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1. Escobar-Páramo  P, Giudicelli  C, Parsot  C, Denamur  E,     ( 2003 )

The evolutionary history of Shigella and enteroinvasive Escherichia coli revised.

Journal of molecular evolution 57 (2)
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.
KeywordMeSH Terms
Evolution, Molecular
Phylogeny
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.

Infection and immunity 71 (11)
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.
KeywordMeSH Terms
3. 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.

Infection and immunity 71 (10)
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.
KeywordMeSH Terms
4. Loudon  JA, Loughlin  RE,     ( 1992 )

Mutagenesis and regulation of the cysJ promoter of Escherichia coli K-12.

Gene 122 (1)
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.
KeywordMeSH Terms
Promoter Regions, Genetic
5. 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.

The Journal of biological chemistry 267 (32)
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.
KeywordMeSH Terms
Genes, Bacterial
Mutagenesis, Site-Directed
6. 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.

Journal of bacteriology 174 (21)
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.
KeywordMeSH Terms
Amino Acid Transport Systems, Neutral
Biological Evolution
7. Csitkovits  VC, Zechner  EL,     ( 2003 )

Extent of single-stranded DNA required for efficient TraI helicase activity in vitro.

The Journal of biological chemistry 278 (49)
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.
KeywordMeSH Terms
8. Biskri  L, Mazel  D,     ( 2003 )

Erythromycin esterase gene ere(A) is located in a functional gene cassette in an unusual class 2 integron.

Antimicrobial agents and chemotherapy 47 (10)
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.
KeywordMeSH Terms
9. 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.

FEBS letters 553 (1��2��)
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.
KeywordMeSH Terms
10. 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.

Molecular & general genetics : MGG 234 (3)
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.
KeywordMeSH Terms
F Factor
Genes, Bacterial
Transduction, Genetic
11. 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.

Molecular microbiology 6 (7)
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.
KeywordMeSH Terms
Escherichia coli Proteins
Gene Expression Regulation, Bacterial
R Factors
12. 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.

Journal of the American Chemical Society 125 (41)
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.
KeywordMeSH Terms
13. 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.

Journal of bacteriology 174 (20)
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.
KeywordMeSH Terms
Escherichia coli Proteins
Membrane Proteins
14. Raha  M, Kawagishi  I, Müller  V, Kihara  M, Macnab  RM,     ( 1992 )

Escherichia coli produces a cytoplasmic alpha-amylase, AmyA.

Journal of bacteriology 174 (20)
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.
KeywordMeSH Terms
15. 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.

Journal of the American Chemical Society 125 (41)
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.
KeywordMeSH Terms
16. 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.

Journal of the American Chemical Society 125 (41)
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.
KeywordMeSH Terms
17. Ivanova  A, Renshaw  M, Guntaka  RV, Eisenstark  A,     ( 1992 )

DNA base sequence variability in katF (putative sigma factor) gene of Escherichia coli.

Nucleic acids research 20 (20)
PMID : 1437569  :   DOI  :   10.1093/nar/20.20.5479     PMC  :   PMC334364    
Abstract >>
N/A
KeywordMeSH Terms
18. 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.

Antimicrobial agents and chemotherapy 36 (9)
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.
KeywordMeSH Terms
19. Kim  SR, Komano  T,     ( 1992 )

Nucleotide sequence of the R721 shufflon.

Journal of bacteriology 174 (21)
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.
KeywordMeSH Terms
Integrases
20. 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.

Journal of molecular biology 228 (1)
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.
KeywordMeSH Terms
Chromosomal Proteins, Non-Histone
Endoribonucleases
Escherichia coli Proteins
Genes, Bacterial
21. 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.

Journal of bacteriology 174 (20)
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.
KeywordMeSH Terms
Escherichia coli Proteins
Fimbriae Proteins
Molecular Chaperones
22. Bockmann  J, Heuel  H, Lengeler  JW,     ( 1992 )

Characterization of a chromosomally encoded, non-PTS metabolic pathway for sucrose utilization in Escherichia coli EC3132.

Molecular & general genetics : MGG 235 (1)
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.
KeywordMeSH Terms
23. Salomón  RA, Farías  RN,     ( 1992 )

Microcin 25, a novel antimicrobial peptide produced by Escherichia coli.

Journal of bacteriology 174 (22)
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.
KeywordMeSH Terms
24. 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.

Proceedings of the National Academy of Sciences of the United States of America 89 (20)
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.
KeywordMeSH Terms
Genes, Bacterial
25. 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.

FEMS microbiology letters 226 (2)
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.
KeywordMeSH Terms
Integrons
26. 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.

The EMBO journal 11 (7)
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.
KeywordMeSH Terms
Escherichia coli Proteins
Fimbriae Proteins
Gene Expression Regulation, Bacterial
27. 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.

Molecular microbiology 6 (17)
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.
KeywordMeSH Terms
Transcription Factors
28. 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.

Molecular microbiology 6 (16)
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.
KeywordMeSH Terms
Biological Evolution
Fimbriae, Bacterial
Operon
Transfection
29. 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.

The Journal of infectious diseases 187 (12)
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.
KeywordMeSH Terms
30. Shaikh  N, Tarr  PI,     ( 2003 )

Escherichia coli O157:H7 Shiga toxin-encoding bacteriophages: integrations, excisions, truncations, and evolutionary implications.

Journal of bacteriology 185 (12)
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.
KeywordMeSH Terms
31. Klann  AG, Hull  RA, Hull  SI,     ( 1992 )

Sequences of the genes encoding the minor tip components of Pap-3 pili of Escherichia coli.

Gene 119 (1)
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.
KeywordMeSH Terms
Fimbriae, Bacterial
Genes, Bacterial
32. 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.

Journal of general microbiology 138 (7)
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.
KeywordMeSH Terms
33. Stokes  HW, Hall  RM,     ( 1992 )

The integron In1 in plasmid R46 includes two copies of the oxa2 gene cassette.

Plasmid 28 (3)
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.
KeywordMeSH Terms
DNA Transposable Elements
R Factors
34. 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.

Nucleic acids research 20 (22)
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.
KeywordMeSH Terms
35. Herzer  PJ, Inouye  S, Inouye  M,     ( 1992 )

Retron-Ec107 is inserted into the Escherichia coli genome by replacing a palindromic 34bp intergenic sequence.

Molecular microbiology 6 (3)
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.
KeywordMeSH Terms
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.

Infection and immunity 60 (9)
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.
KeywordMeSH Terms
Fimbriae Proteins
Fimbriae, Bacterial
37. 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.

Journal of clinical microbiology 41 (6)
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.
KeywordMeSH Terms
Multigene Family
38. 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.

The Journal of antimicrobial chemotherapy 52 (1)
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.
KeywordMeSH Terms
39. Becker  EC, Meyer  RJ,     ( 2003 )

Relaxed specificity of the R1162 nickase: a model for evolution of a system for conjugative mobilization of plasmids.

Journal of bacteriology 185 (12)
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.
KeywordMeSH Terms
Bacterial Proteins
Evolution, Molecular
40. 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.

Molecular microbiology 6 (2)
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.
KeywordMeSH Terms
Escherichia coli Proteins
Fimbriae Proteins
41. 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.

DNA sequence : the journal of DNA sequencing and mapping 2 (4)
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.
KeywordMeSH Terms
Fimbriae Proteins
42. 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).

Nucleic acids research 20 (22)
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.
KeywordMeSH Terms
43. Clark  CA, Heuzenroeder  MW, Manning  PA,     ( 1992 )

Colonization factor antigen CFA/IV (PCF8775) of human enterotoxigenic Escherichia coli: nucleotide sequence of the CS5 determinant.

Infection and immunity 60 (3)
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.
KeywordMeSH Terms
Fimbriae, Bacterial
44. 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.

The Journal of biological chemistry 267 (6)
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.
KeywordMeSH Terms
Bacterial Proteins
45. Donnenberg  MS, Girón  JA, Nataro  JP, Kaper  JB,     ( 1992 )

A plasmid-encoded type IV fimbrial gene of enteropathogenic Escherichia coli associated with localized adherence.

Molecular microbiology 6 (22)
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.
KeywordMeSH Terms
Escherichia coli Proteins
Fimbriae Proteins
Fimbriae, Bacterial
Genes, Bacterial
46. 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.

The Journal of veterinary medical science 54 (6)
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.
KeywordMeSH Terms
47. Wang  MX, Church  GM,     ( 1992 )

A whole genome approach to in vivo DNA-protein interactions in E. coli.

