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1. Tomoyasu  T, Takaya  A, Sasaki  T, Nagase  T, Kikuno  R, Morioka  M, Yamamoto  T,     ( 2003 )

A new heat shock gene, AgsA, which encodes a small chaperone involved in suppressing protein aggregation in Salmonella enterica serovar typhimurium.

Journal of bacteriology 185 (21)
PMID : 14563868  :   DOI  :   10.1128/jb.185.21.6331-6339.2003     PMC  :   PMC219406    
Abstract >>
We discovered a novel small heat shock protein (sHsp) named AgsA (aggregation-suppressing protein) in the thermally aggregated fraction from a Salmonella enterica serovar Typhimurium dnaK-null strain. The -10 and -35 regions upstream of the transcriptional start site of the agsA gene are characteristic of sigma(32)- and sigma(72)-dependent promoters. AgsA was strongly induced by high temperatures. The similarity between AgsA and the other two sHsps of Salmonella serovar Typhimurium, IbpA and IbpB, is rather low (around 30% amino acid sequence identity). Phylogenetic analysis suggested that AgsA arose from an ancient gene duplication or amplification at an early evolutionary stage of gram-negative bacteria. Here we show that overproduction of AgsA partially complements the DeltadnaK52 thermosensitive phenotype and reduces the amount of heat-aggregated proteins in both DeltadnaK52 and DeltarpoH mutants of Escherichia coli. These data suggest that AgsA is an effective chaperone capable of preventing aggregation of nonnative proteins and maintaining them in a state competent for refolding in Salmonella serovar Typhimurium at high temperatures.
KeywordMeSH Terms
Genes, Bacterial
2. Peng  CF, Chang  SF,     ( 1992 )

Molecular cloning and nucleotide sequencing of a novel aminoglycoside 6'-N-acetyltransferase gene from an R-plasmid of Salmonella typhimurium S24 isolated in Taiwan.

Microbiology and immunology 36 (4)
PMID : 1406363  :   DOI  :   10.1111/j.1348-0421.1992.tb02033.x    
Abstract >>
A conjugative aminoglycoside resistance plasmid pST2 has been isolated from Escherichia coli K-12 14R525, which was mated with a clinical isolate of Salmonella typhimurium S24. A novel resistance gene of aminoglycoside 6'-N-acetyltransferase[AAC(6')] was cloned from plasmid pST2 on a 1,393 kilobase (kb) of SphI-SalI fragment into vector pACYC184 and pUC18. This novel AAC(6') gene in plasmid pST2 acetylated kanamycin, amikacin, dibekacin, tobramycin, gentamicin, netilmicin, and sisomicin. The complete nucleotide sequence of the novel AAC(6') gene and its neighboring sequences were also determined. Minicell experiments detected only one protein of 24.7 kilodaltons (kDa) translated from an open reading frame of the 618 base pairs (bp) gene.
KeywordMeSH Terms
3. 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
4. Ohnishi  K, Kutsukake  K, Suzuki  H, Lino  T,     ( 1992 )

A novel transcriptional regulation mechanism in the flagellar regulon of Salmonella typhimurium: an antisigma factor inhibits the activity of the flagellum-specific sigma factor, sigma F.

Molecular microbiology 6 (21)
PMID : 1453955  :   DOI  :   10.1111/j.1365-2958.1992.tb01771.x    
Abstract >>
We have studied the molecular mechanism of the negative regulation by flgM of the late operons of the flagellar regulon of Salmonella typhimurium. A 7.8 kDa protein that was identified as the flgM gene product was purified to homogeneity; its amino-terminal sequence was identical to the deduced sequence except for the lack of the initiating methionine. The purified FlgM repressed transcription from the fliC promoter, one that is activated by the sigma factor, FliA (sigma F). No DNA-binding activity was detected in FlgM. Chemical cross-linking experiments showed that the purified FlgM bound to sigma F and disturbed its ability to form a complex with RNA polymerase core enzyme. These results indicate that FlgM is a novel type of negative regulator that probably inactivates the flagellum-specific sigma factor through direct interaction, i.e. it is an anti-sigma factor.
KeywordMeSH Terms
Bacterial Proteins
5. Makde  RD, Kumar  V, Gupta  GD, Jasti  J, Singh  TP, Mahajan  SK,     ( 2003 )

Expression, purification, crystallization and preliminary X-ray diffraction studies of recombinant class B non-specific acid phosphatase of Salmonella typhimurium.

Acta crystallographica. Section D, Biological crystallography 59 (Pt 10)
PMID : 14501135  :   DOI  :   10.1107/s0907444903018006    
Abstract >>
The aphA gene of Salmonella enterica sv. Typhimurium strain MD6001 was cloned in the multicopy plasmid pBluescript SK(-). The recombinant AphA protein was purified to homogeneity. The protein crystallized in the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 112.4, b = 130.2, c = 139.6 A. Consistent with the self-rotation function, there are two tetramers in the asymmetric unit, indicating a solvent content of approximately 54%. The crystals are composed of biologically active AphA molecules.
KeywordMeSH Terms
6. Gulig  PA, Caldwell  AL, Chiodo  VA,     ( 1992 )

Identification, genetic analysis and DNA sequence of a 7.8-kb virulence region of the Salmonella typhimurium virulence plasmid.

Molecular microbiology 6 (10)
PMID : 1322485  :   DOI  :   10.1111/j.1365-2958.1992.tb00860.x    
Abstract >>
The 90-kilobase (kb) virulence plasmid of Salmonella typhimurium is responsible for invasion from the intestines to mesenteric lymph nodes and spleens of orally inoculated mice. We used Tn5 and aminoglycoside phosphotransferase (aph) gene insertion mutagenesis and deletion mutagenesis of a previously identified 14-kb virulence region to reduce this virulence region to 7.8kb. The 7.8-kb virulence region subcloned into a low copy-number vector conferred a wild-type level of splenic infection to virulence plasmid-cured S. typhimurium and conferred essentially a wild-type oral LD50. Insertion mutagenesis identified five loci essential for virulence, and DNA sequence analysis of the virulence region identified six open reading frames. Expected protein products were identified from four of the six genes, with three of the proteins identified as doublet bands in Escherichia coli minicells. Three of the five mutated genes were able to be complemented by clones containing only the corresponding wild-type gene. Only one of the five deduced amino acid sequences, that of the positive regulatory element, SpvR, possessed significant homology to other proteins. The codon usage for the virulence genes showed no codon bias, which is consistent with the low levels of expression observed for the corresponding proteins. Consensus promoters for several different sigma factors were identified upstream of several of the genes, whereas only consensus Rho-dependent termination sequences were observed between certain of the genes. The operon structure of this virulence region therefore appears to be complex. The construction of the cloned 7.8-kb virulence region and the determination of the DNA sequence will aid in the further genetic analysis of the five plasmid-encoded virulence genes of S. typhimurium.
KeywordMeSH Terms
7. 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
8. Faldynova  M, Pravcova  M, Sisak  F, Havlickova  H, Kolackova  I, Cizek  A, Karpiskova  R, Rychlik  I,     ( 2003 )

Evolution of antibiotic resistance in Salmonella enterica serovar typhimurium strains isolated in the Czech Republic between 1984 and 2002.

Antimicrobial agents and chemotherapy 47 (6)
PMID : 12760885  :   DOI  :   10.1128/aac.47.6.2002-2005.2003     PMC  :   PMC155862    
Abstract >>
In a collection of 66 Salmonella enterica serovar Typhimurium strains isolated between 1984 and 2002 in the Czech Republic, genes coding for antibiotic resistance were determined by using specific PCRs. We found that the pentadrug-resistant ACSSuT clone first appeared in the Czech Republic in 1990. A new variant of the aadA gene designated aadA21 is described, the 5' end of which was identical to aadA2 and the 3' end of which was identical to aadA1.
KeywordMeSH Terms
9. Cannon  PM, Strike  P,     ( 1992 )

Complete nucleotide sequence and gene organization of plasmid NTP16.

Plasmid 27 (3)
PMID : 1325061  :  
Abstract >>
We have determined the complete nucleotide sequence of the 8.3-kb multicopy plasmid NTP16 and produced a functional map of its gene organization. Sixty percent of the plasmid DNA comprises transposon-derived sequences; in the remaining 3320 bp, we have identified three protein coding regions. NTP16 has a ColE1-type replication system, a cis-acting stability locus and a mobilization system comprising an oriT site and one mobilization protein. The roles of the other two protein products of this plasmid are unknown, but they are possibly involved in the plasmid incompatibility system.
KeywordMeSH Terms
DNA Transposable Elements
Plasmids
10. Belogurov  AA, Delver  EP, Rodzevich  OV,     ( 1992 )

IncN plasmid pKM101 and IncI1 plasmid ColIb-P9 encode homologous antirestriction proteins in their leading regions.

Journal of bacteriology 174 (15)
PMID : 1321121  :   DOI  :   10.1128/jb.174.15.5079-5085.1992     PMC  :   PMC206324    
Abstract >>
The IncN plasmid pKM101 (a derivative of R46), like the IncI1 plasmid ColIb-P9, carries a gene (ardA, for alleviation of restriction of DNA) encoding an antirestriction function. ardA was located about 4 kb from the origin of transfer, in the region transferred early during bacterial conjugation. The nucleotide sequence of ardA was determined, and an appropriate polypeptide with the predicted molecular weight of about 19,500 was identified in maxicells of Escherichia coli. Comparison of the deduced amino acid sequences of the antirestriction proteins of the unrelated plasmids pKM101 and ColIb (ArdA and Ard, respectively) revealed that these proteins have about 60% identity. Like ColIb Ard, pKM101 ArdA specifically inhibits both the restriction and modification activities of five type I systems of E. coli tested and does not influence type III (EcoP1) restriction or the 5-methylcytosine-specific restriction systems McrA and McrB. However, in contrast to ColIb Ard, pKM101 ArdA is effective against the type II enzyme EcoRI. The Ard proteins are believed to overcome the host restriction barrier during bacterial conjugation. We have also identified two other genes of pKM101, ardR and ardK, which seem to control ardA activity and ardA-mediated lethality, respectively. Our findings suggest that ardR may serve as a genetic switch that determines whether the ardA-encoded antirestriction function is induced during mating.
KeywordMeSH Terms
Genes, Bacterial
Plasmids
11. Warner  TG, Harris  R, McDowell  R, Vimr  ER,     ( 1992 )

Photolabelling of Salmonella typhimurium LT2 sialidase. Identification of a peptide with a predicted structural similarity to the active sites of influenza-virus sialidases.

The Biochemical journal 285 (Pt 3) (N/A)
PMID : 1295492  :   DOI  :   10.1042/bj2850957     PMC  :   PMC1132888    
Abstract >>
The sialidase from Salmonella typhimurium LT2 was characterized by using photoaffinity-labelling techniques. The well-known sialidase inhibitor 5-acetamido-2,6-anhydro-3,5-dideoxy-D-glycero-D-galacto-non- 2-enonic acid (Neu5Ac2en) was modified to contain an amino group at C-9, which permitted the incorporation of 4-azidosalicylic acid in amide linkage at this position. Labelling of the purified protein with the radioactive (125I) photoprobe was determined to be highly specific for a region within the active-site cavity. This conclusion was based on the observation that the competitive inhibitor Neu5Ac2en in the photolysis mixture prevented labelling of the protein. In contrast, compounds with structural and chemical features similar to the probe and Neu5Ac2en, but which were not competitive enzyme inhibitors, did not affect the photolabelling of the protein. The peptide interacting with the probe was identified by CNBr treatment of the labelled protein, followed by N-terminal sequence analysis. Inspection of the primary structure of the protein, predicted from the cloned structural gene for the sialidase [Hoyer, Hamilton, Steenbergen & Vimr (1992) Mol. Microbiol. 6, 873-884] revealed that the label was incorporated into a 9.6 kDa fragment situated within the terminal third of the molecule near the C-terminal end. Secondary-structural predictions using the Garnier-Robson algorithm [Garnier, Osguthorpe & Robson (1978) J. Mol. Biol. 120, 97-120] of the labelled peptide revealed a structural similarity to the active site of influenza-A- and Sendai-HN-virus sialidases with a repetitive series of alternating beta-sheets connected with loops.
KeywordMeSH Terms
Affinity Labels
12. Schlumberger  MC, Friebel  A, Buchwald  G, Scheffzek  K, Wittinghofer  A, Hardt  WD,     ( 2003 )

Amino acids of the bacterial toxin SopE involved in G nucleotide exchange on Cdc42.

The Journal of biological chemistry 278 (29)
PMID : 12719429  :   DOI  :   10.1074/jbc.M302475200    
Abstract >>
RhoGTPases are central switches in all eukaryotic cells. There are at least two known families of guanine nucleotide exchange factors that can activate RhoGTPases: the Dbl-like eukaryotic G nucleotide exchange factors and the SopE-like toxins of pathogenic bacteria, which are injected into host cells to manipulate signaling. Both families have strikingly different sequences, structures, and catalytic core elements. This suggests that they have emerged by convergent evolution. Nevertheless, both families of G nucleotide exchange factors also share some similarities: (a) both rearrange the G nucleotide binding site of RhoGTPases into virtually identical conformations, and (b) two SopE residues (Gln-109SopE and Asp-124SopE) engage Cdc42 in a similar way as equivalent residues of Dbl-like G nucleotide exchange factors (i.e. Asn-810Dbs and Glu-639Dbs). The functional importance of these observations has remained unclear. Here, we have analyzed the effect of amino acid substitutions at selected SopE residues implicated in catalysis (Asp-124SopE, Gln-109SopE, Asp-103SopE, Lys-198SopE, and Gly-168SopE) on in vitro catalysis of G nucleotide release from Cdc42 and on in vivo activity. Substitutions at Asp-124SopE, Gln-109SopE, and Gly-168SopE severely reduced the SopE activity. Slight defects were observed with Asp-103SopE variants, whereas Lys-198SopE was not found to be required in vitro or in vivo. Our results demonstrate that G nucleotide exchange by SopE involves both catalytic elements unique to the SopE family (i.e. 166GAGA169 loop, Asp-103SopE) and amino acid contacts resembling those of key residues of Dbl-like guanine nucleotide exchange factors. Therefore, besides all of the differences, the catalytic mechanisms of the SopE and the Dbl families share some key functional aspects.
KeywordMeSH Terms
13. Pelludat  C, Mirold  S, Hardt  WD,     ( 2003 )

The SopEPhi phage integrates into the ssrA gene of Salmonella enterica serovar Typhimurium A36 and is closely related to the Fels-2 prophage.

Journal of bacteriology 185 (17)
PMID : 12923091  :   DOI  :   10.1128/jb.185.17.5182-5191.2003     PMC  :   PMC181011    
Abstract >>
Salmonella spp. are enteropathogenic gram-negative bacteria that use a large array of virulence factors to colonize the host, manipulate host cells, and resist the host's defense mechanisms. Even closely related Salmonella strains have different repertoires of virulence factors. Bacteriophages contribute substantially to this diversity. There is increasing evidence that the reassortment of virulence factor repertoires by converting phages like the GIFSY phages and SopEPhi may represent an important mechanism in the adaptation of Salmonella spp. to specific hosts and to the emergence of new epidemic strains. Here, we have analyzed in more detail SopEPhi, a P2-like phage from Salmonella enterica serovar Typhimurium DT204 that encodes the virulence factor SopE. We have cloned and characterized the attachment site (att) of SopEPhi and found that its 47-bp core sequence overlaps the 3' terminus of the ssrA gene of serovar Typhimurium. Furthermore, we have demonstrated integration of SopEPhi into the cloned attB site of serovar Typhimurium A36. Sequence analysis of the plasmid-borne prophage revealed that SopEPhi is closely related to (60 to 100% identity over 80% of the genome) but clearly distinct from the Fels-2 prophage of serovar Typhimurium LT2 and from P2-like phages in the serovar Typhi CT18 genome. Our results demonstrate that there is considerable variation among the P2-like phages present in closely related Salmonella spp.
KeywordMeSH Terms
Virus Integration
14. Cano  DA, Pucciarelli  MG, Martínez-Moya  M, Casadesús  J, García-del Portillo  F,     ( 2003 )

Selection of small-colony variants of Salmonella enterica serovar typhimurium in nonphagocytic eucaryotic cells.

Infection and immunity 71 (7)
PMID : 12819049  :   DOI  :   10.1128/iai.71.7.3690-3698.2003     PMC  :   PMC161971    
Abstract >>
Salmonella enterica strains are enteropathogenic bacteria that survive and proliferate within vacuolar compartments of epithelial and phagocytic cells. Recently, it has been reported that fibroblast cells are capable of restricting S. enterica serovar Typhimurium intracellular growth. Here, we show that prolonged residence of bacteria in the intracellular environment of fibroblasts results in the appearance of genetically stable small-colony variants (SCV). A total of 103 SCV isolates, obtained from four independent infections, were subjected to phenotypic analysis. The following phenotypes were observed: (i) delta-aminolevulinic acid auxotrophy; (ii) requirement for acetate or succinate for growth in glucose minimal medium; (iii) auxotrophy for aromatic amino acids; and (iv) reduced growth rate under aerobic conditions not linked to nutrient auxotrophy. The exact mutations responsible for the SCV phenotype in three representative isolates were mapped in the lpd, hemL, and aroD genes, which code for dihydrolipoamide dehydrogenase, glutamate-1-semyaldehyde aminotransferase, and 3-dehydroquinate dehydratase, respectively. The lpd, hemL, and aroD mutants had intracellular persistence rates in fibroblasts that were 3 to 4 logs higher than that of the parental strain and decreased susceptibility to aminoglycoside antibiotics. All three of these SCV isolates were attenuated in the BALB/c murine typhoid model. Complementation with lpd(+), hem(+), and aroD(+) genes restored the levels of intracellular persistence and antibiotic susceptibility to levels of the wild-type strain. However, virulence was not exhibited by any of the complemented strains. Altogether, our data demonstrate that similar to what it has been reported for SCV isolates of other pathogens, S. enterica SCV display enhanced intracellular persistence in eucaryotic cells and are impaired in the ability to cause overt disease. In addition, they also suggest that S. enterica SCV may be favored in vivo.
KeywordMeSH Terms
15. Makde  RD, Kumar  V, Rao  AS, Yadava  VS, Mahajan  SK,     ( 2003 )

Purification, crystallization and preliminary X-ray diffraction studies of recombinant class A non-specific acid phosphatase of Salmonella typhimurium.

Acta crystallographica. Section D, Biological crystallography 59 (Pt 3)
PMID : 12595712  :   DOI  :   10.1107/s0907444902022679     DOI  :   10.1107/s0907444902022679    
Abstract >>
The phoN gene of Salmonella enterica sv. Typhimurium strain MD6001 was cloned in the multicopy plasmid pBluescript SK(-). The nucleotide sequence of the cloned gene differs from the corresponding S. typhimurium LT2 sequence at 23 residues, leading to 15 amino-acid differences, but was very close to the S. typhi phoN sequence (only three nucleotide and two amino-acid differences). The recombinant PhoN protein was purified to homogeneity. Two forms of crystals were harvested from a single crystallization condition. Diffraction intensity data were collected using a laboratory X-ray source to resolution limits of 2.5 and 2.8 A for crystals belonging to space group C2 and C222(1), respectively. Based on non-crystallographic symmetry, four monomers of PhoN are expected to be present in the asymmetric unit of the C2 unit cell. Two monomers of a biologically active dimer in the asymmetric unit of the C222(1) unit cell are expected from the Matthews coefficient.
KeywordMeSH Terms
16. Casin  I, Hanau-Berçot  B, Podglajen  I, Vahaboglu  H, Collatz  E,     ( 2003 )

Salmonella enterica serovar Typhimurium bla(PER-1)-carrying plasmid pSTI1 encodes an extended-spectrum aminoglycoside 6'-N-acetyltransferase of type Ib.

Antimicrobial agents and chemotherapy 47 (2)
PMID : 12543680  :   DOI  :   10.1128/aac.47.2.697-703.2003     PMC  :   PMC151738    
Abstract >>
We have studied the aminoglycoside resistance gene, which confers high levels of resistance to both amikacin and gentamicin, that is carried by plasmid pSTI1 in the PER-1 beta-lactamase-producing strain of Salmonella enterica serovar Typhimurium previously isolated in Turkey. This gene, called aac(6')-Ib(11), was found in a class 1 integron and codes for a protein of 188 amino acids, a fusion product between the N-terminal moiety (8 amino acids) of the signal peptide of the beta-lactamase OXA-1 and the acetyltransferase. The gene lacked a plausible Shine-Dalgarno (SD) sequence and was located 45 nucleotides downstream from a small open reading frame, ORF-18, with a coding capacity of 18 amino acids and a properly spaced SD sequence likely to direct the initiation of aac(6')-Ib(11) translation. AAC(6')-Ib(11) had Leu118 and Ser119 as opposed to Gln and Leu or Gln and Ser, respectively, which were observed in all previously described enzymes of this type. We have evaluated the effect of Leu or Gln at position 118 by site-directed mutagenesis of aac(6')-Ib(11) and two other acetyltransferase gene variants, aac(6')-Ib(7) and -Ib(8), which naturally encode Gln118. Our results show that the combination of Leu118 and Ser119 confers an extended-spectrum aminoglycoside resistance, with the MICs of all aminoglycosides in clinical use, including gentamicin, being two to eight times higher for strains with Leu118 and Ser119 than for those with Gln118 and Ser119.
KeywordMeSH Terms
17. Kingsley  RA, Humphries  AD, Weening  EH, De Zoete  MR, Winter  S, Papaconstantinopoulou  A, Dougan  G, Bäumler  AJ,     ( 2003 )

Molecular and phenotypic analysis of the CS54 island of Salmonella enterica serotype typhimurium: identification of intestinal colonization and persistence determinants.

Infection and immunity 71 (2)
PMID : 12540539  :   DOI  :   10.1128/iai.71.2.629-640.2003     PMC  :   PMC145368    
Abstract >>
The shdA gene is carried on a 25-kb genetic island at centisome 54 (CS54 island) of the Salmonella enterica serotype Typhimurium chromosome. In addition to shdA, the CS54 island of Salmonella serotype Typhimurium strain LT2 contains four open reading frames designated ratA, ratB, sivI, and sivH. DNA hybridization analysis revealed that the CS54 island is comprised of two regions with distinct phylogenetic distribution within the genus Salmonella. Homologues of shdA and ratB were detected only in serotypes of Salmonella enterica subsp. I. In contrast, sequences hybridizing with ratA, sivI, and sivH were present in S. enterica subsp. II and S. bongori in addition to S. enterica subsp. I. Deletion of the ratA and sivI genes did not alter the ability of Salmonella serotype Typhimurium to colonize the organs of mice. Insertional inactivation of the sivH gene resulted in defective colonization of the Peyer's patches of the terminal ileum but normal colonization of the cecum, mesenteric lymph nodes, and spleen. Deletion of the shdA gene resulted in decreased colonization of the cecum and Peyer's patches of the terminal ileum and colonization to a lesser degree in the mesenteric lymph nodes and spleen 5 days post-oral inoculation of mice. A strain containing a deletion in the ratB gene exhibited a defect for the colonization of the cecum but not of the Peyer's patches, mesenteric lymph nodes, and spleen. The shdA and ratB deletion strains exhibited a shedding defect in mice, whereas the sivH deletion strain was shed at numbers similar to the wild type. These data suggest that colonization of the murine cecum is required for efficient fecal shedding in mice.
KeywordMeSH Terms
18. Romanova  IuM, Alekseeva  NV, Stepanova  TV, Razumikhin  MV, Shilov  IA, Tomova  AS, Gintsburg  AL,     ( N/A )

[Influence of tumor necrosis factor on the growth of vegetative and nonculturable forms of Salmonella].

Zhurnal mikrobiologii, epidemiologii, i immunobiologii N/A (4)
PMID : 12449692  :  
Abstract >>
The growth rate of the vegetative forms and the recultivation rate of the uncultivable forms of Salmonella isogenous strains, one of these strains carrying mutation in gene pqi, were studied. The multiplication rate of the vegetative and uncultivable forms of Salmonella control strain in the spleen of infected animals at the initial stages of the infectious process was shown (in vivo) to be considerably accelerated after the preliminary incubation of the culture with cytokine (tumor necrosis factor). The multiplication rate, in vivo and in vitro, of Salmonella vegetative and uncultivable forms with mutation in gene pqi did not change after the incubation of the cells with cytokine, which is indicative of an important role played by the product of this gene in the process of the interaction of bacteria with cytokines. The full nucleotide sequence Salmonella gene pqi was determined.
KeywordMeSH Terms
19. Rychlik  I, Martin  G, Methner  U, Lovell  M, Cardova  L, Sebkova  A, Sevcik  M, Damborsky  J, Barrow  PA,     ( 2002 )

Identification of Salmonella enterica serovar Typhimurium genes associated with growth suppression in stationary-phase nutrient broth cultures and in the chicken intestine.

Archives of microbiology 178 (6)
PMID : 12420160  :   DOI  :   10.1007/s00203-002-0458-7    
Abstract >>
Over 2,800 Tn 5 insertion mutants of Salmonella enterica sv. Typhimurium were screened for the loss of ability to suppress the multiplication of a spectinomycin-resistant (Spc(r)) but otherwise isogenic S. enterica sv. Typhimurium strain, when the Spc(r) mutant was added to 24-h LB broth cultures of the mutants. Selected "growth non-suppressive" (GNS) mutants were defective in respiration (insertions in arcA and fnr), amino acid biosynthesis (aroA and aroD), nutrient uptake and its regulation (tdcC and crp), and chemotaxis (fliD). In the last GNS mutant, the transposon inactivated yhjH, an ORF with unknown function which shows sequence similarity to di-guanylate cyclase and to novel two-component signal transduction proteins. In newly hatched chickens, all of the mutants, with the exception of the fliDmutant, were also unable to suppress colonization of the alimentary tract by the parent strain inoculated 1 day later. Defined mutations in luxS or sdiA,genes which contribute to quorum sensing in S. enterica sv. Typhimurium, had no effect on the stationary-phase growth suppression. Analysis of a transcriptional fusion construct indicated that yhjH was moderately expressed in the exponential phase of growth and up-regulated upon entry into stationary phase. Expression of yhjH was also considerably suppressed by the addition of supernatant from a 24-h stationary-phase S. enterica sv. Typhimurium culture, suggesting that the gene belongs to a new sensing and signaling regulatory pathway in S. enterica sv. Typhimurium.
KeywordMeSH Terms
20. Kotewicz  ML, Li  B, Levy  DD, LeClerc  JE, Shifflet  AW, Cebula  TA,     ( 2002 )

Evolution of multi-gene segments in the mutS-rpoS intergenic region of Salmonella enterica serovar Typhimurium LT2.

Microbiology (Reading, England) 148 (Pt 8)
PMID : 12177346  :   DOI  :   10.1099/00221287-148-8-2531    
Abstract >>
The nucleotide sequence of the 12.6 kb region between the mutS and rpoS genes of Salmonella enterica serovar Typhimurium LT2 (S. typhimurium) was compared to other enteric bacterial mutS-rpoS intergenic regions. The mutS-rpoS region is composed of three distinct segments, designated HK, O and S, as defined by sequence similarities to contiguous ORFs in other bacteria. Inverted chromosomal orientations of each of these segments are found between the mutS and rpoS genes in related ENTEROBACTERIACEAE: The HK segment is distantly related to a cluster of seven ORFs found in Haemophilus influenzae and a cluster of five ORFs found between the mutS and rpoS genes in Escherichia coli K-12. The O segment is related to the mutS-rpoS intergenic region found in E. coli O157:H7 and Shigella dysenteriae type 1. The third segment, S, is common to diverse Salmonella species, but is absent from E. coli. Despite the extensive collinearity and conservation of the overall genetic maps of S. typhimurium and E. coli K-12, the insertions, deletions and inversions in the mutS-rpoS region provide evidence that this region of the chromosome is an active site for horizontal gene transfer and rearrangement.
KeywordMeSH Terms
DNA-Binding Proteins
Evolution, Molecular
Genes, Bacterial
21. Worlock  AJ, Smith  RL,     ( 2002 )

ZntB is a novel Zn2+ transporter in Salmonella enterica serovar Typhimurium.

Journal of bacteriology 184 (16)
PMID : 12142406  :   DOI  :   10.1128/jb.184.16.4369-4373.2002     PMC  :   PMC135258    
Abstract >>
A Zn2+ transport system encoded by the zntB locus of Salmonella enterica serovar Typhimurium has been identified. The protein encoded by this locus is homologous to the CorA family of Mg2+ transport proteins and is widely distributed among the eubacteria. Mutations at zntB confer an increased sensitivity to the cytotoxic effects of Zn2+ and Cd2+, a phenotype that suggests that the encoded protein mediates the efflux of both cations. A direct analysis of transport activity identified a capacity for Zn2+ efflux. These data identify ZntB as a zinc efflux pathway in the enteric bacteria and assign a new function to the CorA family of cation transporters.
KeywordMeSH Terms
22. Buchwald  G, Friebel  A, Galán  JE, Hardt  WD, Wittinghofer  A, Scheffzek  K,     ( 2002 )

Structural basis for the reversible activation of a Rho protein by the bacterial toxin SopE.

The EMBO journal 21 (13)
PMID : 12093730  :   DOI  :   10.1093/emboj/cdf329     PMC  :   PMC126081    
Abstract >>
The bacterial enteropathogen Salmonella typhimurium employs a type III secretion system to inject bacterial toxins into the host cell cytosol. These toxins transiently activate Rho family GTP-binding protein-dependent signaling cascades to induce cytoskeletal rearrangements. One of these translocated Salmonella toxins, SopE, can activate Cdc42 in a Dbl-like fashion despite its lack of sequence similarity to Dbl-like proteins, the Rho-specific eukaryotic guanine nucleotide exchange factors. To elucidate the mechanism of SopE-mediated guanine nucleotide exchange, we have analyzed the structure of the complex between a catalytic fragment of SopE and Cdc42. SopE binds to and locks the switch I and switch II regions of Cdc42 in a conformation that promotes guanine nucleotide release. This conformation is strikingly similar to that of Rac1 in complex with the eukaryotic Dbl-like exchange factor Tiam1. However, the catalytic domain of SopE has an entirely different architecture from that of Tiam1 and interacts with the switch regions via different amino acids. Therefore, SopE represents the first example of a non-Dbl-like protein capable of inducing guanine nucleotide exchange in Rho family proteins.
KeywordMeSH Terms
23. 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
24. Malorny  B, Schroeter  A, Bunge  C, Helmuth  R,     ( 2002 )

Prevalence of Escherichia coli O157:H7 prophage-like sequences among German Salmonella enterica serotype Typhimurium phage types and their use in detection of phage type DT104 by the polymerase chain reaction.

Veterinary microbiology 87 (3)
PMID : 12052335  :   DOI  :   10.1016/s0378-1135(02)00064-0    
Abstract >>
A 1.6kb DNA fragment identified by random amplifiable polymorphic DNA differentiation (RAPD) from a Salmonella enterica serotype Typhimurium phage type DT104 isolate was used to investigate the prevalence of the region in 160 DT104 isolates, 83 other epidemiological important S. Typhimurium phage types and 20 strains selected from 17 other Salmonella serotypes. PCR screening tests using two different primer-sets derived from the RAPD fragment's nucleotide sequence showed that 76% of the 160 DT104 isolates investigated, including subtypes DT104A, DT104B, DT104B low, DT104H and DT104L, reacted positively. High sensitivity was shown for DT104 strains expressing at least the penta-resistance pattern ACSSuT (97% of 104 strains tested). DT104 susceptible strains showed only a sensitivity of 35% (17 strains tested). In contrast, 83% of the 83 strains from the other S. Typhimurium phage types reacted negatively. Strains from five out of the 17 other serotypes showed a positive signal with one primer-set. The other primer-set exhibited only a positive reaction with one S. Dublin isolate. The analysis of a 2415bp extended sequence revealed homologies to genes encoded by Escherichia coli O157:H7 prophages, suggesting that the described region contains genes of a prophage specific for DT104 and related phage types.
KeywordMeSH Terms
25. Morty  RE, Fülöp  V, Andrews  NW,     ( 2002 )

Substrate recognition properties of oligopeptidase B from Salmonella enterica serovar Typhimurium.

Journal of bacteriology 184 (12)
PMID : 12029050  :   DOI  :   10.1128/jb.184.12.3329-3337.2002     PMC  :   PMC135088    
Abstract >>
Oligopeptidase B (OpdB) is a serine peptidase broadly distributed among unicellular eukaryotes, gram-negative bacteria, and spirochetes which has emerged as an important virulence factor and potential therapeutic target in infectious diseases. We report here the cloning and expression of the opdB homologue from Salmonella enterica serovar Typhimurium and demonstrate that it exhibits amidolytic activity exclusively against substrates with basic residues in P(1). While similar to its eukaryotic homologues in terms of substrate specificity, Salmonella OpdB differs significantly in catalytic power and inhibition and activation properties. In addition to oligopeptide substrates, restricted proteolysis of histone proteins was observed, although no cleavage was seen at or near residues that had been posttranslationally modified or at defined secondary structures. This supports the idea that the catalytic site of OpdB may be accessible only to unstructured oligopeptides, similar to the closely related prolyl oligopeptidase (POP). Salmonella OpdB was employed as a model enzyme to define determinants of substrate specificity that distinguish OpdB from POP, which hydrolyzes substrates exclusively at proline residues. Using site-directed mutagenesis, nine acidic residues that are conserved in OpdBs but absent from POPs were converted to their corresponding residues in POP. In this manner, we identified a pair of glutamic acid residues, Glu(576) and Glu(578), that define P(1) specificity and direct OpdB cleavage C terminal to basic residues. We have also identified a second pair of residues, Asp(460) and Asp(462), that may be involved in defining P(2) specificity and thus direct preferential cleavage by OpdB after pairs of basic residues.
KeywordMeSH Terms
Bacterial Proteins
26. Villa  L, Visca  P, Tosini  F, Pezzella  C, Carattoli  A,     ( 2002 )

Composite integron array generated by insertion of an ORF341-type integron within a Tn21-like element.

Microbial drug resistance (Larchmont, N.Y.) 8 (1)
PMID : 12002644  :   DOI  :   10.1089/10766290252913692    
Abstract >>
Two class 1 integrons, In-t1 and In-t2, were previously identified in IncFI plasmids of Salmonella enterica serotype Typhimurium. Molecular analysis revealed a close physical link between the two integrons. In-t1 is preceded by the transposase genes of Tn21, whereas In-t2 is located downstream the 3'-conserved segment (3'-CS) of In-t1, in a head-to-tail configuration. In-t1 shows a peculiar sequence downstream the 3'-CS, containing an extended version of the open reading frame known as ORF341 (referred to as ORF341E) and a novel trimethoprim resistance gene, designated dfrA18. Retrospective analysis provided evidence for In-t1 insertion within Tn1935, a Tn21-related transposon identified in IncFI plasmids circulating among epidemic clones of multidrug-resistant S. enterica during the 1970s. Structural comparison between Tn21 derivatives from recent and ancestor IncFI plasmids showed that In-t2 has been conserved by these replicons. In-t1 belongs to a novel family of class 1 integrons containing the ORF341E sequence, and appears to have been acquired by IncFI plasmids after the assembly of Tn1935. In-t1 insertion occurred within the 5'-conserved segment (5'-CS) proximal region of the resident In-t2.
KeywordMeSH Terms
27. Wong  RS, Chow  AW,     ( 2002 )

Identification of enteric pathogens by heat shock protein 60 kDa (HSP60) gene sequences.

FEMS microbiology letters 206 (1)
PMID : 11786265  :   DOI  :   10.1111/j.1574-6968.2002.tb10994.x    
Abstract >>
A highly specific and reproducible approach for the simultaneous detection of enteric pathogenic bacteria was developed using bacterial hsp60 gene and molecular biological tools. A single pair of universal primers was derived from the highly conserved sequence of hsp60 genes encompassing a 600-bp hypervariable region. PCR amplification followed by either dot blot hybridization or restriction enzyme digestion performed on 38 enteric bacteria indicated that this approach could differentiate not only different genera such as Campylobacter, Yersinia and Vibrio, but also species that are closely related genetically, such as between C. jejuni and C. coli, or between Salmonella and Shigella or Escherichia coli.
KeywordMeSH Terms
Amino Acid Sequence
28. Chu  C, Chiu  CH, Chu  CH, Ou  JT,     ( 2002 )

Nucleotide and amino acid sequences of oriT-traM-traJ-traY-traA-traL regions and mobilization of virulence plasmids of Salmonella enterica serovars enteritidis, gallinarum-pullorum, and typhimurium.

Journal of bacteriology 184 (11)
PMID : 12003924  :   DOI  :   10.1128/jb.184.11.2857-2862.2002     PMC  :   PMC135071    
Abstract >>
The virulence plasmid of Salmonella enterica serovar Gallinarum-Pullorum (pSPV) but not those of Salmonella enterica serovars Enteritidis (pSEV) and Typhimurium (pSTV) can be readily mobilized by an F or F-like conjugative plasmid. To investigate the reason for the difference, the oriT-traM-traJ-traY-traA-traL regions of the three salmonella virulence plasmids (pSVs) were cloned and their nucleotide and deduced amino acid sequences were examined. The cloned fragments were generally mobilized more readily than the corresponding full-length pSVs, but the recombinant plasmid containing the oriT of pSPV was, as expected, more readily mobilized, with up to 100-fold higher frequency than the recombinant plasmids containing the oriT of the other two pSVs. The nucleotide sequences of the oriT-traM-traJ-traY-traA-traL region of pSEV and pSTV were almost identical (only 4 bp differences), but differed from that of pSPV. Major nucleotide sequence variations were found in traJ, traY, and the Tra protein binding sites sby and sbm. sby of pSPV showed higher similarity than that of pSEV or pSTV to that of the F plasmid. The reverse was true for sbm: similarity was higher with pSEV and pSTV than with pSPV. In the deduced amino acid sequences of the five Tra proteins, major differences were found in TraY: pSEV's TraY was 75 amino acids, pSTV's was 106 amino acids, and pSPV's was 133 amino acids; and there were duplicate consensus betaalphaalpha fragments in the TraY of pSPV and F plasmid, whereas there was only a single betaalphaalpha fragment in that of pSEV and pSTV.
KeywordMeSH Terms
Escherichia coli Proteins
Genes, Bacterial
29. Carattoli  A, Tosini  F, Giles  WP, Rupp  ME, Hinrichs  SH, Angulo  FJ, Barrett  TJ, Fey  PD,     ( 2002 )

Characterization of plasmids carrying CMY-2 from expanded-spectrum cephalosporin-resistant Salmonella strains isolated in the United States between 1996 and 1998.

Antimicrobial agents and chemotherapy 46 (5)
PMID : 11959555  :   DOI  :   10.1128/aac.46.5.1269-1272.2002     PMC  :   PMC127137    
Abstract >>
Sequencing of DNA from 15 expanded-spectrum cephalosporin (e.g., ceftriaxone)-resistant Salmonella isolates obtained in the United States revealed that resistance to ceftriaxone in all isolates was mediated by cmy-2. Hybridization patterns revealed three plasmid structures containing cmy-2 in these 15 isolates. These data suggest that the spread of cmy-2 among Salmonella strains is occurring through mobilization of the cmy-2 gene into different plasmid backbones and consequent horizontal transfer by conjugation.
KeywordMeSH Terms
30. van der Straaten  T, van Diepen  A, Kwappenberg  K, van Voorden  S, Franken  K, Janssen  R, Kusters  JG, Granger  DL, van Dissel  JT,     ( 2001 )

Novel Salmonella enterica serovar Typhimurium protein that is indispensable for virulence and intracellular replication.

Infection and immunity 69 (12)
PMID : 11705915  :   DOI  :   10.1128/IAI.69.12.7413-7418.2001     PMC  :   PMC98829    
Abstract >>
Upon contact with host cells, the intracellular pathogen Salmonella enterica serovar Typhimurium promotes its uptake, targeting, and survival in intracellular niches. In this process, the bacterium evades the microbicidal effector mechanisms of the macrophage, including oxygen intermediates. This study reports the phenotypic and genotypic characterization of an S. enterica serovar Typhimurium mutant that is hypersusceptible to superoxide. The susceptible phenotype is due to a MudJ insertion-inactivation of a previously undescribed Salmonella gene designated sspJ that is located between 54.4 and 64 min of the Salmonella chromosome and encodes a 392-amino-acid protein. In vivo, upon intraperitoneal injection of 10(4) to 10(7) bacteria in C3H/HeN and 10(1) to 10(4) bacteria in BALB/c mice, the mutant strain was less virulent than the wild type. Consistent with this finding, during the first hour after ingestion by macrophage-like J774 and RAW264.7 cells in vitro, the intracellular killing of the strain carrying sspJ::MudJ is enhanced fivefold over that of wild-type microorganisms. Wild-type salmonellae displayed significant intracellular replication during the first 24 h after uptake, but sspJ::MudJ mutants failed to do so. This phenotype could be restored to that of the wild type by sspJ complementation. The SspJ protein is found in the cytoplasmic membrane and periplasmic space. Amino acid sequence homology analysis did reveal a leader sequence and putative pyrroloquinoline quinone-binding domains, but no putative protein function. We excluded the possibility that SspJ is a scavenger of superoxide or has superoxide dismutase activity.
KeywordMeSH Terms
31. Figueroa-Bossi  N, Coissac  E, Netter  P, Bossi  L,     ( 1997 )

Unsuspected prophage-like elements in Salmonella typhimurium.

Molecular microbiology 25 (1)
PMID : 11902718  :   DOI  :   10.1046/j.1365-2958.1997.4451807.x    
Abstract >>
We present evidence for the existence of two large (approximately 50 kb) excisable segments in the chromosome of Salmonella typhimurium. The two elements--designated Gifsy-1 and Gifsy-2--cover, respectively, the 57 units and the 24 units of the genetic map where they contribute indicative rare restriction sites. The two elements are closely interrelated and both contain a region of sequence similarity to the recE locus of the Rac prophage of Escherichia coli. Mutations within this region of Gifsy-1 yield the classical 'Sbc' phenotype: they suppress the recombination defect of recB mutants, apparently by activating a normally silent recE-like gene. At the same time, these 'sbcE' mutations activate a Xis-type function that promotes excision of one or other of the two elements. Predictably, curing of Gifsy-1 results in the loss of recB mutant suppression. Surprisingly, the suppressor phenotype is also lost in cells cured for Gifsy-2 even though the Gifsy-1-associated sbcE mutation is still present. Moreover, the excision frequency of Gifsy-1 drops dramatically in Gifsy-2-cured cells. Thus, both elements must co-operate in the activation of recombination and excision functions. Overall, the data presented here suggest that Gifsy-1 and Gifsy-2 are cryptic prophages. They are distinct from previously described Fels prophages. Unlike Fels, they are not specific to S. typhimurium strain LT2 since they are both also found in a virulent S. typhimurium isolate (ATCC 14028s).
KeywordMeSH Terms
Escherichia coli Proteins
Genes, Suppressor
32. Mulec  J, Starcic  M, Zgur-Bertok  D,     ( 2002 )

F-like plasmid sequences in enteric bacteria of diverse origin, with implication of horizontal transfer and plasmid host range.

Current microbiology 44 (4)
PMID : 11910490  :   DOI  :   10.1007/s00284-001-0039-7    
Abstract >>
Seventy-eight bacterial isolates from human, animal, and plant hosts, representing eight species of the family Enterobacteriaceae, were screened for F-like plasmid sequences. Of the examined human Escherichia coli strains, 28% harbored one or two of the three F-like, RepFI replication regions, while 35% of the examined animal and all phytopathogenic strains harbored RepFIA-specific sequences. Comparative analysis of Salmonella, Shigella, Erwinia, and E. coli plasmid RepFI sequences showed 100% or very high homology, indicating frequent and recent interspecies gene transfer. The high incidence of RepFIA sequences in enteric bacterial species, including Klebsiella and Erwinia, showed that F-like plasmids are successful in avoiding natural barriers to establishment of horizontally transferred DNA and that in the natural environment conjugal transfer is efficient in diverse ecological niches.
KeywordMeSH Terms
Escherichia coli Proteins
33. Fahlen  TF, Wilson  RL, Boddicker  JD, Jones  BD,     ( 2001 )

Hha is a negative modulator of transcription of hilA, the Salmonella enterica serovar Typhimurium invasion gene transcriptional activator.

Journal of bacteriology 183 (22)
PMID : 11673432  :   DOI  :   10.1128/JB.183.22.6620-6629.2001     PMC  :   PMC95493    
Abstract >>
An early step in the establishment of Salmonella enterica serovar Typhimurium murine infection is the penetration of the intestinal mucosa of the small intestine. The majority of the genes responsible for the Salmonella invasive phenotype are encoded on Salmonella pathogenicity island 1, and their transcription is controlled by the hilA transcriptional activator. The expression of hilA is regulated by environmental signals including oxygen, osmolarity, pH, and growth phase such that the presence of any one suboptimal condition results in repression of hilA expression and the invasive phenotype. We have conducted a search for negative regulators of hilA by introduction of a Salmonella enterica serovar Typhimurium chromosomal DNA gene bank into a Salmonella enterica serovar Typhimurium hilA::Tn5lacZY reporter strain. This screen has identified the hha gene as a regulator that exerts a negative influence on hilA expression. Plasmid-encoded hha significantly reduces hilA::Tn5lacZY chromosomal expression, as well as expression of the invasion genes invF, prgH, and sipC. An hha null mutation results in substantial derepression of both chromosomally encoded and plasmid-encoded hilA::Tn5lacZY expression. Introduction of plasmid-encoded hha into strain SL1344 results in attenuation of invasion using in vitro and in vivo assays. Importantly, purified Hha protein was found to bind to a hilA DNA promoter fragment, suggesting that the regulatory activity of the Hha protein occurs at the hilA promoter. These data add detail to the developing model of the regulation of Salmonella invasion genes.
KeywordMeSH Terms
DNA-Binding Proteins
Escherichia coli Proteins
34. Calhoun  DH, Bonner  CA, Gu  W, Xie  G, Jensen  RA,     ( 2001 )

The emerging periplasm-localized subclass of AroQ chorismate mutases, exemplified by those from Salmonella typhimurium and Pseudomonas aeruginosa.

