylogenetic comparison of character states derived from PCR product data grouped those loci which have merC into one clade; these are locus 1 (including Tn21), locus 3, and locus 4. The mer loci which lack merC grouped into a second clade: locus 6 (including Tn501) and locus 2. Outlying groups lacked merD or possessed merB. While these mer operons are characterized by considerable polymorphism, our ability to discern coherent clades suggests that recombination is not entirely random and indeed may be focused on the immediate 5' and 3' proximal regions of merA. Our observations confirm and extend the idea that the mer operon is a genetic mosaic and has a predominance of insertions and/or deletions of functional genes immediately before and after the merA gene. chi sites are found in several of the sequenced operons and may be involved in the abundant reassortments we observe for mer genes.
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1775. |
Chen CM,
Ye QZ,
Zhu ZM,
Wanner BL,
Walsh CT,
( 1990 ) Molecular biology of carbon-phosphorus bond cleavage. Cloning and sequencing of the phn (psiD) genes involved in alkylphosphonate uptake and C-P lyase activity in Escherichia coli B. PMID : 2155230 : Abstract >>
Whereas bacteria such as Escherichia coli have been known for some time to cleave carbon-phosphorus (C-P) bonds in unactivated alkylphosphonates, the enzymes responsible for C-P lyase activity have resisted detection or purification. Genes from E. coli B that support growth on alkylphosphonates as the sole phosphorus source have now been cloned (B. L. Wanner and J. A. Boline, unpublished data). Deletion analysis demonstrated that at least 13 kilobases of DNA information is required for E. coli to express the phosphonate utilization phenotype (Phn+). The complete nucleotide sequence of 15,611 bases has been determined, and the gene structures were examined. Seventeen open reading frames (phnA to phnQ) were identified in one transcriptional direction and five open reading frames in the divergent direction. Sequence homology searches identify PhnC, PhnK, PhnL, and, possibly, PhnN proteins as members of nucleotide-binding proteins of the binding protein-dependent transport systems. Candidates for other membrane components and regulatory proteins are also identified. A Pho box-like promoter sequence is also found upstream of the gene cluster starting at phnA, which is consistent with the observation of phosphate regulation of the Phn+ response. Fourteen repetitive extragenic palindromic sequences are found in the phn DNA: 10 exist in the extragenic region between phnA and phnB, two between phnD and phnE, and two between phnK and phnL. An unusual finding is that one of the repetitive extragenic palindromic sequences actually overlaps with the reading frame of the phnE gene.
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1776. |
Jobling MG,
Holmes RK,
( 2012 ) Type II heat-labile enterotoxins from 50 diverse Escherichia coli isolates belong almost exclusively to the LT-IIc family and may be prophage encoded. PMID : 22242186 : DOI : 10.1371/journal.pone.0029898 PMC : PMC3252337 Abstract >>
Some enterotoxigenic Escherichia coli (ETEC) produce a type II heat-labile enterotoxin (LT-II) that activates adenylate cyclase in susceptible cells but is not neutralized by antisera against cholera toxin or type I heat-labile enterotoxin (LT-I). LT-I variants encoded by plasmids in ETEC from humans and pigs have amino acid sequences that are ? 95% identical. In contrast, LT-II toxins are chromosomally encoded and are much more diverse. Early studies characterized LT-IIa and LT-IIb variants, but a novel LT-IIc was reported recently. Here we characterized the LT-II encoding loci from 48 additional ETEC isolates. Two encoded LT-IIa, none encoded LT-IIb, and 46 encoded highly related variants of LT-IIc. Phylogenetic analysis indicated that the predicted LT-IIc toxins encoded by these loci could be assigned to 6 subgroups. The loci corresponding to individual toxins within each subgroup had DNA sequences that were more than 99% identical. The LT-IIc subgroups appear to have arisen by multiple recombinational events between progenitor loci encoding LT-IIc1- and LT-IIc3-like variants. All loci from representative isolates encoding the LT-IIa, LT-IIb, and each subgroup of LT-IIc enterotoxins are preceded by highly-related genes that are between 80 and 93% identical to predicted phage lysozyme genes. DNA sequences immediately following the B genes differ considerably between toxin subgroups, but all are most closely related to genomic sequences found in predicted prophages. Together these data suggest that the LT-II loci are inserted into lambdoid type prophages that may or may not be infectious. These findings raise the possibility that production of LT-II enterotoxins by ETEC may be determined by phage conversion and may be activated by induction of prophage, in a manner similar to control of production of Shiga-like toxins by converting phages in isolates of enterohemmorhagic E. coli.
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1777. |
Nordmann P,
Boulanger AE,
Poirel L,
( 2012 ) NDM-4 metallo-�]-lactamase with increased carbapenemase activity from Escherichia coli. PMID : 22252797 : DOI : 10.1128/AAC.05961-11 PMC : PMC3318389 Abstract >>
A clinical Escherichia coli isolate resistant to all �]-lactams, including carbapenems, expressed a novel metallo-�]-lactamase (MBL), NDM-4, differing from NDM-1 by a single amino acid substitution (Met154Leu). NDM-4 possessed increased hydrolytic activity toward carbapenems and several cephalosporins compared to that of NDM-1. This amino acid substitution was not located in the known active sites of NDM-1, indicating that remote amino acid substitutions might also play a role in the extended activity of this MBL.
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1778. |
Sundström L,
Sköld O,
( 1990 ) The dhfrI trimethoprim resistance gene of Tn7 can be found at specific sites in other genetic surroundings. PMID : 2188588 : DOI : 10.1128/aac.34.4.642 PMC : PMC171658 Abstract >>
The dhfrI gene, mediating high-level trimethoprim resistance, was earlier found only on Tn7. Evidence is given here for an alternative location of this gene at a site identical to sites observed earlier for dhfrII on plasmid R388, dhfrV on pLMO20, and aadA on Tn21. All these genes and dhfrI are precisely inserted as discrete GTTA-flanked elements at distinct loci in very conserved surrounding sequences. One of these dhfrI insertions was observed to occur in association with a similarly inserted aadA nucleotidyltransferase gene, which mediates streptomycin and spectinomycin resistance. Close to the insertion site, there is an open reading frame translating into a 337-amino-acid peptide which shows striking similarities to recombinases of the integrase family, sulI, the sulfonamide resistance gene, is very often found close to the insertion point forming a genetic surrounding, originally observed as a part of Tn21-like transposons. The alleged integration mechanism thus provides a recombination pathway for the genetic linkage of sulfonamide and other antibiotic resistance genes, including the most frequently encountered gene for trimethoprim resistance, dhfrI. Furthermore, the newly observed location of dhfrI could shed light on the evolution of the antibiotic resistance region of Tn7, which could be able to take up genes by the same mechanism as that of Tn21-like transposons.
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1779. |
Gebru E,
Choi MJ,
Lee SJ,
Damte D,
Park SC,
( 2011 ) Mutant-prevention concentration and mechanism of resistance in clinical isolates and enrofloxacin/marbofloxacin-selected mutants of Escherichia coli of canine origin. PMID : 21596912 : DOI : 10.1099/jmm.0.028654-0 Abstract >>
The antibacterial activity and selection of resistant bacteria, along with mechanisms of fluoroquinolone resistance, were investigated by integrating the static [MIC or mutant-prevention concentration (MPC)] and in vitro dynamic model approaches using Escherichia coli isolates from diseased dogs. Using the dynamic models, selected E. coli strains and enrofloxacin and marbofloxacin at a range of simulated area under concentration-time curve over a 24 h interval (AUC(24 h))/MIC ratios were investigated. Our results indicated increasing losses in susceptibility of E. coli upon continuous exposure to enrofloxacin and marbofloxacin in vitro. This effect was transferable to other fluoroquinolones, as well as to structurally unrelated drugs. Our results also confirmed an AUC(24 h)/MIC (AUC(24 h)/MPC)-dependent antibacterial activity and selection of resistant E. coli mutants, in which maximum losses in fluoroquinolone susceptibility occurred at simulated AUC(24 h)/MIC ratios of 40-60. AUC(24 h)/MPC ratios of 39 (enrofloxacin) and 32 (marbofloxacin) were considered protective against the selection of resistant mutants of E. coli. Integrating our MIC and MPC data with published pharmacokinetic information in dogs revealed a better effect of the conventional dosing regimen of marbofloxacin than that of enrofloxacin in restricting the selection of resistant mutants of E. coli. Target mutations, especially at codon 83 (serine to leucine) of gyrA, and overexpression of efflux pumps contributed to resistance development in both clinically resistant and in vitro-selected mutants of E. coli. We also report here a previously undescribed mutation at codon 116 of parC in two laboratory-derived resistant mutants of E. coli. Additional studies would determine the exact role of this mutation in fluoroquinolone susceptibility, as well as establish the importance of our findings in the clinical setting.