Nature 360 (6404)
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.
KeywordMeSH Terms
Genes, Bacterial
Site-Specific DNA-Methyltransferase (Adenine-Specific)
48. 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.

Molecular & general genetics : MGG 235 (1)
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.
KeywordMeSH Terms
DNA Transposable Elements
Genetic Linkage
R Factors
49. 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.

Molecular & general genetics : MGG 234 (1)
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.
KeywordMeSH Terms
ATP-Binding Cassette Transporters
Escherichia coli Proteins
Genes, Lethal
Operon
50. 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.

Microbial pathogenesis 13 (2)
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.
KeywordMeSH Terms
Fimbriae, Bacterial
51. 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.

Journal of bacteriology 174 (16)
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.
KeywordMeSH Terms
Escherichia coli Proteins
Genes, Bacterial
52. 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.

Infection and immunity 60 (5)
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.
KeywordMeSH Terms
Fimbriae, Bacterial
Genes, Bacterial
53. Prentki  P,     ( 1992 )

Nucleotide sequence of the classical lacZ deletion delta M15.

Gene 122 (1)
PMID : 1339377  :   DOI  :   10.1016/0378-1119(92)90056-u    
Abstract >>
N/A
KeywordMeSH Terms
Genes, Bacterial
Sequence Deletion
54. van Biesen  T, Frost  LS,     ( 1992 )

Differential levels of fertility inhibition among F-like plasmids are related to the cellular concentration of finO mRNA.

Molecular microbiology 6 (6)
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.
KeywordMeSH Terms
Escherichia coli Proteins
Repressor Proteins
RNA-Binding Proteins
55. 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.

Journal of molecular biology 224 (3)
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.
KeywordMeSH Terms
56. 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.

The EMBO journal 11 (7)
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)
KeywordMeSH Terms
DNA Helicases
DNA-Binding Proteins
Escherichia coli Proteins
Gene Expression Regulation, Bacterial
Plasmids
Proteins
Trans-Activators
57. Ziegelin  G, Pansegrau  W, Lurz  R, Lanka  E,     ( 1992 )

TraK protein of conjugative plasmid RP4 forms a specialized nucleoprotein complex with the transfer origin.

The Journal of biological chemistry 267 (24)
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.
KeywordMeSH Terms
Conjugation, Genetic
DNA Replication
Escherichia coli Proteins
Genes, Bacterial
Periplasmic Proteins
Plasmids
58. Gamas  P, Craig  NL,     ( 1992 )

Purification and characterization of TnsC, a Tn7 transposition protein that binds ATP and DNA.

Nucleic acids research 20 (10)
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.
KeywordMeSH Terms
Escherichia coli Proteins
59. Pinner  E, Padan  E, Schuldiner  S,     ( 1992 )

Cloning, sequencing, and expression of the nhaB gene, encoding a Na+/H+ antiporter in Escherichia coli.

The Journal of biological chemistry 267 (16)
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.
KeywordMeSH Terms
Genes, Bacterial
60. Collins  CM, Gutman  DM,     ( 1992 )

Insertional inactivation of an Escherichia coli urease gene by IS3411.

Journal of bacteriology 174 (3)
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.
KeywordMeSH Terms
Mutagenesis, Insertional
61. 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.

Infection and immunity 71 (6)
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.
KeywordMeSH Terms
Multigene Family
62. 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.

Gene 111 (1)
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.
KeywordMeSH Terms
DNA Transposable Elements
Escherichia coli Proteins
63. 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.

Infection and immunity 71 (6)
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.
KeywordMeSH Terms
Multigene Family
Plasmids
64. 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.

Journal of bacteriology 174 (3)
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.
KeywordMeSH Terms
65. Mendiola  MV, Jubete  Y, de la Cruz  F,     ( 1992 )

DNA sequence of IS91 and identification of the transposase gene.

Journal of bacteriology 174 (4)
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.
KeywordMeSH Terms
66. 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.

The Journal of biological chemistry 267 (15)
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.
KeywordMeSH Terms
DNA-Binding Proteins
Escherichia coli Proteins
Genes, Bacterial
67. Parent  R, Roy  PH,     ( 1992 )

The chloramphenicol acetyltransferase gene of Tn2424: a new breed of cat.

Journal of bacteriology 174 (9)
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.
KeywordMeSH Terms
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.

Nucleic acids research 20 (13)
PMID : 1321417  :   DOI  :   10.1093/nar/20.13.3521     PMC  :   PMC312521    
Abstract >>
N/A
KeywordMeSH Terms
69. 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.

Microbiology (Reading, England) 149 (Pt 6)
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.
KeywordMeSH Terms
70. Wiegand  TW, Reznikoff  WS,     ( 1992 )

Characterization of two hypertransposing Tn5 mutants.

Journal of bacteriology 174 (4)
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.
KeywordMeSH Terms
71. 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.

Microbiology (Reading, England) 149 (Pt 9)
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.
KeywordMeSH Terms
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.

Applied and environmental microbiology 69 (5)
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.
KeywordMeSH Terms
73. Starcic Erjavec  M, Gaastra  W, van Putten  J, Zgur-Bertok  D,     ( 2003 )

Identification of the origin of replications and partial characterization of plasmid pRK100.

Plasmid 50 (2)
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.
KeywordMeSH Terms
Chromosome Mapping
74. 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.

Journal of microbiological methods 54 (1)
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.
KeywordMeSH Terms
Bacterial Typing Techniques
Polymorphism, Restriction Fragment Length
75. 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.

The Journal of biological chemistry 278 (46)
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.
KeywordMeSH Terms
76. 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.

Journal of clinical microbiology 41 (5)
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.
KeywordMeSH Terms
77. 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).

Journal of clinical microbiology 41 (9)
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.
KeywordMeSH Terms
78. Burian  J, Ausió  J, Phipps  B, Moore  S, Dougan  D, Kay  W,     ( 2003 )

Hexamerization of RepA from the Escherichia coli plasmid pKL1.

Biochemistry 42 (34)
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.
KeywordMeSH Terms
DNA Helicases
DNA-Binding Proteins
Trans-Activators
79. 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.

Journal of clinical microbiology 41 (5)
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.
KeywordMeSH Terms
Genes, Bacterial
80. Volokhov  D, Chizhikov  V, Chumakov  K, Rasooly  A,     ( 2003 )

Microarray analysis of erythromycin resistance determinants.

Journal of applied microbiology 95 (4)
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.
KeywordMeSH Terms
81. Gobius  KS, Higgs  GM, Desmarchelier  PM,     ( 2003 )

Presence of activatable Shiga toxin genotype (stx(2d)) in Shiga toxigenic Escherichia coli from livestock sources.

Journal of clinical microbiology 41 (8)
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.
KeywordMeSH Terms
82. 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.

Infection and immunity 71 (9)
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.
KeywordMeSH Terms
Genes, Bacterial
83. 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.

Canadian journal of microbiology 49 (5)
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.
KeywordMeSH Terms
Cloning, Molecular
Genes, Bacterial
84. Wang  L, Rothemund  D, Curd  H, Reeves  PR,     ( 2003 )

Species-wide variation in the Escherichia coli flagellin (H-antigen) gene.

Journal of bacteriology 185 (9)
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.
KeywordMeSH Terms
85. 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.

Journal of molecular biology 331 (4)
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.
KeywordMeSH Terms
Bacterial Adhesion
86. 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.

Microbial pathogenesis 13 (3)
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.
KeywordMeSH Terms
Genes, Bacterial
Sudden Infant Death
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.

Antimicrobial agents and chemotherapy 47 (9)
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.
KeywordMeSH Terms
88. 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.

The Journal of infectious diseases 188 (5)
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.
KeywordMeSH Terms
89. Betteridge  T, Yang  J, Pittard  AJ, Praszkier  J,     ( 2003 )

Interaction of the initiator protein of an IncB plasmid with its origin of DNA replication.

Journal of bacteriology 185 (7)
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.
KeywordMeSH Terms
DNA Replication
Replication Origin
90. 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.

Molecular microbiology 48 (1)
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.
KeywordMeSH Terms
91. 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.

FEMS microbiology letters 225 (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.
KeywordMeSH Terms
Genes, Bacterial
92. 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.

Molecular microbiology 48 (2)
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.
KeywordMeSH Terms
Conjugation, Genetic
93. 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.

Journal of applied microbiology 94 (4)
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.
KeywordMeSH Terms
94. Yaron  S, White  DG, Matthews  KR,     ( 2003 )

Characterization of an Escherichia coli O157:H7 marR mutant.

International journal of food microbiology 85 (3)
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).
KeywordMeSH Terms
95. 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.