Genome biology 2 (8)
PMID : 11532214  :   DOI  :   10.1186/gb-2001-2-8-research0030     PMC  :   PMC55327    
Abstract >>
Chorismate mutases of the AroQ homology class are widespread in the Bacteria and the Archaea. Many of these exist as domains that are fused with other aromatic-pathway catalytic domains. Among the monofunctional AroQ proteins, that from Erwinia herbicola was previously shown to have a cleavable signal peptide and located in the periplasmic compartment. Whether or not this might be unique to E. herbicola was unknown. The gene coding for the AroQ protein was cloned from Salmonella typhimurium, and the AroQ protein purified from both S. typhimurium and Pseudomonas aeruginosa was shown to have a periplasmic location. The periplasmic chorismate mutases (denoted *AroQ) are shown to be a distinct subclass of AroQ, being about twice the size of cytoplasmic AroQ proteins. The increased size is due to a carboxy-terminal extension of unknown function. In addition, a so-far novel aromatic aminotransferase was shown to be present in the periplasm of P. aeruginosa. Our analysis has detected a number of additional *aroQ genes. The joint presence of *AroQ, cyclohexadienyl dehydratase and aromatic aminotransferase in the periplasmic compartment of P. aeruginosa comprises a complete chorismate-to-phenylalanine pathway and accounts for the hidden overflow pathway" to phenylalanine described previously."
KeywordMeSH Terms
35. Turcot  I, Ponnampalam  TV, Bouwman  CW, Martin  NL,     ( 2001 )

Isolation and characterization of a chromosomally encoded disulphide oxidoreductase from Salmonella enterica serovar Typhimurium.

Canadian journal of microbiology 47 (8)
PMID : 11575497  :  
Abstract >>
In this study, the chromosomally encoded disulphide oxidoreductase dsbA from Salmonella typhimurium was cloned and characterized. A survey of a number of serovars of Salmonella subspecies I showed that dsbA is highly conserved in most, but not all members of this subclass of Salmonella species. Using motility, beta-galactosidase, and alkaline phosphatase assays as indirect indicators of disulphide oxidoreductase activity, we demonstrated that DsbA from S. typhimurium LT2 can only partially complement an Escherichia coli dsbA-null strain. This is surprising considering the high degree of conservation between these two DsbA proteins (87% amino acid identity). To determine the contribution of DsbA to the proper folding and assembly of proteins of S. typhimurium, deletion mutants were created in the avirulent strain LT2 and in the virulent strain SL1344. These null alleles were constructed by partial deletion of the dsbA-coding region and then insertion of an antibiotic resistance marker in the gene. Mutants no longer expressing a functional disulphide oxidoreductase exhibit pleitropic effects, including an increase in colony mucoidy, a dramatic decrease in motility, and an increased susceptibility to the cationic peptide protamine sulphate. The disruption of disulphide bond formation was also shown to specifically affect the stability of several proteins secreted into the extracellular environment.
KeywordMeSH Terms
Cloning, Molecular
36. Bauerfeind  R, Barth  S, Weiss  R, Baljer  G,     ( N/A )

Sequence polymorphism of the Salmonella plasmid virulence factor D (SpvD) in Salmonella enterica isolates of animal origin.

Berliner und Munchener tierarztliche Wochenschrift 114 (9��10��)
PMID : 11570190  :  
Abstract >>
The nucleotide sequence encoding the Salmonella plasmid virulence factor D (SpvD) was determined in 17 Salmonella strains that were different in O and H antigen patterns, animal host and geographical origin, and year of isolation. Nucleotide sequence comparison revealed the existence of at least nine spvD alleles resulting in 8 SpvD protein variants although the nucleotide sequences were highly similar (identity 98.8-100%). The spvD gene products differed from each other in up to 4 amino acid residues only with the exception of the carboxy-terminally truncated SpvD variant of one S. Gallinarum field isolate. The highly conserved primary structure of SpvD in epidemiologically relevant salmonellae suggests that this virulence factor is a promising antigen candidate for diagnostic purposes (i.e. antibody detection in infected animals) but also for immunoprophylaxis in farm animal species.
KeywordMeSH Terms
Antigens, Bacterial
Bacterial Proteins
Polymorphism, Genetic
Virulence Factors
37. Boyd  D, Peters  GA, Cloeckaert  A, Boumedine  KS, Chaslus-Dancla  E, Imberechts  H, Mulvey  MR,     ( 2001 )

Complete nucleotide sequence of a 43-kilobase genomic island associated with the multidrug resistance region of Salmonella enterica serovar Typhimurium DT104 and its identification in phage type DT120 and serovar Agona.

Journal of bacteriology 183 (19)
PMID : 11544236  :   DOI  :   10.1128/JB.183.19.5725-5732.2001     PMC  :   PMC95465    
Abstract >>
This study describes the characterization of the recently described Salmonella genomic island 1 (SGI1) (D. A. Boyd, G. A. Peters, L.-K. Ng, and M. R. Mulvey, FEMS Microbiol. Lett. 189:285-291, 2000), which harbors the genes associated with the ACSSuT phenotype in a Canadian isolate of Salmonella enterica serovar Typhimurium DT104. A 43-kb region has been completely sequenced and found to contain 44 predicted open reading frames (ORFs) which comprised approximately 87% of the total sequence. Fifteen ORFs did not show any significant homology to known gene sequences. A number of ORFs show significant homology to plasmid-related genes, suggesting, at least in part, a plasmid origin for the SGI1, although some with homology to phage-related genes were identified. The SGI1 was identified in a number of multidrug-resistant DT120 and S. enterica serovar Agona strains with similar antibiotic-resistant phenotypes. The G+C content suggests a potential mosaic structure for the SGI1. Emergence of the SGI1 in serovar Agona strains is discussed.
KeywordMeSH Terms
Genome, Bacterial
38. Murray  SR, Bermudes  D, de Felipe  KS, Low  KB,     ( 2001 )

Extragenic suppressors of growth defects in msbB Salmonella.

Journal of bacteriology 183 (19)
PMID : 11544217  :   DOI  :   10.1128/JB.183.19.5554-5561.2001     PMC  :   PMC95446    
Abstract >>
Lipid A, a potent endotoxin which can cause septic shock, anchors lipopolysaccharide (LPS) into the outer leaflet of the outer membrane of gram-negative bacteria. MsbB acylates (KDO)(2)-(lauroyl)-lipid IV-A with myristate during lipid A biosynthesis. Reports of knockouts of the msbB gene describe effects on virulence but describe no evidence of growth defects in Escherichia coli K-12 or Salmonella. Our data confirm the general lack of growth defects in msbB E. coli K-12. In contrast, msbB Salmonella enterica serovar Typhimurium exhibits marked sensitivity to galactose-MacConkey and 6 mM EGTA media. At 37 degrees C in Luria-Bertani (LB) broth, msbB Salmonella cells elongate, form bulges, and grow slowly. msbB Salmonella grow well on LB-no salt (LB-0) agar; however, under specific shaking conditions in LB-0 broth, many msbB Salmonella cells lyse during exponential growth and a fraction of the cells form filaments. msbB Salmonella grow with a near-wild-type growth rate in MSB (LB-0 containing Mg(2+) and Ca(2+)) broth (23 to 42 degrees C). Extragenic compensatory mutations, which partially suppress the growth defects, spontaneously occur at high frequency, and mutants can be isolated on media selective for faster growing derivatives. One of the suppressor mutations maps at 19.8 centisomes and is a recessive IS10 insertional mutation in somA, a gene of unknown function which corresponds to ybjX in E. coli. In addition, random Tn10 mutagenesis carried out in an unsuppressed msbB strain produced a set of Tn10 inserts, not in msbB or somA, that correlate with different suppressor phenotypes. Thus, insertional mutations, in somA and other genes, can suppress the msbB phenotype.
KeywordMeSH Terms
Acyltransferases
DNA Transposable Elements
Escherichia coli Proteins
Gene Expression Regulation, Bacterial
39. Li  YF, Hess  S, Pannell  LK, White Tabor  C, Tabor  H,     ( 2001 )

In vivo mechanism-based inactivation of S-adenosylmethionine decarboxylases from Escherichia coli, Salmonella typhimurium, and Saccharomyces cerevisiae.

Proceedings of the National Academy of Sciences of the United States of America 98 (19)
PMID : 11526206  :   DOI  :   10.1073/pnas.181341198     PMC  :   PMC58508    
Abstract >>
S-adenosylmethionine decarboxylase (AdoMetDC), a key enzyme in the biosynthesis of spermidine and spermine, is first synthesized as a proenzyme, which is cleaved posttranslationally to form alpha and beta subunits. The alpha subunit contains a covalently bound pyruvoyl group derived from serine that is essential for activity. With the use of an Escherichia coli overexpression system, we have purified AdoMetDCs encoded by the E. coli, Saccharomyces cerevisiae, and Salmonella typhimurium genes. Unexpectedly we found by mass spectrometry that these enzymes had been modified posttranslationally in vivo by a mechanism-based "suicide" inactivation. A large percentage of the alpha subunit of each enzyme had been modified in vivo to give peaks with masses m/z = 57 +/- 1 and m/z = 75 +/- 1 daltons higher than the parent peak. AdoMetDC activity decreased markedly during overexpression concurrently with the increase of the additional peaks for the alpha subunit. Sequencing of a tryptic fragment by tandem mass spectrometry showed that Cys-140 was modified with a +75 +/- 1 adduct, which is probably derived from the reaction product. Comparable modification of the alpha subunit was also observed in in vitro experiments after incubation with the substrate or with the reaction product, which is consistent with the in vitro alkylation of E. coli AdoMetDC reported by Diaz and Anton [Diaz, E. & Anton, D. L. (1991) Biochemistry 30, 4078-4081].
KeywordMeSH Terms
Protein Processing, Post-Translational
40. Brown  PK, Dozois  CM, Nickerson  CA, Zuppardo  A, Terlonge  J, Curtiss  R,     ( 2001 )

MlrA, a novel regulator of curli (AgF) and extracellular matrix synthesis by Escherichia coli and Salmonella enterica serovar Typhimurium.

Molecular microbiology 41 (2)
PMID : 11489123  :   DOI  :   10.1046/j.1365-2958.2001.02529.x    
Abstract >>
Production of curli (AgF) adhesins by Escherichia coli and Salmonella enterica serovar Typhimurium (S. typhimurium) is associated with extracellular matrix production and is optimal at low temperature during stationary phase. Curli and extracellular matrix synthesis involves a complex regulatory network that is dependent on the CsgD (AgfD) regulator. We have identified a novel regulator, termed MlrA, that is required for curli production and extracellular matrix formation. Two cosmids from a genomic library of avian pathogenic E. coli chi7122 conferred mannose-resistant haemagglutination (HA) and curli production to E. coli HB101, which is unable to produce curli owing to a defective regulatory pathway. The rpoS gene, encoding a known positive regulator of curli synthesis, and the E. coli open reading frame (ORF) of unknown function, yehV, identified on each of these cosmids, respectively, conferred curli production and HA to E. coli HB101. We have designated yehV as the mlrA gene for MerR-like regulator A because its product shares similarities with regulatory proteins of the MerR family. HA and curli production by strain chi7122 were abolished by disruption of rpoS, mlrA or csgA, the curli subunit gene. Both csgD and csgBA transcription, required for expression of curli, were inactive in an mlrA mutant grown under conditions that promote curli production. An mlrA homologue was identified in S. typhimurium. Analysis of mlrA-lac operon fusions demonstrated that mlrA was positively regulated by rpoS. mlrA mutants of wild-type S. typhimurium SL1344 or SR-11 no longer produced curli or rugose colony morphology, and exhibited enhanced aggregation and extracellular matrix formation when complemented with the mlrA gene from either S. typhimurium or E. coli present on a low-copy-number plasmid. However, inactivation of mlrA did not affect curli production and aggregative morphology in an upregulated curli producing S. typhimurium derivative containing a temperature- and RpoS-independent agfD promoter region. These results indicate that MlrA is a newly defined transcriptional regulator of csgD/agfD that acts as a positive regulator of RpoS-dependent curli and extracellular matrix production by E. coli and S. typhimurium.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
41. Friebel  A, Ilchmann  H, Aepfelbacher  M, Ehrbar  K, Machleidt  W, Hardt  WD,     ( 2001 )

SopE and SopE2 from Salmonella typhimurium activate different sets of RhoGTPases of the host cell.

The Journal of biological chemistry 276 (36)
PMID : 11440999  :   DOI  :   10.1074/jbc.M100609200    
Abstract >>
The bacterial enteropathogen Salmonella typhimurium employs a specialized type III secretion system to inject toxins into host cells, which trigger signaling cascades leading to cell death in macrophages, secretion of pro-inflammatory cytokines, or rearrangements of the host cell cytoskeleton and thus to bacterial invasion. Two of the injected toxins, SopE and the 69% identical protein SopE2, are highly efficient guanine nucleotide exchange factors for the RhoGTPase Cdc42 of the host cell. However, it has been a puzzle why S. typhimurium might employ two toxins with redundant function. We hypothesized that SopE and SopE2 might have different specificities for certain host cellular RhoGTPases. In vitro guanine nucleotide exchange assays and surface plasmon resonance measurements revealed that SopE is an efficient guanine nucleotide exchange factor for Cdc42 and Rac1, whereas SopE2 was interacting efficiently only with Cdc42, but not with Rac1. Affinity precipitation of Cdc42.GTP and Rac1.GTP from lysates and characteristic cytoskeletal rearrangements of infected tissue culture cells confirmed that SopE is highly efficient at activating Cdc42 and Rac1 in vivo, whereas SopE2 was efficiently activating Cdc42, but not Rac1. We conclude that the translocated effector proteins SopE and SopE2 allow S. typhimurium to specifically activate different sets of RhoGTPase signaling cascades.
KeywordMeSH Terms
42. Darwin  KH, Miller  VL,     ( 2001 )

Type III secretion chaperone-dependent regulation: activation of virulence genes by SicA and InvF in Salmonella typhimurium.

The EMBO journal 20 (8)
PMID : 11296219  :   DOI  :   10.1093/emboj/20.8.1850     PMC  :   PMC125432    
Abstract >>
Invasion of the intestinal epithelium by Salmonella sp. requires a type III secretion system (TTSS) common in many bacterial pathogens. TTSS translocate effector proteins from bacteria into eukaryotic cells. These effectors manipulate cellular functions in order to benefit the pathogen. In the human and animal pathogen Salmonella typhimurium, the expression of genes encoding the secreted effector molecules Sip/Ssp ABCD, SigD, SptP and SopE requires both the AraC/XylS-like regulator InvF and the secretion chaperone SICA: In this work, an InvF binding site was identified in the promoter regions of three operons. SicA does not appear to affect InvF stability nor to bind DNA directly. However, SicA could be co-purified with InvF, suggesting that InvF and SicA interact with each other to activate transcription from the effector gene promoters. This is the first demonstration of a contact between a protein cofactor and an AraC/XylS family transcriptional regulator and, moreover, is the first direct evidence of an interaction between a transcriptional regulator and a TTSS chaperone. The regulation of effector genes described here for InvF and SicA may represent a new paradigm for regulation of virulence in a wide variety of pathogens.
KeywordMeSH Terms
Transcription Factors
43. Robbe-Saule  V, Coynault  C, Ibanez-Ruiz  M, Hermant  D, Norel  F,     ( 2001 )

Identification of a non-haem catalase in Salmonella and its regulation by RpoS (sigmaS).

Molecular microbiology 39 (6)
PMID : 11260470  :   DOI  :   10.1046/j.1365-2958.2001.02340.x    
Abstract >>
We report the identification and functional analysis of katN, a gene encoding a non-haem catalase of Salmonella enterica serotype Typhimurium. katN, which is not present in Escherichia coli, is located between the yciGFE and yciD E. coli homologues in the Salmonella genome. Its predicted protein product has a molecular weight of 31 826 Da and is similar to the Mn-catalases of Lactobacillus plantarum and Thermus spp. Its product, KatN, was visualized as a 37 kDa protein in E. coli maxicells. A KatN recombinant protein, containing six histidine residues at its C-terminus, was purified, and its catalase activity was observed on a non-denaturing polyacrylamide gel. KatN was also visualized by catalase activity gel staining of bacterial cell extracts. Its expression was shown to be regulated by growth phase and rpoS. Northern blotting indicated that kat forms an operon with the upstream yciGFE genes. A putative rpoS-regulated promoter was identified upstream of yciG. Southern blotting revealed that katN is conserved within Salmonella serovars. katN homologues were found in Pseudomonas aeruginosa, Klebsiella pneumoniae, Klebsiella oxytoca, Enterobacter cloacae and Serratia marcescens. A katN mutation did not appear to affect the hydrogen peroxide (H2O2) response of Salmonella. However, the expression of katN increased the H2O2 resistance of unadapted cells in the exponential phase and of rpoS mutants in stationary phase. Thus, KatN may contribute to hydrogen peroxide resistance in Salmonella in certain environmental conditions.
KeywordMeSH Terms
44. Zogaj  X, Nimtz  M, Rohde  M, Bokranz  W, Römling  U,     ( 2001 )

The multicellular morphotypes of Salmonella typhimurium and Escherichia coli produce cellulose as the second component of the extracellular matrix.

Molecular microbiology 39 (6)
PMID : 11260463  :   DOI  :   10.1046/j.1365-2958.2001.02337.x    
Abstract >>
Production of cellulose has been thought to be restricted to a few bacterial species such as the model organism Acetobacter xylinus. We show by enzymatic analysis and mass spectrometry that, besides thin aggregative fimbriae, the second component of the extracellular matrix of the multicellular morphotype (rdar) of Salmonella typhimurium and Escherichia coli is cellulose. The bcsA, bcsB, bcsZ and bcsC genes responsible for cellulose biosynthesis are not regulated by AgfD, the positive transcriptional regulator of the rdar morphotype. Transcription of the bcs genes was not co-expressed with the rdar morphotype under any of the environmental conditions examined. However, cellulose biosynthesis was turned on by the sole expression of adrA, a gene encoding a putative transmembrane protein regulated by agfD, indicating a novel pathway for the activation of cellulose synthesis. The co-expression of cellulose and thin aggregative fimbriae leads to the formation of a highly hydrophobic network with tightly packed cells aligned in parallel in a rigid matrix. As the production of cellulose would now appear to be a property widely distributed among bacteria, the function of the cellulose polymer in bacteria will have to be considered in a new light.
KeywordMeSH Terms
Arabidopsis Proteins
Gene Expression Regulation, Bacterial
Transcription Factors
45. Price-Carter  M, Tingey  J, Bobik  TA, Roth  JR,     ( 2001 )

The alternative electron acceptor tetrathionate supports B12-dependent anaerobic growth of Salmonella enterica serovar typhimurium on ethanolamine or 1,2-propanediol.

Journal of bacteriology 183 (8)
PMID : 11274105  :   DOI  :   10.1128/JB.183.8.2463-2475.2001     PMC  :   PMC95162    
Abstract >>
Synthesis of cobalamin de novo by Salmonella enterica serovar Typhimurium strain LT2 and the absence of this ability in Escherichia coli present several problems. This large synthetic pathway is shared by virtually all salmonellae and must be maintained by selection, yet no conditions are known under which growth depends on endogenous B12. The cofactor is required for degradation of 1,2-propanediol and ethanolamine. However, cofactor synthesis occurs only anaerobically, and neither of these carbon sources supports anaerobic growth with any of the alternative electron acceptors tested thus far. This paradox is resolved by the electron acceptor tetrathionate, which allows Salmonella to grow anaerobically on ethanolamine or 1,2-propanediol by using endogenously synthesized B12. Tetrathionate provides the only known conditions under which simple cob mutants (unable to make B12) show a growth defect. Genes involved in this metabolism include the ttr operon, which encodes tetrathionate reductase. This operon is globally regulated by OxrA (Fnr) and induced anaerobically by a two-component system in response to tetrathionate. Salmonella reduces tetrathionate to thiosulfate, which it can further reduce to H2S, by using enzymes encoded by the genes phs and asr. The genes for 1,2-propanediol degradation (pdu) and B12 synthesis (cob), along with the genes for sulfur reduction (ttr, phs, and asr), constitute more than 1% of the Salmonella genome and are all absent from E. coli. In diverging from E. coli, Salmonella acquired some of these genes unilaterally and maintained others that are ancestral but have been lost from the E. coli lineage.
KeywordMeSH Terms
46. Prager  R, Mirold  S, Tietze  E, Strutz  U, Knüppel  B, Rabsch  W, Hardt  WD, Tschäpe  H,     ( 2000 )

Prevalence and polymorphism of genes encoding translocated effector proteins among clinical isolates of Salmonella enterica.

International journal of medical microbiology : IJMM 290 (7)
PMID : 11200542  :   DOI  :   10.1016/S1438-4221(00)80009-0    
Abstract >>
Pathogenic Salmonella enterica strains are capable of causing local and/or systemic infections. They employ two type III secretion systems to translocate an array of virulence-associated proteins (effector proteins) directly into the cytosol of target cells of the host. Earlier data had shown that changes in the repertoire of translocated effector proteins may contribute to the adaptation of Salmonella strains to new hosts and to the emergence of epidemic strains. Using PCR and Southern blot techniques the presence of and the polymorphism among the genes encoding the translocated effector proteins SopB, SopD, SopE, SopE2, SipA, SipB, SipC, AvrA, and SptP was studied in 71 phylogenetically well characterised S. enterica subspecies I (subspecies enterica) strains of the SARB collection and in 209 clinical and epidemic isolates of S. enterica subspecies I belonging to various serovars, phage types, and genotypes. All these Salmonella strains harbour all these respective genes with the exception of sopE and avrA which have been identified in only some of them. Several of the studied genes display genetic polymorphisms (RFLP). These RFLP patterns did not show a strict correlation with the genetic distance, the grouping genes in order to understand their role in the evolution of Salmonella as a pathogen.
KeywordMeSH Terms
Genes, Bacterial
Microfilament Proteins
Polymorphism, Restriction Fragment Length
47. Tezcan-Merdol  D, Nyman  T, Lindberg  U, Haag  F, Koch-Nolte  F, Rhen  M,     ( 2001 )

Actin is ADP-ribosylated by the Salmonella enterica virulence-associated protein SpvB.

Molecular microbiology 39 (3)
PMID : 11169102  :   DOI  :   10.1046/j.1365-2958.2001.02258.x    
Abstract >>
The Salmonella enterica virulence-associated protein SpvB was recently shown to contain a carboxy-terminal mono(ADP-ribosyl)transferase domain. We demonstrate here that the catalytic domain of SpvB as well bacterial extracts containing full-length SpvB modifies a 43 kDa protein from macrophage-like J774-A.1 and epithelial MDCK cells as shown by label transfer from [32P]-nicotinamide adenine dinucleotide (NAD) to the 43 kDa protein. When analysed by two-dimensional gel electrophoresis, the same protein was modified in cells infected with S. enterica serovariant Dublin strain SH9325, whereas infection with an isogenic spvB mutant strain did not result in modification. Immunoprecipitation and immunoblotting experiments using SH9325-infected cells identified the modified protein as actin. The isolated catalytic domain of SpvB mediated transfer of 32P from [32P]-NAD to actins from various sources in vitro, whereas isolated eukaryotic control proteins or bacterial proteins were not modified. In an in vitro actin polymerization assay, the isolated catalytic SpvB domain prevented the conversion of G actin into F actin. Microscopic examination of MDCK cells infected with SH9325 revealed morphological changes and loss of filamentous actin content, whereas cells infected with the spvB mutant remained virtually unaffected. We conclude that actin is a target for an SpvB-mediated modification, most probably ADP-ribosylation, and that the modification of G actin interferes with actin polymerization.
KeywordMeSH Terms
Virulence Factors
48. Figueroa-Bossi  N, Uzzau  S, Maloriol  D, Bossi  L,     ( 2001 )

Variable assortment of prophages provides a transferable repertoire of pathogenic determinants in Salmonella.

Molecular microbiology 39 (2)
PMID : 11136448  :   DOI  :   10.1046/j.1365-2958.2001.02234.x    
Abstract >>
Gene transfer between separate lineages of a bacterial pathogen can promote recombinational divergence and the emergence of new pathogenic variants. Temperate bacteriophages, by virtue of their ability to carry foreign DNA, are potential key players in this process. Our previous work has shown that representative strains of Salmonella typhimurium (LT2, ATCC14028 and SL1344) are lysogenic for two temperate bacteriophages: Gifsy-1 and Gifsy-2. Several lines of evidence suggested that both elements carry genes that contribute to Salmonella virulence. One such gene, on the Gifsy-2 prophage, codes for the [Cu, Zn] superoxide dismutase SodCI. Other putative pathogenicity determinants were uncovered more recently. These include genes for known or presumptive type III-translocated proteins and a locus, duplicated on both prophages, showing sequence similarity to a gene involved in Salmonella enteropathogenesis (pipA). In addition to Gifsy-1 and Gifsy-2, each of the above strains was found to harbour a specific set of prophages also carrying putative pathogenicity determinants. A phage released from strain LT2 and identified as phage Fels-1 carries the nanH gene and a novel sodC gene, which was named sodCIII. Strain ATCC14028 releases a lambdoid phage, named Gifsy-3, which contains the phoP/phoQ-activated pagJ gene and the gene for the secreted leucine-rich repeat protein SspH1. Finally, a phage specifically released from strain SL1344 was identified as SopEPhi. Most phage-associated loci transferred efficiently between Salmonella strains of the same or different serovars. Overall, these results suggest that lysogenic conversion is a major mechanism driving the evolution of Salmonella bacteria.
KeywordMeSH Terms
49. Koch  WH, Fernández de Henestrosa  AR, Woodgate  R,     ( 2000 )

Identification of mucAB-like homologs on two IncT plasmids, R394 and Rts-1.

Mutation research 457 (1��2��)
PMID : 11106794  :   DOI  :   10.1016/s0027-5107(00)00134-2    
Abstract >>
Recent phylogenetic analysis of the superfamily of lesion-replicating DNA polymerases suggest that they can be broadly divided into four sub-groups comprised of UmuC-like, DinB-like, Rev1-like and Rad30-like proteins. The UmuC-like sub-family is best characterized at the genetic level and sequence analysis of eleven umu orthologs, residing on bacterial chromosomes or on self-transmissible R-plasmids allows further subdivision into five sub-groups (UmuDC, MucAB, ImpAB, RumAB and RulAB) based on amino acid sequence conservation. Some of these orthologs are apparently inactive in situ, but may promote increased mutagenesis and survival when subcloned and expressed from high-copy number plasmids. We were, therefore, interested in devising an assay that would identify umuC-like genes in situ in the absence of a functional assay. To this end, degenerate primers directed towards conserved amino acid regions within the UmuC-like sub-family of DNA polymerases were designed and used to identify mucAB-like operons on the IncT plasmids, R394 and Rts-1.Interestingly, DNA sequence analysis of an approximately 7kb region of R394 identified two LexA-regulated genes immediately downstream of mucAB((R394)) that are similar to the chromosomally-encoded Escherichia coli tus gene and the IncI plasmid-encoded impC gene, respectively. Analysis of the R394 and Rts-1 mucB genes revealed that both contain insertions which result in the expression of a truncated inactive MucB protein. While R394 was unable to restore mutagenesis functions to a DeltaumuDC E. coli strain, Rts-1 surprisingly promoted significant levels of MMS-induced SOS mutagenesis, raising the possibility that Rts-1 encodes another, yet unidentified, umu-like homolog.
KeywordMeSH Terms
Escherichia coli Proteins
Genes, Bacterial
50. Dailly  YP, Bunch  P, Clark  DP,     ( 2000 )

Comparison of the fermentative alcohol dehydrogenases of Salmonella typhimurium and Escherichia coli.

Microbios 103 (406)
PMID : 11131810  :  
Abstract >>
The adhE gene, encoding the fermentative alcohol dehydrogenase, from Salmonella typhimurium (Genbank accession number U68173) was cloned and sequenced. The Salmonella AdhE protein has 619/878 (70%) amino acid residues identical to the AdhE protein of Escherichia coli. Salmonella AdhE was synthesized only anaerobically. It was present in higher amounts when cells were grown on reduced substrates such as sorbitol, instead of glucose. Growth on glucuronate, which generated no net nicotinamide-adenine dinucleotide reduced (NADH) during metabolism, showed the lowest AdhE levels. Analysis of fermentation products by in vivo nuclear magenetic resonance showed that the proportion of ethanol was highest with sorbitol, intermediate with glucose and negligible with glucuronate. The Salmonella enzyme had a lower Michaelis-Menten constant (Km) for alcohol substrates than AdhE of E. coli although both enzymes displayed a similar Km for nicotinamide-adenine dinucleotide (NAD+). Although AdhE of E. coli was inactive with alcohols longer than four carbons, the Salmonella enzyme was still active with alcohols up to eight carbons.
KeywordMeSH Terms
51. 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
52. El Hanafi  D,     ( 2000 )

Activation and silencing of leu-500 promoter by transcription-induced DNA supercoiling in the Salmonella chromosome.

Molecular microbiology 37 (3)
PMID : 10931352  :   DOI  :   10.1046/j.1365-2958.2000.02015.x    
Abstract >>
The notion that transcription can generate supercoils in the DNA template largely stems from work with small circular plasmids. In the present work, we tested this model in the bacterial chromosome using a supercoiling-sensitive promoter as a functional sensor of superhelicity changes. The leu-500 promoter of Salmonella typhimurium is a mutant and inactive variant of the leucine operon promoter that regains activity if negative DNA supercoiling rises above normal levels, typically as a result of mutations affecting DNA topoisomerase I (topA mutants). Activation of the leu-500 promoter was analysed in topA mutant cells harbouring transcriptionally inducible tet or cat gene cassettes inserted in the region upstream from the leu operon. Some insertions inhibited leu-500 promoter activation in the absence of inducer. This effect is dramatic in the interval between 1.7 kb and 0.6 kb from the leu operon, suggesting that the insertions physically interfere with the mechanism responsible for activation. Superimposed on these effects, transcription of the inserted gene stimulated or inhibited leu-500 promoter activity depending on whether this gene was oriented divergently from the leu operon or in the same direction respectively. Interestingly, transcription-mediated inhibition of leu-500 promoter was observed with inserts as far as 5 kb from the leu operon, and it could be relieved by the introduction of a strong gyrase site between the inserted element and the leu-500 promoter. These results are consistent with the idea that transcriptionally generated positive and negative supercoils can diffuse along chromosomal DNA and, depending on their topological sign, elicit opposite responses from the leu-500 promoter.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
53. Peters  GA, Boyd  DA,     ( 2000 )

Partial characterization of a genomic island associated with the multidrug resistance region of Salmonella enterica Typhymurium DT104.

FEMS microbiology letters 189 (2)
PMID : 10930753  :   DOI  :   10.1111/j.1574-6968.2000.tb09245.x    
Abstract >>
This study describes the identification of the insertion site and partial characterization of a 43-kb region harboring the genes associated with the penta-resistant phenotype of a Canadian isolate of Salmonella enterica Typhymurium DT104 labelled 96-5227. The 43-kb fragment, here referred to as Salmonella genomic island I (SgiI), was found in the genome of S. enterica Typhymurium between the thdf and a prophage CP-4-like integrase (int2) gene and is flanked by an imperfect 18-bp direct repeat. A region downstream of sulI in the right end of SgiI contained four open reading frames which includes an IS6100 element, and a 2-kb region from the left end contained two open reading frames which showed homology to an integrase and an excisionase. Furthermore, a 1.9-kb retron sequence located between int2 and yidY was identified which may be unique to the S. enterica Typhymurium genome. The int-retron sequence is flanked by a 27-bp imperfect direct repeat.
KeywordMeSH Terms
Genome, Bacterial
54. Dubé  F, Chénier  I,     ( 2000 )

Mutations permitting fdhF (formate dehydrogenase H) expression in a selD mutant of Salmonella typhimurium.

Current microbiology 41 (1)
PMID : 10919397  :  
Abstract >>
Translation of fdhF mRNA, encoding formate dehydrogenase H, requires the context-specific insertion of selenocysteine at an opal codon. We have cloned the Tn10 insertion previously shown to block fdhF transcription in Salmonella typhimurium and shown that it lies in selD, which encodes phosphoselenate synthetase. A spontaneous mutant of the selD::Tn10 strain that expresses anfdhF::lacZ protein fusion (where lacZ is fused after the opal codon) was isolated and characterized. Suppression showed the same context requirement as previously reported for SelB insertion of selenocysteine. The suppressing mutation was mapped by P22 co-transduction to the 74-75 min of the Salmonella typhimurium genome.
KeywordMeSH Terms
Drosophila Proteins
55. Lin  S, Gibbons  HS,     ( 2000 )

Oxygen requirement for the biosynthesis of the S-2-hydroxymyristate moiety in Salmonella typhimurium lipid A. Function of LpxO, A new Fe2+/alpha-ketoglutarate-dependent dioxygenase homologue.

The Journal of biological chemistry 275 (42)
PMID : 10903325  :   DOI  :   10.1074/jbc.M005779200    
Abstract >>
Lipid A molecules of certain Gram-negative bacteria, including Salmonella typhimurium and Pseudomonas aeruginosa, may contain secondary S-2-hydroxyacyl chains. S. typhimurium has recently been shown to synthesize its S-2-hydroxymyristate-modified lipid A in a PhoP/PhoQ-dependent manner, suggesting a possible role for the 2-OH group in pathogenesis. We postulated that 2-hydroxylation might be catalyzed by a novel dioxygenase. Lipid A was extracted from a PhoP-constitutive mutant of S. typhimurium grown in the presence or absence of O(2). Under anaerobic conditions, no 2-hydroxymyristate-containing lipid A was formed. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of lipid A from cells grown in the presence of (18)O(2) confirmed the direct incorporation of molecular oxygen into 2-hydroxyacyl-modified lipid A. Using several well characterized dioxygenase protein sequences as probes, tBLASTn searches revealed unassigned open reading frame(s) with similarity to mammalian aspartyl/asparaginyl beta-hydroxylases in bacteria known to make 2-hydroxyacylated lipid A molecules. The S. typhimurium aspartyl/asparaginyl beta-hydroxylase homologue (designated lpxO) was cloned into pBluescriptSK and expressed in Escherichia coli K-12, which does not contain lpxO. Analysis of the resulting construct revealed that lpxO expression is sufficient to induce O(2)-dependent formation of 2-hydroxymyristate-modified lipid A in E. coli. LpxO very likely is a novel Fe(2+)/alpha-ketoglutarate-dependent dioxygenase that catalyzes the hydroxylation of lipid A (or of a key precursor). The S. typhimurium lpxO gene encodes a polypeptide of 302 amino acids with predicted membrane-anchoring sequences at both ends. We hypothesize that 2-hydroxymyristate chains released from lipopolysaccharide inside infected macrophages might be converted to 2-hydroxymyristoyl coenzyme A, a well characterized, potent inhibitor of protein N-myristoyl transferase.
KeywordMeSH Terms
56. Miao  EA,     ( 2000 )

A conserved amino acid sequence directing intracellular type III secretion by Salmonella typhimurium.

Proceedings of the National Academy of Sciences of the United States of America 97 (13)
PMID : 10861017  :   DOI  :   10.1073/pnas.97.13.7539     PMC  :   PMC16581    
Abstract >>
Type III secretion systems (TTSS) are important virulence factors that Gram-negative bacteria use to translocate proteins into the cytoplasm of eukaryotic host cells. Salmonellae encode two virulence-associated TTSS. The Salmonella pathogenicity island 1 (SPI1)-encoded TTSS is active on contact with host cells, whereas the Salmonella pathogenicity island 2 (SPI2)-encoded TTSS is expressed after phagocytosis of bacteria by host cells. Previously, no consensus signal sequence for translocation has been identified among TTSS effector proteins. In this work, seven proteins, termed Salmonella-translocated effectors (STE), are described that contain conserved amino acid sequences that direct translocation by TTSS in Salmonella typhimurium. STE that are coordinately regulated with SPI2 gene expression contain translocation signals that are recognized by the SPI2 but not by the SPI1 TTSS. STE that are constitutively expressed contain signals that direct translocation through both SPI1 and SPI2 TTSS. Of the seven STE examined, SspH1 and SspH2 have been previously shown to be translocated and involved in virulence; SlrP and SifA were identified as virulence factors, but were not previously known to be associated with TTSS; and SseI, SseJ, and SifB were previously unidentified. Three STE genes (sspH1, sspH2, and sseI) are located within temperate bacteriophages, suggesting a common mechanism for the dissemination of more recently evolved STE.
KeywordMeSH Terms
57. Daly  M, Fanning  S,     ( 2000 )

Characterization and chromosomal mapping of antimicrobial resistance genes in Salmonella enterica serotype typhimurium.

Applied and environmental microbiology 66 (11)
PMID : 11055933  :   DOI  :   10.1128/aem.66.11.4842-4848.2000     PMC  :   PMC92389    
Abstract >>
Two hundred and twenty-six Salmonella enterica serotype Typhimurium isolates were examined for the presence of integron-associated gene cassettes. All but two of the non-DT104 isolates, together with DT104 isolates, contained gene cassettes. Amplicons of 1.5 kbp each were found in two non-DT104 isolates, encoding a dhfrI gene (trimethoprim resistance) linked to an aadA gene (streptomycin and spectinomycin resistance), by site-specific recombination. DT104 isolates of resistance (R) type ACSSuT possessed the recently described 1.0- and 1.2-kbp gene cassettes. Macrorestriction analysis with XbaI and DNA probing mapped ant(3")-1a, bla(PSE-1), and dhfrI genes to large multiresistant gene clusters in a DT170a isolate and a DT193 isolate. In contrast, all DT104 isolates (R-type ACSSuT) possessed a conserved 10-kbp Xba1 DNA fragment. Our study highlights the occurrence of integrons (and antimicrobial resistance determinants) among serotype Typhimurium isolates other than DT104. Larger and previously unrecognized multiresistance gene clusters were identified in these isolates by DNA probing.
KeywordMeSH Terms
Genes, Bacterial
58. Piddock  LJ, White  DG, Gensberg  K, Pumbwe  L, Griggs  DJ,     ( 2000 )

Evidence for an efflux pump mediating multiple antibiotic resistance in Salmonella enterica serovar Typhimurium.

Antimicrobial agents and chemotherapy 44 (11)
PMID : 11036033  :   DOI  :   10.1128/aac.44.11.3118-3121.2000     PMC  :   PMC101613    
Abstract >>
The mechanism of multiple antibiotic resistance in six isolates of Salmonella enterica serovar Typhimurium recovered from a patient treated with ciprofloxacin was studied to investigate the role of efflux in the resistance phenotype. Compared to the patient's pretherapy isolate (L3), five of six isolates accumulated less ciprofloxacin, three of six isolates accumulated less chloramphenicol, and all six accumulated less tetracycline. The accumulation of one or more antibiotics was increased by carbonyl cyanide m-chlorophenylhydrazone to concentrations similar to those accumulated by L3 for all isolates except one, in which accumulation of all three agents remained approximately half that of L3. All isolates had the published wild-type sequences of marO and marR. No increased expression of marA, tolC, or soxS was observed by Northern blotting; however, three isolates showed increased expression of acrB, which was confirmed by quantitative competitive reverse transcription-PCR. However, there were no mutations within acrR or the promoter region of acrAB in any of the isolates.
KeywordMeSH Terms
Carrier Proteins
Drug Resistance, Microbial
Escherichia coli Proteins
59. Stevenson  G, Lan  R, Reeves  PR,     ( 2000 )

The colanic acid gene cluster of Salmonella enterica has a complex history.

FEMS microbiology letters 191 (1)
PMID : 11004393  :   DOI  :   10.1111/j.1574-6968.2000.tb09312.x    
Abstract >>
The colanic acid gene cluster of Salmonella enterica LT2 was sequenced and compared with that of Escherichia coli K-12. The two clusters are similar with divergence slightly higher than average for genes of the two species. The cluster was divided into four blocks by GC content and seems likely to have transferred from a higher GC content species to the ancestor of E. coli and S. enterica. All 19 genes of K-12 and 13 genes of LT2 appear to have undergone random genetic drift with amelioration of the GC content. However, in the case of S. enterica, we believe that the six genes of the GDP-fucose pathway group were replaced relatively recently by genes closely related to those of the original donor species. Two repetitive elements were observed: a bacterial interspersed mosaic element in the intergenic region between wzx and wcaK in K-12 only and a RSA (repetitive sequence element) sequence between wcaJ and wzx in LT2 only.
KeywordMeSH Terms
Evolution, Molecular
Genes, Bacterial
60. Villa  L, Pezzella  C, Tosini  F, Visca  P, Petrucca  A, Carattoli  A,     ( 2000 )

Multiple-antibiotic resistance mediated by structurally related IncL/M plasmids carrying an extended-spectrum beta-lactamase gene and a class 1 integron.

Antimicrobial agents and chemotherapy 44 (10)
PMID : 10991889  :   DOI  :   10.1128/aac.44.10.2911-2914.2000     PMC  :   PMC90180    
Abstract >>
A conjugative IncL/M plasmid (pSEM) conferring resistance to gentamicin, amikacin, kanamycin, sulfonamides, and expanded-spectrum cephalosporins was found in pathogenic strains of Salmonella enterica serotype Typhimurium. Resistance to aminoglycosides was encoded by a sul1-type class 1 integron (In-t3). An extended-spectrum beta-lactamase gene, bla(SHV-5), was identified 3. 5 kb downstream of the integrase (intI1) gene of In-t3. Nucleotide sequence analysis of the 5.3-kb bla(SHV-5)-In-t3 region of pSEM highlighted striking similarities with IncL/M plasmids isolated from nosocomial gram-negative pathogens, conferring resistance to expanded-spectrum cephalosporins and aminoglycosides.
KeywordMeSH Terms
61. Ching  KH, Worley  MJ,     ( 2000 )

Salmonella SsrB activates a global regulon of horizontally acquired genes.

Molecular microbiology 36 (3)
PMID : 10844662  :   DOI  :   10.1046/j.1365-2958.2000.01902.x    
Abstract >>
Salmonella enterica is a bacterial pathogen of humans that can proliferate within epithelial cells as well as professional phagocytes of the immune system. This ability requires an S. enterica specific locus termed Salmonella pathogenicity island 2 (SPI-2). SPI-2 encodes a type III secretion system that injects effectors encoded within the island into host cell cytosol to promote virulence. SsrAB is a two-component regulator encoded within SPI-2 that was assumed to activate SPI-2 genes exclusively. Here, it is shown that SsrB in fact activates a global regulon. At least 10 genes outside SPI-2 are SsrB regulated within epithelial and macrophage cells. Nine of these 10 SsrB-regulated genes outside SPI-2 reside within previously undescribed regions of the Salmonella genome. Most share no sequence homology with current database entries. However, one is remarkably homologous to human glucosyl ceramidase, an enzyme involved in the ceramide signalling pathway. The SsrB regulon is modulated by the two-component regulatory systems PhoP/PhoQ and OmpR/EnvZ, and is upregulated in the intracellular microenvironment.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
Transcription Factors
62. Otto  H, Tezcan-Merdol  D, Girisch  R, Haag  F, Rhen  M, Koch-Nolte  F,     ( 2000 )

The spvB gene-product of the Salmonella enterica virulence plasmid is a mono(ADP-ribosyl)transferase.

Molecular microbiology 37 (5)
PMID : 10972829  :   DOI  :   10.1046/j.1365-2958.2000.02064.x    
Abstract >>
A number of well-known bacterial toxins ADP-ribosylate and thereby inactivate target proteins in their animal hosts. Recently, several vertebrate ecto-enzymes (ART1-ART7) with activities similar to bacterial toxins have also been cloned. We show here that PSIBLAST, a position-specific-iterative database search program, faithfully connects all known vertebrate ecto-mono(ADP-ribosyl)transferases (mADPRTs) with most of the known bacterial mADPRTs. Intriguingly, no matches were found in the available public genome sequences of archaeabacteria, the yeast Saccharomyces cerevisiae or the nematode Caenorhabditis elegans. Significant new matches detected by PSIBLAST from the public sequence data bases included only one open reading frame (ORF) of previously unknown function: the spvB gene contained in the virulence plasmids of Salmonella enterica. Structure predictions of SpvB indicated that it is composed of a C-terminal ADP-ribosyltransferase domain fused via a poly proline stretch to a N-domain resembling the N-domain of the secretory toxin TcaC from nematode-infecting enterobacteria. We produced the predicted catalytic domain of SpvB as a recombinant fusion protein and demonstrate that it, indeed, acts as an ADP-ribosyltransferase. Our findings underscore the power of the PSIBLAST program for the discovery of new family members in genome databases. Moreover, they open a new avenue of investigation regarding salmonella pathogenesis.
KeywordMeSH Terms
Virulence Factors
63. 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
64. Ibanez-Ruiz  M, Robbe-Saule  V, Hermant  D, Labrude  S, Norel  F,     ( 2000 )

Identification of RpoS (sigma(S))-regulated genes in Salmonella enterica serovar typhimurium.