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1780. |
Haugum K,
Lindstedt BA,
Løbersli I,
Kapperud G,
Brandal LT,
( 2012 ) Identification of the anti-terminator qO111:H)- gene in Norwegian sorbitol-fermenting Escherichia coli O157:NM. PMID : 22268961 : DOI : 10.1111/j.1574-6968.2012.02505.x Abstract >>
Sorbitol-fermenting Escherichia coli O157:NM (SF O157) is an emerging pathogen suggested to be more virulent than nonsorbitol-fermenting Escherichia coli O157:H7 (NSF O157). Important virulence factors are the Shiga toxins (stx), encoded by stx1 and/or stx2 located within prophages integrated in the bacterial genome. The stx genes are expressed from p(R) (') as a late protein, and anti-terminator activity from the Q protein is necessary for read through of the late terminator t(R) (') and activation of p(R) (') . We investigated the regulation of stx2(EDL933) expression at the genomic level in 17 Norwegian SF O157. Sequencing of three selected SF O157 strains revealed that the anti-terminator q gene and genes upstream of stx2(EDL933) were identical or similar to the ones observed in the E. coli O111:H- strain AP010960, but different from the ones observed in the NSF O157 strain EDL933 (AE005174). This suggested divergent stx2(EDL933) -encoding bacteriophages between NSF O157 and the SF O157 strains (FR874039-41). Furthermore, different DNA structures were detected in the SF O157 strains, suggesting diversity among bacteriophages also within the SF O157 group. Further investigations are needed to elucidate whether the q(O111:H) (-) gene observed in all our SF O157 contributes to the increased virulence seen in SF O157 compared to NSF O157. An assay for detecting q(O111:H) (-) was developed.
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1781. |
Diwan V,
Chandran SP,
Tamhankar AJ,
Stålsby Lundborg C,
Macaden R,
( 2012 ) Identification of extended-spectrum �]-lactamase and quinolone resistance genes in Escherichia coli isolated from hospital wastewater from central India. PMID : 22267239 : DOI : 10.1093/jac/dkr564 Abstract >>
To investigate the presence of antibiotic resistance genes (ARGs), related to commonly used �]-lactams and quinolones, in Escherichia coli present in hospital wastewater in central India. Cefotaxime- and ciprofloxacin-resistant E. coli isolates from hospital-associated wastewater samples were collected from two tertiary care hospitals in the Ujjain district of India during 2008-09. The presence of bla(CTX-M), bla(TEM,) bla(SHV), qnrA, qnrB, qnrS, aac(6')-Ib-cr and qepA genes was detected by PCR and sequencing. Twenty-five E. coli isolates were extended-spectrum �]-lactamase (ESBL) positive. bla(CTX-M-15) and bla(TEM-1) genes were identified in 21 and 16 ESBL-positive isolates by PCR, respectively. Amongst 30 fluoroquinolone-resistant E. coli isolates, aac(6')-Ib-cr was detected in 27 isolates, qnrA in 1 isolate, qnrB in 2 isolates and qepA in 3 isolates. bla(CTX-M-15,) bla(TEM-1), aac(6')-Ib-cr, qnrA, qnrB and qepA genes are present in E. coli occurring in hospital wastewater in central India.
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1782. |
( 1997 ) Evolutionary relationship among rfb gene clusters synthesizing mannose homopolymer as O-specific polysaccharides in Escherichia coli and Klebsiella. PMID : 9370271 : DOI : 10.1016/s0378-1119(97)00300-4 Abstract >>
In order to clarify the evolutionary relationship among rfb gene clusters synthesizing mannose homopolymer as O-specific polysaccharides in Escherichia coli and Klebsiella, we studied the DNA sequence of the boundary region between the rfb and his genes in a series of strains possessing mannose homopolymer as O-specific polysaccharide. All had a characteristic gene organization carrying no gene between the rfb and his genes. Further, the recombination event was suggested to occur at the same site of the hisI gene in those strains. It was suggested that there was a close evolutionary relationship among rfb gene clusters synthesizing mannose homopolymer as O-specific polysaccharide in E. coli and Klebsiella.
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1783. |
Eberle W,
Klaus W,
Cesareni G,
Sander C,
Rösch P,
( 1990 ) Proton nuclear magnetic resonance assignments and secondary structure determination of the ColE1 rop (rom) protein. PMID : 2223771 : DOI : 10.1021/bi00484a007 Abstract >>
The complete resonance assignment of the ColE1 rop (rom) protein at pH 2.3 was obtained by two-dimensional (2D) proton nuclear magnetic resonance spectroscopy (1H NMR) at 500 and 600 MHz using through-bond and through-space connectivities. Sequential assignments and elements of regular secondary structure were deduced by analysis of nuclear Overhauser enhancement spectroscopy (NOESY) experiments and 3JHN alpha coupling constants. One 7.2-kDa monomer of the homodimer consists of two antiparallel helices connected by a hairpin loop at residue 31. The C-terminal peptide consisting of amino acids 59-63 shows no stable conformation. The dimer forms a four-helix bundle with opposite polarization of neighboring elements in agreement with the X-ray structure.
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1784. |
Hou J,
Huang X,
Deng Y,
He L,
Yang T,
Zeng Z,
Chen Z,
Liu JH,
( 2012 ) Dissemination of the fosfomycin resistance gene fosA3 with CTX-M �]-lactamase genes and rmtB carried on IncFII plasmids among Escherichia coli isolates from pets in China. PMID : 22232290 : DOI : 10.1128/AAC.05104-11 PMC : PMC3318358 Abstract >>
The presence and characterization of plasmid-mediated fosfomycin resistance determinants among Escherichia coli isolates collected from pets in China between 2006 and 2010 were investigated. Twenty-nine isolates (9.0%) were positive for fosA3, and all of them were CTX-M producers. The fosA3 genes were flanked by IS26 and were localized on F2:A-:B- plasmids or on very similar F33:A-:B- plasmids carrying both bla(CTX-M-65) and rmtB. These findings indicate that the fosA3 gene may be coselected by antimicrobials other than fosfomycin.
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1785. |
( 1997 ) Pathogenicity island sequences of pyelonephritogenic Escherichia coli CFT073 are associated with virulent uropathogenic strains. PMID : 9199454 : PMC : PMC175396 Abstract >>
Urinary tract infection is the most frequently diagnosed kidney and urologic disease, and Escherichia coli is by far the most common etiologic agent. Defined blocks of DNA termed pathogenicity islands have been found in uropathogenic strains to carry genes not generally found in fecal strains. We have identified one of these regions of DNA within the chromosome of the highly virulent E. coli CFT073, isolated from the blood and urine of a woman with acute pyelonephritis. This strain, which is cytotoxic for cultured renal cells and causes acute pyelonephritis in transurethrally infected CBA mice, contains two distinct copies of the pap operon and is hemolytic. One pap operon was localized on a cosmid clone which was used to identify three overlapping cosmid clones. By using restriction mapping, DNA hybridization, sequencing, and PCR amplification, a region of approximately 50 kb was found to be present in this uropathogenic strain and to have no corresponding sequences in E. coli K-12. This gene block also carries hemolysin genes hlyCABD. The pathogenicity island begins 7 bp downstream of dadX (catabolic alanine racemase; 26.55 min) and ends at a position in the K-12 genome 75 bp downstream of the metV tRNA gene (62.74 min); this suggests that a chromosomal rearrangement has occurred relative to the K-12 linkage map. The junctions of the pathogenicity island were verified by PCR amplification directly from the genomic DNA of strain CFT073. DNA sequencing within the boundaries of the junctions revealed genes not previously identified in E. coli or in some cases bearing no known homologs. When used as probes for DNA hybridization, these sequences were found significantly more often in strains associated with the clinical syndromes of cystitis (82%) and acute pyelonephritis (79%) than in fecal strains (19%; P < 0.001).