Molecular microbiology 49 (3)
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.
KeywordMeSH Terms
96. 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.

Cellular microbiology 5 (8)
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.
KeywordMeSH Terms
97. 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).

International journal of medical microbiology : IJMM 292 (7��8��)
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.
KeywordMeSH Terms
Gene Transfer, Horizontal
Phosphoproteins
Phylogeny
98. 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.

Infection and immunity 71 (8)
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.
KeywordMeSH Terms
99. 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.

Journal of bacteriology 185 (7)
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.
KeywordMeSH Terms
Gene Transfer, Horizontal
Genes, Bacterial
100. 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).

Veterinary research 34 (2)
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.
KeywordMeSH Terms
101. 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.

Journal of bacteriology 185 (6)
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.
KeywordMeSH Terms
Evolution, Molecular
Genetic Variation
Genome, Bacterial
102. 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.

Journal of bacteriology 185 (6)
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).
KeywordMeSH Terms
DNA Replication
Gene Expression Regulation, Bacterial
103. 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.

Antimicrobial agents and chemotherapy 47 (7)
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.
KeywordMeSH Terms
104. Lim  D,     ( 1992 )

Structure and biosynthesis of unbranched multicopy single-stranded DNA by reverse transcriptase in a clinical Escherichia coli isolate.

Molecular microbiology 6 (23)
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.
KeywordMeSH Terms
105. Rahn  A, Whitfield  C,     ( 2003 )

Transcriptional organization and regulation of the Escherichia coli K30 group 1 capsule biosynthesis (cps) gene cluster.

Molecular microbiology 47 (4)
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.
KeywordMeSH Terms
Escherichia coli Proteins
Genes, Bacterial
Multigene Family
Transcription Factors
106. 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.

Antimicrobial agents and chemotherapy 47 (7)
PMID : 12821505  :   DOI  :   10.1128/aac.47.7.2380-2381.2003     PMC  :   PMC161840    
Abstract >>
N/A
KeywordMeSH Terms
107. 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.

FEMS microbiology letters 77 (1��3��)
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.
KeywordMeSH Terms
Genes, Bacterial
108. 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.

Journal of molecular biology 330 (2)
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.
KeywordMeSH Terms
109. 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.

The Journal of biological chemistry 278 (17)
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.
KeywordMeSH Terms
110. Perreten  V, Boerlin  P,     ( 2003 )

A new sulfonamide resistance gene (sul3) in Escherichia coli is widespread in the pig population of Switzerland.

Antimicrobial agents and chemotherapy 47 (3)
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.
KeywordMeSH Terms
111. 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.

Antimicrobial agents and chemotherapy 47 (3)
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.
KeywordMeSH Terms
112. 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.

Infection and immunity 71 (3)
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.
KeywordMeSH Terms
113. 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).

Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases 3 (2)
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.
KeywordMeSH Terms
Genes, Bacterial
114. 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.

Journal of bacteriology 185 (5)
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.
KeywordMeSH Terms
115. 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.

Journal of clinical microbiology 41 (7)
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.
KeywordMeSH Terms
Bacterial Proteins
Multigene Family
116. Partridge  SR, Hall  RM,     ( 2003 )

In34, a complex In5 family class 1 integron containing orf513 and dfrA10.

Antimicrobial agents and chemotherapy 47 (1)
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.
KeywordMeSH Terms
117. Rodríguez  E, Laviña  M,     ( 2003 )

The proton channel is the minimal structure of ATP synthase necessary and sufficient for microcin h47 antibiotic action.

Antimicrobial agents and chemotherapy 47 (1)
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.
KeywordMeSH Terms
Peptides
Plasmids
118. Blank  TE, Lacher  DW, Scaletsky  IC, Zhong  H, Whittam  TS, Donnenberg  MS,     ( 2003 )

Enteropathogenic Escherichia coli O157 strains from Brazil.

Emerging infectious diseases 9 (1)
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.
KeywordMeSH Terms
119. 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.

Protein science : a publication of the Protein Society 12 (1)
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.
KeywordMeSH Terms
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.

The Journal of antimicrobial chemotherapy 51 (1)
PMID : 12493806  :   DOI  :   10.1093/jac/dkf253    
Abstract >>
N/A
KeywordMeSH Terms
Genes, Bacterial
121. Dozois  CM, Daigle  F, Curtiss  R,     ( 2003 )

Identification of pathogen-specific and conserved genes expressed in vivo by an avian pathogenic Escherichia coli strain.

Proceedings of the National Academy of Sciences of the United States of America 100 (1)
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.
KeywordMeSH Terms
122. 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.

Journal of bacteriology 185 (3)
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.
KeywordMeSH Terms
Conjugation, Genetic
Escherichia coli Proteins
Virulence Factors
123. 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.

Journal of clinical microbiology 40 (12)
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.
KeywordMeSH Terms
Escherichia coli Proteins
Genetic Variation
124. 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.

The Journal of infectious diseases 186 (11)
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.
KeywordMeSH Terms
Disease Models, Animal
Disease Outbreaks
125. 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.

Infection and immunity 70 (12)
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.
KeywordMeSH Terms
Cloning, Molecular
126. 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.

The Journal of biological chemistry 278 (7)
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.
KeywordMeSH Terms
Bacterial Proteins
127. 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.

Infection and immunity 70 (12)
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.
KeywordMeSH Terms
Escherichia coli Proteins
Evolution, Molecular
128. D'Souza  JM, Wang  L, Reeves  P,     ( 2002 )

Sequence of the Escherichia coli O26 O antigen gene cluster and identification of O26 specific genes.

Gene 297 (1��2��)
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.
KeywordMeSH Terms
129. 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.

Infection and immunity 70 (12)
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.
KeywordMeSH Terms
Genes, Bacterial
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.

The Journal of biological chemistry 277 (52)
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.
KeywordMeSH Terms
Mutation
Peptides
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.

The EMBO journal 21 (24)
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.
KeywordMeSH Terms
132. 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.

Journal of applied microbiology 93 (5)
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.
KeywordMeSH Terms
133. Lanz  R, Kuhnert  P, Boerlin  P,     ( 2003 )

Antimicrobial resistance and resistance gene determinants in clinical Escherichia coli from different animal species in Switzerland.

Veterinary microbiology 91 (1)
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.
KeywordMeSH Terms
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.

Genetics 162 (2)
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.
KeywordMeSH Terms
Bacterial Proteins
Biological Evolution
DNA-Binding Proteins
Mutation
Selection, Genetic
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.

Journal of bacteriology 184 (21)
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.
KeywordMeSH Terms
136. 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.

Antimicrobial agents and chemotherapy 46 (10)
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.
KeywordMeSH Terms
Animals, Domestic
137. 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.

Infection and immunity 70 (11)
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.
KeywordMeSH Terms
138. Dai  L, Zimmerly  S,     ( 2002 )

The dispersal of five group II introns among natural populations of Escherichia coli.

RNA (New York, N.Y.) 8 (10)
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.
KeywordMeSH Terms
Introns
139. 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.

Journal of clinical microbiology 40 (10)
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.
KeywordMeSH Terms
140. 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'.

Molecular medicine (Cambridge, Mass.) 8 (7)
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.
KeywordMeSH Terms
Genes, Bacterial
141. 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.

Antimicrobial agents and chemotherapy 46 (11)
PMID : 12384395  :   DOI  :   10.1128/aac.46.11.3688-3689.2002     PMC  :   PMC128722    
Abstract >>
N/A
KeywordMeSH Terms
beta-Lactamase Inhibitors
142. Steiniger-White  M, Bhasin  A, Lovell  S, Rayment  I, Reznikoff  WS,     ( 2002 )

Evidence for "unseen" transposase--DNA contacts.

Journal of molecular biology 322 (5)
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.
KeywordMeSH Terms
Nucleic Acid Conformation
143. 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.

Structure (London, England : 1993) 10 (10)
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.
KeywordMeSH Terms
Plasmids
144. Thanassi  DG, Stathopoulos  C, Dodson  K, Geiger  D, Hultgren  SJ,     ( 2002 )

Bacterial outer membrane ushers contain distinct targeting and assembly domains for pilus biogenesis.

Journal of bacteriology 184 (22)
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.
KeywordMeSH Terms
Escherichia coli Proteins
145. 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.

Journal of bacteriology 184 (19)
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.
KeywordMeSH Terms
146. 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.

Applied and environmental microbiology 68 (10)
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.
KeywordMeSH Terms
Escherichia coli Proteins
147. 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.

Infection and immunity 70 (8)
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.
KeywordMeSH Terms
Escherichia coli Proteins
Fimbriae Proteins
Fimbriae, Bacterial
Multigene Family
Operon
148. 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.