Journal of bacteriology 182 (20)
PMID : 11004173  :   DOI  :   10.1128/jb.182.20.5749-5756.2000     PMC  :   PMC94696    
Abstract >>
The rpoS gene encodes the alternative sigma factor sigma(S) (RpoS) and is required for survival of bacteria under starvation and stress conditions. It is also essential for Salmonella virulence in mice. Most work on the RpoS regulon has been in the closely related enterobacterial species Escherichia coli. To characterize the RpoS regulon in Salmonella, we isolated 38 unique RpoS-activated lacZ gene fusions from a bank of Salmonella enterica serovar Typhimurium mutants harboring random Tn5B21 mutations. Dependence on RpoS varied from 3-fold to over 95-fold, and all gene fusions isolated were regulated by growth phase. The identities of 21 RpoS-dependent fusions were determined by DNA sequence analysis. Seven of the fusions mapped to DNA regions in Salmonella serovar Typhimurium that do not match any known E. coli sequence, suggesting that the composition of the RpoS regulon differs markedly in the two species. The other 14 fusions mapped to 13 DNA regions very similar to E. coli sequences. None of the insertion mutations in DNA regions common to both species appeared to affect Salmonella virulence in BALB/c mice. Of these, only three (otsA, katE, and poxB) are located in known members of the RpoS regulon. Ten insertions mapped in nine open reading frames of unknown function (yciF, yehY, yhjY, yncC, yjgB, yahO, ygaU, ycgB, and yeaG) appear to be novel members of the RpoS regulon. One insertion, that in mutant C52::H87, was in the noncoding region upstream from ogt, encoding a O(6)-methylguanine DNA methyltransferase involved in repairing alkylation damage in DNA. The ogt coding sequence is very similar to the E. coli homolog, but the ogt 5' flanking regions were found to be markedly different in the two species, suggesting genetic rearrangements. Using primer extension assays, a specific ogt mRNA start site was detected in RNAs of the Salmonella serovar Typhimurium wild-type strains C52 and SL1344 but not in RNAs of the mutant strains C52K (rpoS), SL1344K (rpoS), and C52::H87. In mutant C52::H87, Tn5B21 is inserted at the ogt mRNA start site, with lacZ presumably transcribed from the identified RpoS-regulated promoter. These results indicate that ogt gene expression in Salmonella is regulated by RpoS in stationary phase of growth in rich medium, a finding that suggests a novel role for RpoS in DNA repair functions.
KeywordMeSH Terms
Open Reading Frames
65. Rohde  M, Olsén  A, Römling  U,     ( 2000 )

AgfD, the checkpoint of multicellular and aggregative behaviour in Salmonella typhimurium regulates at least two independent pathways.

Molecular microbiology 36 (1)
PMID : 10760159  :   DOI  :   10.1046/j.1365-2958.2000.01822.x    
Abstract >>
The regulatory programme of multicellular behaviour in Salmonella typhimurium is determined by mutations in the agfD promoter. AgfD has already been identified to regulate the extracellular matrix associated with the multicellular morphotype composed of thin aggregative fimbriae (agf). To detect additional components contributing to the multicellular morphotype in S. typhimurium, we constructed a mutant in agfD, the positive transcriptional regulator of the agfBA(C) operon encoding for fimbrial subunit proteins. The agfD mutant lacked any form of multicellular behaviour as shown by analysis at the macroscopic and microscopic level. In contrast, the agfBA mutant unable to form thin aggregative fimbriae still maintained long-range intercellular adhesion. Promoter and expression analysis revealed that the genes downstream of agfD agfEFG most likely did not contribute to the remaining aggregative behaviour. Screening of transcriptional fusions for agfD dependency uncovered adrA, a homologue of yaiC in Escherichia coli. Environmental factors regulating adrA correspond to the regulation of thin aggregative fimbriae. AdrA is a putative transmembrane protein with a C-terminal GGDEF domain of unknown function although it is present in over 50 bacterial proteins. AdrA mutant cells, which still formed thin aggregative fimbriae with all binding characteristics, exhibited community behaviour but, unlike the wild type, lacked long-range intercellular adhesion. An agfBA adrA double mutant behaved like the agfD mutant. Therefore, it was concluded that agfD regulates at least two independent pathways contributing to the multicellular morphotype in S. typhimurium.
KeywordMeSH Terms
Escherichia coli Proteins
Genes, Bacterial
Genes, Regulator
Transcription Factors
66. Mathur  N, Fahlen  TF,     ( 2000 )

Identification and characterization of mutants with increased expression of hilA, the invasion gene transcriptional activator of Salmonella typhimurium.

FEMS immunology and medical microbiology 28 (1)
PMID : 10767604  :   DOI  :   10.1111/j.1574-695X.2000.tb01453.x    
Abstract >>
Induction of invasion gene transcription and expression of the invasive phenotype of Salmonella strains are regulated by environmental conditions. Experimental evidence indicates that oxygen, pH, and osmotic conditions need to closely resemble those of the host intestinal lumen for invasion gene activation. The hilA gene, encoded on Salmonella pathogenicity island 1 (SPI-1), is a transcriptional activator which is required for invasion and whose expression is modulated by oxygen, pH, and osmolarity. Additionally, hilA is regulated by genetic elements encoded on SPI-1 (hilC/sirC/sprA and hilD), as well as by elements which reside outside of SPI-1 (phoP/phoQ and sirA), although how environmental signals modulate hilA is unknown. In an effort to further characterize the Salmonella invasion gene regulon, we have created and preliminarily characterized 18 Tn5 insertions which result in upregulation of a hilA::lacZY fusion. We have classified the mutations based on location and phenotype into three classes. Six class 1 and six class 2 mutants have insertions in SPI-1 near the invasion gene orgA or the invasion gene regulator hilD, respectively. Six class 3 mutants reside outside of SPI-1 in four different loci. The class 2 and 3 mutations induce overexpression of an episomal hilA::lacZY fusion and significantly increase S. typhimurium invasion of HEp-2 cells in a standard invasion assay. These data implicate new regions of SPI-1 as being involved in the regulation of invasion by S. typhimurium and identify new invasion gene regulators located outside of SPI-1.
KeywordMeSH Terms
Mutation
67. Yoshida  T, Komano  T,     ( 2000 )

The transfer region of IncI1 plasmid R64: similarities between R64 tra and legionella icm/dot genes.

Molecular microbiology 35 (6)
PMID : 10760136  :   DOI  :   10.1046/j.1365-2958.2000.01769.x    
Abstract >>
The entire nucleotide sequence of the transfer region of IncI1 plasmid R64 was determined together with previously reported sequences. Twenty-two transfer genes, traE-Y and nuc, were newly identified in the present study. The protein products of 17 genes were detected by maxicell experiments or by the T7 RNA polymerase expression system. Mutagenesis experiments indicated that 16 genes were indispensable for R64 transfer both in liquid and on surfaces. In summary, the R64 transfer region located within an approximately 54 kb DNA segment was shown to encode the most complex transfer system so far studied. It contains at least 49 genes and may produce 58 different proteins as a result of shufflon DNA rearrangement and overlapping genes. Among the 49 genes, 23 tra, trb and nik genes have been shown to be indispensable for R64 conjugal transfer in liquid and on surfaces. Twelve additional pil genes are required only for liquid matings. The amino acid sequences of 10 R64 tra/trb products share similarity with those of the icm/dot products of Legionella pneumophila that are responsible for its virulence, suggesting that the R64 transfer and L. pneumophila icm/dot systems have evolved from a common ancestral genetic system.
KeywordMeSH Terms
Escherichia coli Proteins
68. Suyemoto  M, Ruiz  AI, Altier  C,     ( 2000 )

Characterization of two novel regulatory genes affecting Salmonella invasion gene expression.

Molecular microbiology 35 (3)
PMID : 10672185  :   DOI  :   10.1046/j.1365-2958.2000.01734.x    
Abstract >>
A Salmonella typhimurium chromosomal deletion removing approximately 19 kb of DNA at centisome 65 reduces invasion of cultured epithelial cells as well as the expression of lacZY operon fusions to several genes required for the invasive phenotype. As the deleted region contains no genes previously known to affect Salmonella invasion, we investigated the roles of individual genes in the deleted region using a combination of cloning, complementation and directed mutation. We find that the deletion includes two unrelated regulatory genes. One is the Salmonella homologue of Escherichia coli barA (airS), which encodes a member of the multistep phosphorelay subgroup of two-component sensor kinases. The action of BarA is coupled to that of SirA, a member of the phosphorylated response regulator family of proteins, and includes both HilA-dependent and HilA-independent components. The other regulatory gene removed by the deletion is the Salmonella homologue of E. coli csrB, which specifies a regulatory RNA implicated in controlling specific message turnover in E. coli. These results identify a protein that is likely to play a key role in the environmental control of Salmonella invasion gene expression, and they also suggest that transcriptional control of invasion genes could be subject to refinement at the level of message turnover.
KeywordMeSH Terms
Escherichia coli Proteins
Phosphotransferases
Repressor Proteins
RNA, Untranslated
69. O'Hare  C, Cormican  M, Power  E, Buckley  J, Daly  M,     ( 2000 )

Molecular characterization of Irish Salmonella enterica serotype typhimurium: detection of class I integrons and assessment of genetic relationships by DNA amplification fingerprinting.

Applied and environmental microbiology 66 (2)
PMID : 10653725  :   DOI  :   10.1128/aem.66.2.614-619.2000     PMC  :   PMC91870    
Abstract >>
Salmonella enterica is among the principal etiological agents of food-borne illness in humans. Increasing antimicrobial resistance in S. enterica is a cause for worldwide concern. There is concern at present in relation to the increasing incidence of human infection with antimicrobial agent-resistant strains of S. enterica serotype Typhimurium, in particular of phage type DT104. Integrons appear to play an important role in the dissemination of antimicrobial resistance genes in many Enterobacteriaceae including S. enterica. In this study the antimicrobial susceptibilities and phage types of 74 randomly collected strains of S. enterica serotype Typhimurium from the Cork region of southern Ireland, obtained from human, animal (clinical), and food sources, were determined. Each strain was examined for integrons and typed by DNA amplification fingerprinting (DAF). Phage type DT104 predominated (n = 48). Phage types DT104b (n = 3), -193 (n = 9), -195 (n = 6), -208 (n = 3), -204a (n = 2), PT U302 (n = 1), and two nontypeable strains accounted for the remainder. All S. enterica serotype Typhimurium DT104 strains were resistant to ampicillin, chloramphenicol, streptomycin, Sulfonamide Duplex, and tetracycline, and one strain was additionally resistant to trimethoprim. All DT104 strains but one were of a uniform DAF type (designated DAF-I) and showed a uniform pattern of integrons (designated IP-I). The DT104b and PT U302 strains also exhibited the same resistance phenotype, and both had the DAF-I and IP-I patterns. The DAF-I pattern was also observed in a single DT193 strain in which no integrons were detectable. Greater diversity of antibiograms and DAF and IP patterns among non-DT104 phage types was observed. These data indicate a remarkable degree of homogeneity at a molecular level among contemporary isolates of S. enterica serotype Typhimurium DT104 from animal, human, and food sources in this region.
KeywordMeSH Terms
70. Tsui  HC, Björk  GR, Leung  HC, Esberg  B,     ( 1999 )

Identification of the miaB gene, involved in methylthiolation of isopentenylated A37 derivatives in the tRNA of Salmonella typhimurium and Escherichia coli.

Journal of bacteriology 181 (23)
PMID : 10572129  :   PMC  :   PMC103688    
Abstract >>
The tRNA of the miaB2508::Tn10dCm mutant of Salmonella typhimurium is deficient in the methylthio group of the modified nucleoside N(6)-(4-hydroxyisopentenyl)-2-methylthioadenosine (ms(2)io(6)A37). By sequencing, we found that the Tn10dCm of this strain had been inserted into the f474 (yleA) open reading frame, which is located close to the nag locus in both S. typhimurium and Escherichia coli. By complementation of the miaB2508::Tn10dCm mutation with a minimal subcloned f474 fragment, we showed that f474 could be identified as the miaB gene, which is transcribed in the counterclockwise direction on the bacterial chromosome. Transcriptional studies revealed two promoters upstream of miaB in E. coli and S. typhimurium. A Rho-independent terminator was identified downstream of the miaB gene, at which the majority (96%) of the miaB transcripts terminate in E. coli, showing that the miaB gene is part of a monocistronic operon. A highly conserved motif with three cysteine residues was present in MiaB. This motif resembles iron-binding sites in other proteins. Only a weak similarity to an AdoMet-binding site was found, favoring the idea that the MiaB protein is involved in the thiolation step and not in the methylating reaction of ms(2)i(o)(6)A37 formation.
KeywordMeSH Terms
71. Altier  C,     ( 1999 )

A recombinase-based selection of differentially expressed bacterial genes.

Gene 240 (1)
PMID : 10564816  :   DOI  :   10.1016/s0378-1119(99)00427-8    
Abstract >>
Bacterial genes are often differentially expressed in response to specific environmental conditions. We have devised a method to identify regulated bacterial promoters, such that transient promoter expression leads to a permanent and selectable change in bacterial phenotype. This system consists of a promoterless derivative of cre, the phage P1 recombinase, carried on a plasmid, and two chromosomal loxP sites, the targets of the Cre recombinase. The loxP sites flank npt, conferring kanamycin resistance, and sacB, which confers sensitivity to sucrose, allowing positive selection for both the presence and absence of this chromosomal cassette. Fusion of active promoters to cre induces recombination of the loxP sites and deletion of intervening DNA, allowing selection on media containing sucrose, while inactive promoters fail to induce recombination and so remain resistant to kanamycin. We tested the system in Salmonella typhimurium using a known regulated promoter, that from the araBAD operon, and found it to be a sensitive indicator of gene expression over a wide range of promoter induction. We then used this system to identify S. typhimurium genes that are specifically expressed when bacteria interact with cultured epithelial cells and identified a novel DNA fragment, not found in E. coli, which might represent part of a new pathogenicity island.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
Viral Proteins
72. Miao  EA, Miller  SI, Ficht  TA, Townsend  SM, Tsolis  RM,     ( 1999 )

Identification of a putative Salmonella enterica serotype typhimurium host range factor with homology to IpaH and YopM by signature-tagged mutagenesis.

Infection and immunity 67 (12)
PMID : 10569754  :   PMC  :   PMC97046    
Abstract >>
The genetic basis for the host adaptation of Salmonella serotypes is currently unknown. We have explored a new strategy to identify Salmonella enterica serotype Typhimurium (S. typhimurium) genes involved in host adaptation, by comparing the virulence of 260 randomly generated signature-tagged mutants during the oral infection of mice and calves. This screen identified four mutants, which were defective for colonization of only one of the two host species tested. One mutant, which only displayed a colonization defect during the infection of mice, was further characterized. During competitive infection experiments performed with the S. typhimurium wild type, the mutant was defective for colonization of murine Peyer's patches but colonized bovine Peyer's patches at the wild-type level. No difference in virulence between wild type and mutant was observed when calves were infected orally with 10(10) CFU/animal. In contrast, the mutant possessed a sixfold increase in 50% lethal morbidity dose when mice were infected orally. The transposon in this mutant was inserted in a 2.9-kb pathogenicity islet, which is located between uvrB and yphK on the S. typhimurium chromosome. This pathogenicity islet contained a single gene, termed slrP, with homology to ipaH of Shigella flexneri and yopM of Yersinia pestis. These data show that comparative screening of signature-tagged mutants in two animal species can be used for scanning the S. typhimurium genome for genes involved in host adaptation.
KeywordMeSH Terms
Antigens, Bacterial
73. Shen  P, Samsel  L, Liu  R, Allen  T,     ( 1999 )

Phylogenetic analysis of L4-mediated autogenous control of the S10 ribosomal protein operon.

Journal of bacteriology 181 (19)
PMID : 10498727  :   PMC  :   PMC103642    
Abstract >>
We investigated the regulation of the S10 ribosomal protein (r-protein) operon among members of the gamma subdivision of the proteobacteria, which includes Escherichia coli. In E. coli, this 11-gene operon is autogenously controlled by r-protein L4. This regulation requires specific determinants within the untranslated leader of the mRNA. Secondary structure analysis of the S10 leaders of five enterobacteria (Salmonella typhimurium, Citrobacter freundii, Yersinia enterocolitica, Serratia marcescens, and Morganella morganii) and two nonenteric members of the gamma subdivision (Haemophilus influenzae and Vibrio cholerae) shows that these foreign leaders share significant structural homology with the E. coli leader, particularly in the region which is critical for L4-mediated autogenous control in E. coli. Moreover, these heterologous leaders produce a regulatory response to L4 oversynthesis in E. coli. Our results suggest that an E. coli-like L4-mediated regulatory mechanism may operate in all of these species. However, the mechanism is not universally conserved among the gamma subdivision members, since at least one, Pseudomonas aeruginosa, does not contain the required S10 leader features, and its leader cannot provide the signals for regulation by L4 in E. coli. We speculate that L4-mediated autogenous control developed during the evolution of the gamma branch of proteobacteria.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
Operon
Regulatory Sequences, Nucleic Acid
74. Wei  Y,     ( 1999 )

Characterization of a group of anaerobically induced, fnr-dependent genes of Salmonella typhimurium.

Journal of bacteriology 181 (19)
PMID : 10498722  :   PMC  :   PMC103637    
Abstract >>
We have previously reported the isolation of a group of anaerobically regulated, fnr-dependent lac fusions in Salmonella typhimurium and have grouped these oxd genes into classes based on map position. In order to identify these genes, we have replaced the original Mud-lac fusion in a member of each oxd class with the much smaller Mud-cam element, cloned the fusion, and determined DNA sequence sufficient to define the oxd gene. Several of the fusions correspond to previously known genes from S. typhimurium or Escherichia coli: oxd-4 = cbiA and oxd-11 = cbiK, oxd-5 = hybB, oxd-7 = dcuB, oxd-8 = moaB, oxd-12 = dmsA, and oxd-14 = napB (aeg-46. 5). Two other fusions correspond to previously unknown loci: oxd-2 encodes an acetate/propionate kinase, and oxd-6 encodes a putative ABC transporter present in S. typhimurium but not in E. coli.
KeywordMeSH Terms
Carrier Proteins
Dicarboxylic Acid Transporters
Escherichia coli Proteins
Gene Expression Regulation, Bacterial
Genes, Bacterial
75. 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
76. Havemann  GD, Busch  RJ, Bobik  TA,     ( 1999 )

The propanediol utilization (pdu) operon of Salmonella enterica serovar Typhimurium LT2 includes genes necessary for formation of polyhedral organelles involved in coenzyme B(12)-dependent 1, 2-propanediol degradation.

Journal of bacteriology 181 (19)
PMID : 10498708  :   PMC  :   PMC103623    
Abstract >>
The propanediol utilization (pdu) operon of Salmonella enterica serovar Typhimurium LT2 contains genes needed for the coenzyme B(12)-dependent catabolism of 1,2-propanediol. Here the completed DNA sequence of the pdu operon is presented. Analyses of previously unpublished pdu DNA sequence substantiated previous studies indicating that the pdu operon was acquired by horizontal gene transfer and allowed the identification of 16 hypothetical genes. This brings the total number of genes in the pdu operon to 21 and the total number of genes at the pdu locus to 23. Of these, six encode proteins of unknown function and are not closely related to sequences of known function found in GenBank. Two encode proteins involved in transport and regulation. Six probably encode enzymes needed for the pathway of 1,2-propanediol degradation. Two encode proteins related to those used for the reactivation of adenosylcobalamin (AdoCbl)-dependent diol dehydratase. Five encode proteins related to those involved in the formation of polyhedral organelles known as carboxysomes, and two encode proteins that appear distantly related to those involved in carboxysome formation. In addition, it is shown that S. enterica forms polyhedral bodies that are involved in the degradation of 1,2-propanediol. Polyhedra are formed during either aerobic or anaerobic growth on propanediol, but not during growth on other carbon sources. Genetic tests demonstrate that genes of the pdu operon are required for polyhedral body formation, and immunoelectron microscopy shows that AdoCbl-dependent diol dehydratase is associated with these polyhedra. This is the first evidence for a B(12)-dependent enzyme associated with a polyhedral body. It is proposed that the polyhedra consist of AdoCbl-dependent diol dehydratase (and perhaps other proteins) encased within a protein shell that is related to the shell of carboxysomes. The specific function of these unusual polyhedral bodies was not determined, but some possibilities are discussed.
KeywordMeSH Terms
Genes, Bacterial
77. Marcus  SL, Steele-Mortimer  O, Pfeifer  CG,     ( 1999 )

Salmonella typhimurium virulence genes are induced upon bacterial invasion into phagocytic and nonphagocytic cells.

Infection and immunity 67 (11)
PMID : 10531217  :   PMC  :   PMC96943    
Abstract >>
Survival and growth of salmonellae within host cells are important aspects of bacterial virulence. We have developed an assay to identify Salmonella typhimurium genes that are induced inside Salmonella-containing vacuoles within macrophage and epithelial cells. A promoterless luciferase gene cassette was inserted randomly into the Salmonella chromosome, and the resulting mutants were screened for genes upregulated in intracellular bacteria compared to extracellular bacteria. We identified four genes in S. typhimurium that were upregulated upon bacterial invasion of both phagocytic and nonphagocytic cells. Expression of these genes was not induced by factors secreted by host cells or media alone. All four genes were induced at early time points (2 to 4 h) postinvasion and continued to be upregulated within host cells at later times (5 to 7 h). One mutant contained an insertion in the ssaR gene, within Salmonella pathogenicity island 2 (SPI-2), which abolished bacterial virulence in a murine typhoid model. Two other mutants contained insertions within SPI-5, one in the sopB/sigD gene and the other in a downstream gene, pipB. The insertions within SPI-5 resulted in the attenuation of S. typhimurium in the mouse model. The fourth mutant contained an insertion within a previously undescribed region of the S. typhimurium chromosome, iicA (induced intracellularly A). We detected no effect on virulence as a result of this insertion. In conclusion, all but one of the genes identified in this study were virulence factors within pathogenicity islands, illustrating the requirement for specific gene expression inside mammalian cells and indicating the key role that virulence factor regulation plays in Salmonella pathogenesis.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
Genes, Bacterial
78. Goebel  W, Emmerth  M,     ( 1999 )

Genomic subtraction identifies Salmonella typhimurium prophages, F-related plasmid sequences, and a novel fimbrial operon, stf, which are absent in Salmonella typhi.

Journal of bacteriology 181 (18)
PMID : 10482505  :   PMC  :   PMC94084    
Abstract >>
Salmonella typhimurium causes systemic and fatal infection in inbred mice, while the related serotype Salmonella typhi is avirulent for mammals other than humans. In order to identify genes from the virulent strain S. typhimurium ATCC 14028 that are absent in S. typhi Ty2, and therefore might be involved in S. typhimurium mouse virulence, a PCR-supported genomic subtractive hybridization procedure was employed. We have identified a novel putative fimbrial operon, stfACDEFG, located at centisome 5 of the S. typhimurium chromosome, which is absent in S. typhi, Salmonella arizonae, and Salmonella bongori but was detected in several other Salmonella serotypes. The fimbrial genes represent a genomic insertion in S. typhimurium compared to the respective region between fhuB and hemL in Escherichia coli K-12. In addition, the subtraction procedure yielded F plasmid-related sequences from the S. typhimurium virulence plasmid, a number of DNA fragments representing parts of lambdoid prophages and putative sugar transporters, and several fragments with unknown sequences. The majority of subtracted chromosomal sequences map to three distinct locations, around centisomes 5, 27, and 57.
KeywordMeSH Terms
Operon
79. Graham  JE, Morrow  BJ,     ( 1999 )

Genomic subtractive hybridization and selective capture of transcribed sequences identify a novel Salmonella typhimurium fimbrial operon and putative transcriptional regulator that are absent from the Salmonella typhi genome.

Infection and immunity 67 (10)
PMID : 10496884  :   PMC  :   PMC96859    
Abstract >>
Salmonella typhi, the etiologic agent of typhoid fever, is adapted to the human host and unable to infect nonprimate species. The genetic basis for host specificity in S. typhi is unknown. The avirulence of S. typhi in animal hosts may result from a lack of genes present in the broad-host-range pathogen Salmonella typhimurium. Genomic subtractive hybridization was successfully employed to isolate S. typhimurium genomic sequences which are absent from the S. typhi genome. These genomic subtracted sequences mapped to 17 regions distributed throughout the S. typhimurium chromosome. A positive cDNA selection method was then used to identify subtracted sequences which were transcribed by S. typhimurium following macrophage phagocytosis. A novel putative transcriptional regulator of the LysR family was identified as transcribed by intramacrophage S. typhimurium. This putative transcriptional regulator was absent from the genomes of the human-adapted serovars S. typhi and Salmonella paratyphi A. Mutations within this gene did not alter the level of S. typhimurium survival within macrophages or virulence within mice. A subtracted genomic fragment derived from the ferrichrome operon also hybridized to the intramacrophage cDNA. Nucleotide sequence analysis of S. typhimurium and S. typhi chromosomal sequences flanking the ferrichrome operon identified a novel S. typhimurium fimbrial operon with a high level of similarity to sequences encoding Proteus mirabilis mannose-resistant fimbriae. The novel fimbrial operon was absent from the S. typhi genome. The absence of specific genes may have allowed S. typhi to evolve as a highly invasive, systemic human pathogen.
KeywordMeSH Terms
Genes, Regulator
Genome, Bacterial
Operon
80. Igwe  E, Rüssmann  H, Stender  S, Rabsch  W, Rohde  M,     ( 1999 )

Isolation of a temperate bacteriophage encoding the type III effector protein SopE from an epidemic Salmonella typhimurium strain.

Proceedings of the National Academy of Sciences of the United States of America 96 (17)
PMID : 10449782  :   DOI  :   10.1073/pnas.96.17.9845     PMC  :   PMC22298    
Abstract >>
Salmonella typhimurium employs the specialized type III secretion system encoded in pathogenicity island 1 (SPI1) to translocate effector proteins into host cells and to modulate host cell signal transduction. The SPI1 type III system and the effector proteins are conserved among all salmonellae and are thought to be acquired by horizontal gene transfer. The genetic mechanisms mediating this horizontal transfer are unknown. Here, we describe that SopE, a SPI1-dependent translocated effector protein, is present in relatively few S. typhimurium isolates. We have isolated a temperate phage that encodes SopE. Phage morphology and DNA hybridization, as well as partial sequence information, suggest that this phage (SopEPhi) is a new member of the P2 family of bacteriophages. By lysogenic conversion this phage can horizontally transfer genes between different S. typhimurium strains. Strikingly, most of the isolates harboring SopEPhi belong to the small group of epidemic strains of S. typhimurium that have been responsible for a large percentage of human and animal salmonellosis and have persisted for a long period of time. Our data suggest that horizontal transfer of type III dependent effector proteins by lysogenic infection with bacteriophages (lysogenic conversion) may provide an efficient mechanism for fine-tuning the interaction of Salmonella spp. with their hosts.
KeywordMeSH Terms
81. Klimke  WA, Manchak  J, Frost  LS,     ( 1999 )

Comparison of proteins involved in pilus synthesis and mating pair stabilization from the related plasmids F and R100-1: insights into the mechanism of conjugation.

Journal of bacteriology 181 (17)
PMID : 10464182  :   PMC  :   PMC94017    
Abstract >>
F and R100-1 are closely related, derepressed, conjugative plasmids from the IncFI and IncFII incompatibility groups, respectively. Heteroduplex mapping and genetic analyses have revealed that the transfer regions are extremely similar between the two plasmids. Plasmid specificity can occur at the level of relaxosome formation, regulation, and surface exclusion between the two transfer systems. There are also differences in pilus serology, pilus-specific phage sensitivity, and requirements for OmpA and lipopolysaccharide components in the recipient cell. These phenotypic differences were exploited in this study to yield new information about the mechanism of pilus synthesis, mating pair stabilization, and surface and/or entry exclusion, which are collectively involved in mating pair formation (Mpf). The sequence of the remainder of the transfer region of R100-1 (trbA to traS) has been completed, and the complete sequence is compared to that of F. The differences between the two transfer regions include insertions and deletions, gene duplications, and mosaicism within genes, although the genes essential for Mpf are conserved in both plasmids. F+ cells carrying defined mutations in each of the Mpf genes were complemented with the homologous genes from R100-1. Our results indicate that the specificity in recipient cell recognition and entry exclusion are mediated by TraN and TraG, respectively, and not by the pilus.
KeywordMeSH Terms
Conjugation, Genetic
F Factor
Fimbriae, Bacterial
Genes, Bacterial
Pili, Sex
R Factors
82. Löfdahl  S, Advani  A, Sukupolvi  S, Pfeifer  JD, Normark  S,     ( 1999 )

Multiple insertions of fimbrial operons correlate with the evolution of Salmonella serovars responsible for human disease.

Molecular microbiology 33 (3)
PMID : 10417651  :   DOI  :   10.1046/j.1365-2958.1999.01508.x    
Abstract >>
On centisome 7, Salmonella spp. contain a large region not present in the corresponding region of Escherichia coli. This region is flanked by sequences with significant homology to the E. coli tRNA gene aspV and the hypothetical E. coli open reading frame yafV. The locus consists of a mosaic of differentially acquired inserts forming a dynamic cs7 region of horizontally transferred inserts. Salmonella enterica subspecies I, responsible for most Salmonella infections in warm-blooded animals, carries a fimbrial gene cluster (saf) in this region as well as a regulatory gene (sinR). These genes are flanked by inverted repeats and are inserted in another laterally transferred region present in most members of Salmonella spp. encoding a putative invasin (pagN). S. enterica subspecies I serovar Typhi, the Salmonella serovar that causes the most severe form of human salmonellosis, contains an additional insert of at least 8 kb in the sinR-pagN intergenic region harbouring a novel fimbrial operon (tcf) similar to the coo operon encoding the CS1 fimbrial adhesin expressed by human-specific enterotoxigenic E. coli. It is suggested that the multiple insertions of fimbrial genes that have occurred in the cs7 region have contributed to phylogenetic diversity and host adaptation of Salmonella spp.
KeywordMeSH Terms
Genes, Bacterial
83. Hardt  WD, Galán  JE,     ( 1999 )

Characterization of SprA, an AraC-like transcriptional regulator encoded within the Salmonella typhimurium pathogenicity island 1.

Molecular microbiology 33 (1)
PMID : 10411731  :   DOI  :   10.1046/j.1365-2958.1999.01458.x    
Abstract >>
Pathogenicity island 1 (SPI-1) located at centisome 63 of the Salmonella chromosome encodes a type III protein secretion system that is essential for its pathogenicity. The translocation of effector proteins through this system results in the stimulation of signalling events, leading to actin cytoskeletal rearrangements and nuclear responses. These cellular responses ultimately lead to bacterial uptake, production of proinflammatory cytokines in non-phagocytic cells and the initiation of programmed cell death in macrophages. The regulation of expression of components and substrates of this type III secretion system is complex and involves the activity of several specific transcriptional regulatory proteins encoded within SPI-1. Here, we describe two additional regulatory proteins, SprA and SprB, which are encoded within SPI-1. SprA and SprB exhibit significant sequence similarity to the AraC/XylS and the LuxR/UhaP family of transcriptional regulatory proteins respectively. Insertion mutations in sprA and sprB did not significantly affect the transcription of invasion-associated genes and, consequently, did not affect the ability of Salmonella typhimurium to gain access into host cells. However, expression of sprA from an inducible heterologous promoter resulted in increased expression of genes associated with the centisome 63 type III secretion system and increased the ability of S. typhimurium to enter into host cells. Further analysis demonstrated that SprA acts either upstream or at the same level as HilA in the SPI-1 transcriptional regulatory cascade.
KeywordMeSH Terms
DNA-Binding Proteins
Gene Expression Regulation, Bacterial
Genes, Bacterial
Transcription Factors
Transcription, Genetic
84. Marqués  S, Casadesús  J,     ( 1999 )

Synthesis of FinP RNA by plasmids F and pSLT is regulated by DNA adenine methylation.

Genetics 152 (1)
PMID : 10408954  :   PMC  :   PMC1460579    
Abstract >>
DNA adenine methylase mutants of Salmonella typhimurium contain reduced amounts of FinP, an antisense RNA encoded by the virulence plasmid pSLT. Lowered FinP levels are detected in both Dam- FinO+ and Dam- FinO- backgrounds, suggesting that Dam methylation regulates FinP production rather than FinP half-life. Reduced amounts of F-encoded FinP RNA are likewise found in Dam- mutants of Escherichia coli. A consequence of FinP RNA scarcity in the absence of DNA adenine methylation is that Dam- mutants of both S. typhimurium and E. coli show elevated levels of F plasmid transfer. Inhibition of F fertility by the S. typhimurium virulence plasmid is also impaired in a Dam- background.
KeywordMeSH Terms
F Factor
Plasmids
85. Bolton  LF, Maurer  JJ, Fedorka-Cray  PJ,     ( 1999 )

Detection of multidrug-resistant Salmonella enterica serotype typhimurium DT104 based on a gene which confers cross-resistance to florfenicol and chloramphenicol.

Journal of clinical microbiology 37 (5)
PMID : 10203484  :   PMC  :   PMC84772    
Abstract >>
Salmonella enterica serotype typhimurium (S. typhimurium) DT104 (DT104) first emerged as a major pathogen in Europe and is characterized by its pentadrug-resistant pattern. It has also been associated with outbreaks in the United States. The organism typically carries resistance to ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline. The mechanism of chloramphenicol resistance in DT104 was determined by producing antibiotic-resistant Escherichia coli host strain clones from DT104 DNA. DNA from chloramphenicol-resistant clones was sequenced, and probes specific for the genes floS. typhimurium (floSt), int, invA, and spvC were produced for colony blot hybridizations. One hundred nine Salmonella isolates, including 44 multidrug-resistant DT104 isolates, were tested to evaluate the specificities of the probes. The gene floSt, reported in this study, confers chloramphenicol and florfenicol resistance on S. typhimurium DT104. Florfenicol resistance is unique to S. typhimurium DT104 and multidrug-resistant S. typhimurium isolates with the same drug resistance profile among all isolates evaluated. Of 44 DT104 isolates tested, 98% were detected based on phenotypic florfenicol resistance and 100% had the floSt-positive genotype. Resistances to florfenicol and chloramphenicol are conferred by the gene floSt, described in this paper. Presumptive identification of S. typhimurium DT104 can be made rapidly based on the presence of the floSt gene or its resulting phenotype.
KeywordMeSH Terms
86. Hilbert  F, García-del Portillo  F,     ( 1999 )

A periplasmic D-alanyl-D-alanine dipeptidase in the gram-negative bacterium Salmonella enterica.

Journal of bacteriology 181 (7)
PMID : 10094694  :   PMC  :   PMC93629    
Abstract >>
The VanX protein is a D-alanyl-D-alanine (D-Ala-D-Ala) dipeptidase essential for resistance to the glycopeptide antibiotic vancomycin. While this enzymatic activity has been typically associated with vancomycin- and teicoplainin-resistant enterococci, we now report the identification of a D-Ala-D-Ala dipeptidase in the gram-negative species Salmonella enterica. The Salmonella enzyme is only 36% identical to VanX but exhibits a similar substrate specificity: it hydrolyzes D-Ala-D-Ala, DL-Ala-DL-Phe, and D-Ala-Gly but not the tripeptides D-Ala-D-Ala-D-Ala and DL-Ala-DL-Lys-Gly or the dipeptides L-Ala-L-Ala, N-acetyl-D-Ala-D-Ala, and L-Leu-Pro. The Salmonella dipeptidase gene, designated pcgL, appears to have been acquired by horizontal gene transfer because pcgL-hybridizing sequences were not detected in related bacterial species and the G+C content of the pcgL-containing region (41%) is much lower than the overall G+C content of the Salmonella chromosome (52%). In contrast to wild-type Salmonella, a pcgL mutant was unable to use D-Ala-D-Ala as a sole carbon source. The pcgL gene conferred D-Ala-D-Ala dipeptidase activity upon Escherichia coli K-12 but did not allow growth on D-Ala-D-Ala. The PcgL protein localizes to the periplasmic space of Salmonella, suggesting that this dipeptidase participates in peptidoglycan metabolism.
KeywordMeSH Terms
Bacterial Proteins
87. Hensel  M, Berks  BC, Sawers  G, Nikolaus  T,     ( 1999 )

The genetic basis of tetrathionate respiration in Salmonella typhimurium.

Molecular microbiology 32 (2)
PMID : 10231485  :   DOI  :   10.1046/j.1365-2958.1999.01345.x    
Abstract >>
A range of bacteria are able to use tetrathionate as a terminal respiratory electron acceptor. Here we report the identification and characterization of the ttrRSBCA locus required for tetrathionate respiration in Salmonella typhimurium LT2a. The ttr genes are located within Salmonella pathogenicity island 2 at centisome 30.5. ttrA, ttrB and ttrC are the tetrathionate reductase structural genes. Sequence analysis suggests that TtrA contains a molybdopterin guanine dinucleotide cofactor and a [4Fe-4S] cluster, that TtrB binds four [4Fe-4S] clusters, and that TtrC is an integral membrane protein containing a quinol oxidation site. TtrA and TtrB are predicted to be anchored by TtrC to the periplasmic face of the cytoplasmic membrane implying a periplasmic site for tetrathionate reduction. It is inferred that the tetrathionate reductase, together with thiosulphate and polysulphide reductases, make up a previously unrecognized class of molybdopterin-dependent enzymes that carry out the reductive cleavage of sulphur-sulphur bonds. Cys-256 in TtrA is proposed to be the amino acid ligand to the molybdopterin cofactor. TtrS and TtrR are the sensor and response regulator components of a two-component regulatory system that is absolutely required for transcription of the ttrBCA operon. Expression of an active tetrathionate reduction system also requires the anoxia-responsive global transcriptional regulator Fnr. The ttrRSBCA gene cluster confers on Escherichia coli the ability to respire with tetrathionate as electron acceptor.
KeywordMeSH Terms
88. Kallipolitis  BH, Valentin-Hansen  P, Pedersen  M, Nørregaard-Madsen  M,     ( 1999 )

Protein-ligand interaction: grafting of the uridine-specific determinants from the CytR regulator of Salmonella typhimurium to Escherichia coli CytR.

Journal of molecular biology 288 (1)
PMID : 10329134  :   DOI  :   10.1006/jmbi.1999.2668    
Abstract >>
Members of the LacI family of transcriptional repressors respond to the presence of small effector molecules. The binding of the ligands affect the proteins ability to repress transcription by stabilizing a conformation that, in most cases, is unfavorable for high-affinity DNA binding. The CytR anti-activator diverges from the other family members by relying on the cooperative DNA binding with the global regulator CRP. The inducers of CytR do not affect CytR-DNA binding per se, but alleviate repression by interrupting protein-protein interactions between the two regulators. Here, we have studied of the CytR-inducer interaction by exploring a discrepancy in the inducer response observed for the homologous CytR regulators of Escherichia coli and Salmonella typhimurium. CytR of S. typhimurium (CytRSt) appears to respond to the presence of both uridine and cytidine nucleosides, whereas E. coli CytR (CytREc) responds to cytidine only. We have used a combination of genetic and structural modeling studies to provide detailed information regarding the nature of this discrepancy. By analysis of hybrid CytR proteins followed by site-directed mutagenesis, we have successfully transferred the specificity determinants for uridine from CytRSt to CytREc, revealing that serine substitutions of only two residues (G131 and A152) in CytREc is required to make CytREc sensitive to uridine. In addition, by employing a genetic screen for induction of defective mutants, we have identified four amino acid residues in CytRSt that appear to be important for the response to uridine. The implications of these findings for the understanding of the ligand binding and induction of CytR are discussed in the context of the structural knowledge of CytR and homologous protein-ligand complexes.
KeywordMeSH Terms
Protein Binding
Protein Conformation
89. Stuckey  JA,     ( 1999 )

Crystal structure of a phospholipase D family member.

Nature structural biology 6 (3)
PMID : 10074947  :   DOI  :   10.1038/6716    
Abstract >>
The first crystal structure of a phospholipase D (PLD) family member has been determined at 2.0 A resolution. The PLD superfamily is defined by a common sequence motif, HxK(x)4D(x)6GSxN, and includes enzymes involved in signal transduction, lipid biosynthesis, endonucleases and open reading frames in pathogenic viruses and bacteria. The crystal structure suggests that residues from two sequence motifs form a single active site. A histidine residue from one motif acts as a nucleophile in the catalytic mechanism, forming a phosphoenzyme intermediate, whereas a histidine residue from the other motif appears to function as a general acid in the cleavage of the phosphodiester bond. The structure suggests that the conserved lysine residues are involved in phosphate binding. Large-scale genomic sequencing revealed that there are many PLD family members. Our results suggest that all of these proteins may possess a common structure and catalytic mechanism.
KeywordMeSH Terms
90. Hensel  M, Nikolaus  T,     ( 1999 )

Molecular and functional analysis indicates a mosaic structure of Salmonella pathogenicity island 2.

Molecular microbiology 31 (2)
PMID : 10027966  :   DOI  :   10.1046/j.1365-2958.1999.01190.x    
Abstract >>
Two large virulence loci encoding type III secretion systems are present on the chromosome of Salmonella typhimurium. Salmonella pathogenicity island 2 (SPI2) is important for the survival of S. typhimurium in host organs and forms an insertion of about 40 kb at the tRNA(Val) gene. However, several indications suggested that SPI2 was not the result of a single event of horizontal gene transfer. We characterized the portion of SPI2 towards the 30 cs boundary and performed mutational analysis to investigate the contribution of this region to S. enterica virulence. This analysis indicates that SPI2 may be composed of at least two different genetic elements. About 15 kb of the 40 kb of SPI2 contain genes without a significant contribution to systemic infections in the model of murine salmonellosis. Our study allowed us to define genes in SPI2 important for virulence further and indicated that this locus has a complex mosaic structure.
KeywordMeSH Terms
Mosaicism
91. Lu  CD,     ( 1999 )

Role of ArgR in activation of the ast operon, encoding enzymes of the arginine succinyltransferase pathway in Salmonella typhimurium.

Journal of bacteriology 181 (6)
PMID : 10074092  :   PMC  :   PMC93598    
Abstract >>
The ast operon, encoding enzymes of the arginine succinyltransferase (AST) pathway, was cloned from Salmonella typhimurium, and the nucleotide sequence for the upstream flanking region was determined. The control region contains several regulatory consensus sequences, including binding sites for NtrC, cyclic AMP receptor protein (CRP), and ArgR. The results of DNase I footprintings and gel retardation experiments confirm binding of these regulatory proteins to the identified sites. Exogenous arginine induced AST under nitrogen-limiting conditions, and this induction was abolished in an argR derivative. AST was also induced under carbon starvation conditions; this induction required functional CRP as well as functional ArgR. The combined data are consistent with the hypothesis that binding of one or more ArgR molecules to a region between the upstream binding sites for NtrC and CRP and two putative promoters plays a pivotal role in modulating expression of the ast operon in response to nitrogen or carbon limitation.
KeywordMeSH Terms
Genes, Bacterial
Operon
Trans-Activators
Transcription Factors
92. Zhou  D, Hardt  WD,     ( 1999 )

Salmonella typhimurium encodes a putative iron transport system within the centisome 63 pathogenicity island.

Infection and immunity 67 (4)
PMID : 10085045  :   PMC  :   PMC96555    
Abstract >>
Upon entry into the host, Salmonella enterica strains are presumed to encounter an iron-restricted environment. Consequently, these bacteria have evolved a variety of often-redundant high-affinity acquisition systems to obtain iron in this restricted environment. We have identified an iron transport system that is encoded within the centisome 63 pathogenicity island of Salmonella typhimurium. The nucleotide composition of this locus is significantly different from that of the rest of this pathogenicity island, suggesting a different ancestry and a mosaic structure for this region of the S. typhimurium chromosome. This locus, designated sit, consists of four open reading frames which encode polypeptides with extensive homology to the yfe ABC iron transport system of Yersinia pestis, as well as other ABC transporters. The sitA gene encodes a putative periplasmic binding protein, sitB encodes an ATP-binding protein, and sitC and sitD encode two putative permeases (integral membrane proteins). This operon is capable of complementing the growth defect of the enterobactin-deficient Escherichia coli strain SAB11 in iron-restricted minimal medium. Transcription of the sit operon is repressed under iron-rich growth conditions in a fur-dependent manner. Introduction of a sitBCD deletion into wild-type S. typhimurium resulted in no apparent growth defect in either nutrient-rich or minimal medium and no measurable virulence phenotype. These results further support the existence of redundant iron uptake systems in S. enterica.
KeywordMeSH Terms
93. Majtánová  L, Majtán  T, Majtán  V,     ( 2007 )

Molecular characterization of class 1 integrons in clinical strains of Salmonella typhimurium isolated in Slovakia.