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1786. |
( 1997 ) Distribution of drb genes coding for Dr binding adhesins among uropathogenic and fecal Escherichia coli isolates and identification of new subtypes. PMID : 9169726 : PMC : PMC175278 Abstract >>
The Dr family of related adherence structures, some fimbriated and others afimbriated, bind to decay-accelerating factor molecules on human cells. Dr is associated with recurring urinary tract infection (UTI), but the distribution of Dr subtypes among uropathogenic Escherichia coli causing UTI among otherwise healthy women has yet to be described. A total of 787 UTI and fecal E. coli isolates from college women were screened for the presence of Dr sequences (drb). Fifteen percent of UTI strains were drb positive, compared to 5% of fecal strains. The adhesin (E gene) subtype of each drb-positive strain was determined by type-specific PCR followed by restriction enzyme analysis. Among 78 drb-positive strains, we found 14 (18%) afaE1, 1 (1.3%) afaE2, 1 (1.3%) afaE3, 9 (12%) draE, 9 (12%) draE-afaE3 hybrid, 1 (1.3%) daaE, 32 (41%) afaE5, 4 (5.1%) F131 E gene-like, and 7 untypeable strains. All untypeable E genes were cloned and sequenced, revealing four additional new classes of E genes, including two similar to the previously identified nonfimbrial E series. While a great range of diversity exists among the E genes, restriction fragment length polymorphism analysis demonstrated that all of these drb operons share a highly conserved gene structure. The most common subtype, afaE5, occurred three times as often among UTI than fecal strains. Over half of the drb-positive strains and 80% of those positive for afaE5 have the same virulence signature (positive for aer, kpsMT, ompT, and fim), suggesting an association of this profile with UTI pathogenesis.
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1787. |
( 1996 ) Mapping of the H7-serospecific domain of Escherichia coli flagellin. PMID : 8877129 : PMC : PMC170400 Abstract >>
The amino acid sequences responsible for H7 and H23 flagellum serology have been identified by using a genetic approach. The H7-specific domain was located between amino acids 352 and 374 of the H7 flagellin. The sequencing data also demonstrated that the difference between the H7 and H23 flagellins in this region results from a single substitution at amino acid 366 (Ser-->Thr). The common epitopes for H7 and H23 were located between amino acids 284 and 366.
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1788. |
( 1996 ) The clp (CS31A) operon is negatively controlled by Lrp, ClpB, and L-alanine at the transcriptional level. PMID : 8858583 : DOI : 10.1046/j.1365-2958.1996.00651.x Abstract >>
Biosynthesis of the Escherichia coli CS31A surface antigen was subject to phase-variation control and repressed by L-alanine. Nucleotide sequence analysis of the clp operon, encoding the biosynthesis of CS31A, revealed the presence of a regulatory gene, clpB. The amino acid sequence of the regulatory protein ClpB showed similarity to the primary structure of PapB, FaeB and AfaA, involved in the regulation of expression of Pap, K88, and Afa-3 fimbriae, respectively. The clp regulatory region contained two deoxyadenosine methylase sites (GATC-I and GATC-II). The leucine-responsive regulatory protein (Lrp) was required for specific methylation inhibition of the GATC-II site. The activity of the clp promoter was monitored in a clp-lacZYA single-copy fusion. The cloned DNA used in this study did not contain a related papl gene. In these conditions, we showed, as expected, that phase variation did not occur. However, transcription of the clp operon was negatively controlled by ClpB and Lrp, and was maximal in the absence of Dam methylase. In the presence of AfaF, a Papl equivalent, the phase-variation control was restored. We concluded that two regulatory mechanisms were superimposed to control the clp expression. Phase variation, mediated by Lrp and a Papl equivalent, controlled the number of cells producing CS31A in a single colony. The second mechanism, described in this report, was mediated by ClpB and Lrp and controls the level of CS31A produced by a single cell. Furthermore, we showed that L-alanine reduced, by about twofold, the clp promoter activity independently of a Papl equivalent, ClpB, Lrp or Dam methylase. In addition, the presence of L-alanine prevented the phase-variation control mediated by AfaF.
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1789. |
( 1997 ) Identification of a new porin, RafY, encoded by raffinose plasmid pRSD2 of Escherichia coli. PMID : 9294435 : DOI : 10.1128/jb.179.18.5783-5788.1997 PMC : PMC179467 Abstract >>
The conjugative plasmid pRSD2 carries a raf operon that encodes a peripheral raffinose metabolic pathway in enterobacteria. In addition to the previously known raf genes, we identified another gene, rafY, which in Escherichia coli codes for an outer membrane protein (molecular mass, 53 kDa) similar in function to the known glycoporins LamB (maltoporin) and ScrY (sucrose porin). Sequence comparisons with LamB and ScrY revealed no significant similarities; however, both lamB and scrY mutants are functionally complemented by RafY. Expressed from the tac promoter, RafY significantly increases the uptake rates for maltose, sucrose, and raffinose at low substrate concentrations; in particular it shifts the apparent K(m) for raffinose transport from 2 mM to 130 microM. Moreover, RafY permits diffusion of the tetrasaccharide stachyose and of maltodextrins up to maltoheptaose through the outer membrane of E. coli. A comparison of all three glycoporins in regard to their substrate selectivity revealed that both ScrY and RafY have a broad substrate range which includes alpha-galactosides while LamB seems to be restricted to malto-oligosaccharides. It supports growth only on maltodextrins but not, like the others, on raffinose and stachyose.
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1790. |
( 1997 ) A third secreted protein that is encoded by the enteropathogenic Escherichia coli pathogenicity island is required for transduction of signals and for attaching and effacing activities in host cells. PMID : 9169753 : PMC : PMC175305 Abstract >>
Enteropathogenic Escherichia coli strains are able to signal host cells, cause dramatic cytoskeletal rearrangements, and adhere intimately to the cell surface in a process known as the attaching and effacing effect. A pathogenicity island of 35 kb known as the locus of enterocyte effacement (LEE) is necessary and sufficient for this effect. The LEE encodes an outer membrane adhesin called intimin, a type III secretion apparatus, and the EspA and EspB secreted proteins. The DNA sequence of the region between espA and espB revealed a new gene, espD. The product of espD was demonstrated by using a T7 expression system. We constructed a nonpolar mutation in espD and found that the mutant is incapable of the signal transduction events that lead to activation of the putative intimin receptor in host cells and that the mutant fails to induce the attaching and effacing effect. These phenotypes were restored to the mutant by complementation with a plasmid containing the cloned espD locus. We demonstrated by immunoblotting and microsequencing that the EspD protein is secreted via the type III apparatus. Thus, we describe a novel locus encoding a secreted protein that is required for attaching and effacing activity.
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1791. |
( 1997 ) Escherichia coli proteome analysis using the gene-protein database. PMID : 9298644 : DOI : 10.1002/elps.1150180805 Abstract >>
The gene-protein database of Escherichia coli is a collection of data, largely generated from the separation of complex mixtures of cellular proteins on two-dimensional (2-D) polyacrylamide gel electrophoresis. The database currently contains about 1600 protein spots. The data are comprised of both identification information for many of these proteins and data on how the level or synthesis rates of proteins vary under different growth conditions. Three projects are underway to further elucidate the E. coli proteome including a project to localize on 2-D gels all of the open reading framed encoded by the E. coli chromosome, a project to determine the condition(s) under which each open reading frame is expressed and a project to determine the abundance and location of each protein in the cell. Applications for proteome databases for cell modeling are discussed and examples of applications in therapeutic drug discovery are given.
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1792. |
( 1997 ) Molecular cloning and characterization of Dr-II, a nonfimbrial adhesin-I-like adhesin isolated from gestational pyelonephritis-associated Escherichia coli that binds to decay-accelerating factor. PMID : 9317041 : PMC : PMC175617 Abstract >>
Bacterial adhesins play an important role in the colonization of the human urogenital tract. Escherichia coli Dr family adhesins have been found to be frequently expressed in strains associated with pyelonephritis in pregnant females. The tissue receptor for known Dr adhesins has been localized to the short consensus repeat-3 (SCR-3) domain of decay accelerating factor (DAF), a complement regulatory protein. In this report, we identified and cloned draE2, a gene encoding a novel 17-kDa DAF-binding adhesin, Dr-II, from a strain of E. coli associated with acute gestational pyelonephritis. Despite the significant sequence diversity between Dr-II and Dr family adhesins, the receptor of Dr-II was found to be the SCR-3 domain of DAF. Sequence analysis of the 186-amino-acid Dr-II open reading frame revealed significant diversity from other members of the Dr adhesin family, including Dr, AFA-I, AFA-III, and F1845, but only an 8-amino-acid difference in sequence from that of the 17-kDa nonfimbrial adhesin NFA-I of unknown receptor specificity. N-terminal peptide sequencing of the purified adhesin confirmed the identity of the open reading frame and indicated cleavage of a 28-amino-acid signal peptide. Antibodies raised against purified Dr-II adhesin exhibited little or no cross-reactivity to Dr adhesin. Characterization of the biological properties demonstrated that like the Dr adhesins, Dr-II was associated with the ability of E. coli to bind to tubular basement membranes and Bowman's capsule and to be internalized into HeLa cells.