Infection and immunity 70 (8)
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.
KeywordMeSH Terms
Genes, Bacterial
149. 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).

Journal of applied microbiology 93 (2)
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.
KeywordMeSH Terms
Random Amplified Polymorphic DNA Technique
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.

Microbiology (Reading, England) 148 (Pt 8)
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.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
151. 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.

FEBS letters 524 (1��3��)
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.
KeywordMeSH Terms
Operon
152. Brown  EW, Kotewicz  ML, Cebula  TA,     ( 2002 )

Detection of recombination among Salmonella enterica strains using the incongruence length difference test.

Molecular phylogenetics and evolution 24 (1)
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.
KeywordMeSH Terms
DNA-Binding Proteins
Models, Genetic
Recombination, Genetic
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.

Antimicrobial agents and chemotherapy 46 (8)
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.
KeywordMeSH Terms
154. 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).

Antimicrobial agents and chemotherapy 46 (8)
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.
KeywordMeSH Terms
Escherichia coli Proteins
Saccharomyces cerevisiae Proteins
Transcription Factors
155. 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.

FEMS microbiology letters 213 (2)
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.
KeywordMeSH Terms
156. 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.

Molecular genetics and genomics : MGG 267 (5)
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.
KeywordMeSH Terms
Genome, Bacterial
157. Fukushima  M, Kakinuma  K, Kawaguchi  R,     ( 2002 )

Phylogenetic analysis of Salmonella, Shigella, and Escherichia coli strains on the basis of the gyrB gene sequence.

Journal of clinical microbiology 40 (8)
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.
KeywordMeSH Terms
Phylogeny
158. 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.

Molecular genetics and genomics : MGG 266 (5)
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.
KeywordMeSH Terms
Genes, Bacterial
159. Heroven  AK, Dersch  P,     ( 2002 )

Two different open reading frames named slyA in the E. coli sequence databases.

Trends in microbiology 10 (6)
PMID : 12088660  :  
Abstract >>
N/A
KeywordMeSH Terms
Bacterial Proteins
Open Reading Frames
Transcription Factors
160. Campbell  JW, Cronan  JE,     ( 2002 )

The enigmatic Escherichia coli fadE gene is yafH.

Journal of bacteriology 184 (13)
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.
KeywordMeSH Terms
161. 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.

The Journal of antimicrobial chemotherapy 49 (2)
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).
KeywordMeSH Terms
Bacterial Proteins
162. 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.

Journal of bacteriology 184 (13)
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
KeywordMeSH Terms
163. 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.

Journal of clinical microbiology 40 (7)
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.
KeywordMeSH Terms
Escherichia coli Proteins
164. 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.

Molecular microbiology 44 (6)
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.
KeywordMeSH Terms
Phosphoproteins
165. 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.

Molecular microbiology 44 (4)
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.
KeywordMeSH Terms
Escherichia coli Proteins
Topoisomerase II Inhibitors
166. Subbarayan  PR, Deutscher  MP,     ( 2001 )

Escherichia coli RNase M is a multiply altered form of RNase I.

RNA (New York, N.Y.) 7 (12)
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.
KeywordMeSH Terms
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.

Functional & integrative genomics 1 (5)
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.
KeywordMeSH Terms
Escherichia coli Proteins
168. D'Haeze  W, Verplancke  C, Mironov  V, Holsters  M,     ( 2002 )

pMH11, A tool for gene disruption and expression analysis in Azorhizobium caulinodans.

Plasmid 47 (2)
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.
KeywordMeSH Terms
Mutagenesis
169. 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?

Journal of bacteriology 184 (10)
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.
KeywordMeSH Terms
Conjugation, Genetic
Escherichia coli Proteins
Membrane Proteins
170. 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.

Infection and immunity 70 (6)
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.
KeywordMeSH Terms
Disease Outbreaks
DNA, Bacterial
Plasmids
171. 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.

Proceedings of the National Academy of Sciences of the United States of America 99 (10)
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.
KeywordMeSH Terms
Escherichia coli Proteins
172. Brinkkötter  A, Shakeri-Garakani  A, Lengeler  JW,     ( 2002 )

Two class II D-tagatose-bisphosphate aldolases from enteric bacteria.

Archives of microbiology 177 (5)
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.
KeywordMeSH Terms
173. 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.

Infection and immunity 70 (1)
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.
KeywordMeSH Terms
174. 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.

FEMS microbiology letters 209 (2)
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.
KeywordMeSH Terms
Escherichia coli Proteins
Hospitals
175. Tarr  CL, Whittam  TS,     ( 2002 )

Molecular evolution of the intimin gene in O111 clones of pathogenic Escherichia coli.

Journal of bacteriology 184 (2)
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.
KeywordMeSH Terms
Escherichia coli Proteins
Evolution, Molecular
Genes, Bacterial
176. 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.

Research in microbiology 152 (9)
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.
KeywordMeSH Terms
Environmental Microbiology
177. 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.

Environmental and molecular mutagenesis 38 (2��3��)
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.
KeywordMeSH Terms
Biological Evolution
DNA Repair
DNA Replication
Recombination, Genetic
178. Sundin  GW,     ( 2002 )

Distinct recent lineages of the strA- strB streptomycin-resistance genes in clinical and environmental bacteria.

Current microbiology 45 (1)
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.
KeywordMeSH Terms
Genes, Bacterial
179. Partridge  SR, Brown  HJ, Hall  RM,     ( 2002 )

Characterization and movement of the class 1 integron known as Tn2521 and Tn1405.

Antimicrobial agents and chemotherapy 46 (5)
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.
KeywordMeSH Terms
Recombination, Genetic
180. Smeds  A, Hemmann  K, Jakava-Viljanen  M, Pelkonen  S, Imberechts  H, Palva  A,     ( 2001 )

Characterization of the adhesin of Escherichia coli F18 fimbriae.

Infection and immunity 69 (12)
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.
KeywordMeSH Terms
Adhesins, Bacterial
181. 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.

Infection and immunity 69 (12)
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.
KeywordMeSH Terms
182. 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.

Journal of bacteriology 183 (23)
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.
KeywordMeSH Terms
183. 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.

Journal of bacteriology 184 (10)
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.
KeywordMeSH Terms
Multigene Family
184. 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.

Antimicrobial agents and chemotherapy 45 (12)
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.
KeywordMeSH Terms
185. 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.

Applied and environmental microbiology 68 (5)
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.
KeywordMeSH Terms
186. 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.

FEMS microbiology letters 204 (2)
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.
KeywordMeSH Terms
Peptides
187. 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.

The Journal of biological chemistry 277 (24)
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.
KeywordMeSH Terms
188. 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.

Infection and immunity 70 (5)
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.
KeywordMeSH Terms
189. Seah  JN, Kwang  J,     ( 2001 )

Mapping of Escherichia coli H27-specific epitope from H-specific polypeptides.

Clinical and diagnostic laboratory immunology 8 (6)
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.
KeywordMeSH Terms
190. Naumann  TA, Reznikoff  WS,     ( 2002 )

Tn5 transposase active site mutants.

The Journal of biological chemistry 277 (20)
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.
KeywordMeSH Terms
191. Lovell  S, Goryshin  IY, Reznikoff  WR, Rayment  I,     ( 2002 )

Two-metal active site binding of a Tn5 transposase synaptic complex.

Nature structural biology 9 (4)
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.
KeywordMeSH Terms
192. 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.

FEMS microbiology letters 204 (1)
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.
KeywordMeSH Terms
DNA Transposable Elements
193. 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.

Biochemistry 41 (13)
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.
KeywordMeSH Terms
Escherichia coli Proteins
Oxidation-Reduction
194. 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.

Journal of clinical microbiology 40 (4)
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.
KeywordMeSH Terms
Genetic Variation
195. 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.

Biochemistry 40 (43)
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.
KeywordMeSH Terms
Escherichia coli Proteins
196. Tame  JR, Namba  K, Dodson  EJ, Roper  DI,     ( 2002 )

The crystal structure of HpcE, a bifunctional decarboxylase/isomerase with a multifunctional fold.

Biochemistry 41 (9)
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.
KeywordMeSH Terms
197. 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.

Journal of molecular biology 313 (1)
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.
KeywordMeSH Terms
198. 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.

Infection and immunity 69 (11)
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.
KeywordMeSH Terms
199. 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.

Chemistry & biology 9 (2)
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.
KeywordMeSH Terms
Escherichia coli Proteins
200. Pai  H, Jacoby  GA,     ( 2001 )

Sequences of the NPS-1 and TLE-1 beta-lactamase genes.