Polish journal of microbiology 56 (1)
PMID : 17419185  :  
Abstract >>
The presence of class 1 integrons was investigated in 156 epidemiologically unrelated Salmonella Typhimurium isolates. Of these 156 isolates, 70 were of definitive phage type DT104 and 86 were strains of various phage type, RDNC and untypable, designated here as non-DT104 strains. Integrons were found in 47 of DT104 isolates (67.1%), while in all strains with characteristic pentaresistance (R-type ACSSuT) two integrons 1.0 kb and 1.2 kb in size were found. Among 86 non-DT104 strains, integrons with sizes of 1.6 kb and 1.9 kb in four multidrug-resistant strains DT193 and U302 were found. The integrons from selected strains were further sequenced and the aadA1, aadA2, dhfr1, dhfr12 and bla(PSE) genes were found embedded in cassettes.
KeywordMeSH Terms
Bacteriophage Typing
94. Hopkins  KL, Wootton  L, Day  MR, Threlfall  EJ,     ( 2007 )

Plasmid-mediated quinolone resistance determinant qnrS1 found in Salmonella enterica strains isolated in the UK.

The Journal of antimicrobial chemotherapy 59 (6)
PMID : 17434878  :   DOI  :   10.1093/jac/dkm081    
Abstract >>
To determine the prevalence of qnr genes in selected Salmonella enterica and Escherichia coli isolated in the UK. One hundred and eighteen S. enterica and 103 E. coli were screened for qnrA, qnrB and qnrS by PCR. Transferability of qnr plasmids was assessed and isolated plasmids compared with previously identified qnr plasmids by restriction fragment length polymorphism analysis and hybridization experiments. PCRs and sequencing identified co-transferred beta-lactamase genes and mutations in the quinolone resistance-determining region of gyrA. Only six S. enterica strains belonging to four serotypes (Stanley, Typhimurium, Virchow and Virginia) were positive for qnrS1. qnrS1 was present on plasmids of 13.5 kb (TPqnrS-1a and -1b) in Typhimurium and Virginia isolates, 44 kb (TPqnrS-2) in two Virchow isolates and >148 kb (TPqnrS-3a and -3b) in two Stanley isolates. bla(TEM-1) and a group 9 bla(CTX-M) were co-transferred on TPqnrS-2 and TPqnrS-3b. Hybridization of a qnrS1 probe to digested qnrS1 plasmids suggested qnrS1 on TPqnrS-2 may be located in a similar genetic environment to Shigella qnrS plasmid pAH0376, but in a different environment in the other plasmids. This is the first report of plasmid-mediated quinolone resistance in a Salmonella isolate from the UK; five isolates were associated with foreign travel to, or food imported from, the Far East. The presence of qnrS1 on different plasmid backbones in several Salmonella serotypes suggests successful dissemination of plasmids or qnrS1. It is of concern that qnrS1 is being identified in Salmonella serotypes that are commonly implicated in human infection in the UK. Coupled with beta-lactam resistance, it may compromise treatment of vulnerable patient groups.
KeywordMeSH Terms
95. Kehrenberg  C, Hopkins  KL, Threlfall  EJ, Schwarz  S,     ( 2007 )

Complete nucleotide sequence of a small qnrS1-carrying plasmid from Salmonella enterica subsp. enterica Typhimurium DT193.

The Journal of antimicrobial chemotherapy 60 (4)
PMID : 17652106  :   DOI  :   10.1093/jac/dkm283    
Abstract >>
N/A
KeywordMeSH Terms
96. García Fernández  A, Cloeckaert  A, Bertini  A, Praud  K, Doublet  B, Weill  FX, Carattoli  A,     ( 2007 )

Comparative analysis of IncHI2 plasmids carrying blaCTX-M-2 or blaCTX-M-9 from Escherichia coli and Salmonella enterica strains isolated from poultry and humans.

Antimicrobial agents and chemotherapy 51 (11)
PMID : 17698627  :   DOI  :   10.1128/AAC.00603-07     PMC  :   PMC2151457    
Abstract >>
Salmonella enterica bla(CTX-M-2) and bla(CTX-M-9) plasmid backbones from isolates from Belgium and France were analyzed. The bla(CTX-M-2-)plasmids from both human and poultry isolates were related to the IncHI2 pAPEC-O1-R plasmid, previously identified in the United States in avian Escherichia coli strains; the bla(CTX-M-9) plasmids were closely related to the IncHI2 R478 plasmid.
KeywordMeSH Terms
97. Bayliss  R, Harris  R, Coutte  L, Monier  A, Fronzes  R, Christie  PJ, Driscoll  PC, Waksman  G,     ( 2007 )

NMR structure of a complex between the VirB9/VirB7 interaction domains of the pKM101 type IV secretion system.

Proceedings of the National Academy of Sciences of the United States of America 104 (5)
PMID : 17244707  :   DOI  :   10.1073/pnas.0609535104     PMC  :   PMC1785264    
Abstract >>
Type IV secretion (T4S) systems translocate DNA and protein effectors through the double membrane of Gram-negative bacteria. The paradigmatic T4S system in Agrobacterium tumefaciens is assembled from 11 VirB subunits and VirD4. Two subunits, VirB9 and VirB7, form an important stabilizing complex in the outer membrane. We describe here the NMR structure of a complex between the C-terminal domain of the VirB9 homolog TraO (TraO(CT)), bound to VirB7-like TraN from plasmid pKM101. TraO(CT) forms a beta-sandwich around which TraN winds. Structure-based mutations in VirB7 and VirB9 of A. tumefaciens show that the heterodimer interface is conserved. Opposite this interface, the TraO structure shows a protruding three-stranded beta-appendage, and here, we supply evidence that the corresponding region of VirB9 of A. tumefaciens inserts in the membrane and protrudes extracellularly. This complex structure elucidates the molecular basis for the interaction between two essential components of a T4S system.
KeywordMeSH Terms
98. Cloeckaert  A, Praud  K, Doublet  B, Bertini  A, Carattoli  A, Butaye  P, Imberechts  H, Bertrand  S, Collard  JM, Arlet  G, Weill  FX,     ( 2007 )

Dissemination of an extended-spectrum-beta-lactamase blaTEM-52 gene-carrying IncI1 plasmid in various Salmonella enterica serovars isolated from poultry and humans in Belgium and France between 2001 and 2005.

Antimicrobial agents and chemotherapy 51 (5)
PMID : 17325216  :   DOI  :   10.1128/AAC.01514-06     PMC  :   PMC1855541    
Abstract >>
We report here the dissemination of a conjugative IncI1 plasmid carrying bla(TEM-52) on a Tn3 transposon conferring resistance to extended-spectrum cephalosporins in Salmonella enterica serovar Agona, Derby, Infantis, Paratyphi B dT(+), and Typhimurium isolates from poultry and humans in Belgium and France from 2001 to 2005. The most prevalent serovar spreading this resistance was serovar Infantis.
KeywordMeSH Terms
Plasmids
99. Makde  RD, Mahajan  SK, Kumar  V,     ( 2007 )

Structure and mutational analysis of the PhoN protein of Salmonella typhimurium provide insight into mechanistic details.

Biochemistry 46 (8)
PMID : 17263560  :   DOI  :   10.1021/bi062180g    
Abstract >>
The Salmonella typhimurium PhoN protein is a nonspecific acid phosphatase and belongs to the phosphatidic acid phosphatase type 2 (PAP2) superfamily. We report here the crystal structures of phosphate-bound PhoN, the PhoN-tungstate complex, and the T159D mutant of PhoN along with functional characterization of three mutants: L39T, T159D, and D201N. Invariant active site residues, Lys-123, Arg-130, Ser-156, Gly-157, His-158, and Arg-191, interact with phosphate and tungstate oxyanions. Ser-156 also accepts a hydrogen bond from Thr-159. The T159D mutation, surprisingly, severely diminishes phosphatase activity, apparently by disturbing the active site scaffold: Arg-191 is swung out of the active site resulting in conformational changes in His-158 and His-197 residues. Our results reveal a hitherto unknown functional role of Arg-191, namely, restricting the active conformation of catalytic His-158 and His-197 residues. Consistent with the conserved nature of Asp-201 in the PAP2 superfamily, the D201N mutation completely abolished phosphatase activity. On the basis of this observation and in silico analysis we suggest that the crucial mechanistic role of Asp-201 is to stabilize the positive charge on the phosphohistidine intermediate generated by the transfer of phosphoryl to the nucleophile, His-197, located within hydrogen bond distance to the invariant Asp-201. This is in contrast to earlier suggestions that Asp-201 stabilizes His-197 and the His197-Asp201 dyad facilitates formation of the phosphoenzyme intermediate through a charge-relay system. Finally, the L39T mutation in the conserved polyproline motif (39LPPPP43) of dimeric PhoN leads to a marginal reduction in activity, in contrast to the nearly 50-fold reduction observed for monomeric Prevotella intermedia acid phosphatase, suggesting that the varying quaternary structure of PhoN orthologues may have functional significance.
KeywordMeSH Terms
100. Antunes  P, Machado  J, Peixe  L,     ( 2007 )

Dissemination of sul3-containing elements linked to class 1 integrons with an unusual 3' conserved sequence region among Salmonella isolates.

Antimicrobial agents and chemotherapy 51 (4)
PMID : 17283193  :   DOI  :   10.1128/AAC.01275-06     PMC  :   PMC1855504    
Abstract >>
A sul3 domain (IS440-sul3-orf1-IS26) was found linked to an unusual 3' conserved sequence region (qacH) of class 1 integrons and detected among nontyphoid Salmonella isolates (n=47) from different sources. Three types of integrons differing in the gene cassette array (dfrA12-orfF-aadA2-cmlA1-aadA1, dfrA12-orfF-aadA2/1, and estX-psp-aadA2-cmlA1-aadA1) were found associated with this sul3 domain. They were associated with particular clones and specific high-molecular-weight plasmids.
KeywordMeSH Terms
Conserved Sequence
101. Murphy  BP, O'Mahony  R, Buckley  JF, Shine  P, Boyd  EF, Gilroy  D, Fanning  S,     ( 2007 )

Investigation of a global collection of nontyphoidal Salmonella of various serotypes cultured between 1953 and 2004 for the presence of class 1 integrons.

FEMS microbiology letters 266 (2)
PMID : 17233727  :   DOI  :   10.1111/j.1574-6968.2006.00537.x    
Abstract >>
In this study, antibiotic resistance profiles, and the presence of class 1 integrons were determined for 108 Salmonella isolates comprising 37 serotypes cultured from a variety of sources between 1953 and 2004. Antibiogram analyses showed that all isolates were resistant to streptomycin/spectinomycin. Molecular analysis revealed that 50% of the collection contained an integrase-encoding gene (int1) and 25% contained class 1 integrons. A Salmonella Wien isolate possessing a complete class 1 integron with a dfrA5-ereA2 gene arrangement within the variable region was characterized.
KeywordMeSH Terms
102. Heffernan  EJ, Harwood  J, Fierer  J, Guiney  D,     ( 1992 )

The Salmonella typhimurium virulence plasmid complement resistance gene rck is homologous to a family of virulence-related outer membrane protein genes, including pagC and ail.

Journal of bacteriology 174 (1)
PMID : 1729227  :   DOI  :   10.1128/jb.174.1.84-91.1992     PMC  :   PMC205680    
Abstract >>
A fragment of the Salmonella typhimurium virulence plasmid containing the rck locus, when cloned in the recombinant cosmid pADE016, was shown previously to confer high-level complement resistance on both rough and smooth Escherichia coli, Salmonella minnesota, and S. typhimurium and was associated with the production of an outer membrane protein. We determined the nucleotide sequence of the fragment containing the rck locus. Mutations in the two major open reading frames confirmed that the complement resistance mediated by pADE016 was due to a single 555-bp rck gene encoding a 17-kDa outer membrane protein. Analysis of the rck gene revealed that the Rck outer membrane protein consisted of 185 amino acid residues, with a calculated postcleavage molecular mass of 17.4 kDa. Rck is homologous to a family of outer membrane proteins expressed in gram-negative bacteria, two of which have been associated with virulence-related phenotypes: PagC, required by S. typhimurium for survival in macrophages and for virulence in mice; and Ail, a product of the Yersinia enterocolitica chromosome capable of mediating bacterial adherence to and invasion of epithelial cell lines. Rck, most closely related to PagC, represents the third outer membrane protein in this five-member family with a distinct virulence-associated phenotype.
KeywordMeSH Terms
103. Vo  AT, van Duijkeren  E, Fluit  AC, Gaastra  W,     ( 2007 )

A novel Salmonella genomic island 1 and rare integron types in Salmonella Typhimurium isolates from horses in The Netherlands.

The Journal of antimicrobial chemotherapy 59 (4)
PMID : 17293368  :   DOI  :   10.1093/jac/dkl531    
Abstract >>
To investigate the genotypic resistance of integron-carrying Salmonella Typhimurium isolates from horses and their genetic relationship. Sixty-one Salmonella isolates were screened for the presence of class 1 integrons by PCR. The gene cassettes of integron-positive isolates were detected by PCR, restriction fragment length polymorphism typing, and sequencing. The potential for the transfer of resistance determinants was investigated by conjugation experiments. The presence of Salmonella genomic island 1 (SGI1) or its variants was studied by PCR and nucleotide sequencing. PFGE was used to genotype the isolates. Eight distinct XbaI-PFGE profiles and seven integron types were observed among 26 integron-carrying Salmonella Typhimurium isolates. The gene cassettes detected were dfrA1, dfrA7, dfrA14, aadA1, aadA2, aadB and bla(PSE). A rare type of integron found in nine isolates carried the dfrA14 and aadA1 gene cassettes. Twelve Salmonella Typhimurium DT104 isolates contained SGI1 or one of its variants (SGI1, SGI1-B and SGI1-C). A novel variant of SGI1, designated SGI1-M, was identified in one isolate in which the aadA2 gene of SGI1 was replaced by the aadB gene. Transfer of integrons and antimicrobial resistance determinants to Escherichia coli K12 via conjugation was possible with nine isolates. Resistance to fluoroquinolones in nine isolates was caused by mutations in the gyrA gene leading to the amino acid changes Ser-83 --> Ala and Asp-87 --> Asn. The integron-positive clinical Salmonella Typhimurium isolates from horses belong to distinct strains. The data demonstrate the capability of Salmonella Typhimurium to acquire additional antibiotic resistance determinants and underline the need for the prudent use of antimicrobials.
KeywordMeSH Terms
104. Malapaka  RR, Adebayo  LO, Tripp  BC,     ( 2007 )

A deletion variant study of the functional role of the Salmonella flagellin hypervariable domain region in motility.

Journal of molecular biology 365 (4)
PMID : 17109884  :   DOI  :   10.1016/j.jmb.2006.10.054    
Abstract >>
The eubacterial flagellum is a complex structure with an elongated extracellular filament that is composed primarily of many subunits of a flagellin protein. The highly conserved N and C termini of flagellin are important in its export and self-assembly, whereas the middle sequence region varies greatly in size and composition in different species and is known to be deletion-tolerant. In Salmonella typhimurium phase 1 flagellin, this "hypervariable" region encodes two solvent-exposed domains, D2 and D3, that form a knob-like feature on flagella fibers. The functional role of this structural feature in motility remains unclear. We investigated the structural and physiological role of the hypervariable region in flagella assembly, stability and cellular motility. A library of random internal deletion variants of S. typhimurium flagellin was constructed and screened for functional variants using a swarming agar motility assay. The relative cellular motility and propulsive force of ten representative variants were determined in semi-solid and liquid medium using colony swarming motility assays, video microscopy and optical trapping of single cells. All ten variants exhibited diminished motility, with varying extents of motility observed for internal deletions less than 75 residues and nearly complete loss of motility for deletions greater than 100 residues. The mechanical stability of the variant flagella fibers also decreased with increasing size of deletion. Comparison of the variant sequences with the wild-type sequence and structure indicated that all deletions involved loss of hydrophobic core residues, and removal of both partial and complete segments of secondary structure in the D2 and D3 domains. Homology modeling predicted disruptions of secondary structures in each variant. The hypervariable region D2 and D3 domains appear to stabilize the folded conformation of the flagellin protein and contribute to the mechanical stability and propulsive force of the flagella fibers.
KeywordMeSH Terms
Gene Deletion
105. Tankouo-Sandjong  B, Sessitsch  A, Liebana  E, Kornschober  C, Allerberger  F, Hächler  H, Bodrossy  L,     ( 2007 )

MLST-v, multilocus sequence typing based on virulence genes, for molecular typing of Salmonella enterica subsp. enterica serovars.

Journal of microbiological methods 69 (1��1��)
PMID : 17208323  :   DOI  :   10.1016/j.mimet.2006.11.013    
Abstract >>
Salmonella enterica subsp. enterica is one of the main causative agents of food-borne disease in man, and can also be the cause of serious systemic illness. Organisms belonging to this genus have traditionally been classified on the basis of the antigenic properties of the cell-surface lipopolysaccharide and of the phase 1 and phase 2 flagellar proteins. Primary isolation, biochemical identification, and serotyping are laborious and time consuming. Molecular identification based on suitable marker genes could be an attractive alternative to conventional bacteriological and serological methods. We have assessed the applicability of two housekeeping genes, gyrB, atpD, in combination with the flagellin genes fliC and fljB in multilocus sequence typing of Salmonella. Sequencing and comparative analysis of sequence data was performed on multiple strains from Austria, the United Kingdom, and Switzerland, representing all subspecies and 22 of the more prevalent non-typhoid S. enterica subsp. enterica serovars. A combination of these four marker genes allowed for a clear differentiation of all the strains analysed, indicating their applicability in molecular typing. The term MLST-v, for multilocus sequence typing based on virulence genes, is proposed to distinguish this approach from MLST based solely on housekeeping genes. An assortative recombination of the fliC gene was found in seven of the analysed serovars indicating multiple phylogenetic origin of these serovars.
KeywordMeSH Terms
Genes, Bacterial
Sequence Analysis, DNA
106. Tarr  CL, Patel  JS, Puhr  ND, Sowers  EG, Bopp  CA, Strockbine  NA,     ( 2007 )

Identification of Vibrio isolates by a multiplex PCR assay and rpoB sequence determination.

Journal of clinical microbiology 45 (1)
PMID : 17093013  :   DOI  :   10.1128/JCM.01544-06     PMC  :   PMC1828960    
Abstract >>
Vibrio, a diverse genus of aquatic bacteria, currently includes 72 species, 12 of which occur in human clinical samples. Of these 12, three species--Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus-account for the majority of Vibrio infections in humans. Rapid and accurate identification of Vibrio species has been problematic because phenotypic characteristics are variable within species and biochemical identification requires 2 or more days to complete. To facilitate the identification of human-pathogenic species, we developed a multiplex PCR that uses species-specific primers to amplify gene regions in four species (V. cholerae, V. parahaemolyticus, V. vulnificus, and V. mimicus). The assay was tested on a sample of 309 Vibrio isolates representing 26 named species (including 12 human pathogens) that had been characterized by biochemical methods. A total of 190 isolates that had been identified as one of the four target species all yielded results consistent with the previous classification. The assay identified an additional four V. parahaemolyticus isolates among the other 119 isolates. Sequence analysis based on rpoB was used to validate the multiplex results for these four isolates, and all clustered with other V. parahaemolyticus sequences. The rpoB sequences for 12 of 15 previously unidentified isolates clustered with other Vibrio species in a phylogenetic analysis, and three isolates appeared to represent unnamed Vibrio species. The PCR assay provides a simple, rapid, and reliable tool for identification of the major Vibrio pathogens in clinical samples, and rpoB sequencing provides an additional identification tool for other species in the genus Vibrio.
KeywordMeSH Terms
Sequence Analysis, DNA
107. Chen  CY, Nace  GW, Solow  B, Fratamico  P,     ( 2007 )

Complete nucleotide sequences of 84.5- and 3.2-kb plasmids in the multi-antibiotic resistant Salmonella enterica serovar Typhimurium U302 strain G8430.

Plasmid 57 (1)
PMID : 16828159  :   DOI  :   10.1016/j.plasmid.2006.05.005    
Abstract >>
The multi-antibiotic resistant (MR) Salmonella enterica serovar Typhimurium phage type U302 strain G8430 exhibits the penta-resistant ACSSuT-phenotype (ampicillin, chloramphenicol, streptomycin, sulfonamides and tetracycline), and is also resistant to carbenicillin, erythromycin, kanamycin, and gentamicin. Two plasmids, 3.2- and 84.5-kb in size, carrying antibiotic resistance genes were isolated from this strain, and the nucleotide sequences were determined and analyzed. The 3.2-kb plasmid, pU302S, belongs to the ColE1 family and carries the aph(3')-I gene (Kan(R)). The 84.5-kb plasmid, pU302L, is an F-like plasmid and contains 14 complete IS elements and multiple resistance genes including aac3, aph(3')-I, sulII, tetA/R, strA/B, bla(TEM-1), mph, and the mer operon. Sequence analyses of pU302L revealed extensive homology to various plasmids or transposons, including F, R100, pHCM1, pO157, and pCTX-M3 plasmids and TnSF1 transposon, in regions involved in plasmid replication/maintenance functions and/or in antibiotic resistance gene clusters. Though similar to the conjugative plasmids F and R100 in the plasmid replication regions, pU302L does not contain oriT and the tra genes necessary for conjugal transfer. This mosaic pattern of sequence similarities suggests that pU302L acquired the resistance genes from a variety of enteric bacteria and underscores the importance of a further understanding of horizontal gene transfer among the enteric bacteria.
KeywordMeSH Terms
Drug Resistance, Multiple, Bacterial
108. Tracz  DM, Tabor  H, Jerome  M, Ng  LK, Gilmour  MW,     ( 2006 )

Genetic determinants and polymorphisms specific for human-adapted serovars of Salmonella enterica that cause enteric fever.

Journal of clinical microbiology 44 (6)
PMID : 16757591  :   DOI  :   10.1128/JCM.02630-05     PMC  :   PMC1489402    
Abstract >>
Salmonella enterica serovars Typhi, Paratyphi A, and Sendai are human-adapted pathogens that cause typhoid (enteric) fever. The acute prevalence in some global regions and the disease severity of typhoidal Salmonella have necessitated the development of rapid and specific detection tests. Most of the methodologies currently used to detect serovar Typhi do not identify serovars Paratyphi A or Sendai. To assist in this aim, comparative sequence analyses were performed at the loci of core bacterial genetic determinants and Salmonella pathogenicity island 2 genes encoded by clinically significant S. enterica serovars. Genetic polymorphisms specific for serovar Typhi (at trpS), as well as polymorphisms unique to human-adapted typhoidal serovars (at sseC and sseF), were observed. Furthermore, entire coding sequences unique to human-adapted typhoidal Salmonella strains (i.e., serovar-specific genetic loci rather than polymorphisms) were observed in publicly available comparative genomic DNA microarray data sets. These polymorphisms and loci were developed into real-time PCR, standard PCR, and liquid microsphere suspension array-based molecular protocols and tested for with a panel of clinical and reference subspecies I S. enterica strains. A proportion of the nontyphoidal Salmonella strains hybridized with the allele-specific oligonucleotide probes for sseC and sseF; but the trpS allele was unique to serovar Typhi (with a singular serovar Paratyphi B strain as an exception), and the coding sequences STY4220 and STY4221 were unique among serovars Typhi, Paratyphi A, and Sendai. These determinants provided phylogenetic data on the genetic relatedness of serovars Typhi, Paratyphi A, and Sendai; and the protocols developed might allow the rapid identification of these Salmonella serovars that cause enteric fever.
KeywordMeSH Terms
Polymorphism, Genetic
109. Kang  MS, Besser  TE, Hancock  DD, Porwollik  S, McClelland  M, Call  DR,     ( 2006 )

Identification of specific gene sequences conserved in contemporary epidemic strains of Salmonella enterica.

Applied and environmental microbiology 72 (11)
PMID : 16963552  :   DOI  :   10.1128/AEM.01368-06     PMC  :   PMC1636165    
Abstract >>
Genetic elements specific to recent and contemporary epidemic strains of Salmonella enterica were identified using comparative genomic analysis. Two epidemic multidrug-resistant (MDR) strains, MDR Salmonella enterica serovar Typhimurium definitive phage type 104 (DT104) and cephalosporin-resistant MDR Salmonella enterica serovar Newport, and an epidemic pansusceptible strain, Salmonella serovar Typhimurium DT160, were subjected to Salmonella gene microarray and suppression subtractive hybridization analyses. Their genome contents were compared with those of coexisting sporadic strains matched by serotype, geographic and temporal distribution, and host species origin. These paired comparisons revealed that epidemic strains of S. enterica had specific genes and gene regions that were shared by isolates of the same subtype. Most of these gene sequences are related to mobile genetic elements, including phages, plasmids, and plasmid-like and transposable elements, and some genes may encode proteins conferring growth or survival advantages. The emergence of epidemic MDR strains may therefore be associated with the presence of fitness-associated genetic factors in addition to their antimicrobial resistance genes.
KeywordMeSH Terms
Conserved Sequence
110. Reynolds  CM, Ribeiro  AA, McGrath  SC, Cotter  RJ, Raetz  CR, Trent  MS,     ( 2006 )

An outer membrane enzyme encoded by Salmonella typhimurium lpxR that removes the 3'-acyloxyacyl moiety of lipid A.

The Journal of biological chemistry 281 (31)
PMID : 16704973  :   DOI  :   10.1074/jbc.M603527200     PMC  :   PMC2702521    
Abstract >>
The Salmonella and related bacteria modify the structure of the lipid A portion of their lipopolysaccharide in response to environmental stimuli. Some lipid A modifications are required for virulence and resistance to cationic antimicrobial peptides. We now demonstrate that membranes of Salmonella typhimurium contain a novel hydrolase that removes the 3'-acyloxyacyl residue of lipid A in the presence of 5 mM Ca2+. We have identified the gene encoding the S. typhimurium lipid A 3'-O-deacylase, designated lpxR, by screening an ordered S. typhimurium genomic DNA library, harbored in Escherichia coli K-12, for expression of Ca2+-dependent 3'-O-deacylase activity in membranes. LpxR is synthesized with an N-terminal type I signal peptide and is localized to the outer membrane. Mass spectrometry was used to confirm the position of lipid A deacylation in vitro and the release of the intact 3'-acyloxyacyl group. Heterologous expression of lpxR in the E. coli K-12 W3110, which lacks lpxR, resulted in production of significant amounts of 3'-O-deacylated lipid A in growing cultures. Orthologues of LpxR are present in the genomes of E. coli O157:H7, Yersinia enterocolitica, Helicobacter pylori, and Vibrio cholerae. The function of LpxR is unknown, but it could play a role in pathogenesis because it might modulate the cytokine response of an infected animal.
KeywordMeSH Terms
111. Govinden  U, Mocktar  C, Moodley  P, Sturm  AW, Essack  SY,     ( 2006 )

CTX-M-37 in Salmonella enterica serotype Isangi from Durban, South Africa.

International journal of antimicrobial agents 28 (4)
PMID : 16949257  :   DOI  :   10.1016/j.ijantimicag.2006.05.028    
Abstract >>
Beta-lactamase-mediated resistance was investigated in 59 putative extended-spectrum beta-lactamase (ESBL)-positive Salmonella spp. from the paediatric ward of a tertiary hospital in Durban, South Africa. Three Salmonella enterica serotype Isangi cultured from stool samples were multidrug resistant, with susceptibility only to meropenem, piperacillin/tazobactam and cefoxitin. Isoelectric focusing revealed beta-lactamases with isoelectric points of pI 5.8, 6.8 and 7.2. Sequencing identified beta-lactamases CTX-M-37 and TEM-1. To our knowledge, this is the first report of CTX-M-37 from S. enterica serotype Isangi in South Africa.
KeywordMeSH Terms
Drug Resistance, Multiple
112. Foley  SL, White  DG, McDermott  PF, Walker  RD, Rhodes  B, Fedorka-Cray  PJ, Simjee  S, Zhao  S,     ( 2006 )

Comparison of subtyping methods for differentiating Salmonella enterica serovar Typhimurium isolates obtained from food animal sources.

Journal of clinical microbiology 44 (10)
PMID : 17021084  :   DOI  :   10.1128/JCM.00745-06     PMC  :   PMC1594788    
Abstract >>
Molecular characterization (e.g., DNA-based typing methods) of Salmonella isolates is frequently employed to compare and distinguish clinical isolates recovered from animals and from patients with food-borne disease and nosocomial infections. In this study, we compared the abilities of different phenotyping and genotyping methods to distinguish isolates of Salmonella enterica serovar Typhimurium from different food animal sources. One hundred twenty-eight S. enterica serovar Typhimurium strains isolated from cattle, pigs, chickens, and turkeys or derived food products were characterized using pulsed-field gel electrophoresis (PFGE), repetitive element PCR (Rep-PCR), multilocus sequence typing (MLST), plasmid profiling, and antimicrobial susceptibility testing. Among the 128 Salmonella isolates tested, we observed 84 Rep-PCR profiles, 86 PFGE patterns, 89 MLST patterns, 36 plasmid profiles, and 38 susceptibility profiles. The molecular typing methods, i.e., PFGE, MLST, and Rep-PCR, demonstrated the best discriminatory power among Salmonella isolates. However, no apparent correlation was evident between the results of one molecular typing method and those of the others, suggesting that a combination of multiple methods is needed to differentiate S. enterica serovar Typhimurium isolates that genetically cluster according to one particular typing method.
KeywordMeSH Terms
Food Microbiology
113. Hall  RM, Brookes  DE, Stokes  HW,     ( 1991 )

Site-specific insertion of genes into integrons: role of the 59-base element and determination of the recombination cross-over point.

Molecular microbiology 5 (8)
PMID : 1662753  :   DOI  :   10.1111/j.1365-2958.1991.tb00817.x    
Abstract >>
From examination of published DNA sequences of genes found inserted at a specific site in integrons, all genes are shown to be associated, at their 3' ends, with a short imperfect inverted repeat sequence, a 59-base element or relative of this element. The similarity of the arrangement of gene inserts in the integron and in the Tn7 transposon family is described. A refined consensus for the 59-base element is reported. Members of this family are highly diverged and the relationship of a group of longer elements to the 59-base elements is demonstrated. The ability of 59-base elements of different length and sequence to act as sites for recombination catalysed by the integron-encoded DNA integrase is demonstrated, confirming that elements of this family have a common function. The ability of elements located between gene pairs to act as recombination sites has also been demonstrated. The recombination cross-over point has been localized to the GTT triplet which is conserved in the core sites, GTTRRRY, found at the 3' end of 59-base elements. Recombination at the core site found in inverse orientation at the 5' end of the 59-base elements was not detected, and the sequences responsible for orientation of the recombination event appear to reside within the 59-base element. A model for site-specific insertion of genes into integrons and Tn7-like transposons is proposed. Circular units consisting of a gene associated with a 59-base element are inserted into an ancestral element which contains neither a gene nor a 59-base element.(ABSTRACT TRUNCATED AT 250 WORDS)
KeywordMeSH Terms
114. Margarit  SM, Davidson  W, Frego  L, Stebbins  CE,     ( 2006 )

A steric antagonism of actin polymerization by a salmonella virulence protein.

Structure (London, England : 1993) 14 (8)
PMID : 16905096  :   DOI  :   10.1016/j.str.2006.05.022    
Abstract >>
Salmonella spp. require the ADP-ribosyltransferase activity of the SpvB protein for intracellular growth and systemic virulence. SpvB covalently modifies actin, causing cytoskeletal disruption and apoptosis. We report here the crystal structure of the catalytic domain of SpvB, and we show by mass spectrometric analysis that SpvB modifies actin at Arg177, inhibiting its ATPase activity. We also describe two crystal structures of SpvB-modified, polymerization-deficient actin. These structures reveal that ADP-ribosylation does not lead to dramatic conformational changes in actin, suggesting a model in which this large family of toxins inhibits actin polymerization primarily through steric disruption of intrafilament contacts.
KeywordMeSH Terms
Models, Molecular
115. Teplitski  M, Goodier  RI, Ahmer  BM,     ( 2006 )

Catabolite repression of the SirA regulatory cascade in Salmonella enterica.

International journal of medical microbiology : IJMM 296 (7)
PMID : 16949866  :   DOI  :   10.1016/j.ijmm.2006.06.001    
Abstract >>
Orthologs of the Salmonella BarA/SirA two-component system are required for virulence, motility, secondary metabolism and stress survival throughout the gamma-proteobacteria. BarA is a sensor kinase that responds to an unknown signal by phosphorylating the response regulator SirA. SirA increases the expression of genes within Salmonella pathogenicity island 1 (SPI1) that encode a type III secretion system (TTSS-1). SirA does this by directly activating the hilA and hilC regulatory genes encoded within SPI1. SirA also directly activates the csrB regulatory RNA gene. This RNA antagonizes the activity of the post-transcriptional regulatory protein CsrA that binds the mRNA of its targets to regulate SPI1, motility and secondary metabolism. A second regulatory RNA, csrC, is also strongly regulated by SirA, although gel mobility shift assays do not demonstrate a direct interaction. Additionally, we have determined that the sirA gene is activated by crp and cya. The effects of crp and cya were also observed on the downstream members of the SirA regulon, hilA, sopB, csrB, and csrC. However, gel mobility shift experiments and DNA sequence analysis suggest that the regulation of sirA by CRP is probably indirect. Although SirA does not regulate csrA, this gene was also under crp/cya control. Supplementation of a rich medium with phosphate diminished the catabolite control of the csr portion but not the virulence portion of the SirA regulon.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
116. Delmas  J, Breysse  F, Devulder  G, Flandrois  JP, Chomarat  M,     ( 2006 )

Rapid identification of Enterobacteriaceae by sequencing DNA gyrase subunit B encoding gene.

Diagnostic microbiology and infectious disease 55 (4)
PMID : 16626902  :   DOI  :   10.1016/j.diagmicrobio.2006.02.003    
Abstract >>
Real-time polymerase chain reaction and sequencing were used to characterize a 506-bp-long DNA fragment internal to the gyrB gene (gyrBint). The sequences obtained from 32 Enterobacteriaceae-type strains and those available in the Genbank nucleotide sequence database (n = 24) were used as a database to identify 240 clinical enterobacteria isolates. Sequence analysis of the gyrBint fragment of 240 strains showed that gyrBint constitutes a discriminative target sequence to differentiate between Enterobacteriaceae species. Comparison of these identifications with those obtained by phenotypic methods (Vitek 1 system and/or Rapid ID 32E; bioM?rieux, Marcy l'Etoile, France) revealed discrepancies essentially with genera Citrobacter and Enterobacter. Most of the strains identified as Enterobacter cloacae by phenotypic methods were identified as Enterobacter hormaechei strains by gyrBint sequencing. The direct sequencing of gyrBint would be useful as a complementary tool in the identification of clinical Enterobacteriaceae isolates.
KeywordMeSH Terms
117. Vo  AT, van Duijkeren  E, Fluit  AC, Wannet  WJ, Verbruggen  AJ, Maas  HM, Gaastra  W,     ( 2006 )

Antibiotic resistance, integrons and Salmonella genomic island 1 among non-typhoidal Salmonella serovars in The Netherlands.

International journal of antimicrobial agents 28 (3)
PMID : 16911867  :   DOI  :   10.1016/j.ijantimicag.2006.05.027    
Abstract >>
The objective of this study was to investigate the antimicrobial resistance patterns, integron characteristics and gene cassettes as well as the presence of Salmonella genomic island 1 (SGI1) in non-typhoidal Salmonella (NTS) isolates from human and animal origin. Epidemiologically unrelated Dutch NTS strains (n=237) originating from food-producing animals and human cases of salmonellosis were tested for their susceptibility to 15 antimicrobial agents. Resistance to 14 of these antimicrobials, including the third-generation cephalosporins, was detected. Resistance to sulphonamides, ampicillin, tetracycline, streptomycin, trimethoprim and nalidixic acid was common (>/=10% of the strains were resistant). Resistance against three or more antimicrobials was observed in 57 isolates. The same 237 strains were studied for the prevalence of class 1 integrons, their gene cassettes and the presence of SGI1. Thirty-six isolates (15.2%) carried class 1 integrons. These integrons had ten distinct profiles based on the size of the integron and restriction fragment length polymorphism analysis. Integrons were detected for the first time in serovars Indiana and Senftenberg. Multidrug resistance was strongly associated with the presence of class 1 integrons in which the aadA2, aadA1, bla(PSE-1), dfrA1, dfrA5, dfrA14 or sat genes were present, as determined by nucleotide sequence determination. The presence of gene cassettes or combinations of gene cassettes not previously found in integrons in Salmonella was observed. SGI1 or its variants (SGI-B, -C and -F) were present in 16 isolates belonging to either serovar Typhimurium, Derby or Albany. Regardless of whether the isolate was of human or animal origin, the same resistance phenotype, integron profile and SGI1 structure could be observed.
KeywordMeSH Terms
Genomic Islands
Integrons
118. Makde  RD, Dikshit  K, Kumar  V,     ( 2006 )

Protein engineering of class-A non-specific acid phosphatase (PhoN) of Salmonella typhimurium: modulation of the pH-activity profile.

Biomolecular engineering 23 (5)
PMID : 16901752  :   DOI  :   10.1016/j.bioeng.2006.06.004    
Abstract >>
Engineering of the PhoN enzyme of Salmonella typhimurium due to its superior characteristics for bioremediation of heavy metals has been advocated by Macaskie and colleagues [Basnakova, G., Stephens, E.R., Thaller, M.C., Rossolini, G.M., Macaskie, L.E., 1998. The use of Escherichia coli bearing a phoN gene for the removal of uranium and nickel from aqueous flows. Appl. Microbiol. Biotechnol. 50, 266-272]. The native enzyme hydrolyzes disparate organophosphates and exhibits optimal phosphatase activity at pH 5.5, for instance, with substrate p-nitrophenyl phosphate. Structurally guided Ile-78 was mutated using site-directed mutagenesis to Ala, Asp and His residues, with an aim to shift the optimum pH of the PhoN enzyme. Encouragingly, the I78A mutant displays significantly higher (as high as 160%) enzymatic efficiency over a broad pH range of 3.0-9.0, compared to the wild-type PhoN. The higher catalytic efficiency is due to the increase in k(cat), and can be mainly attributed to a deshielding of catalytic His-158 from the bulk-solvent. The I78D mutant possesses nearly twice the specific activity at the optimum pH of 7.0. The alkaline shift of the pH-activity profile agrees well with reasoning based on electrostatics. An increase in K(m), however, lowers the catalytic efficiency of the I78D mutant at the optimum pH. The I78H mutant, counter-intuitively, also exhibits an alkaline shift in the pH-optimum. Nonetheless, the active site scaffold in I78H mutant may not be disturbed, as similar steady-state kinetic parameters are observed for both I78H mutant and wild-type PhoN at their respective pH optima.
KeywordMeSH Terms
119. Octavia  S, Lan  R,     ( 2006 )

Frequent recombination and low level of clonality within Salmonella enterica subspecies I.

Microbiology (Reading, England) 152 (Pt 4)
PMID : 16549673  :   DOI  :   10.1099/mic.0.28486-0    
Abstract >>
The genetic relationship and population structure of Salmonella enterica subspecies I strains were analysed using nucleotide sequences of four genes (mglA, proV, torC and speC). Fifteen strains from the Salmonella reference collection B (SARB), belonging to 13 serovars, were analysed. Sequence data of two housekeeping genes, mdh and mutS, of the same 15 strains reported by Brown et al. (2003) (Proc Natl Acad Sci U S A 100, 15676-15681) were also included in the analyses. Phylogenetic analysis revealed that there was a lack of congruence among the six gene trees. Split decomposition analysis resolved only five strains with a network structure, while others showed a star phylogeny. Compatibility values for the SARB strains were the lowest in comparison to those for strains representing different subspecies of S. enterica. These results showed that the genes studied have undergone frequent recombination, suggesting a low level of clonality within subspecies I of S. enterica.
KeywordMeSH Terms
Genetic Variation
Recombination, Genetic
120. Schmid  K, Ebner  R, Jahreis  K, Lengeler  JW, Titgemeyer  F,     ( 1991 )

A sugar-specific porin, ScrY, is involved in sucrose uptake in enteric bacteria.

Molecular microbiology 5 (4)
PMID : 1649946  :   DOI  :   10.1111/j.1365-2958.1991.tb00769.x    
Abstract >>
During the molecular analysis of a plasmid-coded sucrose metabolic pathway of enteric bacteria, a gene, scrY, was found whose product, ScrY, had all the properties of a bacterial porin (Schmid et al., 1988). Loss of this protein (Mr 58 kDa), localized in the outer membrane, led, as shown here, to an increase in the apparent Km for sucrose transport in whole cells from 10 microM in wild-type cells to 300 microM in mutant cells. This contrasts with the Km for sucrose phosphorylation as measured in membrane vesicles from mutant and wild-type cells, which remained unchanged at about 10 microM, and reflects the activity of the sucrose-specific Enzymell of the phosphoenolpyruvate-dependent carbohydrate:phosphotransferase system (PTS) responsible for uptake through the inner membrane. Furthermore, the presence of ScrY restored growth on maltodextrins in cells devoid of LamB, thus complementing the lack of this maltoporin. The amino acid sequence deduced from the DNA sequence was determined for the plasmid-coded and the ScrY porin coded in the chromosome of Klebsiella pneumoniae. Both show high identity (86%) to each other, and to the channel domain of LamB, further corroborating the conclusion that they constitute porins.
KeywordMeSH Terms
121. Hochmann  H, Pust  S, von Figura  G, Aktories  K, Barth  H,     ( 2006 )

Salmonella enterica SpvB ADP-ribosylates actin at position arginine-177-characterization of the catalytic domain within the SpvB protein and a comparison to binary clostridial actin-ADP-ribosylating toxins.

Biochemistry 45 (4)
PMID : 16430223  :   DOI  :   10.1021/bi051810w    
Abstract >>
The SpvB protein from Salmonella enterica was recently discovered as an actin-ADP-ribosylating toxin. SpvB is most likely delivered via a type-III secretion system into eukaryotic cells and does not have a binding/translocation component. This is in contrast to the family of binary actin-ADP-ribosylating toxins from various Bacillus and Clostridium species. However, there are homologies in amino acid sequences between the C-terminal domain of SpvB and the catalytic domains of the actin-ADP-ribosylating toxins such as C2 toxin from Clostridium botulinum and iota toxin from Clostridium perfringens. We compared the biochemical properties of the catalytic C-terminal domain of SpvB (C/SpvB) with the enzyme components of C2 toxin and iota toxin. The specificity of C/SpvB concerning the modification of G- or F-actin was comparable to the C2 and iota toxins, although there were distinct differences regarding the recognition of actin isoforms. C/SpvB and iota toxin modify both muscle alpha-actin and nonmuscle beta/gamma-actin, whereas C2 toxin only modifies beta/gamma-actin. In contrast to the iota and C2 toxins, C/SpvB possessed no detectable glycohydrolase activity in the absence of a protein substrate. The maximal reaction rates were comparable for all toxins, whereas variable K(m) values for NAD were evident. We identified arginine-177 as the modification site for C/SpvB with the actin homologue protein Act88F from Drosophila.
KeywordMeSH Terms
Catalytic Domain
122. Krause  M, Guiney  DG,     ( 1991 )

Identification of a multimer resolution system involved in stabilization of the Salmonella dublin virulence plasmid pSDL2.

Journal of bacteriology 173 (18)
PMID : 1653217  :   DOI  :   10.1128/jb.173.18.5754-5762.1991     PMC  :   PMC208307    
Abstract >>
The Salmonella dublin virulence plasmid pSDL2 is a low-copy-number plasmid that is highly conserved in its host. Deletion of the 8-kb EcoRI C fragment downstream of the virulence region leads to plasmid instability and formation of multimers. We identified a multimer resolution system in the EcoRI C fragment composed of a trans-acting resolvase gene and a cis-acting resolution site. The resolvase gene, rsd, maps within a 2-kb EcoRV fragment and appears to be part of a multicistronic unit together with at least two other genes of unknown function. The derived protein, 28.7-kDa in size, is almost identical to the D protein of miniF. The C-terminal region was shown to have substantial similarity to the conserved C-terminal domains of the site-specific recombinases of the integrase family. The cis-acting resolution site, crs, is located upstream of rsd within a 628-bp SmaI-HpaI fragment. It contains eight direct incomplete 17-bp repeats followed by a segment rich in indirect repeats, the latter being homologous to the oriV1 sequence of miniF. crs contains the crossover site for specific recombination and mediates bidirectional promoter activity. A replicative function in analogy to that of oriV1 of F could not be demonstrated. The multimer resolution system was shown to stabilize pACYC184 and is dependent on the recA-mediated formation of multimeric plasmids. Screening different Salmonella serovars with a pSDL2-specific recombination assay revealed that only strains harboring a virulence plasmid encode for resolvase activity. Our results suggest that site-specific recombination contributes to the stable inheritance of pSDL2 and other Salmonella virulence plasmids.
KeywordMeSH Terms
Plasmids
123. Mikasová  E, Drahovská  H, Szemes  T, Kuchta  T, Karpísková  R, Sásik  M, Turna  J,     ( 2005 )

Characterization of Salmonella enterica serovar Typhimurium strains of veterinary origin by molecular typing methods.