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1793. |
( 1996 ) Overproduction of three genes leads to camphor resistance and chromosome condensation in Escherichia coli. PMID : 8844142 : PMC : PMC1207417 Abstract >>
We isolated and characterized three genes, crcA, cspE and crcB, which when present in high copy confer camphor resistance on a cell and suppress mutations in the chromosomal partition gene mukB. Both phenotypes require the same genes. Unlike chromosomal camphor resistant mutants, high copy number crcA, cspE and crcB do not result in an increase in the ploidy of the cells. The cspE gene has been previously identified as a cold shock-like protein with homologues in all organisms tested. We also demonstrate that camphor causes the nucleoids to decondense in vivo and when the three genes are present in high copy, the chromosomes do not decondense. Our results implicate camphor and mukB mutations as interfering with chromosome condensation and high copy crcA, cspE and crcB as promoting or protecting chromosome folding.
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1794. |
( 1996 ) A Brevibacterium linens pRBL1 replicon functional in Corynebacterium glutamicum. PMID : 8938050 : DOI : 10.1006/plas.1996.0029 Abstract >>
Brevibacterium linens RBL strain cryptic plasmid pRBL1 (8.0 kb) is described. A region involved in pRBL1 autonomous replication in Corynebacterium glutamicum was identified by insertion and deletion mapping and partially sequenced. This region encodes for a hypothetical 310-amino acid (aa) protein closely related to the replicases of plasmids pXZ10142 (C. glutamicum) and pAL5000 (Mycobacterium fortuitum). The 310-aa protein also shows significant homology to proteins of pColE5-099 (Shigella sonnei) and pJD1 (Neisseria gonorrhoea). At least one of these proteins, the Rep protein of pColE5-099, is known to be involved in theta replication.
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1795. |
( 1997 ) Aggregative adherence fimbria II, a second fimbrial antigen mediating aggregative adherence in enteroaggregative Escherichia coli. PMID : 9317019 : PMC : PMC175595 Abstract >>
Enteroaggregative Escherichia coli (EAEC) has been implicated as an agent of pediatric diarrhea in the developing world. We have shown previously that EAEC adheres to HEp-2 cells by virtue of a plasmid-encoded fimbrial adhesin designated aggregative adherence fimbria I (AAF/I), the genes for which have been cloned and sequenced. However, not all EAEC strains express AAF/I. Using TnphoA mutagenesis, we have characterized a novel fimbria (designated AAF/II) which mediates HEp-2 adherence of the human-pathogenic strain 042. AAF/II is 5 nm in diameter and does not bind AAF/I antiserum, as determined by immunogold transmission electron microscopy. TnphoA identified a gene (designated aafA) which bears significant homology to aggA, the fimbrial subunit of AAF/I (25% identity and 47% similarity at the amino acid level). When hyperexpressed and purified by polyhistidine tagging, the AafA protein assembled into 5-nm-diameter filaments which bound anti-AAF/II antiserum. The cloned aafA gene complemented a mutation in the aggA gene to confer fimbrial expression from the AAF/I gene cluster, manifesting phenotypes characteristic of AAF/II but not AAF/I. The aafA mutant did not adhere to human intestinal tissue in culture, suggesting a role for AAF/II in intestinal colonization. By using DNA probes for AAF/I and AAF/II derived from fimbrial biosynthesis genes, we show that AAF/I and AAF/II are each found in only a minority of EAEC strains, suggesting that still more EAEC adhesins exist. Our data suggest that AAF adhesins represent a new family of fimbrial adhesins which mediate aggregative adherence in EAEC.
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1796. |
( 1996 ) The Tn7 transposase is a heteromeric complex in which DNA breakage and joining activities are distributed between different gene products. PMID : 8947057 : PMC : PMC452457 Abstract >>
The bacterial transposon Tn7 translocates by a cut and paste mechanism: excision from the donor site results from double-strand breaks at each end of Tn7 and target insertion results from joining of the exposed 3' Tn7 tips to the target DNA. Through site-directed mutagenesis of the Tn7-encoded transposition proteins TnsA and TnsB, we demonstrate that the Tn7 transposase is a heteromeric complex of these proteins, each protein executing different DNA processing reactions. TnsA mediates DNA cleavage reactions at the 5' ends of Tn7, and TnsB mediates DNA breakage and joining reactions at the 3' ends of Tn7. Thus the double-strand breaks that underlie Tn7 excision result from a collaboration between two active sites, one in TnsA and one in TnsB; the same (or a closely related) active site in TnsB also mediates the subsequent joining of the 3' ends to the target. Both TnsA and TnsB appear to be members of the retroviral integrase superfamily: mutation of their putative DD(35)E motifs blocks catalytic activity. Recombinases of this class require a divalent metal cofactor that is thought to interact with these acidic residues. Through analysis of the metal ion specificity of a TnsA mutant containing a sulfur (cysteine) substitution, we provide evidence that a divalent metal actually interacts with these acidic amino acids.
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1797. |
( 1997 ) Haem iron-transport system in enterohaemorrhagic Escherichia coli O157:H7. PMID : 9157252 : DOI : 10.1046/j.1365-2958.1997.2641628.x Abstract >>
In this study, we identified the iron-transport systems of Escherichia coli O157:H7 strain EDL933. This strain synthesized and transported enterobactin and had a ferric citrate transport system but lacked the ability to produce or use aerobactin. It used haem and haemoglobin, but not transferrin or lactoferrin, as iron sources. We cloned the gene encoding an iron-regulated haem-transport protein and showed that this E. coli haem-utilization gene (chuA) encoded a 69 kDa outer membrane protein that was synthesized in response to iron limitation. Expression of this protein in a laboratory strain of E. coli was sufficient for utilization of haem or haemoglobin as iron sources. Mutation of the chromosomal chuA and tonB genes in E. coli O157:H7 demonstrated that the utilization of haemin and haemoglobin was ChuA- and TonB-dependent. Nucleotide sequence analysis of chuA revealed features characteristic of TonB-dependent, Fur-regulated, outer membrane iron-transport proteins. It was highly homologous to the shuA gene of Shigella dysenteriae and less closely related to hemR of Yersinia enterocolitica and hmuR of Yersinia pestis. A conserved Fur box was identified upstream of the chuA gene, and regulation by Fur was confirmed.
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1798. |
( 1997 ) The type IC hsd loci of the enterobacteria are flanked by DNA with high homology to the phage P1 genome: implications for the evolution and spread of DNA restriction systems. PMID : 9157244 : DOI : 10.1046/j.1365-2958.1997.2531622.x Abstract >>
EcoR124l, EcoDXXl and Ecoprrl are the known members of the type IC family of DNA restriction and modification systems. The first three are carried on large, conjugative plasmids, while Ecoprrl is chromosomally encoded. The enzymes are coded by three genes, hsdR, hsdM and hsdS. Analysis of the DNA sequences upstream and downstream of the type IC hsd loci shows that all are highly homologous to each other and also to sequences present in the bacteriophage P1 genome. The upstream sequences include functional phd and doc genes, which encode an addiction system that stabilizes the P1 prophage state, and extend to and beyond pac, the site at which phage DNA packaging begins. Downstream of the hsd loci, P1 DNA sequences begin at exactly the same place for all of the systems. For EcoDXXl and Ecoprrl the P1 homology extends for thousands of base pairs while for EcoR124l and IS1 insertion and an associated deletion have removed most of the P1-homologous sequences. The significance of these results for the evolution of DNA restriction and modification systems is discussed.
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1799. |
( 1997 ) Intercontinental spread of promiscuous mercury-resistance transposons in environmental bacteria. PMID : 9159519 : DOI : 10.1046/j.1365-2958.1997.3261688.x Abstract >>
We demonstrate that horizontal spread of mer operons similar to worldwide spread of antibiotic-resistance genes in medically important bacteria occurred in bacteria found in ores, soils and waters. The spread was mediated by different transposons and plasmids. Some of the spreading transposons were damaged in different ways but this did not prevent their further spread. Certain transposons are mosaics composed of segments belonging to distinct sequence types. These mosaics arose as a result of homologous and site-specific recombination. Our data suggest that the mercury-resistance operons of Gram-negative environmental bacteria can be considered as a worldwide population composed of a relatively small number of distinct recombining clones shared, at least partially, by environmental and clinical bacteria.