Antimicrobial agents and chemotherapy 45 (10)
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.
KeywordMeSH Terms
Bacterial Proteins
201. Ishikawa  S, Matsumura  Y, Yoshizako  F, Tsuchido  T,     ( 2002 )

Characterization of a cationic surfactant-resistant mutant isolated spontaneously from Escherichia coli.

Journal of applied microbiology 92 (2)
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.
KeywordMeSH Terms
202. Strader  MB, Smiley  RD, Stinnett  LG, VerBerkmoes  NC, Howell  EE,     ( 2001 )

Role of S65, Q67, I68, and Y69 residues in homotetrameric R67 dihydrofolate reductase.

Biochemistry 40 (38)
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.
KeywordMeSH Terms
203. 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.

Antimicrobial agents and chemotherapy 45 (10)
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.
KeywordMeSH Terms
Bacterial Proteins
204. Wang  L, Qu  W, Reeves  PR,     ( 2001 )

Sequence analysis of four Shigella boydii O-antigen loci: implication for Escherichia coli and Shigella relationships.

Infection and immunity 69 (11)
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.
KeywordMeSH Terms
Bacterial Proteins
Genes, Bacterial
Multigene Family
205. 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.

Infection and immunity 69 (11)
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.
KeywordMeSH Terms
206. 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.

The Journal of infectious diseases 184 (8)
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.
KeywordMeSH Terms
207. 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.

Nucleic acids research 29 (20)
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.
KeywordMeSH Terms
208. 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.

Epidemiology and infection 127 (1)
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.
KeywordMeSH Terms
Genetic Variation
209. 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.

Plasmid 46 (2)
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.
KeywordMeSH Terms
210. 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.

Applied and environmental microbiology 67 (10)
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.
KeywordMeSH Terms
Genetic Variation
Hydrostatic Pressure
211. 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.

Microbiology (Reading, England) 147 (Pt 8)
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.
KeywordMeSH Terms
Adhesins, Bacterial
Carrier Proteins
Escherichia coli Proteins
Genetic Variation
212. Lan  R, Lumb  B, Ryan  D, Reeves  PR,     ( 2001 )

Molecular evolution of large virulence plasmid in Shigella clones and enteroinvasive Escherichia coli.

Infection and immunity 69 (10)
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.
KeywordMeSH Terms
Bacterial Proteins
DNA, Bacterial
Evolution, Molecular
Plasmids
213. Russo  TA, Carlino  UB, Johnson  JR,     ( 2001 )

Identification of a new iron-regulated virulence gene, ireA, in an extraintestinal pathogenic isolate of Escherichia coli.

Infection and immunity 69 (10)
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.
KeywordMeSH Terms
Escherichia coli Proteins
Gene Expression Regulation, Bacterial
214. 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).

The Journal of infectious diseases 184 (6)
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).
KeywordMeSH Terms
Adhesins, Bacterial
Carrier Proteins
Escherichia coli Proteins
Saguinus
215. 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.

The Journal of infectious diseases 184 (7)
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.
KeywordMeSH Terms
Frameshift Mutation
Genes, Bacterial
216. 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.

Journal of clinical microbiology 39 (9)
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.
KeywordMeSH Terms
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.

Infection and immunity 69 (9)
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.
KeywordMeSH Terms
Fimbriae Proteins
Genes, Bacterial
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.

Infection and immunity 69 (9)
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.
KeywordMeSH Terms
Escherichia coli Proteins
219. 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.

Infection and immunity 69 (9)
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.
KeywordMeSH Terms
Escherichia coli Proteins
Gene Transfer, Horizontal
220. 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.

Cell 105 (6)
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.
KeywordMeSH Terms
Fimbriae Proteins
Fimbriae Proteins
221. 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.

FEMS microbiology letters 201 (2)
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.
KeywordMeSH Terms
222. Benz  I, Schmidt  MA,     ( 2001 )

Glycosylation with heptose residues mediated by the aah gene product is essential for adherence of the AIDA-I adhesin.

Molecular microbiology 40 (6)
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.
KeywordMeSH Terms
Escherichia coli Proteins
223. Delgado  MA, Rintoul  MR, Farías  RN, Salomón  RA,     ( 2001 )

Escherichia coli RNA polymerase is the target of the cyclopeptide antibiotic microcin J25.

Journal of bacteriology 183 (15)
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.
KeywordMeSH Terms
Peptides
224. Buetow  L, Flatau  G, Chiu  K, Boquet  P, Ghosh  P,     ( 2001 )

Structure of the Rho-activating domain of Escherichia coli cytotoxic necrotizing factor 1.

Nature structural biology 8 (7)
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.
KeywordMeSH Terms
Escherichia coli Proteins
225. 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.

Antimicrobial agents and chemotherapy 45 (8)
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.
KeywordMeSH Terms
Mutation
226. Kawai  S, Mori  S, Mukai  T, Hashimoto  W, Murata  K,     ( 2001 )

Molecular characterization of Escherichia coli NAD kinase.

European journal of biochemistry 268 (15)
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.
KeywordMeSH Terms
Escherichia coli Proteins
227. 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(-).

Infection and immunity 69 (7)
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(-).
KeywordMeSH Terms
DNA, Bacterial
Escherichia coli Proteins
Periplasmic Proteins
Plasmids
228. 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.

Journal of bacteriology 183 (13)
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.
KeywordMeSH Terms
Adhesins, Escherichia coli
Fimbriae Proteins
229. 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.

Microbiology (Reading, England) 147 (Pt 6)
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.
KeywordMeSH Terms
Amino Acid Transport Systems
Escherichia coli Proteins
Symporters
230. 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.

Infection and immunity 69 (7)
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.
KeywordMeSH Terms
231. 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.

Gene 270 (1��2��)
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.
KeywordMeSH Terms
Antigens, Bacterial
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.

Clinical and diagnostic laboratory immunology 8 (3)
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.
KeywordMeSH Terms
233. Smajs  D, Weinstock  GM,     ( 2001 )

The iron- and temperature-regulated cjrBC genes of Shigella and enteroinvasive Escherichia coli strains code for colicin Js uptake.

Journal of bacteriology 183 (13)
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.
KeywordMeSH Terms
Escherichia coli Proteins
Virulence Factors
234. 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.

Journal of clinical microbiology 39 (5)
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.
KeywordMeSH Terms
235. 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.

Molecular microbiology 39 (5)
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.
KeywordMeSH Terms
Bacterial Adhesion
236. Lee  SH, Jeong  SH, Lee  KJ,     ( 2001 )

Evolution of TEM beta--lactamase genes identified by PCR with newly designed primers in Korean clinical isolates.

Clinical microbiology and infection : the official publication of the European Society of Clinical Microbiology and Infectious Diseases 7 (2)
PMID : 11298153  :  
Abstract >>
N/A
KeywordMeSH Terms
Evolution, Molecular
237. Allen  NL, Hilton  AC, Betts  R, Penn  CW,     ( 2001 )

Use of representational difference analysis to identify Escherichia coli O157-specific DNA sequences.

FEMS microbiology letters 197 (2)
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.
KeywordMeSH Terms
Genome, Bacterial
238. 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.

Journal of clinical microbiology 39 (3)
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.
KeywordMeSH Terms
Adhesins, Bacterial
Carrier Proteins
Escherichia coli Proteins
Simian Immunodeficiency Virus
239. Twining  SS, Goryshin  IY, Bhasin  A, Reznikoff  WS,     ( 2001 )

Functional characterization of arginine 30, lysine 40, and arginine 62 in Tn5 transposase.

The Journal of biological chemistry 276 (25)
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.
KeywordMeSH Terms
240. 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.

Infection and immunity 69 (4)
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.
KeywordMeSH Terms
Chromosome Mapping
Escherichia coli Proteins
241. 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.

Infection and immunity 69 (4)
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.
KeywordMeSH Terms
Alleles
Fimbriae Proteins
242. 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.

Applied and environmental microbiology 67 (4)
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.
KeywordMeSH Terms
243. Stockner  T, Plugariu  C, Koraimann  G, Högenauer  G, Bermel  W, Prytulla  S, Sterk  H,     ( 2001 )

Solution structure of the DNA-binding domain of TraM.

Biochemistry 40 (11)
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.
KeywordMeSH Terms
244. 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.

Applied and environmental microbiology 67 (5)
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.
KeywordMeSH Terms
Escherichia coli Proteins
Mutation
245. Dartigalongue  C, Missiakas  D, Raina  S,     ( 2001 )

Characterization of the Escherichia coli sigma E regulon.