Veterinary microbiology 109 (1��2��)
PMID : 15967599  :   DOI  :   10.1016/j.vetmic.2005.05.006    
Abstract >>
Twenty-eight strains of Salmonella enterica serovar Typhimurium were characterized by three PCR-based methods. Ten strains harbored type I integrons and two different integron profiles were detected. Typing by amplified fragment length polymorphism (AFLP) resulted in observation of 10 profiles that differed by one to six bands. Salmonella strains were screened for presence of phage genes using a PCR-phage typing; five genes from P22 phage and genes encoding putative virulence factors from phages Gifsy-1, Gifsy-2 and Fels-1 were selected for testing. This set of genes was sufficient for dividing the strains into eight different PCR-phage profiles. Similar grouping of strains was observed in case of all the employed DNA techniques and they corresponded well with the phage type and antimicrobial resistance of the strains. The highest discriminating power was achieved with use of the AFLP, yet the detection of integrons and PCR-phage typing also proved to be valuable in typing the S. Typhimurium strains.
KeywordMeSH Terms
124. Latasa  C, Roux  A, Toledo-Arana  A, Ghigo  JM, Gamazo  C, Penadés  JR, Lasa  I,     ( 2005 )

BapA, a large secreted protein required for biofilm formation and host colonization of Salmonella enterica serovar Enteritidis.

Molecular microbiology 58 (5)
PMID : 16313619  :   DOI  :   10.1111/j.1365-2958.2005.04907.x    
Abstract >>
In environmental settings, biofilms represent the common way of life of microorganisms. Salmonella enterica serovar Enteritidis, the most frequent cause of gastroenteritis in developed countries, produces a biofilm whose matrix is mainly composed of curli fimbriae and cellulose. In contrast to other bacterial biofilms, no proteinaceous compound has been reported to participate in the formation of this matrix. Here, we report the discovery of BapA, a large cell-surface protein required for biofilm formation by S. Enteritidis. Deletion of bapA caused the loss of the capacity to form a biofilm whereas the overexpression of a chromosomal copy of bapA increased the biofilm biomass formation. We provide evidence that overproduction of curli fimbriae and not cellulose can compensate for the biofilm deficiency of a bapA mutant strain. BapA is secreted through a type I protein secretion system (BapBCD) situated downstream of the bapA gene and was found to be loosely associated with the cell surface. Experiments with mixed bacterial populations positive or negative for BapA showed that BapA minus cells are not recruited into the biofilm matrix. The expression of bapA is coordinated with that of genes encoding curli fimbriae and cellulose, through the action of csgD. Studies on the contribution of BapA to S. Enteritidis pathogenesis revealed that orally inoculated animals with a bapA-deficient strain survived longer than those inoculated with the wild-type strain. Also, a bapA mutant strain showed a significantly lower colonization rate at the intestinal cell barrier and consequently a decreased efficiency for organ invasion compared with the wild-type strain. Taken together, these data demonstrate that BapA contributes both to biofilm formation and invasion through the regular Salmonella infection route.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
125. Hu  H, Lan  R, Reeves  PR,     ( 2006 )

Adaptation of multilocus sequencing for studying variation within a major clone: evolutionary relationships of Salmonella enterica serovar Typhimurium.

Genetics 172 (2)
PMID : 16204219  :   DOI  :   10.1534/genetics.105.046466     PMC  :   PMC1456234    
Abstract >>
Serovar Typhimurium of Salmonella enterica is a model organism for studies of pathogenesis that exhibits phage-type variation and variation in host range and virulence, but in a recent study showed no sequence variation in four genes, indicating the clonal nature of this serovar. We determined the relationships of 46 Typhimurium isolates of nine phage types using mutational changes detected either by matching AFLP (amplified fragment length polymorphism) fragments to computer-modeled LT2 AFLP fragments or by sequencing intergenic regions. Fifty-one polymorphic sites were detected, which gave a single phylogenetic tree. Comparison with genome sequences of five other serovars, Typhi, Paratyphi A, Gallinarum, Enteritidis, and Pullorum, enabled determination of the root of the tree. Only two parallel events were observed, giving high confidence in the tree branching order. The mutation-based tree provided a high level of consistency and a clear lineage for the Typhimurium isolates studied. This enabled us to show that for seven of the nine phage types used, the isolates studied have a single origin, but that two phage types clearly have more than one independent origin. We found that sequencing intergenic regions provides a good strategy for detection of mutational polymorphisms and study of phylogenetic relationships of closely related isolates and would be applicable to many other species.
KeywordMeSH Terms
Evolution, Molecular
Genetic Variation
Sequence Analysis, DNA
126. Daly  M, Villa  L, Pezzella  C, Fanning  S, Carattoli  A,     ( 2005 )

Comparison of multidrug resistance gene regions between two geographically unrelated Salmonella serotypes.

The Journal of antimicrobial chemotherapy 55 (4)
PMID : 15722395  :   DOI  :   10.1093/jac/dki015    
Abstract >>
The aim of this study was to identify chromosomally integrated genes conferring multidrug resistance to a Salmonella enterica (S.) serotype Typhimurium isolate, phage type DT193, isolated in Ireland and to compare them with resistance genes conferring plasmid-mediated multidrug resistance to a S. Enteritidis isolate from Italy. A complete DNA sequence of the regions containing the resistance genes was obtained from the chromosome of the S. Typhimurium DT193 isolate and from the IncI plasmid of the S. Enteritidis isolate. The plasmid was also characterized by conjugation and incompatibility grouping. Two 10 kb multidrug resistance non-Salmonella Genomic Island 1 type clusters were independently identified in the S. Enteritidis plasmid and in the chromosome of the S. Typhimurium isolate. Detailed characterization identified an IP-type 2 integron containing a dfrA1-aadA1 gene cassette and other common resistance determinants derived from the RSF1010 plasmid. These multidrug resistance regions originate following chromosomal integration of key resistance markers encountered on plasmids circulating in other Salmonella serotypes. This mechanism of marker acquisition may have future implications for the evolution of similar structures in previously susceptible serotypes, leading to an increased public health risk.
KeywordMeSH Terms
127. Villa  L, Carattoli  A,     ( 2005 )

Integrons and transposons on the Salmonella enterica serovar typhimurium virulence plasmid.

Antimicrobial agents and chemotherapy 49 (3)
PMID : 15728925  :   DOI  :   10.1128/AAC.49.3.1194-1197.2005     PMC  :   PMC549245    
Abstract >>
A virulence plasmid was identified in a multidrug-resistant Salmonella enterica serotype Typhimurium strain carrying the spvC, rck, and pefA virulence genes and two class 1 integrons linked to the Tn21 and Tn1696 transposons. A novel trimethoprim resistance gene, designated dfrA23, was also identified within the integron region. The association of multidrug resistance and virulence determinants represents an interesting example of virulence plasmid evolution.
KeywordMeSH Terms
DNA Transposable Elements
Integrons
Plasmids
128. Panutdaporn  N, Kawamoto  K, Asakura  H, Makino  SI,     ( 2006 )

Resuscitation of the viable but non-culturable state of Salmonella enterica serovar Oranienburg by recombinant resuscitation-promoting factor derived from Salmonella Typhimurium strain LT2.

International journal of food microbiology 106 (3)
PMID : 16213054  :   DOI  :   10.1016/j.ijfoodmicro.2005.06.022    
Abstract >>
A gene encoding the resuscitation-promoting factor (Rpf) from Salmonella Typhimurium LT2 was cloned and characterized. The amino acid sequence encoded by S. Typhimurium LT2 rpf gene shares 24.2% homology with Micrococcus luteus Rpf, which is secreted by growing cells, and required to resuscitate from viable but non-culturable (VNC) state. The S. Typhimurium LT2 rpf gene is 696 bp long, and shared a conserved segment with Salmonella enterica serovar Oranienburg (99.4%). Recombinant Rpf (rRpf) proteins of S. Typhimurium LT2 after expression in E. coli BL21 harboring the pET15-b plasmid was approximately 25 kDa. Since S. Oranienburg cells are relatively quick to enter the VNC state just after incubating in the presence of 7% NaCl at 37 degrees C for 3 days, we evaluated the biological effect of rRpf by using S. Oranienburg VNC cells. The rRpf not only promoted proliferation but also induced resuscitation of VNC cells to the culturable state in a dose-dependent manner. Therefore, rRpf may be useful for detection of bacterial contaminants present in the VNC form in food samples and the environment.
KeywordMeSH Terms
Food Microbiology
Gene Expression Regulation, Bacterial
129. Hermans  AP, Abee  T, Zwietering  MH, Aarts  HJ,     ( 2005 )

Identification of novel Salmonella enterica serovar Typhimurium DT104-specific prophage and nonprophage chromosomal sequences among serovar Typhimurium isolates by genomic subtractive hybridization.

Applied and environmental microbiology 71 (9)
PMID : 16151076  :   DOI  :   10.1128/AEM.71.9.4979-4985.2005     PMC  :   PMC1214642    
Abstract >>
Genomic subtractive hybridization was performed between Salmonella enterica serovar Typhimurium LT2 and DT104 to search for novel Salmonella serovar Typhimurium DT104-specific sequences. The subtraction resulted mainly in the isolation of DNA fragments with sequence similarity to phages. Two fragments identified were associated with possible virulence factors. One fragment was identical to irsA of Salmonella serovar Typhimurium ATCC 14028, which is suggested to be involved in macrophage survival. The other fragment was homologous to HldD, an Escherichia coli O157:H7 lipopolysaccharide assembly-related protein. Five selected DNA fragments-irsA, the HldD homologue, and three fragments with sequence similarity to prophages-were tested for their presence in 17 Salmonella serovar Typhimurium DT104 isolates and 27 non-DT104 isolates by PCR. All five selected DNA fragments were Salmonella serovar Typhimurium DT104 specific among the serovar Typhimurium isolates tested. These DNA fragments can be useful for better detection and typing of Salmonella serovar Typhimurium DT104.
KeywordMeSH Terms
Bacteriophage Typing
130. Pasquali  F, Kehrenberg  C, Manfreda  G, Schwarz  S,     ( 2005 )

Physical linkage of Tn3 and part of Tn1721 in a tetracycline and ampicillin resistance plasmid from Salmonella Typhimurium.

The Journal of antimicrobial chemotherapy 55 (4)
PMID : 15731203  :   DOI  :   10.1093/jac/dkh553    
Abstract >>
The complete nucleotide sequence of the 12 656 bp plasmid pFPTB1 from Salmonella enterica subsp. enterica serovar Typhimurium, which mediates resistance to tetracyclines and ampicillin, was determined. The plasmid was analysed for potential reading frames and structural features indicative of transposons and transposon relics. Plasmid pFPTB1 was transformed into Escherichia coli JM109, overlapping restriction fragments were cloned into E. coli plasmid vectors and sequenced. In vitro susceptibility testing was carried out to confirm the resistance phenotype mediated by this plasmid. Plasmid pFPTB1 contains a complete Tn3-like transposon of 4950 bp consisting of the left terminal repeat, Tn3-related tnpR and tnpA genes for transposition functions, a novel gene for ampicillin resistance bla(TEM-135), and the right terminal repeat. Immediately downstream, the terminal 5215 bp at the right end of a Tn1721-like transposon, including the right terminal repeat, a truncated transposase gene, as well as the genes tet(A) and tetR for tetracycline resistance, were detected. A 5 bp direct repeat, TAAAA, was seen immediately upstream of the Tn3 part and immediately downstream of the Tn1721 part. Plasmid pFPTB1 also carries a replication region similar to that of the Klebsiella pneumoniae plasmid pJHCMW1. Plasmid pFPTB1 is one of the few completely sequenced plasmids from S. Typhimurium and harbours a novel transposon-like structure consisting of a Tn3-related part containing the bla(TEM-135) gene for ampicillin resistance and a Tn1721-related part containing the tetR-tet(A) genes for tetracycline resistance.
KeywordMeSH Terms
Genetic Linkage
131. Ahmed  AM, Nakano  H, Shimamoto  T,     ( 2005 )

Molecular characterization of integrons in non-typhoid Salmonella serovars isolated in Japan: description of an unusual class 2 integron.

The Journal of antimicrobial chemotherapy 55 (3)
PMID : 15705645  :   DOI  :   10.1093/jac/dkh534    
Abstract >>
To characterize class 1 and class 2 integrons among non-typhoid Salmonella enterica serovars in Japan, and also to monitor the spread of the multidrug-resistant S. enterica serovar Typhimurium DT104. A total of 105 Salmonella isolates were included in this study. The broth microdilution method was used to determine the MIC values of a range of antibiotics for these isolates. PCR and DNA sequencing were used for screening and characterization of class 1 and class 2 integrons. PCR sequencing analysis revealed the presence of seven profiles of class 1 integrons in addition to a new type of class 2 integron. The identified gene cassettes within class 1 integrons were as follows; aadA1, aadA2 and aadA5, which confer resistance to streptomycin and spectinomycin; aadB, which confers resistance to gentamicin, kanamycin and tobramycin; dfrA1 and dfrA17, which confer resistance to trimethoprim; bla(PSE-1), which confers resistance to ampicillin; catB3, which confers resistance to chloramphenicol; and sat1, which confers resistance to streptothricin. Two strains of the multidrug-resistant S. Typhimurium DT104 were characterized in this study. DNA sequencing of class 2 integrons identified one with an unusual array of gene cassettes, sat, sat1 and aadA1. In this study, we characterized the antibiotic resistance gene cassettes within class 1 integrons in different isolates of non-typhoid Salmonella serovars, and we also identified a new type of class 2 integron.
KeywordMeSH Terms
Integrons
132. Gibbons  HS, Kalb  SR, Cotter  RJ, Raetz  CR,     ( 2005 )

Role of Mg2+ and pH in the modification of Salmonella lipid A after endocytosis by macrophage tumour cells.

Molecular microbiology 55 (2)
PMID : 15659161  :   DOI  :   10.1111/j.1365-2958.2004.04409.x    
Abstract >>
Lipid A of Salmonella typhimurium is covalently modified with additional acyl and/or polar substituents in response to activation of the PhoP/PhoQ and/or PmrA/PmrB signalling systems, which are induced by growth at low Mg2+ concentrations and mild acid pH respectively. Although these conditions are thought to exist within macrophage phagolysosomes, no direct evidence for lipid A modification after endocytosis has been presented. To address this issue, we grew S. typhimurium inside RAW264.7 cells in the presence of 32Pi, and then isolated the labelled lipid A fraction, which was found to be extensively derivatized with phosphoethanolamine, aminoarabinose, 2-hydroxymyristate and/or palmitate moieties. S. typhimurium grown in tissue culture medium synthesized lipid A molecules lacking all these substituents with the exception of the 2-hydroxymyristate chain, which was still present. Using defined minimal media to simulate the intracellular pH and Mg2+ concentrations of endosomes, we found that lipid A of S. typhimurium grown in an acidic, low-Mg2+ medium closely resembled lipid A isolated from bacteria internalized by RAW264.7 cells. A subset of S. typhimurium lipid A modifications were induced by low Mg2+ alone. Escherichia coli K-12 W3110 modified its lipid A molecules in response to growth under acidic but not low-Mg2+ conditions. Growth in a high-Mg2+, mildly alkaline medium resulted in suppression of most lipid A modifications with the exception of the 2-hydroxymyristate in S. typhimurium. Although lpxO transcription was stimulated by growth on low Mg2+, the biosynthesis of lipid A species containing 2-hydroxymyristate was independent of PhoP/PhoQ and PmrA/PmrB in S. typhimurium. Our labelling methods should be applicable to studies of lipid A modifications induced by endocytosis of diverse bacteria.
KeywordMeSH Terms
Endocytosis
Gene Expression Regulation, Bacterial
133. Koski  P, Saarilahti  H, Sukupolvi  S, Taira  S, Riikonen  P, Osterlund  K, Hurme  R, Rhen  M,     ( 1992 )

A new alpha-helical coiled coil protein encoded by the Salmonella typhimurium virulence plasmid.

The Journal of biological chemistry 267 (17)
PMID : 1601892  :  
Abstract >>
A new protein of Salmonella typhimurium was identified and characterized. The gene (tlpA) encoding this protein (TlpA) was isolated from the large virulence-associated plasmid of S. typhimurium and sequenced in order to predict the primary structure of TlpA. tlpA encodes a 371-amino acid soluble protein with a calculated M(r) of 41600 and pI of 4.63. Secondary structure predictions and sequence statistics of TlpA indicated a predominant alpha-helical configuration and presence of heptapeptide repeat motifs characteristic of coiled coil proteins. Purified TlpA was shown to have biochemical properties similar to those of coiled coil proteins, including adoption of an alpha-helical configuration and a tendency to form homodimers. Furthermore, TlpA possessed heat resistance, evidence for a chain register and altered mobility in urea/sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels which are characteristics of tropomyosins. TlpA shows 32% overall sequence similarity with rat cardiac myosin and 36% similarity with horse platelet beta-tropomyosin over 226 residues, whereas selected regions possessed significant sequence identities with myosins, tropomyosins, and alpha-helical surface proteins of Streptococcus pyogenes. Our results indicate that TlpA represents a new member of prokaryotic coiled coil proteins.
KeywordMeSH Terms
Plasmids
134. Miriagou  V, Carattoli  A, Tzelepi  E, Villa  L, Tzouvelekis  LS,     ( 2005 )

IS26-associated In4-type integrons forming multiresistance loci in enterobacterial plasmids.

Antimicrobial agents and chemotherapy 49 (8)
PMID : 16048979  :   DOI  :   10.1128/AAC.49.8.3541-3543.2005     PMC  :   PMC1196216    
Abstract >>
Three distinct multiresistant loci from enterobacterial plasmids each comprised an integron and an IS26-associated sequence. Sequence comparison suggested a common ancestral structure that derived from an IS26 insertion into the 5' conserved segment of an In4-type integron and evolved through acquisition of gene cassettes and IS26-mediated recruitment of additional resistance genes of diverse origin.
KeywordMeSH Terms
135. Li  WC, Huang  FY, Liu  CP, Weng  LC, Wang  NY, Chiu  NC, Chiang  CS,     ( 2005 )

Ceftriaxone resistance of nontyphoidal Salmonella enterica isolates in Northern Taiwan attributable to production of CTX-M-14 and CMY-2 beta-lactamases.

Journal of clinical microbiology 43 (7)
PMID : 16000441  :   DOI  :   10.1128/JCM.43.7.3237-3243.2005     PMC  :   PMC1169146    
Abstract >>
Among 3,027 nontyphoidal Salmonella enterica isolates identified between January 1999 and December 2002 in a medical center in northern Taiwan, 31 were resistant to the extended-spectrum cephalosporin ceftriaxone (1.02% [31/3,027]), including 2 in 1999 (0.36% [2/549]), 13 in 2000 (1.49% [13/870]), 7 in 2001 (0.78% [7/893]), and 9 in 2002 (1.26% [9/715]). Sixteen of these isolates belonged to Salmonella serogroup B, nine belonged to serogroup C, four belonged to serogroup D, and two belonged to serogroup E. The majority were from stool cultures. The mechanism of resistance was investigated for eight isolates, including three S. enterica serovar Typhimurium, one S. enterica serovar Wagenia, one S. enterica serovar Senftenberg, one S. enterica serovar Derby, one S. enterica serovar Panama, and one S. enterica serovar Duesseldorf isolate. All eight patients from whom these isolates were recovered had community-acquired infections. All eight isolates were resistant to ampicillin, ceftriaxone, and cefotaxime but susceptible to imipenem and ciprofloxacin. Ceftriaxone resistance was due to the production of the CMY-2 AmpC beta-lactamase by seven isolates and the CTX-M-14 beta-lactamase by the remaining isolate. Both beta-lactamase genes were carried on conjugative plasmids. In a 2.5-kb region encompassing the bla(CMY-2) gene, at nucleotide 49 upstream of the start codon of bla(CMY-2), three of the seven bla(CMY-2)-positive isolates had an A nucleotide and four had a G nucleotide. In conclusion, the ceftriaxone resistance of nontyphoidal Salmonella isolates in our hospital was attributed to the CTX-M-14 and CMY-2 beta-lactamases.
KeywordMeSH Terms
Cephalosporin Resistance
136. Olliver  A, Vallé  M, Chaslus-Dancla  E, Cloeckaert  A,     ( 2005 )

Overexpression of the multidrug efflux operon acrEF by insertional activation with IS1 or IS10 elements in Salmonella enterica serovar typhimurium DT204 acrB mutants selected with fluoroquinolones.

Antimicrobial agents and chemotherapy 49 (1)
PMID : 15616308  :   DOI  :   10.1128/AAC.49.1.289-301.2005     PMC  :   PMC538886    
Abstract >>
High-level fluoroquinolone (FQ) resistance in Salmonella enterica serovar Typhimurium phage type DT204 has been previously shown to be essentially due to both multiple target gene mutations and active efflux by the AcrAB-TolC efflux system. In this study we show that in intermediatly resistant acrB-inactivated serovar Typhimurium DT204 mutants, high-level resistance to FQs can be restored on in vitro selection with FQs. In each FQ- resistant mutant selected from serovar Typhimurium DT204 acrB mutant strains, an insertion sequence (IS1 or IS10) was found integrated upstream of the acrEF operon, coding for AcrEF, an efflux pump highly homologous to AcrAB. In one of the strains, transposition of IS1 caused partial deletion of acrS, the putative local repressor gene of the acrEF operon. Sequence analysis showed that both IS1 and IS10 elements contain putative promoter sequences that might alter the expression of adjacent acrEF genes. Indeed, reverse transcription-PCR experiments showed an 8- to 10-fold increase in expression of acrF in these insertional mutants, relative to their respective parental strain, which correlated well with the resistance levels observed to FQs and other unrelated drugs. It is noteworthy that AcrEF did not contribute to the intrinsic drug resistance of serovar Typhimurium, since acrF deletion in wild-type strains did not result in any increase in drug susceptibility. Moreover, deletion of acrS did not cause any acrF overexpression or any decrease in drug susceptibility, suggesting that acrEF overexpression is mediated solely by the IS1 and IS10 promoter sequences and not by inactivity of AcrS. Southern blot experiments showed that the number of chromosomal IS1 and IS10 elements in the serovar Typhimurium DT204 genome was about 5 and 15 respectively. None were detected in epidemic serovar Typhimurium DT104 strains or in the serovar Typhimurium reference strain LT2. Carrying IS1 and/or IS10 elements in their chromosome may thus be a selective advantage for serovar Typhimurium DT204 strains as opposed to DT104 strains for which no high-level FQ resistance nor insertional mutations were found. Taken together, the results of the present study indicate that the IS1- or IS10- activated AcrEF efflux pump may relay AcrAB in serovar Typhimurium, and underline the importance of transposable elements in the acquisition of FQ and multidrug resistance.
KeywordMeSH Terms
DNA Transposable Elements
Gene Expression Regulation, Bacterial
Operon
137. Kim  BH, Kim  S, Kim  HG, Lee  J, Lee  IS, Park  YK,     ( 2005 )

The formation of cyclopropane fatty acids in Salmonella enterica serovar Typhimurium.

Microbiology (Reading, England) 151 (Pt 1)
PMID : 15632439  :   DOI  :   10.1099/mic.0.27265-0    
Abstract >>
The formation of cyclopropane fatty acid (CFA) and its role in the acid shock response in Salmonella enterica serovar Typhimurium (S. typhimurium) was investigated. Data obtained by GC/MS demonstrated that the CFA level in S. typhimurium increased upon its entry to the stationary phase, as in other bacteria. The cfa gene encoding CFA synthase was cloned, and mutants of the cfa gene were constructed by allelic exchange. A cfa mutant could not produce CFA and was sensitive to low pH. Introduction of a functional cfa gene into a cfa mutant cell made the mutant convert all unsaturated fatty acids to CFAs and partially restored resistance to low pH. Interestingly, the alternative sigma factor RpoS, which was induced during the stationary phase, affected the production of C(19) CFA but not C(17) CFA. Western blotting analysis showed that the increase in expression of CFA synthase at early stationary phase was due to the alternative sigma factor RpoS.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
Heat-Shock Response
138. Folkesson  A, Eriksson  S, Andersson  M, Park  JT, Normark  S,     ( 2005 )

Components of the peptidoglycan-recycling pathway modulate invasion and intracellular survival of Salmonella enterica serovar Typhimurium.

Cellular microbiology 7 (1)
PMID : 15617530  :   DOI  :   10.1111/j.1462-5822.2004.00443.x    
Abstract >>
beta-Lactam resistance in enteric bacteria is frequently caused by mutations in ampD encoding a cytosolic N-acetylmuramyl- l-alanine amidase. Such mutants are blocked in murein (peptidoglycan) recycling and accumulate cytoplasmic muropeptides that interact with the transcriptional activator ampR, which de-represses beta-lactamase expression. Salmonella enterica serovar Typhimurium, an extensively studied enteric pathogen, was used to show that mutations in ampD decreased the ability of S. typhimurium to enter a macrophage derived cell line and made the bacteria more potent as inducers of inducible nitric oxide synthase (iNOS), as compared with the wild-type. ampG mutants, defective in the transport of recycled muropeptides across the cytoplasmic membrane, behaved essentially as the wild-type in invasion assays and in activation of iNOS. As ampD mutants also have reduced in vivo fitness in a murine model, we suggest that the cytoplasmic accumulation of muropeptides affects the virulence of the ampD mutants.
KeywordMeSH Terms
139. Morgan  E, Campbell  JD, Rowe  SC, Bispham  J, Stevens  MP, Bowen  AJ, Barrow  PA, Maskell  DJ, Wallis  TS,     ( 2004 )

Identification of host-specific colonization factors of Salmonella enterica serovar Typhimurium.

Molecular microbiology 54 (4)
PMID : 15522082  :   DOI  :   10.1111/j.1365-2958.2004.04323.x    
Abstract >>
The severity of infections caused by Salmonella enterica serovar Typhimurium varies depending on the host species. Numerous virulence genes have been identified in S. Typhimurium, largely from studies in mice, but their roles in infections of other species remain unclear. In the most comprehensive survey of its kind, through the use of signature-tagged mutagenesis of S. Typhimurium we have identified mutants that were unable to colonize calf intestines, mutants unable to colonize chick intestines and mutants unable to colonize both species. The type three secretion systems encoded on Salmonella pathogenicity islands (SPIs) 1 and 2 were required for efficient colonization of cattle. However, disruption of these secretion systems only caused a minor defect in S. Typhimurium colonization of chicks. Transposon insertions in SPI-4 compromised S. Typhimurium colonization of cattle, but not chicks. This is the first data confirming a role for SPI-4 in pathogenesis. We have also been able to ascribe a role in colonization for cell surface polysaccharides, cell envelope proteins, and many 'housekeeping' genes and genes of unknown function. We conclude that S. Typhimurium uses different strategies to colonize calves and chicks. This has major implications for vaccine design.
KeywordMeSH Terms
Salmonella Infections
140. Kruger  T, Szabo  D, Keddy  KH, Deeley  K, Marsh  JW, Hujer  AM, Bonomo  RA, Paterson  DL,     ( 2004 )

Infections with nontyphoidal Salmonella species producing TEM-63 or a novel TEM enzyme, TEM-131, in South Africa.

Antimicrobial agents and chemotherapy 48 (11)
PMID : 15504851  :   DOI  :   10.1128/AAC.48.11.4263-4270.2004     PMC  :   PMC525452    
Abstract >>
Salmonella spp. producing extended-spectrum beta-lactamases (ESBLs) have been reported in many countries, but there is no information on their prevalence in Africa. ESBL-producing Salmonella enterica serotype Isangi and S. enterica serotype Typhimurium strains have been noted in South Africa since 2001. A total of 160 consecutive isolates of Salmonella spp. were collected from 13 hospitals located in different cities in South Africa over a 5-month period from December 2002 to April 2003. All strains were screened for production of ESBLs by the double disk diffusion test and for AmpC production by assessing resistance to cefoxitin. bla(SHV), bla(TEM), bla(CTX-M), and bla(CMY-2) were sought from all ESBL-positive and cefoxitin-resistant isolates. A total of 15.6% (25 of 160) isolates produced SHV or TEM ESBLs, and 1.9% (3 of 160) produced CMY-2. Nine S. enterica serotype Typhimurium, eight S. enterica serotype Isangi, and three S. enterica serotype Muenchen strains produced either TEM-63 or a derivative of TEM-63 designated TEM-131. Both TEM-63 and TEM-131 have an isoelectric point of 5.6, and their sequences have the following amino acid substitutions compared to the TEM-1 sequence: Leu21Phe, Glu104Lys, Arg164Ser, and Met182Thr. Additionally, TEM-131 has an Ala237Thr substitution. ESBL-producing Salmonella spp. have become a significant public health problem in South Africa with particular implications for the treatment of serious nontyphoidal Salmonella infections in children, for whom extended-spectrum cephalosporins were the preferred treatment.
KeywordMeSH Terms
141. Malorny  B, Paccassoni  E, Fach  P, Bunge  C, Martin  A, Helmuth  R,     ( 2004 )

Diagnostic real-time PCR for detection of Salmonella in food.

Applied and environmental microbiology 70 (12)
PMID : 15574899  :   DOI  :   10.1128/AEM.70.12.7046-7052.2004     PMC  :   PMC535175    
Abstract >>
A robust 5' nuclease (TaqMan) real-time PCR was developed and validated in-house for the specific detection of Salmonella in food. The assay used specifically designed primers and a probe target within the ttrRSBCA locus, which is located near the Salmonella pathogenicity island 2 at centisome 30.5. It is required for tetrathionate respiration in Salmonella. The assay correctly identified all 110 Salmonella strains and 87 non-Salmonella strains tested. An internal amplification control, which is coamplified with the same primers as the Salmonella DNA, was also included in the assay. The detection probabilities were 70% when a Salmonella cell suspension containing 10(3) CFU/ml was used as a template in the PCR (5 CFU per reaction) and 100% when a suspension of 10(4) CFU/ml was used. A pre-PCR sample preparation protocol including a preenrichment step in buffered peptone water followed by DNA extraction-purification was applied when 110 various food samples (chicken rinses, minced meat, fish, and raw milk) were investigated for Salmonella. The diagnostic accuracy was shown to be 100% compared to the traditional culture method. The overall analysis time of the PCR method was approximately 24 h, in contrast to 4 to 5 days of analysis time for the traditional culture method. This methodology can contribute to meeting the increasing demand of quality assurance laboratories for standard diagnostic methods. Studies are planned to assess the interlaboratory performance of this diagnostic PCR method.
KeywordMeSH Terms
Food Contamination
Food Microbiology
142. Jin  UH, Cho  SH, Kim  MG, Ha  SD, Kim  KS, Lee  KH, Kim  KY, Chung  DH, Lee  YC, Kim  CH,     ( 2004 )

PCR method based on the ogdH gene for the detection of Salmonella spp. from chicken meat samples.

Journal of microbiology (Seoul, Korea) 42 (3)
PMID : 15459651  :  
Abstract >>
In a previous paper, the ogdH gene that encodes 2-oxoglutarate dehydrogenase was isolated from Salmonella typhimurium. The catalytic N-terminal region in the enzyme was found to be very specific for the Salmonella species. Therefore, the aim of the present study was to detect S. typhimurium in food sources using primers designed for OGDH-1 and OGDH-2 which were based on the salmonella-specific region of the ogdH gene. A simple polymerase chain reaction (PCR) detection method was developed to detect low numbers of S. typhimurium in a chicken meat microbial consortium. Using the ogdH-specific primers under stringent amplification conditions and for gene probe analysis, fewer than 100 colony-forming units (CFUs) were detectable when pure cultures were employed. When the PCR assay was run on S. typhimurium-contaminated meat contents, only the positive meat samples containing as few as 200 CFUs reacted to the assay. The method employed for sample processing is simple and it was determined to provide a sensitive means of detecting trace amounts of S. typhimurium-specific sequences in the presence of mixed meat microbial populations. When compared with six representative intestinal gram-negative bacterial strains in foods, including Vibrio parahaemolyticus, V. vulnificus, Enterobacter cloacae, E. coli O157:H7, Pseudomonas aeruginosa, and Proteus sp., S. typhimurium had a unique and distinct PCR product (796 bp). In conclusion, the two OGDH primers were found to be rapid and sensitive detectors of Salmonella spp for the PCR method.
KeywordMeSH Terms
143. Doerrler  WT, Gibbons  HS, Raetz  CR,     ( 2004 )

MsbA-dependent translocation of lipids across the inner membrane of Escherichia coli.

The Journal of biological chemistry 279 (43)
PMID : 15304478  :   DOI  :   10.1074/jbc.M408106200    
Abstract >>
MsbA is an essential ABC transporter in Escherichia coli required for exporting newly synthesized lipids from the inner to the outer membrane. It remains uncertain whether or not MsbA catalyzes trans-bilayer lipid movement (i.e. flip-flop) within the inner membrane. We now show that newly synthesized lipid A accumulates on the cytoplasmic side of the inner membrane after shifting an E. coli msbA missense mutant to the non-permissive temperature. This conclusion is based on the selective inhibition of periplasmic, but not cytoplasmic, covalent modifications of lipid A that occur in polymyxin-resistant strains of E. coli. The accessibility of newly synthesized phosphatidylethanolamine to membrane impermeable reagents, like 2,4,6-trinitrobenzene sulfonic acid, is also reduced severalfold. Our data showed that MsbA facilitates the rapid translocation of some lipids from the cytoplasmic to the periplasmic side of the inner membrane in living cells.
KeywordMeSH Terms
Lipid Metabolism
144. Hopkins  KL, Threlfall  EJ,     ( 2004 )

Frequency and polymorphism of sopE in isolates of Salmonella enterica belonging to the ten most prevalent serotypes in England and Wales.

Journal of medical microbiology 53 (Pt 6)
PMID : 15150335  :   DOI  :   10.1099/jmm.0.05510-0    
Abstract >>
Translocated effector protein, SopE, leads to actin cytoskeletal rearrangements and membrane ruffling. Only a subset of Salmonella enterica serotypes possess sopE, with the majority of sopE-carrying S. enterica serotype Typhimurium associated with epidemics. Using real-time PCR and sequencing, sopE was investigated in the ten most prevalent serotypes of S. enterica in England and Wales in 2001. sopE was identified in S. Typhimurium definitive phage types 29, 44, 49, 204b and 204c, all of which either have been involved in major epidemics or are precursors of epidemic strains. The presence of sopE varied in the remaining nine serotypes, but was more common in the top four (Enteritidis, Virchow, Hadar and Newport). Nucleotide changes were detected throughout sopE and may result in altered specificity for certain signal transduction pathways. Since acquisition of sopE may play a key role in emergence of epidemic strains, detection of sopE could aid identification of those Salmonella strains with the potential for epidemic spread.
KeywordMeSH Terms
Polymorphism, Single Nucleotide
145. Giles  WP, Benson  AK, Olson  ME, Hutkins  RW, Whichard  JM, Winokur  PL, Fey  PD,     ( 2004 )

DNA sequence analysis of regions surrounding blaCMY-2 from multiple Salmonella plasmid backbones.

Antimicrobial agents and chemotherapy 48 (8)
PMID : 15273090  :   DOI  :   10.1128/AAC.48.8.2845-2852.2004     PMC  :   PMC478531    
Abstract >>
The emergence in the United States of resistance to expanded-spectrum cephalosporin (e.g., ceftriaxone) within the salmonellae has been associated primarily with three large (>100-kb) plasmids (designated types A, B, and C) and one 10.1-kb plasmid (type D) that carry the blaCMY-2 gene. In the present study, the distribution of these four known blaCMY-2-carrying plasmids among 35 ceftriaxone-resistant Salmonella isolates obtained from 1998 to 2001 was examined. Twenty-three of these isolates were Salmonella enterica serotype Newport, 10 were Salmonella enterica serotype Typhimurium, 1 was Salmonella enterica serotype Agona, and 1 was Salmonella enterica serotype Reading. All 23 serotype Newport isolates carried a type C plasmid, and 5, 4, and 1 serovar Typhimurium isolate carried type B, A, and C plasmids, respectively. Both the serotype Agona and serotype Reading isolates carried type A plasmids. None of the isolates carried a type D plasmid. Hybridization data suggested that plasmid types A and C were highly related replicons. DNA sequencing revealed that the region surrounding blaCMY-2 was highly conserved in all three plasmid types analyzed (types B, C, and D) and was related to a region surrounding blaCMY-5 from the Klebsiella oxytoca plasmid pTKH11. These findings are consistent with a model in which blaCMY-2 has been disseminated primarily through plasmid transfer, and not by mobilization of the gene itself, to multiple Salmonella chromosomal backbones.
KeywordMeSH Terms
146. Antunes  P, Machado  J, Sousa  JC, Peixe  L,     ( 2004 )

Dissemination amongst humans and food products of animal origin of a Salmonella typhimurium clone expressing an integron-borne OXA-30 beta-lactamase.

The Journal of antimicrobial chemotherapy 54 (2)
PMID : 15243023  :   DOI  :   10.1093/jac/dkh333    
Abstract >>
Characterization of the molecular basis for beta-lactam resistance and evaluation of the clonal relatedness among nine isolates of multidrug-resistant Salmonella typhimurium recovered from seven clinical human samples and two pork end products. The isolates were examined for susceptibility to antimicrobial agents. The relationships between resistance genes, class 1 integrons, plasmids and isolates were screened by molecular methods such as polymerase chain reaction and restriction fragment length analysis. A bla(OXA-30) gene, located in a class 1 integron, was detected in all isolates. This integron was present on a conjugative plasmid in all but one isolate. By pulsed-field gel electrophoresis, it was determined that all strains share the same chromosomal type. This study demonstrates the spread of an OXA-30-producing S. typhimurium in Portugal, suggesting dissemination of a resistant clone through the food chain.
KeywordMeSH Terms
Food Microbiology
147. Hossain  A, Reisbig  MD, Hanson  ND,     ( 2004 )

Plasmid-encoded functions compensate for the biological cost of AmpC overexpression in a clinical isolate of Salmonella typhimurium.

The Journal of antimicrobial chemotherapy 53 (6)
PMID : 15140855  :   DOI  :   10.1093/jac/dkh240    
Abstract >>
In a previous study with a Salmonella typhimurium strain containing cloned ampC-ampR from Enterobacter cloacae, it was suggested that ampC expression must be kept at low levels by AmpR to maintain normal growth and virulent phenotype. The purpose of this study was to determine whether findings obtained with a laboratory model can be extended to a virulent clinical isolate of S. typhimurium expressing the plasmid-encoded bla(CMY-7). Disc induction assays were carried out to investigate inducibility of bla(CMY-7). Primer extension and sequence analyses were carried out to map the transcriptional start site of bla(CMY-7) and determine the relative expression. Growth and invasion potential of Salmonella strains were monitored by optical density, viable counts and cell invasion assays. Sequence analysis confirmed the absence of ampR upstream of bla(CMY-7) therefore confirming the negative results observed using the disc induction assay. Primer extension analysis mapped the start site of bla(CMY-7) transcription within an ISEcp1-like element. The relative expression of bla(CMY-7) was approximately 965-fold higher than the expression of a wild-type Citrobacter freundii chromosomal ampC and approximately 4.1-fold higher than ampC expression from a derepressed mutant of C. freundii. Growth and the capacity to invade mammalian cells were not compromised for either the clinical isolate or the S. typhimurium transconjugant containing bla(CMY-7). However, a Salmonella transformant containing bla(CMY-7) exhibited a compromised phenotype with respect to growth and invasion of mammalian cells. These findings indicate that the biological cost of high-level AmpC production can be compensated by plasmid-encoded factors and not by regulating ampC expression.
KeywordMeSH Terms
148. Guerra  B, Junker  E, Helmuth  R,     ( 2004 )

Incidence of the recently described sulfonamide resistance gene sul3 among German Salmonella enterica strains isolated from livestock and food.

Antimicrobial agents and chemotherapy 48 (7)
PMID : 15215132  :   DOI  :   10.1128/AAC.48.7.2712-2715.2004     PMC  :   PMC434208    
Abstract >>
The sul3 gene recently described in Escherichia coli was found in 22 of 512 (4.3%) German Salmonella isolates from different regions and sources and of different serotypes, antimicrobial resistance phenotypes, and genomic groups. This is the first report on the prevalence of sul3 among Salmonella strains, and the findings support the strong potential of this determinant to spread within bacterial populations.
KeywordMeSH Terms
149. Herrera-León  S, McQuiston  JR, Usera  MA, Fields  PI, Garaizar  J, Echeita  MA,     ( 2004 )

Multiplex PCR for distinguishing the most common phase-1 flagellar antigens of Salmonella spp.

Journal of clinical microbiology 42 (6)
PMID : 15184437  :   DOI  :   10.1128/JCM.42.6.2581-2586.2004     PMC  :   PMC427890    
Abstract >>
Most Salmonella serotypes alternatively express either phase-1 or phase-2 flagellar antigens, encoded by the fliC and fljB genes, respectively. Flagellar phase reversal for the identification of both flagellar antigens is not necessary at the genetic level. Variable internal regions of the fliC genes encoding the H:i, H:r, H:l,v, H:e,h, H:z(10), H:b, and H:d antigens have been sequenced; and the specific sites for each antigen in selected Salmonella serotypes have been determined. These results, together with flagellar G-complex variable internal sequences obtained by the Foodborne and Diarrheal Diseases Branch at the Centers for Disease Control and Prevention in Atlanta, GA, have been used to design a multiplex PCR to identify the G-complex antigens as well as the H:i, H:r, H:l,v, H:e,h, Hz(10), H:b, and H:d first-phase antigens. These antigens are part of the most common Salmonella serotypes possessing first-phase flagellar antigens. Salmonella enterica serotype Enteritidis is identified by adding a specific primer pair published previously. This multiplex PCR includes 13 primers. A total of 161 Salmonella strains associated with 72 different serotypes were tested. Each strain generated one first-phase-specific antigen fragment ranging from 100 to 500 bp; Salmonella serotype Enteritidis, however, generated two amplicons of 500 bp that corresponded to the G complex and a 333-bp serotype-specific amplicon, respectively. Twenty-three strains representing 19 serotypes with flagellar genes different from those targeted in this work did not generate any fragments. The method is quick, specific, and reproducible and is independent of the phase expressed by the bacteria when they are tested.
KeywordMeSH Terms
150. van der Straaten  T, Zulianello  L, van Diepen  A, Granger  DL, Janssen  R, van Dissel  JT,     ( 2004 )

Salmonella enterica serovar Typhimurium RamA, intracellular oxidative stress response, and bacterial virulence.

Infection and immunity 72 (2)
PMID : 14742546  :   DOI  :   10.1128/iai.72.2.996-1003.2004     PMC  :   PMC321585    
Abstract >>
Escherichia coli and Salmonella enterica serovar Typhimurium have evolved genetic systems, such as the soxR/S and marA regulons, to detoxify reactive oxygen species, like superoxide, which are formed as by-products of metabolism. Superoxide also serves as a microbicidal effector mechanism of the host's phagocytes. Here, we investigate whether regulatory genes other than soxR/S and marA are active in response to oxidative stress in Salmonella and may function as virulence determinants. We identified a bacterial gene, which was designated ramA (342 bp) and mapped at 13.1 min on the Salmonella chromosome, that, when overexpressed on a plasmid in E. coli or Salmonella, confers a pleiotropic phenotype characterized by increased resistance to the redox-cycling agent menadione and to multiple unrelated antibiotics. The ramA gene is present in Salmonella serovars but is absent in E. coli. The gene product displays 37 to 52% homology to the transcriptional activators soxR/S and marA and 80 to 100% identity to a multidrug resistance gene in Klebsiella pneumoniae and Salmonella enterica serovar Paratyphi A. Although a ramA soxR/S double null mutant is highly susceptible to intracellular superoxide generated by menadione and displays decreased Mn-superoxide dismutase activity, intracellular survival of this mutant within macrophage-like RAW 264.7 cells and in vivo replication in the spleens in Ityr mice are not affected. We concluded that despite its role in the protective response of the bacteria to oxidative stress in vitro, the newly identified ramA gene, together with soxR/S, does not play a role in initial replication of Salmonella in the organs of mice.
KeywordMeSH Terms
Oxidative Stress
151. Chen  S, Zhao  S, White  DG, Schroeder  CM, Lu  R, Yang  H, McDermott  PF, Ayers  S, Meng  J,     ( 2004 )

Characterization of multiple-antimicrobial-resistant salmonella serovars isolated from retail meats.

Applied and environmental microbiology 70 (1)
PMID : 14711619  :   DOI  :   10.1128/aem.70.1.1-7.2004     PMC  :   PMC321239    
Abstract >>
A total of 133 Salmonella isolates recovered from retail meats purchased in the United States and the People's Republic of China were assayed for antimicrobial susceptibility, the presence of integrons and antimicrobial resistance genes, and horizontal transfer of characterized antimicrobial resistance determinants via conjugation. Seventy-three (82%) of these Salmonella isolates were resistant to at least one antimicrobial agent. Resistance to the following antibiotics was common among the United States isolates: tetracycline (68% of the isolates were resistant), streptomycin (61%), sulfamethoxazole (42%), and ampicillin (29%). Eight Salmonella isolates (6%) were resistant to ceftriaxone. Fourteen isolates (11%) from the People's Republic of China were resistant to nalidixic acid and displayed decreased susceptibility to ciprofloxacin. A total of 19 different antimicrobial resistance genes were identified in 30 multidrug-resistant Salmonella isolates. The bla(CMY-2) gene, encoding a class A AmpC beta-lactamase, was detected in all 10 Salmonella isolates resistant to extended-spectrum beta-lactams. Resistance to ampicillin was most often associated with a TEM-1 family beta-lactamase gene. Six aminoglycoside resistance genes, aadA1, aadA2, aacC2, Kn, aph(3)-IIa, and aac(3)-IVa, were commonly present in the Salmonella isolates. Sixteen (54%) of 30 Salmonella isolates tested had integrons ranging in size from 0.75 to 2.7 kb. Conjugation studies demonstrated that there was plasmid-mediated transfer of genes encoding CMY-2 and TEM-1-like beta-lactamases. These data indicate that Salmonella isolates recovered from retail raw meats are commonly resistant to multiple antimicrobials, including those used for treating salmonellosis, such as ceftriaxone. Genes conferring antimicrobial resistance in Salmonella are often carried on integrons and plasmids and could be transmitted through conjugation. These mobile DNA elements have likely played an important role in transmission and dissemination of antimicrobial resistance determinants among Salmonella strains.
KeywordMeSH Terms
Drug Resistance, Multiple, Bacterial
152. Ehrbar  K, Friebel  A, Miller  SI, Hardt  WD,     ( 2003 )

Role of the Salmonella pathogenicity island 1 (SPI-1) protein InvB in type III secretion of SopE and SopE2, two Salmonella effector proteins encoded outside of SPI-1.