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1800. |
( 1997 ) EspP, a novel extracellular serine protease of enterohaemorrhagic Escherichia coli O157:H7 cleaves human coagulation factor V. PMID : 9194704 : DOI : 10.1046/j.1365-2958.1997.3871751.x Abstract >>
In this study, we identified and characterized a novel secreted protein, the extracellular serine protease EspP, which is encoded by the large plasmid of enterohaemorrhagic Escherichia coli (EHEC) O157:H7. The corresponding espP gene consists of a 3900 bp open reading frame that is able to encode a 1300-amino-acid protein. EspP is synthesized as a large precursor which is then processed at the N- and C-termini during secretion. It can be grouped into the autotransporter protein family. The deduced amino acid sequence of EspP showed homology to several secreted or surface-exposed proteins of pathogenic bacteria, in particular EspC of enteropathogenic E. coli and IgA1 proteases from Neisseria spp. and Haemophilus influenzae. Hybridization experiments and immunoblot analysis of clinical EHEC isolates showed that EspP is widespread among EHEC of the serogroup O157 and that it also exists in serogroup 026. A specific immune response against EspP was detected in sera from patients suffering from EHEC infections. Functional analysis showed that EspP is a protease capable of cleaving pepsin A and human coagulation factor V. Degradation of factor V could contribute to the mucosal haemorrhage observed in patients with haemorrhagic colitis.
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1801. |
( 1997 ) A novel retron that produces RNA-less msDNA in Escherichia coli using reverse transcriptase. PMID : 9281493 : DOI : 10.1006/plas.1997.1298 Abstract >>
Bacterial retroelements, or retrons, use reverse transcriptase (RT) to produce a multicopy single-stranded DNA (msDNA) molecule that is covalently linked to RNA. In these studies we show that a retron from Escherichia coli 110, a clinical isolate, produces a novel RNA-less msDNA with a 5' phosphate residue. The msDNA is a 74-nucleotide single-stranded DNA molecule with a stable stem-loop structure without a mismatched base pair. Only the genes encoding msDNA (msd), msdRNA (msr), and RT (ret) are required to produce the msDNA molecule. The organization of these genes on the retron was similar to that of other elements producing branched msDNA-RNA. The conserved guanine, which is the branched residue in msDNA-RNA complexes and is essential for branch formation, is also present. Site-directed mutagenesis showed that this guanine is essential for the production of RNA-less msDNA. We postulate that the RNA-less msDNA in strain 110 is produced by nucleolytic cleavage of the branched msDNA-RNA compound.
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1802. |
( 1993 ) Identification of the regulatory sequence of anaerobically expressed locus aeg-46.5. PMID : 8432709 : DOI : 10.1128/jb.175.4.1165-1172.1993 PMC : PMC193033 Abstract >>
A newly identified anaerobically expressed locus, aeg-46.5, which is located at min 46.5 on Escherichia coli linkage map, was cloned and analyzed. The phenotype of this gene was studied by using a lacZ operon fusion. aeg-46.5 is induced anaerobically in the presence of nitrate in wild-type and narL cells. It is repressed by the narL gene product, as it showed derepressed anaerobic expression in narL mutant cells. We postulate that aeg-46.5 is subject to multiple regulatory systems, activation as a result of anaerobiosis, narL-independent nitrate-dependent activation, and narL-mediated repression. The regulatory region of aeg-46.5 was identified. A 304-bp DNA sequence which includes the regulatory elements was obtained, and the 5' end of aeg-46.5 mRNA was identified. It was verified that the anaerobic regulation of aeg-46.5 expression is controlled on the transcriptional level. Computer analysis predicted possible control sites for the NarL and FNR proteins. The proposed NarL site was found in a perfect-symmetry element. The aeg-46.5 regulatory elements are adjacent to, but divergent from, those of the eco gene.
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1803. |
( 1996 ) TEM-28 from an Escherichia coli clinical isolate is a member of the His-164 family of TEM-1 extended-spectrum beta-lactamases. PMID : 8787920 : PMC : PMC163097 Abstract >>
TEM-28 (pI 6.1), expressed by an Escherichia coli clinical isolate, is a novel beta-lactamase which hydrolyzed ceftazidime, cefotaxime, and aztreonam with rates of 25, 1.1, and 5.6, respectively, relative to that for benzylpenicillin (100). The nucleotide sequence of blaTEM-28 differed from that of blaTEM-1 by two base changes, resulting in amino acid substitutions of Arg-164 to His and Glu-240 to Lys.
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1804. |
( 1997 ) Small cryptic plasmids of multiplasmid, clinical Escherichia coli. PMID : 9073577 : DOI : 10.1006/plas.1996.1273 Abstract >>
Clinical isolates of Escherichia coli were found to host a multiplicity of plasmids. These were resolved from plasmid gel profiles, from the properties of various transconjugants and transformants of E. coli DH1, by the topoisomerase I relaxation of covalently closed circle plasmid DNA, by electron microscopy, and by the determination of their compatibilities. The majority of these were unusually small, cryptic plasmids (SCPs). From one strain, KL4, 13 electrophoretic bands were resolved to five plasmids, three of which were SCPs. SCPs were phenotypically barren, and the smallest of these, pKL1, contained barely enough information for self-replication. A derivative of pKL1, pKL1Km, in which the transposon was restricted to a small 350-bp region, was stably maintained in Shigella, Salmonella, Serratia, and Citrobacter species and its replication was polA independent. pKL1 encoded only a single protein, RepA (Mr 17960), which specifically bound to pKL1 DNA. No apparent homologies with other RepA protein sequences could be detected. Thus the SCP, pKL1, is a novel minimal plasmid replicon encoding only enough information to ensure perpetuation. A hypothesis is presented describing SCPs as a class of selfish DNA that persists simply due to its ability to replicate and to its stability based on high copy number.
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1805. |
( 1997 ) Copper-inducible transcriptional regulation at two promoters in the Escherichia coli copper resistance determinant pco. PMID : 9141682 : DOI : 10.1099/00221287-143-4-1191 Abstract >>
The pco determinant of Escherichia coli plasmid pRI1004 encodes inducible resistance to the trace element copper. The identification of two copper-dependent transcriptional initiation regions within pco that each contain a similar upstream hyphenated dyad motif is described. Deletion constructs showed that this 'copper box' motif was essential for copper-inducible activity at both pco promoters, PpcoA and PpcoE. The placement of the motif differs in the two promoters, and PpcoA contains an extended -10 nonamer typical of promoters for which RNA polymerase does not bind specifically to -35 sequences. PpcoE does not contain this motif and is the more strongly expressed promoter. The transcript from PpcoA contains the pcoABCDRS genes, while PpcoE expresses only pcoE. The induction profiles for PpcoA- and PpcoE-IacZ fusions were flattened sigmoidal curves with a gradual response to increasing copper concentration. On high-copy-number plasmids, zinc was found also to induce transcription from both promoters in vivo. Both promoters showed inducible activity in the absence of pcoRS, the plasmid-borne two-component regulatory system, indicating that a second trans-acting regulatory system is present on the chromosome. The pcoR product showed repressor action in the absence of pcoS, while still allowing induction, suggesting the chromosome encoded a similar two-component system to pco. TnphoA insertion mutagenesis identified chromosomal genes which affected promoter expression, including ptsH, ptsI (sugar phosphotransferase system) and cya (adenylate cyclase). The results support that idea that pco-encoded copper resistance is an auxiliary mechanism for handling copper, the regulation of which is integrated with the chromosomal regulation of cellular copper metabolism.
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1806. |
( 1997 ) The complete genome sequence of Escherichia coli K-12. PMID : 9278503 : DOI : 10.1126/science.277.5331.1453 Abstract >>
The 4,639,221-base pair sequence of Escherichia coli K-12 is presented. Of 4288 protein-coding genes annotated, 38 percent have no attributed function. Comparison with five other sequenced microbes reveals ubiquitous as well as narrowly distributed gene families; many families of similar genes within E. coli are also evident. The largest family of paralogous proteins contains 80 ABC transporters. The genome as a whole is strikingly organized with respect to the local direction of replication; guanines, oligonucleotides possibly related to replication and recombination, and most genes are so oriented. The genome also contains insertion sequence (IS) elements, phage remnants, and many other patches of unusual composition indicating genome plasticity through horizontal transfer.