The Journal of biological chemistry 276 (24)
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.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
Transcription, Genetic
246. Elwell  C, Chao  K, Patel  K, Dreyfus  L,     ( 2001 )

Escherichia coli CdtB mediates cytolethal distending toxin cell cycle arrest.

Infection and immunity 69 (5)
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.
KeywordMeSH Terms
247. 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.

Microbiology (Reading, England) 147 (Pt 1)
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.
KeywordMeSH Terms
Adhesins, Bacterial
Antigens, Bacterial
Escherichia coli Proteins
Gene Duplication
248. Jensen  SO, Reeves  PR,     ( 2001 )

Molecular evolution of the GDP-mannose pathway genes (manB and manC) in Salmonella enterica.

Microbiology (Reading, England) 147 (Pt 3)
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.
KeywordMeSH Terms
Evolution, Molecular
249. 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.

Canadian journal of microbiology 46 (12)
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.
KeywordMeSH Terms
Antigens, Bacterial
Escherichia coli Proteins
Fimbriae Proteins
Operon
Regulatory Sequences, Nucleic Acid
250. 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.

Journal of bacteriology 183 (5)
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.
KeywordMeSH Terms
Adenosine Triphosphatases
Alleles
DNA-Binding Proteins
Escherichia coli Proteins
Gene Transfer, Horizontal
Phylogeny
251. 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.

Journal of molecular evolution 52 (2)
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.
KeywordMeSH Terms
Evolution, Molecular
Fimbriae Proteins
Sequence Analysis, DNA
252. 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.

Infection and immunity 69 (2)
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.
KeywordMeSH Terms
Multigene Family
Operon
253. 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.

Antimicrobial agents and chemotherapy 45 (3)
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.
KeywordMeSH Terms
Escherichia coli Proteins
254. 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.

Antimicrobial agents and chemotherapy 45 (3)
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.
KeywordMeSH Terms
255. 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.

Infection and immunity 69 (3)
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.
KeywordMeSH Terms
Escherichia coli Proteins
256. 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.

Antimicrobial agents and chemotherapy 45 (2)
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%.
KeywordMeSH Terms
257. 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.

The Journal of infectious diseases 183 (7)
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.
KeywordMeSH Terms
Escherichia coli Proteins
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.

Nature 409 (6820)
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.
KeywordMeSH Terms
Conjugation, Genetic
Escherichia coli Proteins
259. Mellies  JL, Navarro-Garcia  F, Okeke  I, Frederickson  J, Nataro  JP, Kaper  JB,     ( 2001 )

espC pathogenicity island of enteropathogenic Escherichia coli encodes an enterotoxin.

Infection and immunity 69 (1)
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.
KeywordMeSH Terms
Escherichia coli Proteins
260. 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.

Journal of bacteriology 183 (1)
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.
KeywordMeSH Terms
Escherichia coli Proteins
Recombination, Genetic
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.

Cell 103 (5)
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.
KeywordMeSH Terms
Adenosine Triphosphatases
Base Pair Mismatch
DNA Repair
DNA-Binding Proteins
Escherichia coli Proteins
Evolution, Molecular
262. 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.

The Journal of infectious diseases 183 (1)
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.
KeywordMeSH Terms
Escherichia coli Proteins
263. Chen  CC, Fang  M, Majumder  A, Wu  HY,     ( 2001 )

A 72-base pair AT-rich DNA sequence element functions as a bacterial gene silencer.

The Journal of biological chemistry 276 (12)
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.
KeywordMeSH Terms
Gene Silencing
264. 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.

Journal of clinical microbiology 38 (12)
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.
KeywordMeSH Terms
265. Johnson  JR, Delavari  P, Kuskowski  M, Stell  AL,     ( 2001 )

Phylogenetic distribution of extraintestinal virulence-associated traits in Escherichia coli.

The Journal of infectious diseases 183 (1)
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.
KeywordMeSH Terms
Escherichia coli Proteins
266. Liebert  CA, Watson  AL, Summers  AO,     ( 2000 )

The quality of merC, a module of the mer mosaic.

Journal of molecular evolution 51 (6)
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.
KeywordMeSH Terms
Bacterial Proteins
Cation Transport Proteins
267. 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.

Journal of clinical microbiology 39 (1)
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.
KeywordMeSH Terms
268. 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.

Journal of molecular biology 300 (3)
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.
KeywordMeSH Terms
Escherichia coli Proteins
269. Goryshin  IY, Reznikoff  WS, Davies  DR,     ( 2000 )

Three-dimensional structure of the Tn5 synaptic complex transposition intermediate.

Science (New York, N.Y.) 289 (5476)
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.
KeywordMeSH Terms
DNA Transposable Elements
270. Klöss  H, Brinkkötter  A,     ( 2000 )

Pathways for the utilization of N-acetyl-galactosamine and galactosamine in Escherichia coli.

Molecular microbiology 37 (1)
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.
KeywordMeSH Terms
271. Yethon  JA, Whitfield  C,     ( 2001 )

Purification and characterization of WaaP from Escherichia coli, a lipopolysaccharide kinase essential for outer membrane stability.

The Journal of biological chemistry 276 (8)
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.
KeywordMeSH Terms
272. 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.

Infection and immunity 68 (12)
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.
KeywordMeSH Terms
Escherichia coli Proteins
Fimbriae Proteins
273. 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.

The EMBO journal 19 (22)
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.
KeywordMeSH Terms
Bacterial Proteins
Escherichia coli Proteins
Periplasmic Proteins
274. 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.

The Journal of antimicrobial chemotherapy 46 (1)
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.
KeywordMeSH Terms
DNA Transposable Elements
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.

Microbiology (Reading, England) 146 (Pt 7) (N/A)
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.
KeywordMeSH Terms
276. 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.

Journal of bacteriology 182 (21)
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.
KeywordMeSH Terms
277. 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.

Antimicrobial agents and chemotherapy 44 (7)
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.
KeywordMeSH Terms
Escherichia coli Proteins
278. 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.

Journal of bacteriology 182 (21)
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.
KeywordMeSH Terms
Genome, Bacterial
279. Nataro  JP, Poteet-Smith  CE, Steiner  TS,     ( 2000 )

Enteroaggregative Escherichia coli expresses a novel flagellin that causes IL-8 release from intestinal epithelial cells.

The Journal of clinical investigation 105 (12)
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.
KeywordMeSH Terms
280. Herbelin  CJ, Bumbaugh  AC, Reid  SD,     ( 2000 )

Parallel evolution of virulence in pathogenic Escherichia coli.

Nature 406 (6791)
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.
KeywordMeSH Terms
Evolution, Molecular
281. Knoechel  DG, Haynes  CA, Creagh  AL, Pfuetzner  RA, Frey  EA, Luo  Y,     ( 2000 )

Crystal structure of enteropathogenic Escherichia coli intimin-receptor complex.

Nature 405 (6790)
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.
KeywordMeSH Terms
Adhesins, Bacterial
Carrier Proteins
Escherichia coli Proteins
282. 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.

Journal of bacteriology 182 (14)
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.
KeywordMeSH Terms
Adhesins, Escherichia coli
Fimbriae Proteins
283. Gubbins  MJ, Ghetu  AF,     ( 2000 )

Crystal structure of the bacterial conjugation repressor finO.

Nature structural biology 7 (7)
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.
KeywordMeSH Terms
Escherichia coli Proteins
284. Mihara  H, Kurihara  T, Lacourciere  GM,     ( 2000 )

Escherichia coli NifS-like proteins provide selenium in the pathway for the biosynthesis of selenophosphate.

The Journal of biological chemistry 275 (31)
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.
KeywordMeSH Terms
Drosophila Proteins
285. 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.

Biometals : an international journal on the role of metal ions in biology, biochemistry, and medicine 13 (2)
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.
KeywordMeSH Terms
Sequence Analysis, DNA
286. Huang  SH, Stins  MF, Kim  KS,     ( 2000 )

Bacterial penetration across the blood-brain barrier during the development of neonatal meningitis.

Microbes and infection 2 (10)
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.
KeywordMeSH Terms
Blood-Brain Barrier
287. Liesegang  A, Tschäpe  H, Bielaszewska  M, Zhang  WL,     ( 2000 )

Molecular characteristics and epidemiological significance of Shiga toxin-producing Escherichia coli O26 strains.

Journal of clinical microbiology 38 (6)
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.
KeywordMeSH Terms
O Antigens
288. Elwell  CA, Dreyfus  LA,     ( 2000 )

DNase I homologous residues in CdtB are critical for cytolethal distending toxin-mediated cell cycle arrest.