Journal of bacteriology 185 (23)
PMID : 14617659  :   DOI  :   10.1128/jb.185.23.6950-6967.2003     PMC  :   PMC262699    
Abstract >>
Salmonella enterica subspecies 1 serovar Typhimurium encodes a type III secretion system (TTSS) within Salmonella pathogenicity island 1 (SPI-1). This TTSS injects effector proteins into host cells to trigger invasion and inflammatory responses. Effector proteins are recognized by the TTSS via signals encoded in their N termini. Specific chaperones can be involved in this process. The chaperones InvB, SicA, and SicP are encoded in SPI-1 and are required for transport of SPI-1-encoded effectors. Several key effector proteins, like SopE and SopE2, are located outside of SPI-1 but are secreted in an SPI-1-dependent manner. It has not been clear how these effector proteins are recognized by the SPI-1 TTSS. Using pull-down and coimmunoprecipitation assays, we found that SopE is copurified with InvB, the known chaperone for the SPI-1-encoded effector protein Sip/SspA. We also found that InvB is required for secretion and translocation of SopE and SopE2 and for stabilization of SopE2 in the bacterial cytosol. Our data demonstrate that effector proteins encoded within and outside of SPI-1 use the same chaperone for secretion via the SPI-1 TTSS.
KeywordMeSH Terms
153. Gueneau de Novoa  P, Williams  KP,     ( 2004 )

The tmRNA website: reductive evolution of tmRNA in plastids and other endosymbionts.

Nucleic acids research 32 (Database issue)
PMID : 14681369  :   DOI  :   10.1093/nar/gkh102     PMC  :   PMC308836    
Abstract >>
tmRNA combines tRNA- and mRNA-like properties and ameliorates problems arising from stalled ribosomes. Research on the mechanism, structure and biology of tmRNA is served by the tmRNA website (http://www.indiana.edu/~ tmrna), a collection of sequences, alignments, secondary structures and other information. Because many of these sequences are not in GenBank, a BLAST server has been added; another new feature is an abbreviated alignment for the tRNA-like domain only. Many tmRNA sequences from plastids have been added, five found in public sequence data and another 10 generated by direct sequencing; detection in early-branching members of the green plastid lineage brings coverage to all three primary plastid lineages. The new sequences include the shortest known tmRNA sequence. While bacterial tmRNAs usually have a lone pseudoknot upstream of the mRNA segment and a string of three or four pseudoknots downstream, plastid tmRNAs collectively show loss of pseudoknots at both postions. The pseudoknot-string region is also too short to contain the usual pseudoknot number in another new entry, the tmRNA sequence from a bacterial endosymbiont of insect cells, Tremblaya princeps. Pseudoknots may optimize tmRNA function in free-living bacteria, yet become dispensible when the endosymbiotic lifestyle relaxes selective pressure for fast growth.
KeywordMeSH Terms
Databases, Nucleic Acid
Evolution, Molecular
Internet
154. Lee  SH, Galán  JE,     ( 2003 )

InvB is a type III secretion-associated chaperone for the Salmonella enterica effector protein SopE.

Journal of bacteriology 185 (24)
PMID : 14645290  :   DOI  :   10.1128/jb.185.24.7279-7284.2003     PMC  :   PMC296260    
Abstract >>
SopE is a bacteriophage-encoded effector protein of Salmonella enterica serovar Typhimurium that is translocated into the cytosol of eukaryotic cells by a type III secretion system (TTSS) (W.-D. Hardt, H. Urlaub, and J. E. Gal?n, Proc. Natl. Acad. Sci. USA 95:2574-2579, 1998; M. W. Wood, R. Rosqvist, P. B. Mullan, M. H. Edwards, and E. E. Galyov, Mol. Microbiol. 22:327-338, 1996). In this study, we provide evidence that an unlinked gene carried within the Salmonella pathogenicity island 1 (SPI-1), invB (K. Eichelberg, C. Ginocchio, and J. E. Gal?n, J. Bacteriol. 176:4501-4510, 1994), is required for the secretion of SopE through the SPI-1 TTSS. Furthermore, far-Western blotting analysis shows that SopE directly interacts with InvB through a domain located at its amino terminus. We conclude that InvB is the TTSS-associated chaperone for SopE.
KeywordMeSH Terms
155. Sun  Y, Dai  M, Hao  H, Wang  Y, Huang  L, Almofti  YA, Liu  Z, Yuan  Z,     ( 2011 )

The role of RamA on the development of ciprofloxacin resistance in Salmonella enterica serovar Typhimurium.

PloS one 6 (8)
PMID : 21858134  :   DOI  :   10.1371/journal.pone.0023471     PMC  :   PMC3155569    
Abstract >>
Active efflux pump is a primary fluoroquinolone resistant mechanism of clinical isolates of Salmonella enterica serovar Typhimurium. RamA is an essential element in producing multidrug resistant (MDR) S. enterica serovar Typhimurium. The aim of the present study was to elucidate the roles of RamA on the development of ciprofloxacin, the first choice for the treatment of salmonellosis, resistance in S. enterica serovar Typhimurium. Spontaneous mutants were selected via several passages of S. enterica serovar Typhimurium CVCC541 susceptible strain (ST) on M-H agar with increasing concentrations of ciprofloxacin (CIP). Accumulation of ciprofloxacin was tested by the modified fluorometric method. The expression levels of MDR efflux pumps were determined by real time RT-PCR. In ST and its spontaneous mutants, the ramA gene was inactivated by insertion of the kan gene and compensated on a recombinant plasmid pGEX�X(gst-ramA). The mutant prevention concentration (MPC) and mutant frequencies of ciprofloxacin against ST and a spontaneous mutant in the presence, absence and overexpression of RamA were tested. Four spontaneous mutants (SI1-SI4) were obtained. The SI1 (CIP MICs, 0.1 mg/L) without any target site mutation in its quinolone resistant determining regions (QRDRs) and SI3 (CIP MICs, 16 mg/L) harboring the Ser83��Phe mutation in its QRDR of GyrA strains exhibited reduced susceptibility and resistance to multidrugs, respectively. In SI1, RamA was the main factor that controlled the susceptibility to ciprofloxacin by activating MdtK as well as increasing the expression level of acrAB. In SI3, RamA played predominant role in ciprofloxacin resistance via increasing the expression level of acrAB. Likewise, the deficiency of RamA decreased the MPCs and mutant frequencies of ST and SI2 to ciprofloxacin. In conclusion, the expression of RamA promoted the development of ciprofloxacin resistant mutants of S. enterica serovar Typhimurium. The inhibition of RamA could decrease the appearance of the ciprofloxacin resistant mutants.
KeywordMeSH Terms
156. Sukupolvi  S, Vuorio  R, Qi  SY, O'Connor  D, Rhen  M,     ( 1990 )

Characterization of the traT gene and mutants that increase outer membrane permeability from the Salmonella typhimurium virulence plasmid.

Molecular microbiology 4 (1)
PMID : 2181240  :   DOI  :   10.1111/j.1365-2958.1990.tb02014.x    
Abstract >>
The nucleotide sequence of the traT gene present in the virulence-associated plasmid of Salmonella typhimurium was determined. The predicted TraT protein encoded by this gene was found to consist of 243 amino acids and to resemble the known TraT proteins of the plasmids of the F incompatibility group. Thus it contains a signal sequence of 20 amino acids, an amino-terminal lipid attachment site, and two strongly hydrophobic regions close to each other in the mature protein. A mutation leading to increased permeability of the outer membrane to hydrophobic agents, previously localized to the traT gene, was shown to change a glycine residue to arginine within one of these hydrophobic regions. The same principle was found to apply to TraT of R6-5: the introduction, by site-directed mutagenesis, of either positively or negatively charged amino acids or the helix-disrupting proline in the corresponding hydrophobic region led to increased hydrophobic permeability of the outer membrane.
KeywordMeSH Terms
Escherichia coli Proteins
Genes, Bacterial
157. Takahashi  H, Shao  M, Furuya  N, Komano  T,     ( 2011 )

The genome sequence of the incompatibility group I�^ plasmid R621a: evolution of IncI plasmids.

Plasmid 66 (2)
PMID : 21763721  :   DOI  :   10.1016/j.plasmid.2011.06.004    
Abstract >>
We present the complete genome sequence of the tetracycline resistance plasmid R621a isolated from Salmonella typhimurium, which belongs to the incompatibility group I�^. In the 93,185bp circular double-stranded R621a genome, 96 complete ORFs are predicted. In addition, one and six different kinds of proteins are produced by translational reinitiation and shufflon multiple inversions, respectively. The genome consists of four regions: replication, leading, transfer, and miscellaneous regions. The R621a genome is similar to those of IncI1 plasmids such as R64 and ColIb-P9 and particularly to those of pEK204 and pEC_Bactec. Three major differences including inc, parAB, and excA regions were noted between R621a and prototype IncI1 plasmids. Seven nucleotide replacements and one nucleotide deletion in the putative Inc RNA sequence are found between R621a and IncI1 plasmids irrespective of close similarity in the other parts of the rep system. The sequences of R621a parAB and excA genes are significantly different from those of R64 and ColIb-P9, while those of R621a parAB and excA genes exhibit close similarity to those of pEK204 and pEC_Bactec, respectively. The R621a genome is suggested to be formed by acquiring parAB and excA genes from pEK204 and pEC_Bactec genomes, respectively, and then novel inc function by the mutations. The insertions in the R621a, pEK204, and pEC_Bactec genomes are flanked by direct repeats, suggesting that insertions accompanied by long target duplications have also played an important role in the evolution of IncI plasmids.
KeywordMeSH Terms
Evolution, Molecular
Genome, Bacterial
Plasmids
158. Boyle  F, Healy  G, Hale  J, Kariuki  S, Cormican  M, Morris  D,     ( 2011 )

Characterization of a novel extended-spectrum �]-lactamase phenotype from OXA-1 expression in Salmonella Typhimurium strains from Africa and Ireland.

Diagnostic microbiology and infectious disease 70 (4)
PMID : 21767713  :   DOI  :   10.1016/j.diagmicrobio.2011.04.007    
Abstract >>
Salmonella enterica (S. Typhimurium) strains from Kenya, Malawi, and Ireland showing elevated cefepime MIC values carried bla(OXA-1). These strains were not detected by current guidelines for extended-spectrum �]-lactamase production based on MIC values or clavulanate inhibition, since cefepime is a preferred substrate for OXA-1 and this enzyme is reported to be resistant to clavulanate inhibition. bla(OXA-1) was located within a class I integron and an activated P(2) promoter was identified upstream of bla(OXA-1). P(2) was activated by the insertion of a triplet 'GGG' upstream of the -10 signal. All African strains were ST313, prevalent in this continent, and the Irish isolate belonged to ST19.
KeywordMeSH Terms
beta-Lactam Resistance
159. Chen  CY, Strobaugh  TP, Lindsey  RL, Frye  JG, Uhlich  G,     ( 2011 )

Sequence analysis of a group of low molecular-weight plasmids carrying multiple IS903 elements flanking a kanamycin resistance aph gene in Salmonella enterica serovars.

Plasmid 65 (3)
PMID : 21324339  :   DOI  :   10.1016/j.plasmid.2011.02.001    
Abstract >>
A group of low molecular-weight ColE1-like plasmids carrying the aph sequence type aph(ii) from three different Salmonella serovars were sequenced. These plasmids carry two or more copies of IS903 elements, with up to 21bp sequence differences to one another, two of which flank the aph gene. This group of plasmids did not appear to carry any known mobilization genes and instead carry three open reading frames encoding hypothetical proteins of unknown function possibly organized in an operon. The plasmid replication region (RNA I/II--rom) of this plasmid group showed extensive homology to that of pKPN2 plasmid of Klebsiella pneumoniae and pCol-let plasmid of Escherichia coli. Three of the four plasmids had identical sequences, and the fourth had an extra copy of IS903 with target duplication, suggesting a recent divergence in the different Salmonella serovars from a common ancestor.
KeywordMeSH Terms
Genes, Bacterial
Sequence Analysis
160. Shahada  F, Sekizuka  T, Kuroda  M, Kusumoto  M, Ohishi  D, Matsumoto  A, Okazaki  H, Tanaka  K, Uchida  I, Izumiya  H, Watanabe  H, Tamamura  Y, Iwata  T, Akiba  M,     ( 2011 )

Characterization of Salmonella enterica serovar Typhimurium isolates harboring a chromosomally encoded CMY-2 beta-lactamase gene located on a multidrug resistance genomic island.

Antimicrobial agents and chemotherapy 55 (9)
PMID : 21709089  :   DOI  :   10.1128/AAC.00560-11     PMC  :   PMC3165313    
Abstract >>
Since 2004, extended-spectrum cephalosporin (ESC)-resistant Salmonella enterica serovar Typhimurium (S. Typhimurium) isolates have been detected from cattle in the northern major island of Japan, Hokkaido. Resistance to ESCs was found to be mediated by CMY-2 type �]-lactamase among 22 epidemiologically unrelated isolates showing indistinguishable pulsed-field gel electrophoresis patterns. Southern blot analysis using I-CeuI-digested genomic DNA demonstrated that the CMY-2 �]-lactamase gene (bla(CMY-2)) was integrated in a 2.5-Mb chromosomal fragment. Genetic analysis of S. Typhimurium isolate L-3553 indicated that bla(CMY-2) was located on a unique 125-kb genomic island, GI-VII-6, which consists of 140 open reading frames. Pairwise alignment of GI-VII-6 and Escherichia coli plasmid pAR060302 (size, 167 kb) revealed that a large proportion of GI-VII-6 (99%) shows a high sequence similarity (>99%) with pAR060302. GI-VII-6 contains 11 antimicrobial resistance genes including sul1, qacE�G1, aadA2, and dfrA12 in the aadA2 region; sugE1 and bla(CMY-2) in the bla(CMY-2) region; and sul2, strA, strB, tet(A), and floR in the floR region. Two directly repeated IS26 copies were present at both ends of GI-VII-6. Junction regions of GI-VII-6 were flanked by an 8-bp direct repeat, indicating that GI-VII-6 was acquired by transposition involving IS26 transposase. PCR scanning revealed that the overall structure of GI-VII-6 was almost identical in the 22 isolates. Phylogenetic analysis suggested that S. Typhimurium isolates harboring GI-VII-6 belong to a different genomic lineage than other whole-genome-sequenced S. Typhimurium strains. These data indicate that a particular clone of S. Typhimurium harboring GI-VII-6 has spread among the cattle population in Hokkaido, Japan.
KeywordMeSH Terms
161. Smith  NH, Beltran  P, Selander  RK,     ( 1990 )

Recombination of Salmonella phase 1 flagellin genes generates new serovars.

Journal of bacteriology 172 (5)
PMID : 2129534  :   DOI  :   10.1128/jb.172.5.2209-2216.1990     PMC  :   PMC208845    
Abstract >>
To determine the evolutionary mechanisms generating serotypic diversity in Salmonella strains, we sequenced the central, antigen-determining part of the phase 1 flagellin gene (fliC) in strains of several serovars for which estimates of chromosomal genomic relatedness had been obtained by multilocus enzyme electrophoresis. The nucleotide sequence of this region was identical in several chromosomally divergent strains of Salmonella heidelberg (phase 1 antigen r) but differed by 19% from the corresponding and similarly invariant sequence in strains of the closely related serovar Salmonella typhimurium (phase 1 antigen i). Mutational drift of the sequence present in the common ancestor is unlikely to have generated the difference between the phase 1 flagellins of these two serovars, which we attribute instead to a recombination event. This interpretation is supported by evidence that Salmonella strains of very diverse chromosomal backgrounds but similar phase 1 antigens may have closely similar nucleotide sequences for this highly polymorphic region. We suggest that lateral transfer and recombination of phase 1 flagellin genes is a major evolutionary mechanism generating new Salmonella serovars.
KeywordMeSH Terms
Genes, Bacterial
162. Lodwick  D, Owen  D, Strike  P,     ( 1990 )

DNA sequence analysis of the imp UV protection and mutation operon of the plasmid TP110: identification of a third gene.

Nucleic acids research 18 (17)
PMID : 2129552  :   DOI  :   10.1093/nar/18.17.5045     PMC  :   PMC332119    
Abstract >>
The sequence of the imp operon of the plasmid TP110 (which belongs to the Incl1 incompatibility group) has been determined, and is shown to contain three open reading frames. This operon, involved in UV protection and mutation, is functionally analogous to the umuDC operon of E. coli and the mucAB operon of the plasmid pKM101, which belongs to the quite unrelated IncN incompatibility group. The umu and muc operons however contain only two open reading frames, coding for proteins of approximately 16kD and 46kD. The high degree of homology between the two 16kD proteins (UmuD and MucA) and between the two 46kD proteins (UmuC and MucB) clearly shows their relatedness. This is shown also to extend to the imp gene products, with ImpA sharing homology with UmuD and MucA, and ImpB sharing homology with UmuC and MucB. However, the two imp genes are preceded in the operon by a third gene, impC, which encodes a small protein of 9.5kD and which has no equivalent in the umu and muc operons.
KeywordMeSH Terms
Escherichia coli Proteins
Mutation
Operon
Plasmids
Serine Endopeptidases
163. Kang  MS, Kwon  YK, Jung  BY, Kim  A, Lee  KM, An  BK, Song  EA, Kwon  JH, Chung  GS,     ( 2011 )

Differential identification of Salmonella enterica subsp. enterica serovar Gallinarum biovars Gallinarum and Pullorum based on polymorphic regions of glgC and speC genes.

Veterinary microbiology 147 (1��2��)
PMID : 21111918  :   DOI  :   10.1016/j.vetmic.2010.05.039    
Abstract >>
Salmonella enterica subsp. enterica serovar Gallinarum biovars Gallinarum and Pullorum cause fowl typhoid and pullorum disease in avian species, respectively, and have been of considerable economic importance to the poultry industry in parts of the world. The definitive diagnosis of these diseases can be made only by isolation and identification of the causative agent. However, rapid identification of biovars Gallinarum and Pullorum is not easily feasible due to their common antigenic structure and genomic sequence similarity. We developed a duplex polymerase chain reaction (PCR) assay to identify and discriminate between strains of biovars Gallinarum and Pullorum. Duplex PCR primers were designed to target polymorphic regions of glgC and speC genes showing multiple mutations in the sequenced S. enterica subsp. enterica serovar Gallinarum 287/91 genome and were applied to the specific identification of biovars Gallinarum and Pullorum. Boiled lysates of 131 reference and field strains of Salmonella and other related Gram-negative bacteria were tested to validate the duplex PCR assay. All strains of biovars Gallinarum (n=53) and Pullorum (n=21) tested were correctly identified based on this assay (100% sensitivity) while the other strains (n=57) were PCR negative (100% specificity). These results demonstrate that a highly accurate biovar-specific duplex PCR assay can be performed for the rapid identification and discrimination of biovars Gallinarum and Pullorum from field isolates.
KeywordMeSH Terms
Polymerase Chain Reaction
164. Homma  M, Kutsukake  K, Hasebe  M, Iino  T, Macnab  RM,     ( 1990 )

FlgB, FlgC, FlgF and FlgG. A family of structurally related proteins in the flagellar basal body of Salmonella typhimurium.

Journal of molecular biology 211 (2)
PMID : 2129540  :   DOI  :   10.1016/0022-2836(90)90365-S    
Abstract >>
The flagellar basal body of Salmonella typhimurium consists of four rings surrounding a rod. The rod, which is believed to transmit motor rotation to the filament, is not well characterized in terms of its structure and composition. FlgG is known to lie within the distal portion of the rod, in the region where it is surrounded by the L and P rings, just before the rod-hook junction. The FlgC and FlgF proteins are also known to be flagellar basal-body components; by comparison of deduced and experimental N-terminal amino acid sequences we show here that FlgB is a basal-body protein. The flgB, flgC, flgF and flgG gene sequences and the deduced protein sequences are presented. The four proteins are clearly related to each other in primary sequence, especially toward the N and C termini, supporting the hypothesis (based on examination of basal-body subfractions) that FlgB, FlgC and FlgF are, like FlgG, rod proteins. From this and other information we suggest that the rod is the cell-proximal part of a segmented axial structure of the flagellum, with FlgB, FlgC and FlgF located (in unknown order) in successive segments of the proximal rod, followed by FlgG located in the distal rod; the axial structure then continues with the hook, HAPs and filament. Although the rod is external to the cell membrane, none of the four rod proteins contains a consensus signal sequence for the primary export pathway; comparison with the experimentally determined N-terminal amino acid sequence indicates that FlgB has had its N-terminal methionine removed, while the other three are not processed at all. This demonstrates that these proteins are not exported by the primary cellular pathway, and suggests that they are exported by the same flagellum-specific pathway as the flagellar filament protein flagellin. The observed sequence similarities among the rod proteins, especially a six-residue consensus motif about 30 residues in from the N terminus, may constitute a recognition signal for this pathway or they may reflect higher-order structural similarities within the rod.
KeywordMeSH Terms
165. Yau  S, Liu  X, Djordjevic  SP, Hall  RM,     ( 2010 )

RSF1010-like plasmids in Australian Salmonella enterica serovar Typhimurium and origin of their sul2-strA-strB antibiotic resistance gene cluster.

Microbial drug resistance (Larchmont, N.Y.) 16 (4)
PMID : 20617928  :   DOI  :   10.1089/mdr.2010.0033    
Abstract >>
Salmonella enterica serovar Typhimurium phage type 9 isolates resistant to streptomycin and sulfonamide have been recovered from both bovine and human sources in Australia. This study aimed to identify the resistance genes and their location. Polymerase chain reaction was used to screen for resistance genes and sul2 (sulphonamide resistance) and strA and strB (streptomycin resistance) were detected. A small streptomycin and sulfonamide resistance plasmid carrying the three resistance genes was recovered from these isolates by transformation and was shown to be essentially identical to the small IncQ plasmid RSF1010. The sequences of one plasmid, pSRC15, and RSF1010 differed at only a few positions that may be errors in the older sequence. RSF1010 has been recovered from many species and in many countries since its first isolation in the early 1970s. We conclude that this plasmid has persisted unchanged in the environment for over 30 years. The antibiotic resistance gene cluster containing strA, strB, and sul2 genes has clearly arisen from other known entities by a combination of transposition and homologous recombination using a short segment of homology. This resistance gene cluster is now widely distributed in plasmids and genomic islands in a number of contexts.
KeywordMeSH Terms
Multigene Family
166. Bell  RL, González-Escalona  N, Stones  R, Brown  EW,     ( 2011 )

Phylogenetic evaluation of the 'Typhimurium' complex of Salmonella strains using a seven-gene multi-locus sequence analysis.

Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases 11 (1)
PMID : 20970525  :   DOI  :   10.1016/j.meegid.2010.10.005    
Abstract >>
Salmonella enterica comprises over 2500 serovars, many of which are significant foodborne pathogens in humans. The ability to subtype these microbes is difficult due to the highly clonal nature of many Salmonella strains and a lack of congruence among traditional typing approaches. This work examines the phylogenetic utility of a multi-locus sequence typing (MLST) approach to discriminate between members of a closely related collection of salmonellae, the Salmonella reference collection A (SARA). This 72 strain collection, referred to as the 'Typhimurium' complex, consists of S. Typhimurium and its four closest serological relatives. In this analysis, nucleotide sequences from seven housekeeping genes (aroC, dnaN, hemD, hisD, purE, sucA, and thrA) were PCR amplified, sequenced, and combined into a single concatenated character matrix providing 3360bp for cladistic analysis. The resultant most parsimonious tree yielded seven clades of Salmonella strains that partitioned largely along serovar divisions within the collection except for five 'Paratyphi B' strains, two 'Saintpaul' strains, and two 'Typhimurium' strains. Convergence in the SARA tree was approximately 20% indicating that the vast majority of sequence changes were phylogenetically informative. Despite a high consistency among nucleotide substitutions, analysis of congruence identified several SARA strains with recombinant alleles in the concatenated matrix. These findings point to important differences among phylogenetic contributions made by the individual genes comprising this MLST dataset.
KeywordMeSH Terms
Genes, Bacterial
Phylogeny
167. Aramini  JM, Rossi  P, Cort  JR, Ma  LC, Xiao  R, Acton  TB, Montelione  GT,     ( 2011 )

Solution NMR structure of the plasmid-encoded fimbriae regulatory protein PefI from Salmonella enterica serovar Typhimurium.

Proteins 79 (1)
PMID : 20979070  :   DOI  :   10.1002/prot.22869     PMC  :   PMC2995844    
Abstract >>
N/A
KeywordMeSH Terms
168. Hoelzer  K, Soyer  Y, Rodriguez-Rivera  LD, Cummings  KJ, McDonough  PL, Schoonmaker-Bopp  DJ, Root  TP, Dumas  NB, Warnick  LD, Gröhn  YT, Wiedmann  M, Baker  KN, Besser  TE, Hancock  DD, Davis  MA,     ( 2010 )

The prevalence of multidrug resistance is higher among bovine than human Salmonella enterica serotype Newport, Typhimurium, and 4,5,12:i:- isolates in the United States but differs by serotype and geographic region.

Applied and environmental microbiology 76 (17)
PMID : 20639364  :   DOI  :   10.1128/AEM.00377-10     PMC  :   PMC2935064    
Abstract >>
Salmonella represents an important zoonotic pathogen worldwide, but the transmission dynamics between humans and animals as well as within animal populations are incompletely understood. We characterized Salmonella isolates from cattle and humans in two geographic regions of the United States, the Pacific Northwest and the Northeast, using three common subtyping methods (pulsed-field gel electrophoresis [PFGE], multilocus variable number of tandem repeat analysis [MLVA], and multilocus sequence typing [MLST]). In addition, we analyzed the distribution of antimicrobial resistance among human and cattle Salmonella isolates from the two study areas and characterized Salmonella persistence on individual dairy farms. For both Salmonella enterica subsp. enterica serotypes Newport and Typhimurium, we found multidrug resistance to be significantly associated with bovine origin of isolates, with the odds of multidrug resistance for Newport isolates from cattle approximately 18 times higher than for Newport isolates from humans. Isolates from the Northwest were significantly more likely to be multidrug resistant than those from the Northeast, and susceptible and resistant isolates appeared to represent distinct Salmonella subtypes. We detected evidence for strain diversification during Salmonella persistence on farms, which included changes in antimicrobial resistance as well as genetic changes manifested in PFGE and MLVA pattern shifts. While discriminatory power was serotype dependent, the combination of PFGE data with either MLVA or resistance typing data consistently allowed for improved subtype discrimination. Our results are consistent with the idea that cattle are an important reservoir of multidrug-resistant Salmonella infections in humans. In addition, the study provides evidence for the value of including antimicrobial resistance data in epidemiological investigations and highlights the benefits and potential problems of combining subtyping methods.
KeywordMeSH Terms
Drug Resistance, Multiple, Bacterial
169. Hauser  E, Junker  E, Helmuth  R, Malorny  B,     ( 2011 )

Different mutations in the oafA gene lead to loss of O5-antigen expression in Salmonella enterica serovar Typhimurium.

Journal of applied microbiology 110 (1)
PMID : 20961365  :   DOI  :   10.1111/j.1365-2672.2010.04877.x    
Abstract >>
To analyse genetic changes in the oafA gene explaining the loss of O5-antigen expression in Salmonella Typhimurium and Salm. 4,[5],12:i:-. The oafA gene in 52 O5-antigen-negative and 77 O5-antigen-positive Salm. Typhimurium (N = 47) and Salm. 4,[5],12:i:- (monophasic Salm. Typhimurium strains, N = 82) was investigated by a combination of PCR screening and DNA sequencing to identify mutations leading to the suppression of the O5-antigen. Various DNA sequence changes within the open reading frame (ORF) of oafA in O5-antigen-negative strains could be identified. In 77% of the O5-antigen-negative strains, a 7-bp deletion of a duplicated sequence within the functional oafA gene led to a frameshift in the ORF. In four strains, an IS4 element and in two, an IS1 element was inserted at different positions. Four other strains carried at different positions single base pair substitutions causing a premature stop codon. Finally, in two strains, a deletion of the oafA 3'end of undetermined size was responsible for the lack of O5-antigen expression. In none of the strains investigated, the complete ORF of oafA was deleted. Primers were designed and used to detect the most prominent variants. O5-antigen-negative Salm. Typhimurium and Salm. 4,[5],12:i:- strains carry an oafA pseudogene caused by different genetic events indicating that there is a selection for oafA mutations leading to the loss of O5-antigen expression. The loss of O5-antigen expression may be an example of a common evolutionary mechanism to escape host defence or to adapt to environmental changes. The data are the basis for the development of diagnostic PCR assays for the differentiation of O5-antigen-positive and O5-antigen-negative Salm. Typhimurium and its monophasic (Salm. 4,[5],12:i-) strains.
KeywordMeSH Terms
170. Villa  L, García-Fernández  A, Fortini  D, Carattoli  A,     ( 2010 )

Replicon sequence typing of IncF plasmids carrying virulence and resistance determinants.

The Journal of antimicrobial chemotherapy 65 (12)
PMID : 20935300  :   DOI  :   10.1093/jac/dkq347    
Abstract >>
IncF plasmids are frequently encountered in clinical enterobacterial strains associated with the dissemination of relevant antimicrobial resistance and virulence genes. These plasmids are usually heterogeneous in size and carry multiple replicons, and technical difficulties can impair the comparison and detection of related plasmids by restriction fragment length polymorphism analysis. We devised a rapid sequence-based typing scheme to categorize the members of this plasmid family into homogeneous groups. We compared the available IncF replicon sequences, identifying the combination of the different IncF replicon alleles as the discriminating characteristic of these plasmid scaffolds. An IncF typing method based on PCR amplification and sequence typing of the IncF replicons was devised. A collection of IncF plasmids carrying resistance and/or virulence genes, identified in strains from different sources and geographical origins, was tested with this typing system. We devised a replicon sequence typing (RST) scheme discriminating IncF plasmid variants. This system was tested on the collection of IncF plasmids, demonstrating that it was useful for the discrimination of plasmids carrying the same resistance gene (i.e. the bla(CTX-M-15) gene), but also recognized strictly related virulence plasmids (i.e. IncFIme plasmids). The PCR-based replicon typing (PBRT) system was also updated by including new primer pairs to allow the identification of the Salmonella, Klebsiella and Yersinia IncF plasmids. The ability to recognize and sub-categorize IncF plasmids by RST in homogeneous groups on the basis of their phylogenetic relatedness can be helpful in analysing their distribution in nature and discovering their evolutionary origin.
KeywordMeSH Terms
171. Sampei  G, Furuya  N, Tachibana  K, Saitou  Y, Suzuki  T, Mizobuchi  K, Komano  T,     ( 2010 )

Complete genome sequence of the incompatibility group I1 plasmid R64.

Plasmid 64 (2)
PMID : 20594994  :   DOI  :   10.1016/j.plasmid.2010.05.005    
Abstract >>
A streptomycin and tetracycline resistance plasmid R64 isolated from Salmonella enterica serovar Typhimurium belongs to the incompatibility group I1 (IncI1). The DNA sequence of the R64 conjugative transfer region was described previously (Komano et al., 2000). Here, we report the complete genome sequence of R64. In the circular double-stranded R64 genome with 120,826bp, 126 complete ORFs are predicted. In addition, 2 and 6 different kinds of proteins are produced by translational reinitiation and shufflon multiple inversions, respectively. The genome consists of five major regions: replication, drug resistance, stability, transfer leading, and conjugative transfer regions in clockwise order. The nucleotide sequence essential for autonomous replication of R64 is completely identical to that of IncI1 colicinogenic plasmid ColIb-P9, an indication that these two plasmids share the same mechanisms for replication and copy number control. Tetracycline and streptomycin resistance genes are encoded in transposons Tn10 and Tn6082, respectively. These transposons and two insertion elements, IS2 and IS1133, were inserted stepwise into the arsenic-resistant gene, arsA1, present in the drug resistance region. The stability and transfer leading regions contain various important genes such as parAB, resD, ardA, psiAB, or ssb for plasmid maintenance, recombination and transfer reactions. When the genome of R64 was compared with those of other plasmids, varying levels of similarity were observed. It is suggested that genetic recombinations including the site-specific rfsF-ResD system have played an important role in diversity of genomes related to R64. It was found that R64 exhibits highly organized genome structure.
KeywordMeSH Terms
Genome, Bacterial
172. Targant  H, Doublet  B, Aarestrup  FM, Cloeckaert  A, Madec  JY,     ( 2010 )

IS6100-mediated genetic rearrangement within the complex class 1 integron In104 of the Salmonella genomic island 1.

The Journal of antimicrobial chemotherapy 65 (7)
PMID : 20483981  :   DOI  :   10.1093/jac/dkq163    
Abstract >>
N/A
KeywordMeSH Terms
DNA Transposable Elements
Genomic Islands
Integrons
Recombination, Genetic
173. Cain  AK, Liu  X, Djordjevic  SP, Hall  RM,     ( 2010 )

Transposons related to Tn1696 in IncHI2 plasmids in multiply antibiotic resistant Salmonella enterica serovar Typhimurium from Australian animals.

Microbial drug resistance (Larchmont, N.Y.) 16 (3)
PMID : 20701539  :   DOI  :   10.1089/mdr.2010.0042    
Abstract >>
Conjugative IncHI2 plasmids carrying tetracycline, trimethoprim, and sulphonamide resistance genes were recovered from two multiply antibiotic resistant Salmonella enterica serovar Typhimurium isolates from Australian food-producing animals. Transposons related to the mercury resistance transposon Tn1696 were identified in both IncHI2 plasmids. These transposons contained an In4-type class 1 integron that carried a dfrA5 trimethoprim resistance gene cassette and the sul1 sulfonamide resistance gene. These integrons were located in the same position as In4 in Tn1696. The integron from one isolate includes a large transposon-like structure containing four IS26 and the strAB, sul2, bla(TEM), and aphA1 genes conferring resistance to streptomycin, sulphonamides, ampicillin, kanamycin, and neomycin, respectively. This structure is flanked by an 8-bp duplication, but it includes both the aphA1-containing transposon Tn4352 and a transposon, Tn6029, carrying genes derived from RSF1010 and from Tn2. However, Tn4352 and Tn6029 overlap, sharing one IS26 copy. This suggests that they do not move by a standard transpositional mechanism. A circular intermediate, carrying only the region containing the resistance gene(s) and one of the IS26 bounding it, is proposed as an intermediate.
KeywordMeSH Terms
174. Chen  CY, Lindsey  RL, Strobaugh  TP, Frye  JG, Meinersmann  RJ,     ( 2010 )

Prevalence of ColE1-like plasmids and kanamycin resistance genes in Salmonella enterica serovars.

Applied and environmental microbiology 76 (20)
PMID : 20693446  :   DOI  :   10.1128/AEM.00692-10     PMC  :   PMC2953026    
Abstract >>
Multi-antimicrobial-resistant Salmonella enterica strains frequently carry resistance genes on plasmids. Recent studies focus heavily on large conjugative plasmids, and the role that small plasmids play in resistance gene transfer is largely unknown. To expand our previous studies in assessing the prevalence of the isolates harboring ColE1-like plasmids carrying the aph gene responsible for kanamycin resistance (Kan(r)) phenotypes, 102 Kan(r) Salmonella isolates collected through the National Antimicrobial Resistance Monitoring System (NARMS) in 2005 were screened by PCR using ColE1 primer sets. Thirty isolates were found to be positive for ColE1-like replicon. Plasmids from 23 isolates were able to propagate in Escherichia coli and were subjected to further characterization. Restriction mapping revealed three major plasmid groups found in three or more isolates, with each group consisting of two to three subtypes. The aph genes from the Kan(r) Salmonella isolates were amplified by PCR, sequenced, and showed four different aph(3')-I genes. The distribution of the ColE1 plasmid groups in association with the aph gene, Salmonella serovar, and isolate source demonstrated a strong linkage of the plasmid with S. enterica serovar Typhimurium DT104. Due to their high copy number and mobility, the ColE1-like plasmids may play a critical role in transmission of antibiotic resistance genes among enteric pathogens, and these findings warrant a close monitoring of this plasmid incompatibility group.
KeywordMeSH Terms
Kanamycin Resistance
175. Antunes  P, Coque  TM, Peixe  L,     ( 2010 )

Emergence of an IncI�^ plasmid encoding CMY-2 ?-lactamase associated with the international ST19 OXA-30-producing ?-lactamase Salmonella Typhimurium multidrug-resistant clone.

The Journal of antimicrobial chemotherapy 65 (10)
PMID : 20685754  :   DOI  :   10.1093/jac/dkq293    
Abstract >>
To analyse the genetic environment of the bla(CMY-2) gene in a multidrug-resistant isolate belonging to the OXA-30-producing Salmonella enterica serotype Typhimurium clone widespread in European countries. Preliminary characterization of CMY-2 was performed after determining the phenotype against �]-lactams, the pI (isoelectric focusing) and the presence of bla genes (PCR). The genetic context of the bla(CMY-2) gene was identified by PCR mapping and further sequencing. Plasmid analysis included determination of transferability, size and content (S1-PFGE/hybridization), and characterization of the incompatibility group (PCR-based replicon typing, hybridization and sequencing). The �GISEcp1-bla(CMY-2)-blc-sugE cassette identified is located in the pndC-trbA region of an IncI�^ conjugative plasmid designated pSTHV23035. This plasmid differs from previously described European IncI plasmids coding for CMY in the IncI replicon type and in the integration site of the element carrying bla(CMY-2). The emergence of bla(CMY-2) located in a new IncI�^ plasmid in an isolate belonging to the international widespread multidrug-resistant sequence type (ST) 19 OXA-30-producing Salmonella Typhimurium clone is reported for the first time. Acquisition of a new bla(CMY-2)-IncI�^ plasmid by Salmonella multidrug-resistant strains is of concern since these plasmids diminish the activity of therapeutically important broad-spectrum �]-lactams and contribute to the persistence and pathogenicity of widespread clones.
KeywordMeSH Terms
Drug Resistance, Multiple, Bacterial
176. Vo  AT, van Duijkeren  E, Gaastra  W, Fluit  AC,     ( 2010 )

Antimicrobial resistance, class 1 integrons, and genomic island 1 in Salmonella isolates from Vietnam.

PloS one 5 (2)
PMID : 20195474  :   DOI  :   10.1371/journal.pone.0009440     PMC  :   PMC2829082    
Abstract >>
The objective was to investigate the phenotypic and genotypic resistance and the horizontal transfer of resistance determinants from Salmonella isolates from humans and animals in Vietnam. The susceptibility of 297 epidemiologically unrelated non-typhoid Salmonella isolates was investigated by disk diffusion assay. The isolates were screened for the presence of class 1 integrons and Salmonella genomic island 1 by PCR. The potential for the transfer of resistance determinants was investigated by conjugation experiments. Resistance to gentamicin, kanamycin, chloramphenicol, streptomycin, trimethoprim, ampicillin, nalidixic acid, sulphonamides, and tetracycline was found in 13 to 50% of the isolates. Nine distinct integron types were detected in 28% of the isolates belonging to 11 Salmonella serovars including S. Tallahassee. Gene cassettes identified were aadA1, aadA2, aadA5, bla(PSE-1), bla(OXA-30), dfrA1, dfrA12, dfrA17, and sat, as well as open reading frames with unknown functions. Most integrons were located on conjugative plasmids, which can transfer their antimicrobial resistance determinants to Escherichia coli or Salmonella Enteritidis, or with Salmonella Genomic Island 1 or its variants. The resistance gene cluster in serovar Emek identified by PCR mapping and nucleotide sequencing contained SGI1-J3 which is integrated in SGI1 at another position than the majority of SGI1. This is the second report on the insertion of SGI1 at this position. High-level resistance to fluoroquinolones was found in 3 multiresistant S. Typhimurium isolates and was associated with mutations in the gyrA gene leading to the amino acid changes Ser83Phe and Asp87Asn. Resistance was common among Vietnamese Salmonella isolates from different sources. Legislation to enforce a more prudent use of antibiotics in both human and veterinary medicine should be implemented by the authorities in Vietnam.
KeywordMeSH Terms
177. Taira  S, Baumann  M, Riikonen  P, Sukupolvi  S, Rhen  M,     ( 1991 )

Amino-terminal sequence analysis of four plasmid-encoded virulence-associated proteins of Salmonella typhimurium.

FEMS microbiology letters 61 (2��3��)
PMID : 2037236  :   DOI  :   10.1016/0378-1097(91)90573-s    
Abstract >>
We have expressed four virulence-associated proteins encoded by the Salmonella typhimurium plasmid pEX102 in Escherichia coli. The genes coding for the proteins MkaA, MkaB, MkaC and MkaD subcloned in the vectors pUC19 or Bluescript KS+ directed substantial production of these proteins in E. coli. The same host harbouring pEX102 did not produce these proteins in detectable amounts. The proteins were exclusively found in the particulate fraction. The amino-terminal sequence analysis showed that the N-termini of these proteins corresponded to the ones predicted from the open reading frames found previously in the DNA sequence of the virulence determinant and that no N-terminal signal sequence processing of the proteins occurred.
KeywordMeSH Terms
Plasmids
Transcription Factors
178. Heras  B, Totsika  M, Jarrott  R, Shouldice  SR, Guncar  G, Achard  ME, Wells  TJ, Argente  MP, McEwan  AG, Schembri  MA,     ( 2010 )

Structural and functional characterization of three DsbA paralogues from Salmonella enterica serovar typhimurium.

The Journal of biological chemistry 285 (24)
PMID : 20233716  :   DOI  :   10.1074/jbc.M110.101360     PMC  :   PMC2881768    
Abstract >>
In prototypic Escherichia coli K-12 the introduction of disulfide bonds into folding proteins is mediated by the Dsb family of enzymes, primarily through the actions of the highly oxidizing protein EcDsbA. Homologues of the Dsb catalysts are found in most bacteria. Interestingly, pathogens have developed distinct Dsb machineries that play a pivotal role in the biogenesis of virulence factors, hence contributing to their pathogenicity. Salmonella enterica serovar (sv.) Typhimurium encodes an extended number of sulfhydryl oxidases, namely SeDsbA, SeDsbL, and SeSrgA. Here we report a comprehensive analysis of the sv. Typhimurium thiol oxidative system through the structural and functional characterization of the three Salmonella DsbA paralogues. The three proteins share low sequence identity, which results in several unique three-dimensional characteristics, principally in areas involved in substrate binding and disulfide catalysis. Furthermore, the Salmonella DsbA-like proteins also have different redox properties. Whereas functional characterization revealed some degree of redundancy, the properties of SeDsbA, SeDsbL, and SeSrgA and their expression pattern in sv. Typhimurium indicate a diverse role for these enzymes in virulence.
KeywordMeSH Terms
179. Nohmi  T, Hakura  A, Nakai  Y, Watanabe  M, Murayama  SY, Sofuni  T,     ( 1991 )

Salmonella typhimurium has two homologous but different umuDC operons: cloning of a new umuDC-like operon (samAB) present in a 60-megadalton cryptic plasmid of S. typhimurium.

Journal of bacteriology 173 (3)
PMID : 1991707  :   DOI  :   10.1128/jb.173.3.1051-1063.1991     PMC  :   PMC207224    
Abstract >>
Expression of the umuDC operon is required for UV and most chemical mutagenesis in Escherichia coli. The DNA which can restore UV mutability to a umuD44 strain and to a umuC122::Tn5 strain of E. coli has been cloned from Salmonella typhimurium TA1538. DNA sequence analysis indicated that the cloned DNA potentially encoded proteins with calculated molecular weights of 15,523 and 47,726 and was an analog of the E. coli umuDC operon. We have termed this cloned DNA the samAB (for Salmonella mutagenesis) operon and tentatively referred to the umuDC operon of S. typhimurium LT2 (C. M. Smith, W. H. Koch, S. B. Franklin, P. L. Foster, T. A. Cebula, and E. Eisenstadt, J. Bacteriol. 172:4964-4978, 1990; S. M. Thomas, H. M. Crowne, S. C. Pidsley, and S. G. Sedgwick, J. Bacteriol. 172:4979-4987, 1990) as the umuDCST operon. The samAB operon is 40% diverged from the umuDCST operon at the nucleotide level. Among five umuDC-like operons so far sequenced, i.e., the samAB, umuDCST, mucAB, impAB, and E. coli umuDC operons, the samAB operon shows the highest similarity to the impAB operon of TP110 plasmid while the umuDCST operon shows the highest similarity to the E. coli umuDC operon. Southern hybridization experiments indicated that (i) S. typhimurium LT2 and TA1538 had both the samAB and the umuDCST operons and (ii) the samAB operon was located in a 60-MDa cryptic plasmid. The umuDCST operon is present in the chromosome. The presence of the two homologous but different umuDC operons may be involved in the poor mutability of S. typhimurium by UV and chemical mutagens.
KeywordMeSH Terms
Mutagenesis
Operon
Plasmids
180. Wiesner  M, Zaidi  MB, Calva  E, Fernández-Mora  M, Calva  JJ, Silva  C,     ( 2009 )

Association of virulence plasmid and antibiotic resistance determinants with chromosomal multilocus genotypes in Mexican Salmonella enterica serovar Typhimurium strains.