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1807. |
( 1997 ) Polymorphism, duplication, and IS1-mediated rearrangement in the chromosomal his-rfb-gnd region of Escherichia coli strains with group IA and capsular K antigens. PMID : 9150218 : DOI : 10.1128/jb.179.10.3232-3238.1997 PMC : PMC179101 Abstract >>
Individual Escherichia coli strains produce several cell surface polysaccharides. In E. coli E69, the his region of the chromosome contains the rfb (serotype O9 lipopolysaccharide O-antigen biosynthesis) and cps (serotype K30 group IA capsular polysaccharide biosynthesis) loci. Polymorphisms in this region of the Escherichia coli chromosome reflect extensive antigenic diversity in the species. Previously, we reported a duplication of the manC-manB genes, encoding enzymes involved in GDP-mannose formation, upstream of rfb in strain E69 (P. Jayaratne et al., J. Bacteriol. 176:3126-3139, 1994). Here we show that one of the manC-manB copies is flanked by IS1 elements, providing a potential mechanism for the gene duplication. Adjacent to manB1 on the IS1-flanked segment is a further open reading frame (ugd), encoding uridine-5'-diphosphoglucose dehydrogenase. The Ugd enzyme is responsible for the production of UDP-glucuronic acid, a precursor required for K30 antigen synthesis. Construction of a chromosomal ugd::Gm(r) insertion mutation demonstrated the essential role for Ugd in the biosynthesis of the K30 antigen and confirmed that there is no additional functional ugd copy in strain E69. PCR amplification and Southern hybridization were used to examine the distribution of IS1 elements and ugd genes in the vicinity of rfb in other E. coli strains, producing different group IA K antigens. The relative order of genes and, where present, IS1 elements was established in these strains. The regions adjacent to rfb in these strains are highly variable in both size and gene order, but in all cases where a ugd homolog was present, it was found near rfb. The presence of IS1 elements in the rfb regions of several of these strains provides a potential mechanism for recombination and deletion events which could contribute to the antigenic diversity seen in surface polysaccharides.
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1808. |
( 1997 ) Cysteine sulfinate desulfinase, a NIFS-like protein of Escherichia coli with selenocysteine lyase and cysteine desulfurase activities. Gene cloning, purification, and characterization of a novel pyridoxal enzyme. PMID : 9278392 : DOI : 10.1074/jbc.272.36.22417 Abstract >>
Selenocysteine lyase (EC 4.4.1.16) exclusively decomposes selenocysteine to alanine and elemental selenium, whereas cysteine desulfurase (NIFS protein) of Azotobacter vinelandii acts indiscriminately on both cysteine and selenocysteine to produce elemental sulfur and selenium respectively, and alanine. These proteins exhibit some sequence homology. The Escherichia coli genome contains three genes with sequence homology to nifS. We have cloned the gene mapped at 63.4 min in the chromosome and have expressed, purified to homogeneity, and characterized the gene product. The enzyme comprises two identical subunits with 401 amino acid residues (Mr 43,238) and contains pyridoxal 5'-phosphate as a coenzyme. The enzyme catalyzes the removal of elemental sulfur and selenium atoms from L-cysteine, L-cystine, L-selenocysteine, and L-selenocystine to produce L-alanine. Because L-cysteine sulfinic acid was desulfinated to form L-alanine as the preferred substrate, we have named this new enzyme cysteine sulfinate desulfinase. Mutant enzymes having alanine substituted for each of the four cysteinyl residues (Cys-100, Cys-176, Cys-323, and Cys-358) were all active. Cys-358 corresponds to Cys-325 of A. vinelandii NIFS, which is conserved among all NIFS-like proteins and catalytically essential (Zheng, L., White, R. H., Cash, V. L., and Dean, D. R. (1994) Biochemistry 33, 4714-4720), is not required for cysteine sulfinate desulfinase. Thus, the enzyme is distinct from A. vinelandii NIFS in this respect.
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1809. |
( 1997 ) Tn5 transposase mutants that alter DNA binding specificity. PMID : 9268665 : DOI : 10.1006/jmbi.1997.1188 Abstract >>
Tn5 transposase (Tnp) binds to Tn5 and IS50 end inverted repeats, the outside end (OE) and the inside end (IE), to initiate transposition. We report the isolation of four Tnp mutants (YH41, TP47, EK54 and EV54) that increase the OE-mediated transposition frequency and enhance the binding affinity of Tnp for OE DNA. In addition, two of the Tnp mutants (TP47 and EK54) appear to be change-of-specificity mutants, since they alter the recognition of OE versus IE relative to the wild-type Tnp. EK54 enhances OE recognition but decreases IE recognition. TP47 enhances both OE and IE recognition but with a much greater enhancement for IE than for OE. This change-of-specificity effect of TP47 is observed only when TP47 Tnp is synthesized in cis to the DNA that contains the ends. We propose that Lys54 makes a favorable interaction with an OE-specific nucleotide pair(s), while Pro47 may cause a more favorable interaction with an IE-specific nucleotide pair(s) than it does with the corresponding OE-specific nucleotide pair(s). A model to explain the preference of TP47 Tnp for the IE in cis but not in trans is proposed.
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1810. |
( 1996 ) N-acetyl-heparosan lyase of Escherichia coli K5: gene cloning and expression. PMID : 8955411 : DOI : 10.1128/jb.178.24.7260-7264.1996 PMC : PMC178642 Abstract >>
The structure of the capsular polysaccharide of Escherichia coli K5 is identical to that of N-acetyl-heparosan, a nonsulfated precursor of heparin, which makes this E. coli antigen an attractive starting point for the chemical synthesis of analogs of low-molecular-weight heparin. This polysaccharide is synthesized as a high-molecular-weight molecule that can be depolymerized by an enzyme displaying endo-beta-eliminase activity. The eliminase-encoding gene, designated elmA, has been cloned from E. coli K5 by expression in E. coli K-12. The K-12 genome is devoid of the elmA sequence. The elmA gene product is 820 amino acids long. Active recombinant eliminase is produced by K-12 cells in both cell-bound and secreted forms. Deletion analyses have shown that the C terminus and the N terminus are required for activity and secretion, respectively.
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1811. |
( 1997 ) Bacterial interspersed mosaic elements (BIMEs) are a major source of sequence polymorphism in Escherichia coli intergenic regions including specific associations with a new insertion sequence. PMID : 9055066 : PMC : PMC1207841 Abstract >>
A significant fraction of Escherichia coli intergenic DNA sequences is composed of two families of repeated bacterial interspersed mosaic elements (BIME-1 and BIME-2). In this study, we determined the sequence organization of six intergenic regions in 51 E. coli and Shigella natural isolates. Each region contains a BIME in E. coli K-12. We found that multiple sequence variations are located within or near these BIMEs in the different bacteria. Events included excisions of a whole BIME-1, expansion/deletion within a BIME-2 and insertions of non-BIME sequences like the boxC repeat or a new IS element, named IS 1397. Remarkably, 14 out of IS 1397 integration sites correspond to a BIME sequence, strongly suggesting that this IS element is specifically associated with BIMEs, and thus inserts only in extragenic regions. Unlike BIMEs, IS 1397 is not detected in all E. coli isolates. Possible relationships between the presence of this IS element and the evolution of BIMEs are discussed.
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1812. |
( 1996 ) Phylogenetic analysis of tmRNA secondary structure. PMID : 8972778 : PMC : PMC1369456 Abstract >>
The bacterial tmRNA acts with dual tRNA-like and mRNA-like character to tag incomplete translation products for degradation. Comparative analysis of 17 tmRNA genes (including eight new sequences) has allowed us to deduce conserved features of the tmRNA secondary structure. Except in a segment that includes the first codon of the tag reading frame, tmRNA is highly structured, with four pseudoknots and a total of 11 conserved base pairing regions. The previously identified tRNA minihelix structure is connected by a long base paired region to a large structured domain composed of a pseudoknot, followed by the tag reading frame and a string of three rather similar pseudoknots. The conservation of numerous structural elements among diverse eubacterial species indicates that these elements have important function beyond simply forming an endonuclease-resistant link between the reading frame and the tRNA-like domain.