Molecular microbiology 37 (4)
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.
KeywordMeSH Terms
Cell Cycle
289. 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.

Antimicrobial agents and chemotherapy 44 (10)
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.
KeywordMeSH Terms
290. 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.

Antimicrobial agents and chemotherapy 44 (10)
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.
KeywordMeSH Terms
Bacterial Proteins
291. Fang  M, Majumder  A, Tsai  KJ, Wu  HY,     ( 2000 )

ppGpp-dependent leuO expression in bacteria under stress.

Biochemical and biophysical research communications 276 (1)
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.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
292. 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.

Journal of bacteriology 123 (3)
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.
KeywordMeSH Terms
293. 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.

Infection and immunity 68 (10)
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.
KeywordMeSH Terms
DNA Transposable Elements
294. Herbelin  CJ, Chirillo  SC, Melnick  KA, Whittam  TS,     ( 2000 )

Gene conservation and loss in the mutS-rpoS genomic region of pathogenic Escherichia coli.

Journal of bacteriology 182 (19)
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.
KeywordMeSH Terms
Adenosine Triphosphatases
Conserved Sequence
DNA-Binding Proteins
Escherichia coli Proteins
Genes, Bacterial
295. 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.

Infection and immunity 68 (7)
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.
KeywordMeSH Terms
296. Seah  JN, Kwang  J,     ( N/A )

Identification of H-specific determinants in flagellin of four Escherichia coli strains.

Archives of microbiology 174 (1��2��)
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.
KeywordMeSH Terms
297. 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.

FEBS letters 479 (3)
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.
KeywordMeSH Terms
Adhesins, Bacterial
298. 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.

Antimicrobial agents and chemotherapy 44 (9)
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.
KeywordMeSH Terms
Bacterial Proteins
299. 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.

Infection and immunity 68 (9)
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.
KeywordMeSH Terms
300. Pupo  GM, Lan  R, Reeves  PR,     ( 2000 )

Multiple independent origins of Shigella clones of Escherichia coli and convergent evolution of many of their characteristics.

Proceedings of the National Academy of Sciences of the United States of America 97 (19)
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.
KeywordMeSH Terms
Evolution, Molecular
301. Dychinco  B, Mazel  D,     ( 2000 )

Antibiotic resistance in the ECOR collection: integrons and identification of a novel aad gene.

Antimicrobial agents and chemotherapy 44 (6)
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.
KeywordMeSH Terms
Escherichia coli Proteins
Genes, Bacterial
302. 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.

Journal of clinical microbiology 38 (5)
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.
KeywordMeSH Terms
Genetic Variation
Polymorphism, Genetic
303. 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.

Antimicrobial agents and chemotherapy 44 (6)
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.
KeywordMeSH Terms
304. Rosche  TM, Kim  PD,     ( 2000 )

Identification of a potential membrane-targeting region of the replication initiator protein (TrfA) of broad-host-range plasmid RK2.

Plasmid 43 (3)
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.
KeywordMeSH Terms
Escherichia coli Proteins
305. 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).

Journal of bacteriology 182 (18)
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.
KeywordMeSH Terms
Escherichia coli Proteins
Gene Expression Regulation, Bacterial
Transcription, Genetic
306. 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.

Infection and immunity 68 (9)
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.
KeywordMeSH Terms
Genes, Bacterial
307. 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.

Infection and immunity 68 (6)
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.
KeywordMeSH Terms
Fimbriae Proteins
308. Binsztein  N, Pichel  M,     ( 2000 )

CS22, a novel human enterotoxigenic Escherichia coli adhesin, is related to CS15.

Infection and immunity 68 (6)
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.
KeywordMeSH Terms
Enterotoxins
Escherichia coli Proteins
Fimbriae Proteins
309. 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.

Journal of biochemistry 127 (4)
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.
KeywordMeSH Terms
310. 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.

Journal of bacteriology 182 (9)
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.
KeywordMeSH Terms
311. 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.

Infection and immunity 68 (5)
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.
KeywordMeSH Terms
Escherichia coli Proteins
Genes, Bacterial
312. Pfaff-McDonough  SJ, Horne  SM,     ( N/A )

Cloning and sequencing of the iss gene from a virulent avian Escherichia coli.

Avian diseases 44 (1)
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.
KeywordMeSH Terms
Escherichia coli Proteins
313. Miyahara  M,     ( 1999 )

Escherichia coli O157 strains which caused Japanese outbreaks have residues of bacteriophage sequences.

Biological & pharmaceutical bulletin 22 (12)
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.
KeywordMeSH Terms
Disease Outbreaks
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.

Structure (London, England : 1993) 8 (3)
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.
KeywordMeSH Terms
315. 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.

Applied and environmental microbiology 66 (4)
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.
KeywordMeSH Terms
Genetic Variation
316. 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.

Journal of clinical microbiology 38 (4)
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.
KeywordMeSH Terms
Antigens, Bacterial
Escherichia coli Proteins
Fimbriae Proteins
317. 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.

Infection and immunity 68 (4)
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.
KeywordMeSH Terms
Adhesins, Bacterial
Carrier Proteins
Escherichia coli Proteins
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.

Journal of molecular biology 297 (1)
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.
KeywordMeSH Terms
Escherichia coli Proteins
319. McNamara  BP, Lai  LC, Malstrom  C, Scaletsky  IC, Klapproth  JM,     ( 2000 )

A large toxin from pathogenic Escherichia coli strains that inhibits lymphocyte activation.

Infection and immunity 68 (4)
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.
KeywordMeSH Terms
Escherichia coli Proteins
Lymphocyte Activation
320. Aguilar  C, Ayala  G, Estrada  MA, Silva  J,     ( 2000 )

TLA-1: a new plasmid-mediated extended-spectrum beta-lactamase from Escherichia coli.

Antimicrobial agents and chemotherapy 44 (4)
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.
KeywordMeSH Terms
321. Xun  L,     ( 2000 )

Characterization of 4-hydroxyphenylacetate 3-hydroxylase (HpaB) of Escherichia coli as a reduced flavin adenine dinucleotide-utilizing monooxygenase.

Applied and environmental microbiology 66 (2)
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.
KeywordMeSH Terms
322. 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.

Biological chemistry 380 (12)
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.
KeywordMeSH Terms
Plasmids
323. 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.

Microbial pathogenesis 28 (3)
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.
KeywordMeSH Terms
Escherichia coli Proteins
324. Morabito  S, Caprioli  A, Scheef  J, Schmidt  H,     ( 2000 )

A new Shiga toxin 2 variant (Stx2f) from Escherichia coli isolated from pigeons.

Applied and environmental microbiology 66 (3)
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.
KeywordMeSH Terms
325. Kim  PD,     ( 2000 )

Isolation of an inner membrane-derived subfraction that supports in vitro replication of a mini-RK2 plasmid in Escherichia coli.

Journal of bacteriology 182 (6)
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).
KeywordMeSH Terms
DNA Replication
Escherichia coli Proteins
326. Sunde  M,     ( 1999 )

Characterization of integrons in Escherichia coli of the normal intestinal flora of swine.

Microbial drug resistance (Larchmont, N.Y.) 5 (4)
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.
KeywordMeSH Terms
327. 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.

Molecular microbiology 35 (2)
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.
KeywordMeSH Terms
Bacterial Toxins
Escherichia coli Proteins
328. McIver  CJ, White  PA,     ( 2000 )

Characterisation of two new gene cassettes, aadA5 and dfrA17.

FEMS microbiology letters 182 (2)
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.
KeywordMeSH Terms
DNA Transposable Elements
329. Wang  Y,     ( 2000 )

Effect of rpoS mutations on stress-resistance and invasion of brain microvascular endothelial cells in Escherichia coli K1.

FEMS microbiology letters 182 (2)
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.
KeywordMeSH Terms
Mutation
330. 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.

Infection and immunity 68 (3)
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.
KeywordMeSH Terms
Alleles
Escherichia coli Proteins
Polymerase Chain Reaction
331. Schnappinger  D, Hillen  W, Orth  P,     ( 2000 )

Structural basis of gene regulation by the tetracycline inducible Tet repressor-operator system.

Nature structural biology 7 (3)
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.
KeywordMeSH Terms
332. 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.

Journal of bacteriology 182 (3)
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.
KeywordMeSH Terms
333. Thomson  CJ, Adrian  PV,     ( 2000 )

New gene cassettes for trimethoprim resistance, dfr13, and Streptomycin-spectinomycin resistance, aadA4, inserted on a class 1 integron.