BMC microbiology 9 (N/A)
PMID : 19573249  :   DOI  :   10.1186/1471-2180-9-131     PMC  :   PMC2715408    
Abstract >>
Bacterial genomes are mosaic structures composed of genes present in every strain of the same species (core genome), and genes present in some but not all strains of a species (accessory genome). The aim of this study was to compare the genetic diversity of core and accessory genes of a Salmonella enterica subspecies enterica serovar Typhimurium (Typhimurium) population isolated from food-animal and human sources in four regions of Mexico. Multilocus sequence typing (MLST) and macrorestriction fingerprints by pulsed-field gel electrophoresis (PFGE) were used to address the core genetic variation, and genes involved in pathogenesis and antibiotic resistance were selected to evaluate the accessory genome. We found a low genetic diversity for both housekeeping and accessory genes. Sequence type 19 (ST19) was supported as the founder genotype of STs 213, 302 and 429. We found a temporal pattern in which the derived ST213 is replacing the founder ST19 in the four geographic regions analyzed and a geographic trend in the number of resistance determinants. The distribution of the accessory genes was not random among chromosomal genotypes. We detected strong associations among the different accessory genes and the multilocus chromosomal genotypes (STs). First, the Salmonella virulence plasmid (pSTV) was found mostly in ST19 isolates. Second, the plasmid-borne betalactamase cmy-2 was found only in ST213 isolates. Third, the most abundant integron, IP-1 (dfrA12, orfF and aadA2), was found only in ST213 isolates. Fourth, the Salmonella genomic island (SGI1) was found mainly in a subgroup of ST19 isolates carrying pSTV. The mapping of accessory genes and multilocus genotypes on the dendrogram derived from macrorestiction fingerprints allowed the establishment of genetic subgroups within the population. Despite the low levels of genetic diversity of core and accessory genes, the non-random distribution of the accessory genes across chromosomal backgrounds allowed us to discover genetic subgroups within the population. This study provides information about the importance of the accessory genome in generating genetic variability within a bacterial population.
KeywordMeSH Terms
Genetic Variation
Genome, Bacterial
181. Dionisi  AM, Lucarelli  C, Owczarek  S, Luzzi  I, Villa  L,     ( 2009 )

Characterization of the plasmid-borne quinolone resistance gene qnrB19 in Salmonella enterica serovar Typhimurium.

Antimicrobial agents and chemotherapy 53 (9)
PMID : 19528272  :   DOI  :   10.1128/AAC.00294-09     PMC  :   PMC2737868    
Abstract >>
A qnrB19 gene variant, carried by an IncL/M-like plasmid, was detected in a multidrug Salmonella enterica serovar Typhimurium human strain with reduced susceptibility to ciprofloxacin. The genetic environment around the gene was fully sequenced (20 kb). A large gene cluster, containing the aph, qnrB19, and blaSHV-12-like resistance genes, is inserted inside a Tn3 transposon.
KeywordMeSH Terms
182. Kannan  P, Dharne  M, Smith  A, Karns  J, Bhagwat  AA,     ( 2009 )

Motility revertants of opgGH mutants of Salmonella enterica serovar Typhimurium remain defective in mice virulence.

Current microbiology 59 (6)
PMID : 19727946  :   DOI  :   10.1007/s00284-009-9486-8    
Abstract >>
We recently demonstrated that osmoregulated periplasmic glucans (OPGs) of Salmonella enterica serovar Typhimurium are required for optimal mouse virulence. However, lack of OPGs also generated pleiotropic phenotypes such as reduced motility and slower growth rate under hypoosmotic growth conditions. Whether the observed suboptimal virulence of opg mutants was due to reduced motility was investigated by isolating fully motile revertants of opgGH mutants. Motility revertants remained defective in OPGs synthesis and restitution of motility did not restore mouse virulence. In Escherichia coli, inactivation of rcsB, rcsD, and rcsF lead to restoration of motility in opg mutants, while in Salmonella strains, inactivation of the Rcs pathway is known to attenuate virulence. DNA sequence analysis revealed that except for two silent mutations no other changes in the DNA sequences of Rcs pathway genes were detected in the motility-revertant strain. Moreover, transcripts of all the Rcs phosphorelay pathway genes were detected in opgGH mutants and revertant strain. The data suggest that Salmonella may have distinctive regulatory elements in addition to Rcs phosphorelay genes to rescue motility of opg mutants and affecting also mouse virulence.
KeywordMeSH Terms
183. Furuya  N, Komano  T,     ( 1991 )

Determination of the nick site at oriT of IncI1 plasmid R64: global similarity of oriT structures of IncI1 and IncP plasmids.

Journal of bacteriology 173 (20)
PMID : 1917882  :   DOI  :   10.1128/jb.173.20.6612-6617.1991     PMC  :   PMC208999    
Abstract >>
The nick site at the origin of transfer, oriT, of IncI1 plasmid R64 was determined. A site-specific and strand-specific cleavage of the phosphodiester bond was introduced during relaxation of the oriT plasmid DNA. Cleavage occurred between 2'-deoxyguanosine and thymidine residues, within the 44-bp oriT core sequence. The nick site was located 8 bp from the 17-bp repeat. A protein appeared to be associated with the cleaved DNA strand at the oriT site following relaxation. This protein was observed to bind to the 5' end of the cleaved strand, since the 5'-phosphate of the cleaved strand was resistant to the phosphate exchange reaction by polynucleotide kinase. In contrast, the 3' end of the cleaved strand appeared free, since it was susceptible to primer extension by DNA polymerase I. The global similarity of the oriT structures of IncI1 and IncP plasmids is discussed.
KeywordMeSH Terms
Sequence Homology, Nucleic Acid
184. Nelson  K, Whittam  TS, Selander  RK,     ( 1991 )

Nucleotide polymorphism and evolution in the glyceraldehyde-3-phosphate dehydrogenase gene (gapA) in natural populations of Salmonella and Escherichia coli.

Proceedings of the National Academy of Sciences of the United States of America 88 (15)
PMID : 1862091  :   DOI  :   10.1073/pnas.88.15.6667     PMC  :   PMC52149    
Abstract >>
Nucleotide sequences of the gapA gene, encoding the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase, were determined for 16 strains of Salmonella and 13 strains of Escherichia coli recovered from natural populations. Pairs of sequences from strains representing the eight serovar groups of Salmonella differed, on average, at 3.8% of nucleotide sites and 1.1% of inferred amino acids, and comparable values for E. coli were an order of magnitude smaller (0.2% and 0.1%, respectively). The rate of substitution at synonymous sites was significantly higher for codons specifying the catalytic domain of the enzyme than for those encoding the NAD(+)-binding domain, but the nonsynonymous substitution rate showed the opposite relationship. For Salmonella, statistical tests for nonrandom clustering of polymorphic sites failed to provide evidence that intragenic recombination or gene conversion has contributed to the generation of allelic diversity. The topology of a tree constructed from the gapA sequences was generally similar to that of phylogenetic trees of the strains based on multilocus enzyme electrophoresis, but the level of divergence of gapA in Salmonella group V from other Salmonella and E. coli strains is much greater than that indicated by DNA hybridization for the genome as a whole.
KeywordMeSH Terms
Biological Evolution
Genes, Bacterial
Polymorphism, Genetic
185. Rodríguez  I, Rodicio  MR, Herrera-León  S, Echeita  A, Mendoza  MC,     ( 2008 )

Class 1 integrons in multidrug-resistant non-typhoidal Salmonella enterica isolated in Spain between 2002 and 2004.

International journal of antimicrobial agents 32 (2)
PMID : 18571383  :   DOI  :   10.1016/j.ijantimicag.2008.03.005    
Abstract >>
In this study, 119 multidrug-resistant isolates of non-typhoidal Salmonella enterica serovars collected in Spain (2002-2004) were screened for integrons. Among the isolates, 73.1% contained class 1 integrons, however classes 2 and 3 were not detected. Integrons containing gene cassettes were found in S. Enteritidis (16/32), S. Typhimurium biphasic (18/32) and monophasic [4,5,12:i:-] (11/19), S. Virchow (17/18) and S. Brandenburg (8/8), but not in S. Hadar (0/10). Ten complete and four incomplete gene cassettes, combined in 10 variable regions, were identified, one of which (2100 bp/dfrA1-597 bp-aadA24) was a new description. Most integrons mapped on plasmids of ca. 40-340 kb. Exceptions were 1000 bp/aadA2 and 1200 bp/bla PSE-1 found on the chromosome of biphasic S. Typhimurium, probably as part of SGI1-like structures.
KeywordMeSH Terms
186. Wu  JJ, Ko  WC, Chiou  CS, Chen  HM, Wang  LR, Yan  JJ,     ( 2008 )

Emergence of Qnr determinants in human Salmonella isolates in Taiwan.

The Journal of antimicrobial chemotherapy 62 (6)
PMID : 18957397  :   DOI  :   10.1093/jac/dkn426    
Abstract >>
The aim of this study was to determine the prevalence and characteristics of qnr-carrying Salmonella isolates from humans in southern Taiwan. A total of 446 Salmonella isolates collected between 2003 and 2006 were screened for qnrA, qnrB and qnrS by PCR experiments. Genetic structures of qnr were determined by PCR-based methods or direct sequencing of plasmid DNA. qnrB2 and qnrS1 were detected in two serovar Enteritidis isolates and two serovar Typhimurium isolates, respectively. One qnrS1-positive isolate was found to produce the CMY-2 AmpC enzyme. qnrS1 was identified on a 10 kb plasmid, which exhibited >99% nucleotide sequence identity with plasmid TPqnrS-1a reported from the UK. qnrB2 was found in a complex sul1-type class 1 integron on a >100 kb plasmid. This study demonstrated the occurrence of qnrB2 and qnrS1 in Salmonella for the first time in Taiwan and characterized their genetic structures.
KeywordMeSH Terms
Drug Resistance, Bacterial
187. Herrero  A, Mendoza  MC, Rodicio  R, Rodicio  MR,     ( 2008 )

Characterization of pUO-StVR2, a virulence-resistance plasmid evolved from the pSLT virulence plasmid of Salmonella enterica serovar Typhimurium.

Antimicrobial agents and chemotherapy 52 (12)
PMID : 18852276  :   DOI  :   10.1128/AAC.00563-08     PMC  :   PMC2592850    
Abstract >>
pUO-StVR2 is a virulence-resistance plasmid which originated from pSLT of Salmonella enterica serovar Typhimurium through acquisition of a complex resistance island, flanked by regions that provide a toxin-antitoxin system and an iron uptake system. The presence of resistance and virulence determinants on the same plasmid allows coselection of both properties, potentially increasing health risks.
KeywordMeSH Terms
Evolution, Molecular
188. Chang  HW, Nam  YD, Jung  MY, Kim  KH, Roh  SW, Kim  MS, Jeon  CO, Yoon  JH, Bae  JW,     ( 2008 )

Statistical superiority of genome-probing microarrays as genomic DNA-DNA hybridization in revealing the bacterial phylogenetic relationship compared to conventional methods.

Journal of microbiological methods 75 (3)
PMID : 18782592  :   DOI  :   10.1016/j.mimet.2008.08.003    
Abstract >>
The genomic DNA-DNA hybridization (DDH) method has been widely used as a practical method for the determination of phylogenetic relationships between closely related biological strains. Traditional DDH methods have serious limitations including low reproducibility, a high background and a time-consuming procedure. The DDH method using a genome-probing microarray (GPM) has been recently developed to complement conventional methods and could be used to overcome the limitations that are typically encountered. It is necessary to compare the GPM-based DDH method to the conventional methods before using the GPM for the estimation of genomic similarities since all of the previous scientific data have been entirely dependent on conventional DDH methods. In order to address this issue we compared the DDH values obtained using the GPM, microplate and nylon membrane methods to multi-locus sequence typing (MLST) data for 9 Salmonella genomes and an Escherichia coli type strain. The results showed that the genome similarity values and the degrees of standard deviation obtained using the GPM method were lower than those obtained with the microplate and nylon membrane methods. The dendrogram from the cluster analysis of GPM DDH values was consistent with the phylogenetic tree obtained from the multi-locus sequence typing (MLST) data but was not similar to those obtained using the microplate and nylon membrane methods. Although the signal intensity had to be maximal when the targets were hybridized to their own probe, the methods using membranes and microplates frequently produced higher signals in the heterologous hybridizations than those obtained in the homologous hybridizations. Only the GPM method produced the highest signal intensity in homologous hybridizations. These results show that the GPM method can be used to obtain results that are more accurate than those generated by the other methods tested.
KeywordMeSH Terms
Phylogeny
189. Furuya  N, Nisioka  T, Komano  T,     ( 1991 )

Nucleotide sequence and functions of the oriT operon in IncI1 plasmid R64.

Journal of bacteriology 173 (7)
PMID : 1848841  :   DOI  :   10.1128/jb.173.7.2231-2237.1991     PMC  :   PMC207772    
Abstract >>
Two transfer genes of IncI1 plasmid R64, tentatively designated nikA and nikB, were cloned and sequenced. They are located adjacent to the origin of transfer (oriT) and appear to be organized into an operon, which we call the oriT operon. On the basis of the DNA sequence, nikA and nikB were concluded to encode proteins with 110 and 899 amino acid residues, respectively. Complementation analysis indicated that these two genes are indispensable for the transfer of R64 but are not required for the mobilization of ColE1. By the maxicell procedure, the product of nikA was found to be a 15-kDa protein. On treating a cleared lysate prepared from cells harboring a plasmid containing oriT, nikA, and nikB with sodium dodecyl sulfate or proteinase K, superhelical plasmid DNA in the cleared lysate was converted to an open circular form (relaxation). Relaxation of plasmid DNA was found to require the oriT sequence in cis and the nikA and nikB sequences in trans. It would thus follow that the products of nikA and nikB genes form a relaxation complex with plasmid DNA at the oriT site.
KeywordMeSH Terms
Bacterial Proteins
Conjugation, Genetic
Genes, Bacterial
Plasmids
Regulatory Sequences, Nucleic Acid
190. Hardesty  C, Ferran  C, DiRienzo  JM,     ( 1991 )

Plasmid-mediated sucrose metabolism in Escherichia coli: characterization of scrY, the structural gene for a phosphoenolpyruvate-dependent sucrose phosphotransferase system outer membrane porin.

Journal of bacteriology 173 (2)
PMID : 1846143  :   DOI  :   10.1128/jb.173.2.449-456.1991     PMC  :   PMC207032    
Abstract >>
The scrY gene, part of the pUR400-borne sucrose regulon, appeared to be transcribed from its own promoter, with the transcriptional start site located 58 bp upstream from the initiation codon. An open reading frame encoding a polypeptide of 505 amino acid residues (Mr 55,408) was identified. The first 22 amino acid residues formed a leader sequence typical of those found in other procaryotic outer membrane and periplasmic proteins. A frameshift mutation in the scrY gene resulted in a dramatic decrease in sucrose transport with no effect on in vitro phosphorylation activity associated with enzyme IISer. The rate of diffusion of sucrose was 96 times greater than the rate of diffusion of lactose or maltose in liposomes containing the ScrY protein. This increase in sucrose permeability provided strong evidence that the ScrY protein functions as a sucrose porin. There was 23% amino acid sequence identity between the ScrY protein and LamB, a maltose porin from Escherichia coli.
KeywordMeSH Terms
Genes, Bacterial
Plasmids
191. Krause  M, Harwood  J, Fierer  J, Guiney  D,     ( 1991 )

Genetic analysis of homology between the virulence plasmids of Salmonella dublin and Yersinia pseudotuberculosis.

Infection and immunity 59 (5)
PMID : 1840573  :   PMC  :   PMC257928    
Abstract >>
Two segments within the virulence region of Salmonella dublin plasmid pSDL2 that were homologous to regions on Yersinia pseudotuberculosis plasmid pIB1 were located with regard to the four known genes (vsdA, vsdB, vsdC, and vsdD) of pSDL2. One segment mapped upstream of vsdA within an insertion element related to IS630 of Shigella sonnei; the second was confined to a 45-bp sequence containing an inverted repeat between vsdC and vsdD. On pIB1, both areas were located in an intergenic region upstream of yopH. These results indicate that the homology between Yersinia and Salmonella virulence plasmids is not located within structural genes.
KeywordMeSH Terms
Plasmids
Sequence Homology, Nucleic Acid
192. Ruan  P, Xia  XP, Sun  D, Ojcius  DM, Mao  YF, Yue  WY, Yan  J,     ( 2008 )

Recombinant SpaO and H1a as immunogens for protection of mice from lethal infection with Salmonella paratyphi A: implications for rational design of typhoid fever vaccines.

Vaccine 26 (51)
PMID : 18834913  :   DOI  :   10.1016/j.vaccine.2008.09.030    
Abstract >>
The Vi capsular polysaccharide vaccine is one of two vaccines against typhoid recommended worldwide and is the vaccine generally used in China. However, in recent years a Salmonella paratyphi A strain that is naturally devoid of capsule has caused frequent outbreaks of typhoid fever in Southern China, leading to the need for identification of additional antigens that could be incorporated into new vaccines. SpaO acts as a major invasion factor of Salmonella enterica spp. and H1a is the unique flagellin subunit ofS. paratyphi A. In this study, the two prokaryotic recombinant antigens, rSpaO and rH1a, were expressed and their immunogenicity was demonstrated by the slide agglutination test and Western blot assays. Using PCR and sequencing analysis as well as ELISA, we find that the spaO and h1a genes are widely distributed in 196 S. paratyphi A isolates (97.5 and 100%, respectively), with high expression frequencies for the SpaO (98.0%) and H1a (100%) antigens. The two genes also show high sequence conservation (similarities from 99.31 to 99.88% for both genes). In sera from 172 paratyphoid A patients, anti-SpaO and anti-H1a IgGs were detectable by ELISA, in 94.8 and 98.8% of patients, respectively. Furthermore, 41.7-66.7% of mice immunized with rSpaO or rH1a alone were protected against subsequent infection, and the protection rate rose to 75.0-91.7% in mice co-immunized with the two antigens. As the spaO and h1a genes of S. paratyphi A are sequence conserved, extensively distributed and highly expressed, the rSpaO and rH1a immunogens should be considered in the development of novel vaccines to prevent S. paratyphi A-caused typhoid fever.
KeywordMeSH Terms
193. García-Fernández  A, Chiaretto  G, Bertini  A, Villa  L, Fortini  D, Ricci  A, Carattoli  A,     ( 2008 )

Multilocus sequence typing of IncI1 plasmids carrying extended-spectrum beta-lactamases in Escherichia coli and Salmonella of human and animal origin.

The Journal of antimicrobial chemotherapy 61 (6)
PMID : 18367460  :   DOI  :   10.1093/jac/dkn131    
Abstract >>
Plasmids belonging to incompatibility group I1 (IncI1) are widespread in Enterobacteriaceae and are characterized by the presence of a cluster of genes encoding the type IV pili, contributing to the virulence of Shiga-toxigenic Escherichia coli. Recently, IncI1 plasmids were identified in E. coli and Salmonella strains of animal origin as responsible for the dissemination of beta-lactamase genes. Plasmid multilocus sequence typing (pMLST) was developed to discern naturally occurring IncI1 plasmids in homogeneous groups according to their allele assortment. pMLST was developed by selecting multiple target genes on the available complete IncI1 plasmid DNA sequences. Sixteen plasmids, all assigned to the IncI1 group by the PCR-based replicon typing method, were included in this study. They were analysed for beta-lactamase genes and typed by restriction fragment length polymorphism (RFLP) and pMLST. Sixteen plasmids identified in E. coli and Salmonella isolated from animals and humans in different countries carried bla(CMY-2), bla(CTX-M-15), bla(CTX-M-1), bla(CTX-M-14), bla(TEM-52), bla(SHV-12) or bla(TEM-1) beta-lactamase genes. These plasmids were classified by RFLP in nine different groups corresponding to the nine sequence types determined by pMLST. The pMLST method was suitable for rapid and easy subtyping of IncI1 plasmids. This study demonstrates that the pMLST method can contribute to the epidemiological description of circulation of specific resistance plasmids among beta-lactamase producers isolated from animals and humans.
KeywordMeSH Terms
194. Saroj  SD, Shashidhar  R, Karani  M, Bandekar  JR,     ( 2008 )

Distribution of Salmonella pathogenicity island (SPI)-8 and SPI-10 among different serotypes of Salmonella.

Journal of medical microbiology 57 (Pt 4)
PMID : 18349359  :   DOI  :   10.1099/jmm.0.47630-0    
Abstract >>
Many virulence phenotypes of Salmonella enterica are encoded by genes located on pathogenicity islands. Based on genome analysis, it is predicted that Salmonella pathogenicity island (SPI)-8 is restricted to Salmonella serovars Typhi and Paratyphi A, and SPI-10 to Salmonella serovars Typhi, Paratyphi, Enteritidis, Dublin and Gallinarum. This study was conducted to investigate the distribution of SPI-8 and SPI-10 among Salmonella isolates from sprouts, fish, water and blood. A total of 110 Salmonella isolates and 6 Salmonella serovars from the Microbial Type Culture Collection, Chandigarh, India, were screened. All isolates belonging to Salmonella serovars Washington, Enteritidis and Paratyphi A had both SPI-8 and SPI-10. All Salmonella serovar Typhi isolates from water and blood had both SPI-8 and SPI-10, whereas isolates from fish contained only SPI-8. SPI-8 and SPI-10 were also detected in only 3 out of 42 isolates belonging to Salmonella serovar Typhimurium. Both SPI-8 and SPI-10 were absent in Salmonella serovars Worthington, Dublin, Paratyphi B and Paratyphi C. These results contradict the predictions from Salmonella genome sequences available in GenBank and indicate that SPI-8 and SPI-10 are widely distributed among Salmonella serovars and that virulence factors other than those on SPI-8 and SPI-10 may be responsible for host specificity. This is the first report on the distribution of SPIs in Salmonella isolates from India.
KeywordMeSH Terms
195. Gibbons  HS, Reynolds  CM, Guan  Z, Raetz  CR,     ( 2008 )

An inner membrane dioxygenase that generates the 2-hydroxymyristate moiety of Salmonella lipid A.

Biochemistry 47 (9)
PMID : 18254598  :   DOI  :   10.1021/bi702457c     PMC  :   PMC2709818    
Abstract >>
The lipid A residues of certain Gram-negative bacteria, including most strains of Salmonella and Pseudomonas, are esterified with one or two secondary S-2-hydroxyacyl chains. The S-2 hydroxylation process is O 2-dependent in vivo, but the relevant enzymatic pathways have not been fully characterized because in vitro assays have not been developed. We previously reported that expression of the Salmonella lpxO gene confers upon Escherichia coli K-12 the ability to synthesize 2-hydroxymyristate modified lipid A (J. Biol. Chem. (2000) 275, 32940-32949). We now demonstrate that inactivation of lpxO, which encodes a putative Fe (2+)/O 2/alpha-ketoglutarate-dependent dioxygenase, abolishes S-2-hydroxymyristate formation in S. typhimurium. Membranes of E. coli strains expressing lpxO are able to hydroxylate Kdo 2-[4'- (32)P]-lipid A in vitro in the presence of Fe (2+), O 2, alpha-ketoglutarate, ascorbate, and Triton X-100. The Fe (2+) chelator 2,2'-bipyridyl inhibits the reaction. The product generated in vitro is a monohydroxylated Kdo 2-lipid A derivative. The [4'- (32)P]-lipid A released by mild acid hydrolysis from the in vitro product migrates with authentic S-2-hydroxlyated lipid A isolated from (32)P-labeled S. typhimurium cells. Electrospray ionization mass spectrometry and gas chromatography/mass spectrometry of the in vitro product are consistent with the 2-hydroxylation of the 3'-secondary myristoyl chain of Kdo 2-lipid A. LpxO contains two predicted trans-membrane helices (one at each end of the protein), and its active site likely faces the cytoplasm. LpxO is an unusual example of an integral membrane protein that is a member of the Fe (2+)/O 2/alpha-ketoglutarate-dependent dioxygenase family.
KeywordMeSH Terms
196. Alix  E, Blanc-Potard  AB,     ( 2008 )

Peptide-assisted degradation of the Salmonella MgtC virulence factor.

The EMBO journal 27 (3)
PMID : 18200043  :   DOI  :   10.1038/sj.emboj.7601983     PMC  :   PMC2241655    
Abstract >>
MgtC is a virulence factor common to several intracellular pathogens that is required for intramacrophage survival and growth in magnesium-depleted medium. In Salmonella enterica, MgtC is coexpressed with the MgtB magnesium transporter and transcription of the mgtCB operon is induced by magnesium deprivation. Despite the high level of mgtCB transcriptional induction in magnesium-depleted medium, the MgtC protein is hardly detected in a wild-type Salmonella strain. Here, we show that downregulation of MgtC expression is dependent on a hydrophobic peptide, MgtR, which is encoded by the mgtCB operon. Our results suggest that MgtR promotes MgtC degradation by the FtsH protease, providing a negative regulatory feedback. Bacterial two-hybrid assays demonstrate that MgtR interacts with the inner-membrane MgtC protein. We identified mutant derivatives of MgtR and MgtC that prevent both regulation and interaction between the two partners. In macrophages, overexpression of the MgtR peptide led to a decrease of the replication rate of Salmonella. This study highlights the role of peptides in bacterial regulatory mechanisms and provides a natural antagonist of the MgtC virulence factor.
KeywordMeSH Terms
197. Maurice  F, Broutin  I, Podglajen  I, Benas  P, Collatz  E, Dardel  F,     ( 2008 )

Enzyme structural plasticity and the emergence of broad-spectrum antibiotic resistance.

EMBO reports 9 (4)
PMID : 18292754  :   DOI  :   10.1038/embor.2008.9     PMC  :   PMC2288771    
Abstract >>
The emergence of multi-resistant pathogenic bacteria is a worldwide health issue. Recently, clinical variants of a single antibiotic-modifying acetyltransferase, AAC(6')-Ib-a variant of aminoglycoside 6'-N-acetyltransferase-have been identified that confer extended resistance to most aminoglycosides and, more surprisingly, to structurally unrelated fluoroquinolones. The corresponding gene is carried by mobile genetic elements and is present in most multi-resistant pathogenic strains, hence making it a serious threat to current therapies. Here, we report the crystal structures of both narrow- and broad-spectrum resistance variants of this enzyme, which reveal the structural basis for the emergence of extended resistance. The active site shows an important plasticity and has adapted to new substrates by a large-scale gaping process. We have also obtained co-crystals with both substrates, and with a simple transition state analogue, which provides new clues for the design of inhibitors of this resistance mechanism.
KeywordMeSH Terms
Models, Molecular
198. Lee  KE, Lee  Y,     ( 2007 )

Isolation of multidrug-resistant Salmonella typhimurium DT104 from swine in Korea.

Journal of microbiology (Seoul, Korea) 45 (6)
PMID : 18176546  :  
Abstract >>
We report the isolation of Salmonella enterica serotype Typhimurium phage type DT104 (CCARM 8104) from swine in Korea. The CCARM 8104 isolate was resistant to nalidixic acid and showed reduced susceptibility to quinolones. The CCARM 8104 isolate had a missense mutation, Asp87Asn, in the quinolone resistance-determining region in gyrA and produced PSE-1. The CCARM 8104 isolate carried two different class 1 integrons, and the PSE-1 beta-lactamase gene was inserted into a 1,200 bp class 1 integron. The presence of DT104 with pse-1 in an integron located in a plasmid and reduced susceptibility to quinolone in swine pose a significant threat of possible horizontal spread between swine and humans.
KeywordMeSH Terms
199. Hopkins  KL, Threlfall  EJ, Karisik  E, Wardle  JK,     ( 2008 )

Identification of novel plasmid-mediated extended-spectrum beta-lactamase CTX-M-57 in Salmonella enterica serovar Typhimurium.

International journal of antimicrobial agents 31 (1)
PMID : 18054206  :   DOI  :   10.1016/j.ijantimicag.2007.08.017    
Abstract >>
N/A
KeywordMeSH Terms
Plasmids
200. Aulkemeyer  P, Ebner  R, Heilenmann  G, Jahreis  K, Schmid  K, Wrieden  S, Lengeler  JW,     ( 1991 )

Molecular analysis of two fructokinases involved in sucrose metabolism of enteric bacteria.

Molecular microbiology 5 (12)
PMID : 1809835  :   DOI  :   10.1111/j.1365-2958.1991.tb01851.x    
Abstract >>
Sucrose-positive derivatives of Escherichia coli K-12, containing the plasmid pUR400, and of Klebsiella pneumoniae hydrolyse intracellular sucrose 6-phosphate by means of an invertase into D-glucose 6-phosphate and free D-fructose. The latter is phosphorylated by an ATP-dependent fructokinase (gene scrK of an scr regulon) to D-fructose 6-phosphate. The lack of ScrK does not cause any visible phenotype in wild-type strains of both organisms. Using genes and enzymes normally involved in D-arabinitol metabolism from E. coli C and K. pneumoniae, derivatives of E. coli K-12 were constructed which allowed the identification of scrK mutations on conventional indicator plates. Cloning and sequencing of scrK from sucrose plasmid pUR400 and from the chromosome of K. pneumoniae revealed an open reading frame of 924 bp in both cases--the equivalent of a peptide containing 307 amino acid residues (Mr 39 and 34 kDa, respectively, on sodium dodecyl sulphate gels). The sequences showed overall identity among each other (69% identical residues) and to a kinase from Vibrio alginolyticus (57%) also involved in sucrose metabolism, lower overall identity (39%) to a D-ribose-kinase from E. coli, and local similarity to prokaryotic, and eukaryotic phosphofructokinases at the putative ATP-binding sites.
KeywordMeSH Terms
201. Herrero  A, Rodicio  MR, Echeita  MA, Mendoza  MC,     ( 2008 )

Salmonella enterica serotype Typhimurium carrying hybrid virulence-resistance plasmids (pUO-StVR): a new multidrug-resistant group endemic in Spain.

International journal of medical microbiology : IJMM 298 (3��4��)
PMID : 17597002  :   DOI  :   10.1016/j.ijmm.2007.04.008    
Abstract >>
The epidemiological impact in Spain of an emerging group of multidrug-resistant Salmonella enterica serotype Typhimurium, characterized by the presence of virulence-resistance hybrid plasmids (termed pUO-StVR) that are related to the S. Typhimurium virulence plasmid pSLT, was evaluated. Adscription to the group was based on detection of the bla(OXA-1) gene (encoding ampicillin resistance) by PCR, and identification of a pUO-StVR plasmid through hybridization with specific probes for virulence (spvC) and resistance (bla(OXA-1)) genes. In this way, 57 out of 134 ampicillin-resistant clinical isolates of S. Typhimurium, collected over 2002-2004 in 21 Spanish cities, were assigned to the group, which can be already regarded as endemic. Most isolates (>89%) shared the following features: (i) resistance to ampicillin, chloramphenicol, streptomycin/spectinomycin, sulfonamides, and tetracycline, encoded by bla(OXA-1)-catA1-aadA1-sul1-tet(B); (ii) a class 1 integron (InH) with the bla(OXA-1)-aadA1 gene cassettes within its variable region of ca. 2000bp; (iii) the spvC, rck, samA, oriT, traT, traX, repA (RepFIIA), and parA/B genes (but not rsk and pefABCD) of pSLT; (iv) a hybrid plasmid of ca. 125kb, termed pUO-StVR2, where the resistance and virulence genes are located. However, intra-group diversity was also detected, since a total of four resistance phenotypes, five resistance genotypes, two integron profiles, five plasmid variants (pUO-StVR2, 4-7, differing in size, restriction profile and/or resistance pattern), 15 XbaI-BlnI combined macrorestriction profiles, and five phage types were identified. Each hybrid plasmid was revealed as a distinctive BlnI band, through hybridization with pUO-StVR2. The genetic markers used, together with the knowledge generated in the present study, could be applied to epidemiological surveillance of S. Typhimurium pUO-StVR worldwide.
KeywordMeSH Terms
202. Makde  RD, Gupta  GD, Mahajan  SK, Kumar  V,     ( 2007 )

Structural and mutational analyses reveal the functional role of active-site Lys-154 and Asp-173 of Salmonella typhimurium AphA protein.

Archives of biochemistry and biophysics 464 (1)
PMID : 17570338  :   DOI  :   10.1016/j.abb.2007.03.043    
Abstract >>
The Salmonella typhimurium class B nonspecific acid phosphatase (AphA protein) belongs to the L2-haloacid dehalogenase superfamily. The conserved Lys-154 interacts with substrate phosphate, nucleophile Asp-46, and Asp-173 in the wild-type AphA protein. Asp-173 also interacts with Mg(II) water ligand and with main-chain amide of loop-4. We report here the mutational analysis of Lys-154 and Asp-173, the crystal structures of the K154N and K154R mutants, and the results of electrostatic potential calculations. The K154N, K154R and D173N mutants display significant reduction in the phosphatase activity. Lys-154 may not be responsible for a juxtaposition of the substrate phosphate and the aspartyl nucleophile, but has an hitherto unknown functional role of rendering the substrate phosphorous atom electron deficient. Nearly 10,000-fold increase in the K(d) value for dissociation of the cofactor Mg(II) observed for the D173N mutant correlates well with theoretically estimated change in the binding free energy of Mg(II).
KeywordMeSH Terms
203. Fling  ME, Kope  J, Richards  C,     ( 1988 )

Characterization of plasmid pAZ1 and the type III dihydrofolate reductase gene.

Plasmid 19 (1)
PMID : 2840679  :  
Abstract >>
The plasmid pAZ1, which determines trimethoprim and sulfonamide resistance, was characterized by restriction endonuclease mapping. The restriction map was identical to that of the incQ plasmid RSF1010 over a 5.1-kbp region. The type III dihydrofolate reductase gene was cloned, and the DNA sequence was determined. The predicted protein had 162 amino acid residues, and it was more closely related to the gram-negative bacterial chromosomal dihydrofolate reductases than to other plasmid or vertebrate dihydrofolate reductases. Sequence identity was 51% with the Escherichia coli enzyme and 44% with the Neisseria gonorrhoeae enzyme.
KeywordMeSH Terms
Genes
Genes, Bacterial
R Factors
204. Gann  AA, Campbell  AJ, Collins  JF, Coulson  AF, Murray  NE,     ( 1987 )

Reassortment of DNA recognition domains and the evolution of new specificities.

Molecular microbiology 1 (1)
PMID : 2838725  :   DOI  :   10.1111/j.1365-2958.1987.tb00521.x    
Abstract >>
Type I restriction enzymes comprise three subunits only one of which, the S polypeptide, dictates the specificity of the DNA sequence recognized. Recombination between two different hsdS genes, SP and SB, led to the isolation of a system, SQ, which had a different specificity from that of either parent. The finding that the nucleotide sequence recognized by SQ is a hybrid containing components from both the SP and SB target sequences suggested that DNA recognition is carried out by two separable domains within each specificity polypeptide. To test this we have made the recombinant gene of reciprocal structure and demonstrate that it encodes a polypeptide whose recognition sequence, deduced in vivo, is as predicted by this model. We also report the sequence of the SB specificity gene, so that information is now available for the five known members of this family of enzymes. All show a similar organization of conserved and variable regions. Comparisons of the predicted amino acid sequences reveal large non-conserved areas which may not even be structurally similar. This is remarkable since these different S subunits are functionally identical, except for the specificity with respect to the DNA sequence with which they interact. We discuss the correlation of the variation in polypeptide sequence with recognition specificities.
KeywordMeSH Terms
Biological Evolution
Genes
Genes, Viral
205. Cookson  AL, Biggs  PJ, Marshall  JC, Reynolds  A, Collis  RM, French  NP, Brightwell  G,     ( 2017 )

Culture independent analysis using gnd as a target gene to assess Escherichia coli diversity and community structure.

Scientific reports 7 (1)
PMID : 28404985  :   DOI  :   10.1038/s41598-017-00890-6     PMC  :   PMC5429811    
Abstract >>
Current culture methods to investigate changes in Escherichia coli community structure are often slow and laborious. Genes such as gnd (6-phosphogluconate dehydrogenase) have a highly variable nucleotide sequence and may provide a target for E. coli microbiome analysis using culture-independent methods. Metabarcoded PCR primers were used to generate separate libraries from calf faecal samples for high throughput sequencing. Although a total of 348 separate gnd sequence types (gSTs) were identified, 188 were likely to be due to sequencing errors. Of the remaining 160 gSTs, 92 did not match those in a database of 319 separate gnd sequences. 'Animal' was the main determinant of E. coli diversity with limited impact of sample type or DNA extraction method on intra-host E. coli community variation from faeces and recto-anal mucosal swab samples. This culture-independent study has addressed the difficulties of quantifying bacterial intra-species diversity and revealed that, whilst individual animals may harbour >50 separate E. coli strains, communities are dominated by <10 strains alongside a large pool of subdominant strains present at low abundances. This method will be useful for characterising the diversity and population structure of E. coli in experimental studies designed to assess the impact of interventions on the gut microbiome.
KeywordMeSH Terms
Gastrointestinal Microbiome
206. Ouellette  M, Roy  PH,     ( 1987 )

Homology of ORFs from Tn2603 and from R46 to site-specific recombinases.

Nucleic acids research 15 (23)
PMID : 2827107  :   DOI  :   10.1093/nar/15.23.10055     PMC  :   PMC306559    
Abstract >>
N/A
KeywordMeSH Terms
DNA Transposable Elements
207. Xu  C, Kozlov  G, Wong  K, Gehring  K, Cygler  M,     ( 2016 )

Crystal Structure of the Salmonella Typhimurium Effector GtgE.

PloS one 11 (12)
PMID : 27923041  :   DOI  :   10.1371/journal.pone.0166643     PMC  :   PMC5140068    
Abstract >>
Salmonella Typhimurium GtgE is an effector protein contributing to the virulence of this pathogen. It was shown to possess highly selective proteolytic activity against a subset of Rab proteins that helps in evasion of Salmonella-containing vacuole (SCV) fusion with lysosomes. Cys45, His151 and Asp169 are essential for proteolytic activity. The structure of a C-terminal fragment GtgE(79-214) indicated the presence of a papain-like fold. Here, we present the structure of GtgE(17-214) containing the fully assembled active site. The design of a proteolytically active and crystallizable GtgE construct was aided by NMR spectroscopy. The protein indeed displays papain-like fold with an assembled Cys-His-Asp catalytic triad. Like the full-length GtgE, the crystallizable construct showed low activity in vitro for its known substrates, Rab32 and Rab29. NMR titration experiments showed at most very weak binding of GtgE to the peptide encompassing the Rab29 cleavage site. In view of the low in vitro activity and poor substrate binding, we postulate that the function of GtgE in vivo as a proteolytic enzyme is dependent on other factor(s), such as a protein partner or interactions with the SCV membrane, which stimulate(s) GtgE activity in vivo.
KeywordMeSH Terms
208. Hall  RM, Vockler  C,     ( 1987 )

The region of the IncN plasmid R46 coding for resistance to beta-lactam antibiotics, streptomycin/spectinomycin and sulphonamides is closely related to antibiotic resistance segments found in IncW plasmids and in Tn21-like transposons.

Nucleic acids research 15 (18)
PMID : 2821509  :   DOI  :   10.1093/nar/15.18.7491     PMC  :   PMC306263    
Abstract >>
The nucleotide sequence of a 2.5 kb segment of the pKM101 (R46) genome has been determined. The 1.3 kb from a BamHI site at 153 to base 1440 differs by only 2 bases from a part of the published sequence of the aadB (gentamicin resistance) gene region including the coding region for the N-terminal 70 amino acids of the predicted aadB product. The same sequence has been found 5'-to the dhfrII gene of R388 and to the aadA gene of Tn21 (R538-1). Three open reading frames are located in this region, two on the same strand as the resistance genes and one on the complementary strand. The latter predicts a polypeptide of 337 amino acids, whose N-terminal segment is 40% homologous to the predicted product of an open reading frame of 179 amino acids located next to the dhfrI gene of Tn7. The oxa2 (oxacillin resistance) gene predicts a long polypeptide commencing with (the N-terminal) 70 amino acids of the aadB product. A similar arrangement is found in the aadA gene of R538-1. The N-terminal segment of an aadA gene is located 3'- to oxa2, separated by 36 bases. Sequences surrounding the BamHI site are identical to sequences 5'- to the tnpM gene of Tn21 and homology ceases where homology between Tn21 and Tn501 commences. The possibility that this antibiotic resistance segment is a discrete mobile DNA element is discussed.
KeywordMeSH Terms
R Factors
209. Oliva  M, Monno  R, D'Addabbo  P, Pesole  G, Dionisi  AM, Scrascia  M, Chiara  M, Horner  DS, Manzari  C, Luzzi  I, Calia  C, D'Erchia  AM, Pazzani  C,     ( 2017 )

A novel group of IncQ1 plasmids conferring multidrug resistance.

Plasmid 89 (N/A)
PMID : 27916622  :   DOI  :   10.1016/j.plasmid.2016.11.005    
Abstract >>
The IncQ is a group of non-conjugative but mobilisable plasmids that are found and stably maintained in a wide range of bacteria contributing to the spread of antimicrobial resistance genes and to the insurgence of multidrug resistant bacteria. Here we report the identification, in clinical Salmonella Typhimurium strains, of an IncQ1 plasmid (pNUC) which confers resistance to sulfamethoxazole, streptomycin and tetracycline through the presence of sul2, strAB and tetA genes, respectively. pNUC was detected in five multidrug resistant S. Typhimurium strains collected in Southern Italy from various hospitals and years of isolation. Bioinformatics analyses highlighted the presence of pNUC-like plasmids in pathogenic bacteria of various Enterobacteriaceae genera or species. Taken as a whole, these plasmids constitute a novel group of IncQ1 plasmids that might have originated through recombination events between a tetR-tetA gene cluster (possibly derived from a Tn1721) and a recipient IncQ1 plasmid related to RSF1010. Our findings raise concerns regarding the possible contribution of the newly identified group of IncQ1 plasmids to the spread of tetracycline resistance.
KeywordMeSH Terms
IncQ1
Multidrug Resistance
Plasmid
tetA
Drug Resistance, Multiple, Bacterial
210. Abraham  S, O'Dea  M, Trott  DJ, Abraham  RJ, Hughes  D, Pang  S, McKew  G, Cheong  EYL, Merlino  J, Saputra  S, Malik  R, Gottlieb  T,     ( 2016 )

Isolation and plasmid characterization of carbapenemase (IMP-4) producing Salmonella enterica Typhimurium from cats.

Scientific reports 6 (N/A)
PMID : 27767038  :   DOI  :   10.1038/srep35527     PMC  :   PMC5073282    
Abstract >>
Carbapenem-resistant Enterobacteriaceae (CRE) are a pressing public health issue due to limited therapeutic options to treat such infections. CREs have been predominantly isolated from humans and environmental samples and they are rarely reported among companion animals. In this study we report on the isolation and plasmid characterization of carbapenemase (IMP-4) producing Salmonella enterica Typhimurium from a companion animal. Carbapenemase-producing S. enterica Typhimurium carrying blaIMP-4 was identified from a systemically unwell (index) cat and three additional cats at an animal shelter. All isolates were identical and belonged to ST19. Genome sequencing revealed the acquisition of a multidrug-resistant IncHI2 plasmid (pIMP4-SEM1) that encoded resistance to nine antimicrobial classes including carbapenems and carried the blaIMP-4-qacG-aacA4-catB3 cassette array. The plasmid also encoded resistance to arsenic (MIC-150 mM). Comparative analysis revealed that the plasmid pIMP4-SEM1 showed greatest similarity to two blaIMP-8 carrying IncHI2 plasmids from Enterobacter spp. isolated from humans in China. This is the first report of CRE carrying a blaIMP-4 gene causing a clinical infection in a companion animal, with presumed nosocomial spread. This study illustrates the broader community risk entailed in escalating CRE transmission within a zoonotic species such as Salmonella, and in a cycle that encompasses humans, animals and the environment.
KeywordMeSH Terms
Bacterial Proteins
beta-Lactamases
Drug Resistance, Multiple, Bacterial
Plasmids
Salmonella typhimurium
211. Norel  F, Pisano  MR, Nicoli  J, Popoff  MY,     ( N/A )

A plasmid-borne virulence region (2.8 kb) from Salmonella typhimurium contains two open reading frames.

Research in microbiology 140 (9)
PMID : 2697048  :  
Abstract >>
N/A
KeywordMeSH Terms
212. Mani  N, Bhandari  S, Moreno  R, Hu  L, Prasad  BVV, Suguna  K,     ( 2016 )

Multiple oligomeric structures of a bacterial small heat shock protein.