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1813. |
( 1996 ) Molecular analysis of the gat genes from Escherichia coli and of their roles in galactitol transport and metabolism. PMID : 8955298 : DOI : 10.1128/jb.178.23.6790-6795.1996 PMC : PMC178577 Abstract >>
In enteric bacteria, the hexitol galactitol (Gat) (formerly dulcitol) is taken up through enzyme II (II(Gat)) of the phosphoenolpyruvate-dependent phosphotransferase system (PTS), and accumulated as galactitol 1-phosphate (Gat1P). The gat genes involved in galactitol metabolism have been isolated from the wild-type isolate Escherichia coli EC3132 and cloned on a 7.8-kbp PstI DNA fragment. They comprise six complete open reading frames and one truncated open reading frame in the order gatYZABCDR'. The genes gatABC code for the proteins GatA (150 residues) and GatB (94 residues), which correspond to the hydrophilic domains IIA(Gat) and IIB(Gat), and GatC, which represents a membrane-bound transporter domain IIC(Gat) (35 kDa, 427 residues). The three polypeptides together constitute a II(Gat) of average size (671 residues). Gene gatD codes for a Gat1P-specific NAD-dependent dehydrogenase (38 kDa, 346 residues), gatZ codes for a protein (42 kDa, 378 residues) of unknown function, and gatY (31 kDa, 286 residues) codes for a D-tagatose-1,6-bisphosphate aldolase with similarity to other known ketose-bisphosphate aldolases. The truncated gatR' gene, whose product shows similarity to the glucitol repressor GutR, closely resembles a gatR gene fragment from E. coli K-12. The gat genes map in both organisms at similar positions, in E. coli K-12, where they are transcribed counterclockwise at precisely 46.7 min or 2,173 to 2,180 kbp. The genes are expressed constitutively in both strains, probably due to a mutation(s) in gatR. Transcription initiation sites for the gatYp and the gatRp promoters were determined by primer extension analysis.
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1814. |
( 1997 ) Association of mercury resistance with antibiotic resistance in the gram-negative fecal bacteria of primates. PMID : 9361435 : PMC : PMC168768 Abstract >>
Gram-negative fecal bacterial from three longitudinal Hg exposure experiments and from two independent survey collections were examined for their carriage of the mercury resistance (mer) locus. The occurrence of antibiotic resistance was also assessed in both mercury-resistant (Hgr) and mercury-susceptible (Hgs) isolates from the same collections. The longitudinal studies involved exposure of the intestinal flora to Hg released from amalgam "silver" dental restorations in six monkeys. Hgr strains were recovered before the installation of amalgams, and frequently these became the dominant strains while amalgams were installed. Such persistent Hgr strains always carried the same mer locus throughout the experiments. In both the longitudinal and survey collections, certain mer loci were preferentially associated with one genus, whereas other mer loci were recovered from many genera. In general, strains with any mer locus were more likely to be multiresistant than were strains without mer loci; this clustering tendency was also seen for antibiotic resistance genes. However, the association of antibiotic multiresistance with mer loci was not random; regardless of source, certain mer loci occurred in highly multiresistant strains (with as many as seven antibiotic resistances), whereas other mer loci were found in strains without any antibiotic resistance. The majority of highly multiresistant Hgr strains also carried genes characteristic of an integron, a novel genetic element which enables the formation of tandem arrays of antibiotic resistance genes. Hgr strains lacking antibiotic resistance showed no evidence of integron components.
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1815. |
( 1997 ) Identification of a family of intimins common to Escherichia coli causing attaching-effacing lesions in rabbits, humans, and swine. PMID : 8975932 : PMC : PMC174596 Abstract >>
Intimin, an outer membrane protein encoded by eaeA that mediates close attachment of enteropathogenic bacteria to apical surfaces of epithelial cells, is required for formation of the attaching-effacing lesions and for full pathogenesis of the bacteria. Analysis of the eaeA sequence indicates that there is a high degree of homology at the N termini but less at the C termini of intimins. Antisera specific for the C-terminal third of RDEC-1 intimin, used to screen outer membrane proteins from 50 rabbit enteropathogenic Escherichia coli (EPEC), human EPEC, and human enterohemorrhagic E. coli (EHEC) strains, identified cross-reactive intimins from 24 isolates. Sequence analysis of the eaeA genes from human EPEC O111 and EHEC O26 isolates indicates that their intimins have C termini nearly identical to that of RDEC-1 intimin. Our results suggest that there are at least three families of related intimins and that the presence of intimin similar to that of RDEC-1 is not restricted by serogroup or host specificity.
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1816. |
( 1997 ) Detection and sequences of the enteroaggregative Escherichia coli heat-stable enterotoxin 1 gene in enterotoxigenic E. coli strains isolated from piglets and calves with diarrhea. PMID : 8968912 : PMC : PMC229543 Abstract >>
Enterotoxigenic Escherichia coli (ETEC) strains isolated from piglets and calves with diarrhea were tested for the presence of the enteroaggregative E. coli enterotoxin 1 (EAST1) gene sequences by PCR and colony hybridization. The EAST1 gene was found in most porcine ETEC strains with adherence factor K88, especially in those elaborating heat-labile enterotoxin. One porcine ETEC strain with adherence factor K99 was also positive for the EAST1 gene. In contrast, 987P-positive (987P+) ETEC strains from piglets, K99+ ETEC strains from calves, and K99+ F41+ or F41+ ETEC strains from piglets and calves were negative for the EAST1 gene. The K88ab+ or K88ac+ ETEC strains tested possessed the EAST1 gene on a plasmid that was distinct from a K88-encoding plasmid. The EAST1 gene sequences of the K88+ ETEC strains were identical to each other and 99.1 and 98.3% homologous to the previously reported sequences of ETEC strains colonizing humans and enteroaggregative E. coli strains, respectively. The data indicate that the EAST1 gene is distributed among porcine ETEC strains in association with the adherence factor type.
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1817. |
( 1997 ) Comparison of the nucleotide sequence of enteroaggregative Escherichia coli heat-stable enterotoxin 1 genes among diarrhea-associated Escherichia coli. PMID : 9037769 : DOI : 10.1111/j.1574-6968.1997.tb10225.x Abstract >>
The presence of the enteroaggregative Escherichia coli (EAggEC) heat-stable enterotoxin 1 (EAST1) gene was investigated in 15 strains each of EAggEC, enteropathogenic E. coli (EPEC), EPEC-related strains of non-EPEC serotypes, diffusely adhering E. coli (type 1 DAEC) that carries F1845 adhesive pili (or a related adhesin), and enteroinvasive E. coli (EIEC) by PCR and colony hybridization. The EAST1 gene or its homologue was present in 53.3% of EAggEC, 20% of EPEC, 13.3% of the EPEC-related strains, and 6.7% of type 1 DAEC, EIEC and E. coli unrelated with diarrhea had no gene with sequence similarity to the EAST1 gene. Comparison of the EAST1 gene sequences analyzed in this study as well as those reported previously showed that EAggEC (including strain O42, which was shown to be pathogenic in volunteer experiments), EPEC, type 1 DAEC, type 2 DAEC (which carries the 57-kDa outer membrane protein as an adhesin), and enterotoxigenic E. coli shared a common sequence. A variant type of the EAST1 gene sequence was present in the EAggEC strain 17-2 (initially characterized for the EAST1 gene) and in an EPEC-related strain of a non-EPEC serotype. These data suggest that the EAST1 gene or its variant is a virulence gene widely distributed among diarrhea-associated E. coli.
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1818. |
( 1997 ) Molecular diversity and evolution of blaTEM genes encoding beta-lactamases resistant to clavulanic acid in clinical E. coli. PMID : 9010136 : Abstract >>
The molecular diversity of inhibitor-resistant TEM (IRT) enzymes was explored using a strategy which involved DNA amplification by polymerase chain reaction (PCR), analysis of restriction fragment length polymorphism (RFLP), and direct nucleotide sequencing. The study of plasmid-borne genes from 27 strains, resistant to amoxicillin and beta-lactamase-inhibitor combinations, identified mutations resulting in amino acid change at positions 69, 244, 275, and 276 known to be associated with the IRT phenotype and a mutation at nucleotide position 162 in the promoter region. These mutations were found to lie on two different gene sequences, described here as "TEM-1B like" and "TEM-2 like" restriction linkage groups. Further analysis, of nucleotide sequences of promoter and coding regions of the beta-lactamases, confirmed that a given mutation causing IRT phenotype could be associated with two different gene sequence frameworks and two different causal mutations could lie on identical gene sequence framework. These data argue in favor of convergent phenotypic evolution of IRT enzymes under the selective pressure imposed by the intensive clinical use of beta-lactam-beta-lactamase inhibitor combinations.