Antimicrobial agents and chemotherapy 44 (2)
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.
KeywordMeSH Terms
Escherichia coli Proteins
334. 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.

Folia microbiologica 44 (3)
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.
KeywordMeSH Terms
335. 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.

Infection and immunity 68 (1)
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.
KeywordMeSH Terms
Adhesins, Bacterial
Carrier Proteins
Escherichia coli Proteins
Genes, Bacterial
Genetic Variation
336. Sandvang  D,     ( 1999 )

Novel streptomycin and spectinomycin resistance gene as a gene cassette within a class 1 integron isolated from Escherichia coli.

Antimicrobial agents and chemotherapy 43 (12)
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.
KeywordMeSH Terms
337. Li  B, LeClerc  JE,     ( 1999 )

Promiscuous origin of a chimeric sequence in the Escherichia coli O157:H7 genome.

Journal of bacteriology 181 (24)
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.
KeywordMeSH Terms
Adenosine Triphosphatases
DNA-Binding Proteins
Escherichia coli Proteins
Genome, Bacterial
338. Bradford  PA,     ( 1999 )

Automated thermal cycling is superior to traditional methods for nucleotide sequencing of bla(SHV) genes.

Antimicrobial agents and chemotherapy 43 (12)
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.
KeywordMeSH Terms
Escherichia coli Proteins
339. Kuhar  I,     ( 1999 )

Transcription regulation of the colicin K cka gene reveals induction of colicin synthesis by differential responses to environmental signals.

Journal of bacteriology 181 (23)
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.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
340. Stuchlík  S, Burian  J,     ( 1999 )

Replication control of a small cryptic plasmid of Escherichia coli.

Journal of molecular biology 294 (1)
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.
KeywordMeSH Terms
DNA Helicases
DNA Replication
Trans-Activators
341. Wang  SY, Lai  XH,     ( 1999 )

Expression of cytotoxicity by potential pathogens in the standard Escherichia coli collection of reference (ECOR) strains.

Microbiology (Reading, England) 145 (Pt 11) (N/A)
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.
KeywordMeSH Terms
Escherichia coli Proteins
342. 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.

Microbiology and immunology 43 (9)
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.
KeywordMeSH Terms
Endopeptidases
Fimbriae Proteins
Genes, Bacterial
343. Caprioli  A, Huter  V, Zanial  M, Knutton  S, Batchelor  M,     ( 1999 )

Development of a universal intimin antiserum and PCR primers.

Journal of clinical microbiology 37 (12)
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.
KeywordMeSH Terms
Adhesins, Bacterial
Carrier Proteins
DNA Primers
Escherichia coli Proteins
344. Zhao  D, Li  C,     ( 1999 )

Crystal structure of colicin E3 immunity protein: an inhibitor of a ribosome-inactivating RNase.

Structure (London, England : 1993) 7 (11)
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.
KeywordMeSH Terms
Colicins
Escherichia coli Proteins
345. Jose  J, Maurer  J,     ( 1999 )

Characterization of the essential transport function of the AIDA-I autotransporter and evidence supporting structural predictions.

Journal of bacteriology 181 (22)
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.
KeywordMeSH Terms
346. Hall  RM, Liebert  CA,     ( 1999 )

Transposon Tn21, flagship of the floating genome.

Microbiology and molecular biology reviews : MMBR 63 (3)
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.
KeywordMeSH Terms
Genome, Bacterial
347. Tsuda  J, Kato  T, Kita  K,     ( 1999 )

Evidence of horizontal transfer of the EcoO109I restriction-modification gene to Escherichia coli chromosomal DNA.

Journal of bacteriology 181 (21)
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.
KeywordMeSH Terms
Genes, Bacterial
348. Staendner  LH, Manning  PA, Duthy  TG,     ( 1999 )

CS5 pilus biosynthesis genes from enterotoxigenic Escherichia coli O115:H40.

Journal of bacteriology 181 (18)
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.
KeywordMeSH Terms
Genes, Bacterial
Multigene Family
349. 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.

DNA sequence : the journal of DNA sequencing and mapping 9 (3)
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.
KeywordMeSH Terms
DNA Transposable Elements
Escherichia coli Proteins
Genes, Bacterial
Operon
350. 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.

Infection and immunity 67 (11)
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.
KeywordMeSH Terms
Bacterial Proteins
351. Paton  AW,     ( 1999 )

Molecular characterization of the locus encoding biosynthesis of the lipopolysaccharide O antigen of Escherichia coli serotype O113.

Infection and immunity 67 (11)
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.
KeywordMeSH Terms
Chromosome Mapping
352. Feldman  MF, Marolda  CL,     ( 1999 )

Genetic organization of the O7-specific lipopolysaccharide biosynthesis cluster of Escherichia coli VW187 (O7:K1).

Microbiology (Reading, England) 145 (Pt 9) (N/A)
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.
KeywordMeSH Terms
Bacterial Proteins
353. Rakin  A, Fischer  D, Schubert  S,     ( 1999 )

Characterization of the integration site of Yersinia high-pathogenicity island in Escherichia coli.

FEMS microbiology letters 179 (2)
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.
KeywordMeSH Terms
Genes, Bacterial
354. Czeczulin  J, Eslava  C, Henderson  IR,     ( 1999 )

Characterization of pic, a secreted protease of Shigella flexneri and enteroaggregative Escherichia coli.

Infection and immunity 67 (11)
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.
KeywordMeSH Terms
355. Althorpe  NJ, Roscoe  RA, Bates  S,     ( 1999 )

Expression of leading region genes on IncI1 plasmid ColIb-P9: genetic evidence for single-stranded DNA transcription.

Microbiology (Reading, England) 145 (Pt 10) (N/A)
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.
KeywordMeSH Terms
DNA-Binding Proteins
Escherichia coli Proteins
Gene Expression Regulation, Bacterial
356. Carlino  UB, Russo  TA,     ( 1999 )

Identification of genes in an extraintestinal isolate of Escherichia coli with increased expression after exposure to human urine.

Infection and immunity 67 (10)
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.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
Genes, Bacterial
357. 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.

Microbiology (Reading, England) 145 (Pt 7) (N/A)
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.
KeywordMeSH Terms
Bacterial Toxins
Escherichia coli Proteins
Fimbriae Proteins
Genes, Bacterial
358. Kim  KS, Huang  SH, Wass  CA, Stins  MF,     ( 1999 )

The gene locus yijP contributes to Escherichia coli K1 invasion of brain microvascular endothelial cells.

Infection and immunity 67 (9)
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.
KeywordMeSH Terms
Escherichia coli Proteins
Membrane Proteins
359. Lengyel  Z, Tsunekawa  H,     ( 1999 )

Cloning, sequencing, and expression of cscA invertase from Escherichia coli B-62.

Canadian journal of microbiology 45 (5)
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.
KeywordMeSH Terms
360. 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.

Biochemistry 38 (29)
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.
KeywordMeSH Terms
Acyltransferases
Conserved Sequence
Escherichia coli Proteins
Mutagenesis, Site-Directed
361. 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.

Biochemistry 38 (30)
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.
KeywordMeSH Terms
beta-Lactamase Inhibitors
362. 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.

Infection and immunity 67 (10)
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.
KeywordMeSH Terms
Genes, Bacterial
Multigene Family
363. 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.

Research in microbiology 150 (5)
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.
KeywordMeSH Terms
Adhesins, Bacterial
Carrier Proteins
Escherichia coli Proteins
Genetic Variation
364. 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.

Nucleic acids research 27 (17)
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.
KeywordMeSH Terms
Mutation
365. 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.

Molecular microbiology 33 (4)
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.
KeywordMeSH Terms
Adhesins, Bacterial
Carrier Proteins
Escherichia coli Proteins
366. Gaggero  C, Laviña  M,     ( 1999 )

The structural gene for microcin H47 encodes a peptide precursor with antibiotic activity.

Antimicrobial agents and chemotherapy 43 (9)
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.
KeywordMeSH Terms
Peptides
367. Waksman  G, Hultgren  SJ, Dodson  KW, Fütterer  K, Pinkner  JS,     ( 1999 )

Structural basis of chaperone function and pilus biogenesis.

Science (New York, N.Y.) 285 (5430)
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.
KeywordMeSH Terms
Escherichia coli Proteins
Periplasmic Proteins
368. 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.

FEMS microbiology letters 179 (1)
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.
KeywordMeSH Terms
Gene Deletion
Operon
369. 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.

FEMS microbiology letters 175 (2)
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.
KeywordMeSH Terms
DNA Transposable Elements
Escherichia coli Proteins
370. 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.

Biochemistry 38 (27)
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 opti