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1819. |
( 1997 ) Evolutionary genetics of the isocitrate dehydrogenase gene (icd) in Escherichia coli and Salmonella enterica. PMID : 9352899 : DOI : 10.1128/jb.179.21.6551-6559.1997 PMC : PMC179578 Abstract >>
Sequences of the icd gene, encoding isocitrate dehydrogenase (IDH), were obtained for 33 strains representing the major phylogenetic lineages of Escherichia coli and Salmonella enterica. Evolutionary relationships of the strains based on variation in icd are generally similar to those previously obtained for several other housekeeping and for invasion genes, but the sequences of S. enterica subspecies V strains are unusual in being almost intermediate between those of the other S. enterica subspecies and E. coli. For S. enterica, the ratio of synonymous (silent) to nonsynonymous (replacement) nucleotide substitutions between pairs of strains was larger than comparable values for 12 other housekeeping and invasion genes, reflecting unusually strong purifying selection against amino acid replacement in the IDH enzyme. All amino acids involved in the catalytic activity and conformational changes of IDH are strictly conserved within and between species. In E. coli, the level of variation at the 3' end of the gene is elevated by the presence in some strains of a 165-bp replacement sequence supplied by the integration of either lambdoid phage 21 or defective prophage element e14. The 72 members of the E. coli Reference Collection (ECOR) and five additional E. coli strains were surveyed for the presence of phage 21 (as prophage) by PCR amplification of a phage 21-specific fragment in and adjacent to the host icd, and the sequence of the phage 21 segment extending from the 3' end of icd through the integrase gene (int) was determined in nine strains of E. coli. Phage 21 was found in 39% of E. coli strains, and its distribution among the ECOR strains is nonrandom. In two ECOR strains, the phage 21 int gene is interrupted by a 1,313-bp insertion element that has 99.3% nucleotide sequence identity with IS3411 of E. coli. The phylogenetic relationships of phage 21 strains derived from sequences of two different genomic regions were strongly incongruent, providing evidence of frequent recombination.
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1820. |
( 1997 ) Molecular and functional analysis of genes required for expression of group IB K antigens in Escherichia coli: characterization of the his-region containing gene clusters for multiple cell-surface polysaccharides. PMID : 9383197 : DOI : 10.1046/j.1365-2958.1997.5631930.x Abstract >>
Escherichia coli group I capsular K antigens are found in two forms on the cell surface. The K(LPS) form is linked to lipopolysaccharide lipid A core, whereas the high-molecular-weight capsular form is assembled independently of lipid A core. Subgroup IB K antigens are generally co-expressed with either the O8 or O9 antigen and, under the appropriate conditions, with the exopolysaccharide, colanic acid. To examine the relationships between the genetic loci and the synthetic pathways for these various cell-surface polymers, the gene cluster responsible for expression of a prototype group IB K antigen (serotype K40) was cloned and the flanking chromosomal regions characterized. Analysis of the six orfs within the cluster indicates features typical of Wzy (Rfc)-dependent O antigens. Synthesis of group IB K antigens is initiated by WecA (Rfe), a UDP-GlcNAc::undecaprenylphosphate GlcNAc-1-phosphate transferase, and the chain length of K40LPS is determined by the wzz gene product. The his-region of the E. coli O8:K40 prototype is almost exclusively devoted to the expression of three different surface polysaccharides. The rfbK40 cluster is located adjacent to the cps (colanic acid synthesis) and rfbO8 (O8 antigen synthesis) loci in the gene order: his-rfbO8/O9-wzz-ugd-gnd-rfbK40-galF-cps. Thus, rfbK40 is in the location occupied by other Wzy-dependent rfb gene clusters, and rfbO8/O9 represents an additional locus.
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1821. |
( 1996 ) Role of the Escherichia coli O157:H7 O side chain in adherence and analysis of an rfb locus. PMID : 8890241 : PMC : PMC174447 Abstract >>
Shiga-toxigenic Escherichia coli strains belonging to serotype O157 are important human pathogens, but the genetic basis of expression of the O157 antigen and the role played by the lipopolysaccharide O side chain in the adherence of this organism to epithelial cells are not understood. We performed TnphoA mutagenesis on E. coli O157:H7 strain 86-24 to identify a mutant (strain F12) deficient in O-antigen expression. Nucleotide sequence analysis demonstrated that the transposon inserted within an open reading frame with significant homology to rfbE of Vibrio cholerae O1 (U. H. Stroeher, L. E. Karageorgos, R. Morona, and P. A. Manning, Proc. Natl. Acad. Sci. USA 89:2566-2570, 1992), which is postulated to encode perosamine synthetase. This open reading frame was designated rfbE(EcO157:H7). The guanine-plus-cytosine fraction (0.35) suggests that rfbE(EcO157:H7) may have originated in a species other than E. coli. rfbE(EcO157:H7) is conserved in nontoxigenic E. coli O157 strains expressing a variety of other flagellar antigens but is not found in E. coli O55:H7 strains, which are more closely related to E. coli O157:H7. Strain F12 was significantly more adherent to HeLa cells in a quantitative adherence assay than was its E. coli O157:H7 parent, but they did not differ in other phenotypes. Restoration of the expression of the O side chain by complementation of the TnphoA mutation in strain F12 by a plasmid expressing intact rfbE(EcO157:H7) reduced the adherence of the hyperadherent strain F12. We conclude that rfbE(EcO157:H7) is necessary for the expression of the O157 antigen, that acquisition of E. coli rfb genes occurred independently in E. coli O157:H7 and unrelated O157 strains, and that the O side chain of E. coli O157:H7 lipopolysaccharide interferes with the adherence of E. coli O157:H7 to epithelial cells.
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1822. |
( 1996 ) Temperature- and medium-dependent secretion of proteins by Shiga toxin-producing Escherichia coli. PMID : 8890194 : PMC : PMC174400 Abstract >>
Infections due to Shiga toxin-producing Escherichia coli (STEC) are responsible for severe diarrheal disease in humans and livestock, and these bacteria have recently emerged as a leading cause of renal failure in children. In this study, we have examined medium- and temperature-dependent production of secreted proteins from a STEC O26 serotype strain. Growth of bacteria in Luria broth led to the detection of secreted polypeptides of 104, 55, 54, and 37 kDa (p104, p55, p54, and p37, respectively). When grown in serum-free tissue culture medium, only p104, p37 and two additional polypeptides of 25 and 22 kDa (p25 and p22) were present in supernatant fluids. Production of these polypeptides was growth temperature dependent and induced in cultures grown at 37 degrees C. N-terminal amino acid sequencing revealed that p104 was homologous to the secreted p110 of enteropathogenic Escherichia coli (EPEC), and both proteins belong to a family of secreted proteins in pathogenic bacteria of which the immunoglobulin A protease of Neisseria gonorrhoeae is the prototype. The N-terminal amino acid sequences of p55 and p54 were unique to the STEC strain, while p37 and p25 were found to be highly homologous to the similarly sized EspA and EspB proteins, previously detected in culture supernatants of EPEC. Molecular cloning and sequencing of STEC espB alleles from two different serotypes showed that the encoded polypeptides were about 80% homologous. A monoclonal antibody raised against STEC EspB also cross-reacted with its EPEC analog and allowed us to demonstrate medium- and temperature-dependent production of this important virulence factor in STEC and EPEC strains of differing serotypes.
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1823. |
( 1996 ) Cloning and characterization of bfpTVW, genes required for the transcriptional activation of bfpA in enteropathogenic Escherichia coli. PMID : 8885267 : DOI : 10.1046/j.1365-2958.1996.531415.x Abstract >>
Expression of the bundle-forming pilus (BFP) of enteropathogenic Escherichia coli (EPEC) is regulated at the transcriptional level by growth phase, temperature, calcium and ammonium. Genes required for the transcriptional activation of bfpA were localized to a 1.8 kb fragment of the enteroadherent factor (EAF) plasmid of EPEC that is separated from the bfp operon by 6 kb. Within this fragment three identically oriented and closely spaced open reading frames (ORFs) were identified and designated bfpT, bfpV and bfpW. bfpT is predicted to encode a 31.8 kDa protein that shares homology with the AraC family of transcriptional regulators, including the presence of a conserved C-terminal DNA-binding helix-turn-helix motif. Insertional inactivation of bfpT led to the loss of bfpA transcription, BfpA protein production and the localized adherence (LA) phenotype; this mutant phenotype could be complemented by introduction of bfpTVW and, on separate plasmids, bfpT + bfpW. However, introduction of bfpT + bfpV, bfpV alone, bfpW alone, or bfpV + bfpW did not enable recovery of the wild-type phenotype. Maximal efficiency of bfpA transcription required all three genes, but bfpV and bfpW each enhanced transcription providing bfpT was also present. A series of deletions of the bfpA upstream promoter region was prepared; with respect to the bfpA transcription start site, sequence between nucleotides -94 and -55 was found to bind bfpT. BfpT also bound a DNA fragment containing the eaeA promoter region on the EPEC chromosome. From these results we conclude that bfpTV W causes transcriptional activation of bfpA, and possibly eaeA, by a trans-acting mech |