Scientific reports 6 (N/A)
PMID : 27053150  :   DOI  :   10.1038/srep24019     PMC  :   PMC4823740    
Abstract >>
Small heat shock proteins are ubiquitous molecular chaperones that form the first line of defence against the detrimental effects of cellular stress. Under conditions of stress they undergo drastic conformational rearrangements in order to bind to misfolded substrate proteins and prevent cellular protein aggregation. Owing to the dynamic nature of small heat shock protein oligomers, elucidating the structural basis of chaperone action and oligomerization still remains a challenge. In order to understand the organization of sHSP oligomers, we have determined crystal structures of a small heat shock protein from Salmonella typhimurium in a dimeric form and two higher oligomeric forms: an 18-mer and a 24-mer. Though the core dimer structure is conserved in all the forms, structural heterogeneity arises due to variation in the terminal regions.
KeywordMeSH Terms
Protein Conformation
Protein Multimerization
213. Lopes  GV, Michael  GB, Cardoso  M, Schwarz  S,     ( 2016 )

Antimicrobial resistance and class 1 integron-associated gene cassettes in Salmonella enterica serovar Typhimurium isolated from pigs at slaughter and abattoir environment.

Veterinary microbiology 194 (N/A)
PMID : 27142182  :   DOI  :   10.1016/j.vetmic.2016.04.020    
Abstract >>
Forty-five multi-resistant Salmonella enterica subsp. enterica serovar (S.) Typhimurium isolates obtained at five pig abattoirs in Southern Brazil were characterized. Their relatedness was determined by XbaI-macrorestriction analysis. Resistance genes, integrons and plasmid-mediated quinolone resistance genes (PMQR) were investigated by PCR. Amplicons for the variable part of class 1 integrons and the quinolone resistance-determining regions (QRDR) were sequenced. Plasmids were characterized by conjugation assays and replicon typing. Eighteen XbaI-macrorestriction patterns and 19 plasmid profiles were seen. Resistance to ampicillin (blaTEM), chloramphenicol (catA1 and floR), streptomycin (strA-strB), streptomycin/spectinomycin (aadA variants), sulphonamides (sul1, sul2, sul3) and tetracyclines [tet(A) and tet(B)] were commonly found. A trimethoprim resistance gene, dfrA8, was identified on a 100-kb plasmid. Single substitutions in the QRDR of GyrA but no PMQR genes were found. Twenty-five isolates carried class 1 integrons with an aadA23 gene cassette or unusual class 1 integrons with a dfrA12-orfF-aadA27 gene cassette array. Both integrons were found on large conjugative plasmids. Salmonella plasmid-located virulence genes spvR, spvA, spvB, rck and pefA were found on an IncFIB resistance plasmid. Hybrid virulence-resistance plasmids or plasmids harbouring class 1 integrons may play a role in the maintenance and dissemination of antimicrobial resistance among S. Typhimurium in this pig production system.
KeywordMeSH Terms
Co-selection
Food-producing animals
Hybrid virulence-resistance plasmids
Unusual class 1 integron
Abattoirs
214. Kariuki  S, Okoro  C, Kiiru  J, Njoroge  S, Omuse  G, Langridge  G, Kingsley  RA, Dougan  G, Revathi  G,     ( 2015 )

Ceftriaxone-resistant Salmonella enterica serotype typhimurium sequence type 313 from Kenyan patients is associated with the blaCTX-M-15 gene on a novel IncHI2 plasmid.

Antimicrobial agents and chemotherapy 59 (6)
PMID : 25779570  :   DOI  :   10.1128/AAC.00078-15     PMC  :   PMC4432211    
Abstract >>
Multidrug-resistant bacteria pose a major challenge to the clinical management of infections in resource-poor settings. Although nontyphoidal Salmonella (NTS) bacteria cause predominantly enteric self-limiting illness in developed countries, NTS is responsible for a huge burden of life-threatening bloodstream infections in sub-Saharan Africa. Here, we characterized nine S. Typhimurium isolates from an outbreak involving patients who initially failed to respond to ceftriaxone treatment at a referral hospital in Kenya. These Salmonella enterica serotype Typhimurium isolates were resistant to ampicillin, chloramphenicol, cefuroxime, ceftriaxone, aztreonam, cefepime, sulfamethoxazole-trimethoprim, and cefpodoxime. Resistance to �]-lactams, including to ceftriaxone, was associated with carriage of a combination of blaCTX-M-15, blaOXA-1, and blaTEM-1 genes. The genes encoding resistance to heavy-metal ions were borne on the novel IncHI2 plasmid pKST313, which also carried a pair of class 1 integrons. All nine isolates formed a single clade within S. Typhimurium ST313, the major clone of an ongoing invasive NTS epidemic in the region. This emerging ceftriaxone-resistant clone may pose a major challenge in the management of invasive NTS in sub-Saharan Africa.
KeywordMeSH Terms
215. Gharieb  RM, Tartor  YH, Khedr  MH,     ( 2015 )

Non-Typhoidal Salmonella in poultry meat and diarrhoeic patients: prevalence, antibiogram, virulotyping, molecular detection and sequencing of class I integrons in multidrug resistant strains.

Gut pathogens 7 (N/A)
PMID : 26705426  :   DOI  :   10.1186/s13099-015-0081-1     PMC  :   PMC4690223    
Abstract >>
The worldwide increase of food-borne infections with antibiotic resistant pathogens constitutes a major public health problem. Therefore, this study aimed to determine the prevalence, antibiogram, virulence genes profiles and integron characteristics of non-typhoidal Salmonella spp. isolated from poultry meat and diarrhoeic patients in Egypt. A total of 150 samples comprising (100 poultry meat and 50 diarrhoeic patients' stool) were examined for the presence of Salmonella spp. using culture methods followed by biochemical and serological identification of the isolates. All Salmonella strains were tested for their susceptibility to the antibiotics using disk diffusion method and screened for the presence of virulence genes and class I integrons using PCR. The overall prevalence of Salmonella spp. in poultry meat samples was 10 % compared to 4 % in diarrhoeic patients. All the isolates were serologically identified into Salmonella Typhimurium (seven isolates), S. Derby, S. Kiel, S. Rubislaw (one isolate, each) and untypable strains (two isolates). Antibiotic susceptibility testing showed a higher resistance of the total isolates to erythromycin and tetracycline (100 %, each), followed by amoxicillin-clavulanic acid (91.7 %), trimethoprim-sulfamethoxazole (83.3 %), streptomycin, nalidixic acid, ampicillin-sulbactam (75 %, each), gentamycin, ampicillin (66.7 %, each), chloramphenicol (58.3 %), ciprofloxacin (25 %) and ceftriaxone (16.7 %). Virulence genes profiles revealed the presence of sopB gene in five Salmonella strains isolated from poultry meat (n = 3) and humans (n = 2). Moreover, pefA was only identified in three isolates from poultry meat. On the other hand, S. Kiel and S. Typhimurium (one isolate, each) were harboring hilA and stn genes, respectively. Class 1 integrons were detected in all Salmonella spp. with variable amplicon sizes ranged from 650-3000 bp. Sequencing of these amplicons revealed the presence of gene cassettes harboring aac(3)-Id, aadA2, aadA4, aadA7, sat, dfrA15, lnuF and estX resistance genes. Nucleotide sequence analysis showed point mutations in the aac(3)-Id of S. Derby, aadA2, estX-sat genes of S. Typhimurium. Meanwhile, frame shift mutation was observed in aadA7 genes of S. Typhimurium. Increasing rate of antimicrobial resistance and class 1 integrons among multidrug resistant Salmonella spp. has prompted calls for the reduction of antimicrobial use in livestock to prevent future emergence of resistance.
KeywordMeSH Terms
Class I integrons
Salmonella spp.
Virulence
Zoonoses, multidrug resistance
216. Wei  W, Sun  Y, Zhu  M, Liu  X, Sun  P, Wang  F, Gui  Q, Meng  W, Cao  Y, Zhao  J,     ( 2015 )

Structural Insights and the Surprisingly Low Mechanical Stability of the Au-S Bond in the Gold-Specific Protein GolB.

Journal of the American Chemical Society 137 (49)
PMID : 26636614  :   DOI  :   10.1021/jacs.5b09895    
Abstract >>
The coordination bond between gold and sulfur (Au-S) has been widely studied and utilized in many fields. However, detailed investigations on the basic nature of this bond are still lacking. A gold-specific binding protein, GolB, was recently identified, providing a unique opportunity for the study of the Au-S bond at the molecular level. We probed the mechanical strength of the gold-sulfur bond in GolB using single-molecule force spectroscopy. We measured the rupture force of the Au-S bond to be 165 pN, much lower than Au-S bonds measured on different gold surfaces (?1000 pN). We further solved the structures of apo-GolB and Au(I)-GolB complex using X-ray crystallography. These structures showed that the average Au-S bond length in GolB is much longer than the reported average value of Au-S bonds. Our results highlight the dramatic influence of the unique biological environment on the stability and strength of metal coordination bonds in proteins.
KeywordMeSH Terms
Models, Molecular
217. Nücken  EJ, Henschke  RB, Schmidt  FR,     ( 1989 )

Nucleotide sequence of an OXA-2 beta-lactamase gene from the R-plasmid R1767 derived plasmid pBP11 and comparison to closely related resistance determinants found in R46 and Tn2603.

Journal of general microbiology 135 (4)
PMID : 2689593  :   DOI  :   10.1099/00221287-135-4-761    
Abstract >>
Plasmid pBP11 contains a sequence homologous to Tn21-like element Tn2410 encoding dihydropteroate synthetase and beta-lactamase OXA-2. The nucleotide sequence of a 1.5 kb segment of this region has been determined including the bla gene. It reveals strong sequence homology with the OXA-2 operon of plasmid R46. The implications of an additional 319 bp segment in pBP11 for the different evolution of R46/pKM101 and pBP11 are discussed.
KeywordMeSH Terms
Genes, Bacterial
218. Inoue  YH, Kutsukake  K, Iino  T, Yamaguchi  S,     ( 1989 )

Sequence analysis of operator mutants of the phase-1 flagellin-encoding gene, fliC, in Salmonella typhimurium.

Gene 85 (1)
PMID : 2695399  :   DOI  :   10.1016/0378-1119(89)90485-x    
Abstract >>
In phase-2 cells of diphasic Salmonella strains, expression of the phase-1 flagellin-encoding gene, fliC, is repressed by the repressor encoded by the fljA gene. Nine operator-constitutive (Oc) mutants of fliC were isolated from S. typhimurium by selecting those which could express fliC in the presence of the repressor. Among them, eight mutants could express fliC both in the presence and the absence of the repressor, whereas the ninth one could express only in the presence of the repressor. Nucleotide sequence analysis revealed that the Oc mutations of the former type were all located between bp 7 and 20 upstream from the coding region of fliC, which suggests that this region may correspond to the operator for fliC. The latter mutant was found to have a tandem duplication of 28 bp which contains a part of the operator sequence, and seems to require the repressor to activate fliC expression.
KeywordMeSH Terms
Genes, Bacterial
Mutation
Operon
219. Taira  S, Rhen  M,     ( 1989 )

Identification and genetic analysis of mkaA--a gene of the Salmonella typhimurium virulence plasmid necessary for intracellular growth.

Microbial pathogenesis 7 (3)
PMID : 2693884  :  
Abstract >>
Salmonella typhimurium, like many other Salmonella serovars, harbours a large plasmid required for mouse virulence and growth of bacteria in host cells. The nature of one virulence-abolishing Tn5 insertion (zzx-2556::Tn5) in the plasmid was characterized. A 3.7 kb insert harbouring this region was cloned in pBR325. Plasmid-directed protein synthesis in minicells indicated that the transposon had eliminated the expression of a 70 kDa protein encoded by the virulence plasmid. Nucleotide sequence analysis of the corresponding wild type DNA region showed a 1773 bp open reading frame (mkaA), encoding a protein of 591 amino acid residues and a predicted molecular mass of 60.6 kDa; zzx-2556::Tn5 was situated within mkaA.
KeywordMeSH Terms
Genes, Bacterial
220. Taira  S, Rhen  M,     ( 1989 )

Molecular organization of genes constituting the virulence determinant on the Salmonella typhimurium 96 kilobase pair plasmid.

FEBS letters 257 (2)
PMID : 2684688  :   DOI  :   10.1016/0014-5793(89)81551-0    
Abstract >>
The ability of intracellular growth is plasmid-dependent in Salmonella typhimurium. Only a small portion of this 96 kilobase pair plasmid appears essential for intracellular growth. The genetic organization of this region (the essential virulence determinant) was resolved. Fragments of the virulence determinant were cloned from the 96-kb plasmid pEX102 and transformed into minicell-producing E. coli. Plasmid-directed protein synthesis was investigated in metabolically labeled minicells. This analysis indicated the presence of at least four genes, mkaA, mkaB, mkaC and mkaD, within the virulence determinant encoding proteins of 70, 31, 30 and 29 kDa, respectively. The genes were positioned on the restriction map of the 96-kb virulence plasmid and the map locations confirmed by nucleotide sequence analysis of two new virulence genes (mkaB and mkaC).
KeywordMeSH Terms
Genes, Bacterial
Plasmids
Transcription Factors
221. Calva  E, Silva  C, Zaidi  MB, Sanchez-Flores  A, Estrada  K, Silva  GG, Soto-Jiménez  LM, Wiesner  M, Fernández-Mora  M, Edwards  RA, Vinuesa  P,     ( 2015 )

Complete Genome Sequencing of a Multidrug-Resistant and Human-Invasive Salmonella enterica Serovar Typhimurium Strain of the Emerging Sequence Type 213 Genotype.

Genome announcements 3 (3)
PMID : 26089426  :   DOI  :   10.1128/genomeA.00663-15     PMC  :   PMC4472903    
Abstract >>
Salmonella enterica subsp. enterica serovar Typhimurium strain YU39 was isolated in 2005 in the state of Yucat?n, Mexico, from a human systemic infection. The YU39 strain is representative of the multidrug-resistant emergent sequence type 213 (ST213) genotype. The YU39 complete genome is composed of a chromosome and seven plasmids.
KeywordMeSH Terms
222. McWhorter  AR, Chousalkar  KK,     ( 2015 )

Comparative phenotypic and genotypic virulence of Salmonella strains isolated from Australian layer farms.

Frontiers in microbiology 6 (N/A)
PMID : 25667583  :   DOI  :   10.3389/fmicb.2015.00012     PMC  :   PMC4304256    
Abstract >>
There are over 2500 Salmonella enterica serovars that circulate globally. Of these, serovars those classified into subspecies I are the most common cause of human salmonellosis. Many subspecies I Salmonella serovars are routinely isolated from egg farm environments but are not frequently associated with causing disease in humans. In this study, virulence profiles were generated for 10 strains of Salmonella enterica isolated directly from egg farm environments to investigate their potential public health risk. Three virulence parameters were assessed including in vitro invasion, in vivo pathogenicity and characterization of genomic variation within five specific pathogenicity islands. These 10 Salmonella strains exhibited significant differences in invasion into the human intestinal epithelial cell line, Caco2. Low, moderate, and high invasion patterns were observed and the degree of invasion was dependent on bacterial growth in a nutritive environment. Interestingly, two Salmonella strains, S. Adelaide and S. Bredeney had consistently low invasion. The S. Typhimurium definitive types and S. Virchow exhibited the greatest cell invasion following growth in Luria Bertani broth. Only the S. Typhimurium strains caused disease in BALB/c mice, yet the majority of serovars were consistently detected in feces over the 21 day experiment. Genomic comparison of the five specific pathogenicity islands has shown that variation in virulence is likely multifactorial. Sequence variability was observed primarily in strains with low virulence. In particular, genes involved in forming the structures of the SPI-1 and SPI-2 type 3 secretion systems as well as multiple effector proteins were among the most variable. This variability suggest that serovars with low virulence are likely to have both invasion and within host replication defects that ultimately limit their pathogenicity.
KeywordMeSH Terms
BALB/c mice
Caco2
Salmonella
Salmonella pathogenicity islands
cell invasion
eggs
223. Octavia  S, Sara  J, Lan  R,     ( 2015 )

Characterization of a large novel phage-like plasmid in Salmonella enterica serovar Typhimurium.

FEMS microbiology letters 362 (8)
PMID : 25795590  :   DOI  :   10.1093/femsle/fnv044    
Abstract >>
Salmonella enterica serovar Typhimurium is a food-borne pathogen and a leading cause of gastroenteritis in humans. Recently, we sequenced a phage-type DT108 strain (L945) and found reads with high similarity to both Salmonella typhi strain CT18 plasmid pHCM2 and bacteriophage SSU5. In this study, we completely sequenced the novel phage-like plasmid which was designated as pSTM_�X. The presence of this phage-like plasmid was examined in a collection of 284 Salmonella Typhimurium isolates using PCR of the parB gene and only one other isolate (L946) was found to carry the phage-like plasmid suggesting that it is infrequently present amongst Salmonella Typhimurium isolates. pSTM_�X is a circular phage-like plasmid of 107.7 kb encoding 132 coding regions (ORFs) with the majority of the ORFs encoding hypothetical proteins. Comparative analysis with other closely related phage-like plasmids and the SSU5 phage revealed that there were four divergent lineages of phage-like plasmids found in the family of Enterobacteriaceae. In conclusion, pSTM_�X is a new member of an emerging family of phage-like plasmids.
KeywordMeSH Terms
Salmonella Typhimurium
phage-like elements
phage-type DT108
plasmid
Plasmids
Salmonella Phages
224. Kubasova  T, Matiasovicova  J, Rychlik  I, Juricova  H,     ( 2014 )

Complete sequence of multidrug resistance p9134 plasmid and its variants including natural recombinant with the virulence plasmid of Salmonella serovar Typhimurium.

Plasmid 76 (N/A)
PMID : 25195837  :   DOI  :   10.1016/j.plasmid.2014.08.003    
Abstract >>
In this study we determined the complete nucleotide sequence of multidrug-resistance plasmid p9134, and its variants p9134dT and p9134dAT which spontaneously lost either tetracycline or both tetracycline and ampicillin resistance, respectively. The plasmids were 133,802 bp, 109,512 bp and 127,291 bp in size, respectively, and their basic backbone was similar to that of IncI plasmids. Genes coding for ampicillin (blaTEM), chloramphenicol (catA1), streptomycin (strA, strB), tetracycline (tetA(A)) and gentamicin (aac(3)-IV) resistance were confirmed in wild-type p9134. Moreover, a gene for hygromycine resistance (hph) and a putative gene for apramycin resistance were newly determined. In p9134dAT, a continuous sequence coding for ampicillin and tetracycline resistances was lost. Genetic rearrangements in p9134dT were more complex and 2 recombination events must have occurred. During the first one, the tetracycline resistance locus was replaced with rck, srgB, srgA, orf7 and pefI originating from Salmonella virulence plasmid pSLT. During the second one, ydjA, pifA and repC genes from p9134 were replaced with repA2, PSLT025 and PSLT026 genes from pSLT. Our findings indicate that recombination event between unrelated plasmids might be quite common and may lead to the generation and selection of plasmids both transferring antibiotic resistance and increasing virulence of their host.
KeywordMeSH Terms
Antibiotic resistance
Conjugation
Plasmid sequence
Recombination
Salmonella
225. Herrero-Fresno  A, Rodicio  R, Montero  I, García  P, Rodicio  MR,     ( 2015 )

Transposition and homologous recombination drive evolution of pUO-StVR2, a multidrug resistance derivative of pSLT, the virulence plasmid specific of Salmonella enterica serovar Typhimurium.

Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases 29 (N/A)
PMID : 25461846  :   DOI  :   10.1016/j.meegid.2014.11.010    
Abstract >>
Five variants of a resistant derivative of pSLT (termed pUO-StVR2) were detected in clinical isolates of Salmonella enterica serovar Typhimurium recovered in Spain. The structure of these variants revealed the involvement of IS1, IS26 and Tn21-like transposition, as well as homologous recombination in the generation of deletions, inversions and insertions which, depending on the variant, affected an orf of unknown function, genes encoding a possible iron acquisition system, and/or resistance properties. These variants, which appeared at a relatively low frequency, can be used as a model to understand the co-selection mechanisms that are helping to maintain multidrug resistance in bacterial pathogens, despite the structural instability of the responsible DNA.
KeywordMeSH Terms
Class 1 integron
Complex integron
Insertion sequence
Iron acquisition
Multidrug resistance
Transposon
Evolution, Molecular
226. Mossakowska  D, Ali  NA, Dale  JW,     ( 1989 )

Oxacillin-hydrolysing beta-lactamases. A comparative analysis at nucleotide and amino acid sequence levels.

European journal of biochemistry 180 (2)
PMID : 2538329  :   DOI  :   10.1111/j.1432-1033.1989.tb14649.x    
Abstract >>
We have extended the sequence of the OXA-2 beta-lactamase which together with S1 mapping has enabled us to identify the promoter site for this gene. This lies in a region that is found upstream from a variety of resistance genes on different plasmids; each gene appears to have been inserted at the same specific site and to be expressed from the same promoter. The ancestral plasmid thus appears to function as a natural expression vector. The sequence of the recombination site at the 5' end of the OXA-2 gene shows a marked similarity with the attP sequence of lambda. DNA-probe analysis confirmed that the OXA-2 and OXA-3 beta-lactamases are related, and indicated no similarity with other beta-lactamase genes. However, a comparison of amino acid sequences demonstrates that the OXA-2, OXA-1 and PSE-2 beta-lactamases show some similarities to the typical class A enzymes, especially in the central helical domain of the latter, which is largely responsible for forming the active site of the enzyme. The three oxacillinases also show marked amino acid sequence similarity with the product of a regulatory gene, blaR1, required for beta-lactamase induction in Bacillus licheniformis.
KeywordMeSH Terms
Genes
Genes, Bacterial
Promoter Regions, Genetic
227. Carattoli  A, Seiffert  SN, Schwendener  S, Perreten  V, Endimiani  A,     ( 2015 )

Differentiation of IncL and IncM Plasmids Associated with the Spread of Clinically Relevant Antimicrobial Resistance.

PloS one 10 (5)
PMID : 25933288  :   DOI  :   10.1371/journal.pone.0123063     PMC  :   PMC4416936    
Abstract >>
blaOXA-48, blaNDM-1 and blaCTX-M-3 are clinically relevant resistance genes, frequently associated with the broad-host range plasmids of the IncL/M group. The L and M plasmids belong to two compatible groups, which were incorrectly classified together by molecular methods. In order to understand their evolution, we fully sequenced four IncL/M plasmids, including the reference plasmids R471 and R69, the recently described blaOXA-48-carrying plasmid pKPN-El.Nr7 from a Klebsiella pneumoniae isolated in Bern (Switzerland), and the blaSHV-5 carrying plasmid p202c from a Salmonella enterica from Tirana (Albania). Sequencing was performed using 454 Junior Genome Sequencer (Roche). Annotation was performed using Sequin and Artemis software. Plasmid sequences were compared with 13 fully sequenced plasmids belonging to the IncL/M group available in GenBank. Comparative analysis of plasmid genomes revealed two distinct genetic lineages, each containing one of the R471 (IncL) and R69 (IncM) reference plasmids. Conjugation experiments demonstrated that plasmids representative of the IncL and IncM groups were compatible with each other. The IncL group is constituted by the blaOXA-48-carrying plasmids and R471. The IncM group contains two sub-types of plasmids named IncM1 and IncM2 that are each incompatible. This work re-defines the structure of the IncL and IncM families and ascribes a definitive designation to the fully sequenced IncL/M plasmids available in GenBank.
KeywordMeSH Terms
228. Wong  MH, Chan  EW, Liu  LZ, Chen  S,     ( 2014 )

PMQR genes oqxAB and aac(6')Ib-cr accelerate the development of fluoroquinolone resistance in Salmonella typhimurium.

Frontiers in microbiology 5 (N/A)
PMID : 25324840  :   DOI  :   10.3389/fmicb.2014.00521     PMC  :   PMC4183184    
Abstract >>
Emergence of multidrug-resistant Salmonella typhimurium strains, especially the ACSSuT and nalidixic acid R types, has significantly compromised the effectiveness of current strategies to control Salmonella infections, resulting in increased morbidity and mortality. Clinical S. typhimurium isolates recovered in Hong Kong during the period of 2005-2011 were increasingly resistant to ciprofloxacin (CIP) and antibiotics of the ACSSuT group. Our data revealed that oqxAB and aac(6')Ib-cr were encoded on plasmids of various sizes and the presence of these two elements together with a single gyrA mutation in S. typhimurium were sufficient to mediate resistance to CIP. Acquisition of the oqxAB and aac(6')Ib-cr encoding plasmids by S. typhimurium caused a fourfold increase in CIP minimal inhibitory concentration. Furthermore, the presence of oqxAB and aac(6')Ib-cr in Salmonella dramatically increased the mutation prevention concentration of CIP which may due to mutational changes in the drug target genes. In conclusion, possession of oqxAB and aac(6')Ib-cr encoding plasmid facilitate the selection of CIP resistant S. typhimurium, thereby causing a remarkable increase of CIP resistance among clinical Salmonella strains in Hong Kong.
KeywordMeSH Terms
ACSSuT R type
S. typhimurium
aac(6′)Ib-cr
ciprofloxacin resistance
oqxAB
229. Hiley  L, Fang  NX, Micalizzi  GR, Bates  J,     ( 2014 )

Distribution of Gifsy-3 and of variants of ST64B and Gifsy-1 prophages amongst Salmonella enterica Serovar Typhimurium isolates: evidence that combinations of prophages promote clonality.

PloS one 9 (1)
PMID : 24475087  :   DOI  :   10.1371/journal.pone.0086203     PMC  :   PMC3901673    
Abstract >>
Salmonella isolates harbour a range of resident prophages which can influence their virulence and ability to compete and survive in their environment. Phage gene profiling of a range of phage types of Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium) indicates a significant level of correlation of phage gene profile with phage type as well as correlation with genotypes determined by a combination of multi-locus variable-number tandem repeat (VNTR) typing and clustered regularly interspaced short palindromic repeats (CRISPR) typing. Variation in phage gene profiles appears to be partly linked to differences in composition of variants of known prophages. We therefore conducted a study of the distribution of variants of ST64B and Gifsy-1 prophages and coincidently the presence of Gifsy-3 prophage in a range of S. Typhimurium phage types and genotypes. We have discovered two variants of the DT104 variant of ST64B and at least two new variants of Gifsy-1 as well as variants of related phage genes. While there is definite correlation between phage type and the prophage profile based on ST64B and Gifsy-1 variants we find stronger correlation between the VNTR/CRISPR genotype and prophage profile. Further differentiation of some genotypes is obtained by addition of the distribution of Gifsy-3 and a sequence variant of the substituted SB26 gene from the DT104 variant of ST64B. To explain the correlation between genotype and prophage profile we propose that suites of resident prophages promote clonality possibly through superinfection exclusion systems.
KeywordMeSH Terms
230. Wiesner  M, Fernández-Mora  M, Cevallos  MA, Zavala-Alvarado  C, Zaidi  MB, Calva  E, Silva  C,     ( BMC microbiology )


Conjugative transfer of an IncA/C plasmid-borne blaCMY-2 gene through genetic re-arrangements with an IncX1 plasmid. 2013 (13)
PMID : 24262067  :   DOI  :   10.1186/1471-2180-13-264     PMC  :   PMC4222815    
Abstract >>
Our observation that in the Mexican Salmonella Typhimurium population none of the ST19 and ST213 strains harbored both the Salmonella virulence plasmid (pSTV) and the prevalent IncA/C plasmid (pA/C) led us to hypothesize that restriction to horizontal transfer of these plasmids existed. We designed a conjugation scheme using ST213 strain YU39 as donor of the blaCMY-2 gene (conferring resistance to ceftriaxone; CRO) carried by pA/C, and two E. coli lab strains (DH5�\ and HB101) and two Typhimurium ST19 strains (SO1 and LT2) carrying pSTV as recipients. The aim of this study was to determine if the genetic background of the different recipient strains affected the transfer frequencies of pA/C. YU39 was able to transfer CRO resistance, via a novel conjugative mechanism, to all the recipient strains although at low frequencies (10-7 to 10-10). The presence of pSTV in the recipients had little effect on the conjugation frequency. The analysis of the transconjugants showed that three different phenomena were occurring associated to the transfer of blaCMY-2: 1) the co-integration of pA/C and pX1; 2) the transposition of the CMY region from pA/C to pX1; or 3) the rearrangement of pA/C. In addition, the co-lateral mobilization of a small (5 kb) ColE1-like plasmid was observed. The transconjugant plasmids involving pX1 re-arrangements (either via co-integration or ISEcp1-mediated transposition) obtained the capacity to conjugate at very high levels, similar to those found for pX1 (10-1). Two versions of the region containing blaCMY-2 were found to transpose to pX1: the large version was inserted into an intergenic region located where the genetic load" operons are frequently inserted into pX1, while the short version was inserted into the stbDE operon involved in plasmid addiction system. This is the first study to report the acquisition of an extended spectrum cephalosporin (ESC)-resistance gene by an IncX1 plasmid.
KeywordMeSH Terms
Conjugation, Genetic
Gene Transfer, Horizontal
Plasmids
231. Hawkey  J, Edwards  DJ, Dimovski  K, Hiley  L, Billman-Jacobe  H, Hogg  G, Holt  KE,     ( 2013 )

Evidence of microevolution of Salmonella Typhimurium during a series of egg-associated outbreaks linked to a single chicken farm.

BMC genomics 14 (N/A)
PMID : 24245509  :   DOI  :   10.1186/1471-2164-14-800     PMC  :   PMC3870983    
Abstract >>
The bacterium Salmonella enterica serovar Typhimurium (S. Typhimurium) is one of the most frequent causes of foodborne outbreaks of gastroenteritis. Between 2005-2008 a series of S. Typhimurium outbreaks occurred in Tasmania, Australia, that were all traced to eggs originating from a single chicken farm. We sequenced the genomes of 12 isolates linked to these outbreaks, in order to investigate the microevolution of a pathogenic S. Typhimurium clone in a natural, spatiotemporally restricted population. The isolates, which shared a phage type similar to DT135 known locally as 135@ or 135a, formed a clade within the S. Typhimurium population with close similarity to the reference genome SL1334 (160 single nucleotide polymorphisms, or SNPs). Ten of the isolates belonged to a single clone (<23 SNPs between isolate pairs) which likely represents the population of S. Typhimurium circulating at the chicken farm; the other two were from sporadic cases and were genetically distinct from this clone. Divergence dating indicated that all 12 isolates diverged from a common ancestor in the mid 1990 s, and the clone began to diversify in 2003-2004. This clone spilled out into the human population several times between 2005-2008, during which time it continued to accumulate SNPs at a constant rate of 3-5 SNPs per year or 1x10-6 substitutions site-1 year-1, faster than the longer-term (~50 year) rates estimated previously for S. Typhimurium. Our data suggest that roughly half of non-synonymous substitutions are rapidly removed from the S. Typhimurium population, after which purifying selection is no longer important and the remaining substitutions become fixed in the population. The S. Typhimurium 135@ isolates were nearly identical to SL1344 in terms of gene content and virulence plasmids. Their phage contents were close to SL1344, except that they carried a different variant of Gifsy-1, lacked the P2 remnant found in SL1344 and carried a novel P2 phage, P2-Hawk, in place SL1344's P2 phage SopE?. DT135 lacks P2 prophage. Two additional plasmids were identified in the S. Typhimurium 135@ isolates, pSTM2 and pSTM7. Both plasmids were IncI1, but phylogenetic analysis of the plasmids and their bacterial hosts shows these plasmids are genetically distinct and result from independent plasmid acquisition events. This study provides a high-resolution insight into short-term microevolution of the important human pathogen S. Typhimurium. It indicates that purifying selection occurs rapidly in this population (? 6 years) and then declines, and provides an estimate for the short-term substitution rate. The latter is likely to be more relevant for foodborne outbreak investigation than previous estimates based on longer time scales.
KeywordMeSH Terms
Evolution, Molecular
232. Woegerbauer  M, Zeinzinger  J, Springer  B, Hufnagl  P, Indra  A, Korschineck  I, Hofrichter  J, Kopacka  I, Fuchs  R, Steinwider  J, Fuchs  K, Nielsen  KM, Allerberger  F,     ( 2014 )

Prevalence of the aminoglycoside phosphotransferase genes aph(3')-IIIa and aph(3')-IIa in Escherichia coli, Enterococcus faecalis, Enterococcus faecium, Pseudomonas aeruginosa, Salmonella enterica subsp. enterica and Staphylococcus aureus isolates in Austria.

Journal of medical microbiology 63 (Pt 2)
PMID : 24194558  :   DOI  :   10.1099/jmm.0.065789-0    
Abstract >>
The aminoglycoside phosphotransferase aph(3')-IIa primarily inactivates kanamycin and neomycin, whilst aph(3')-IIIa also inactivates amikacin. The aim of this study was to determine the frequency of both resistance genes in major human pathogens to obtain their baseline prevalence in the gene pool of these bacterial populations in Austria. In total, 10 541 Escherichia coli, Enterococcus faecalis, Enterococcus faecium, Pseudomonas aeruginosa, Salmonella enterica subsp. enterica and Staphylococcus aureus isolates were collected representatively without selection bias between 2008 and 2011. Isolates were analysed by aph(3')-IIIa/nptIII- and aph(3')-IIa/nptII-specific TaqMan real-time PCR. For positive strains, MICs using Etests were performed and resistance gene sequences were determined. The overall prevalence of aph(3')-IIIa/nptIII was 1.62 % (95 % confidence interval: 1.38-1.88 %). In Escherichia coli, enterococci, Staphylococcus aureus, P. aeruginosa and Salmonella spp., the aph(3')-IIIa/nptIII prevalence was 0.47 % (0-1.47 %), 37.53 % (32.84-42.40 %), 2.90 % (1.51-5.02 %), 0 % (0-0.32 %) and 0 % (0-0.037 %), respectively. Eleven of a total of 169 carriers showed single-nucleotide polymorphisms in the resistance allele. The overall prevalence of aph(3')-IIa/nptII was 0.0096 % (0-0.046 %). Escherichia coli (0-0.70 %), enterococci (0-0.75 %), Staphylococcus aureus (0-0.73 %) and P. aeruginosa (0-0.32 %) did not carry aph(3')-IIa. A single Salmonella isolate was positive, resulting in an aph(3')-IIa prevalence of 0.013 % (0-0.058 %). aph(3')-IIIa/nptIII carriers were moderately prevalent in the strains tested except for in enterococci, which appeared to be an important reservoir for aph(3')-IIIa. aph(3')-IIa/nptII genes were detected at clinically irrelevant frequencies and played no significant role in the aminoglycoside resistance gene pool during the observation period.
KeywordMeSH Terms
Drug Resistance, Bacterial
233. Kozyreva  VK, Ilina  EN, Malakhova  MV, Carattoli  A, Azizov  IS, Tapalski  DV, Kozlov  RS, Edelstein  MV,     ( 2014 )

Long-term dissemination of CTX-M-5-producing hypermutable Salmonella enterica serovar typhimurium sequence type 328 strains in Russia, Belarus, and Kazakhstan.

Antimicrobial agents and chemotherapy 58 (9)
PMID : 24957829  :   DOI  :   10.1128/AAC.02506-14     PMC  :   PMC4135874    
Abstract >>
In this paper, we present evidence of long-term circulation of cefotaxime-resistant clonally related Salmonella enterica serovar Typhimurium strains over a broad geographic area. The genetic relatedness of 88 isolates collected from multiple outbreaks and sporadic cases of nosocomial salmonellosis in various parts of Russia, Belarus, and Kazakhstan from 1996 to 2009 was established by multilocus tandem-repeat analysis (MLVA) and multilocus sequence typing (MLST). The isolates belong to sequence type 328 (ST328) and produce CTX-M-5 �]-lactamase, whose gene is carried by highly related non-self-conjugative but mobilizable plasmids. Resistance to nalidixic acid and low-level resistance to ciprofloxacin is present in 37 (42%) of the isolates and in all cases is determined by various single point mutations in the gyrA gene quinolone resistance-determining region (QRDR). Isolates of the described clonal group exhibit a hypermutable phenotype that probably facilitates independent acquisition of quinolone resistance mutations.
KeywordMeSH Terms
234. Schnierow  BJ, Yamada  M, Saier  MH,     ( 1989 )

Partial nucleotide sequence of the pts operon in Salmonella typhimurium: comparative analyses in five bacterial genera.

Molecular microbiology 3 (1)
PMID : 2497295  :   DOI  :   10.1111/j.1365-2958.1989.tb00110.x    
Abstract >>
The nucleotide sequence of a Salmonella typhimurium DNA segment of 549 base pairs which encompasses the operator-promoter of the pts operon, the entirety of the ptsH gene, encoding HPr of the phosphotransferase system (PTS), the first 29 nucleotides of the ptsI gene, encoding Enzyme I of the PTS, and the intercistronic region between the ptsH and ptsI genes was determined and compared with the corresponding sequence from Escherichia coli (De Reuse et al., 1985). The two sequences showed 91% overall identity, with some regions showing sequence conservation and others exhibiting relative divergence. Two open reading frames were identified in both species: one encoded HPr on the 'sense' strand (255 nucleotides; 12 nucleotide differences, no amino acid differences); the other, on the anti-sense strand, consisted of 291 nucleotides (13 nucleotide differences, 13 amino acid differences). While HPr bears a net negative charge, the putative protein encoded by the open reading frame on the anti-sense strand is strongly basic. Computer analyses of HPr proteins from five different bacterial genera revealed four regions which show strong sequence identity and therefore are presumed to be critical for maintenance of biological activity. Two of these regions were specific to Gram-positive bacteria. Proposed functions for each of these regions are discussed. Relative evolutionary distances between the HPr proteins were also computed.
KeywordMeSH Terms
Bacterial Proteins
Base Sequence
Genes, Bacterial
Operon
Sequence Homology, Nucleic Acid
235. Tamamura  Y, Tanaka  K, Akiba  M, Kanno  T, Hatama  S, Ishihara  R, Uchida  I,     ( 2013 )

Complete nucleotide sequences of virulence-resistance plasmids carried by emerging multidrug-resistant Salmonella enterica Serovar Typhimurium isolated from cattle in Hokkaido, Japan.

PloS one 8 (10)
PMID : 24155970  :   DOI  :   10.1371/journal.pone.0077644     PMC  :   PMC3796477    
Abstract >>
In the present study, we have shown that virulence-resistance plasmids from emerging multidrug-resistant isolates of Salmonella enterica serovar Typhimurium were derived from a virulence-associated plasmid, essential for systematic invasiveness of S. Typhimurium in mice (pSLT), through acquisition of a large insert containing a resistance island flanked by IS1294 elements. A bla CMY-2-carrying plasmid from a cefotaxime-resistant isolate comprised a segment of Escherichia coli plasmid pAR060302 and the replication region (IncFIB) of a virulence-resistance plasmid. These results provide insights into the evolution of drug resistance in emerging clones of S. Typhimurium.
KeywordMeSH Terms
236. Shiroda  M, Pratt  ZL, Döpfer  D, Wong  AC, Kaspar  CW,     ( 2014 )

RpoS impacts the lag phase of Salmonella enterica during osmotic stress.

FEMS microbiology letters 357 (2)
PMID : 24985365  :   DOI  :   10.1111/1574-6968.12523    
Abstract >>
Salmonella enterica can survive harsh environmental conditions, including hyperosmotic stress. It is well established that the alternative sigma factor, �m(s) (RpoS), is required for maximal survival of enteric pathogens, including S. enterica. Although RpoS levels are greatest during stationary phase or stress conditions, RpoS can be found in S. enterica during growth. However, its activity during growth is poorly characterized. In this study, the impact of RpoS levels on the growth of S. enterica in LB supplemented with 6% NaCl (LB-NaCl) was examined. Cells in stationary phase prior to inoculation into LB-NaCl had a shorter lag phase than did exponential-phase cells. In addition, the deletion of rpoS from S. enterica Typhimurium M-09 (M-09 �GrpoS) increased the length of lag phase in LB-NaCl relative to the parental strain. Complementation of M-09 �GrpoS in trans by an inducible plasmid encoding rpoS reduced the length of lag phase. The length of lag phase in both the rpoS mutant and complemented strain was independent of their growth phase prior to inoculation of LB-NaCl. The results from this study demonstrate that the level of RpoS influences the length of lag phase and the growth of S. enterica in hyperosmotic growth conditions.
KeywordMeSH Terms
RpoS
Salmonella
desiccation
lag phase
osmotic stress
stress response
Osmotic Pressure
237. Shariat  N, Sandt  CH, DiMarzio  MJ, Barrangou  R, Dudley  EG,     ( 2013 )

CRISPR-MVLST subtyping of Salmonella enterica subsp. enterica serovars Typhimurium and Heidelberg and application in identifying outbreak isolates.

BMC microbiology 13 (N/A)
PMID : 24219629  :   DOI  :   10.1186/1471-2180-13-254     PMC  :   PMC3840669    
Abstract >>
Salmonella enterica subsp. enterica serovars Typhimurium (S. Typhimurium) and Heidelberg (S. Heidelberg) are major causes of foodborne salmonellosis, accounting for a fifth of all annual salmonellosis cases in the United States. Rapid, efficient and accurate methods for identification are required for routine surveillance and to track specific strains during outbreaks. We used Pulsed-field Gel Electrophoresis (PFGE) and a recently developed molecular subtyping approach termed CRISPR-MVLST that exploits the hypervariable nature of virulence genes and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) to subtype clinical S. Typhimurium and S. Heidelberg isolates. We analyzed a broad set of 175 S. Heidelberg and S. Typhimurium isolates collected over a five-year period. We identified 21 Heidelberg Sequence Types (HSTs) and 37 Typhimurium STs (TSTs) that were represented by 27 and 45 PFGE pulsotypes, respectively, and determined the discriminatory power of each method. For S. Heidelberg, our data shows that combined typing by both CRISPR-MVLST and PFGE provided a discriminatory power of 0.9213. Importantly, CRISPR-MVLST was able to separate common PFGE patterns such as JF6X01.0022 into distinct STs, thus providing significantly greater discriminatory power. Conversely, we show that subtyping by either CRISPR-MVLST or PFGE independently provides a sufficient discriminatory power (0.9345 and 0.9456, respectively) for S. Typhimurium. Additionally, using isolates from two S. Typhimurium outbreaks, we demonstrate that CRISPR-MVLST provides excellent epidemiologic concordance.
KeywordMeSH Terms
Disease Outbreaks
238. Li  L, Liao  X, Yang  Y, Sun  J, Li  L, Liu  B, Yang  S, Ma  J, Li  X, Zhang  Q, Liu  Y,     ( 2013 )

Spread of oqxAB in Salmonella enterica serotype Typhimurium predominantly by IncHI2 plasmids.

The Journal of antimicrobial chemotherapy 68 (10)
PMID : 23737490  :   DOI  :   10.1093/jac/dkt209    
Abstract >>
To investigate the prevalence and genetic environment of the multiresistance gene oqxAB in Salmonella enterica serotype Typhimurium isolated from food-producing animals. In this study, 63 Salmonella enterica serotype Typhimurium isolates were analysed for the presence of plasmid-mediated quinolone resistance determinants and mutations in the quinolone resistance-determining region by molecular methods (PCR/sequencing). The oqxAB-positive isolates were typed by pulsed-field gel electrophoresis (PFGE). Plasmids carrying oqxAB were studied by conjugation/transformation, replicon typing, Southern hybridization, long-range PCR and restriction fragment length polymorphism (RFLP). The oqxAB, aac(6')-Ib-cr and qnrS1 genes were present alone or in combination in 20 (31.7%), 23 (36.5%) and 1 (1.6%) isolate, respectively. The oqxAB-positive isolates were clonally related, as determined by PFGE. All of the oqxAB-aac(6')-Ib-cr-positive isolates carried transferable IncHI2-type plasmids containing an oqxAB cassette and an incomplete class 1 integron harbouring aac(6')-Ib-cr, blaOXA-1, catB3, arr3, qacE�G1 and sul1. Meanwhile, 6 of 15 plasmids carrying both oqxAB and aac(6')-Ib-cr showed identical RFLP patterns. The results suggest that both clonal expansion and horizontal transmission of IncHI2-type plasmids containing oqxAB and aac(6')-Ib-cr may be involved in the spread of oqxAB in Salmonella Typhimurium isolates in food-producing animals in China. There is a great need to monitor the potential dissemination of this multiresistance gene.
KeywordMeSH Terms
Enterobacteriaceae
PFGE
aac(6′)-Ib-cr
plasmid-mediated quinolone resistance
plasmids
Drug Resistance, Multiple, Bacterial
Gene Transfer, Horizontal
239. Jean-Francois  FL, Dai  J, Yu  L, Myrick  A, Rubin  E, Fajer  PG, Song  L, Zhou  HX, Cross  TA,     ( 2014 )

Binding of MgtR, a Salmonella transmembrane regulatory peptide, to MgtC, a Mycobacterium tuberculosis virulence factor: a structural study.

Journal of molecular biology 426 (2)
PMID : 24140750  :   DOI  :   10.1016/j.jmb.2013.10.014     PMC  :   PMC3947350    
Abstract >>
MgtR, a highly hydrophobic peptide expressed in Salmonella enterica serovar Typhimurium, inhibits growth in macrophages through binding to the membrane protein MgtC that has been identified as essential for replication in macrophages. While the Mycobacterium tuberculosis MgtC is highly homologous to its S. Typhi analogue, there does not appear to be an Mtb homologue for MgtR, raising significant pharmacological interest in this system. Here, solid-state NMR and EPR spectroscopy in lipid bilayer preparations were used to demonstrate the formation of a heterodimer between S. Typhi MgtR and the transmembrane helix 4 of Mtb MgtC. Based on the experimental restraints, a structural model of this heterodimer was developed using computational techniques. The result is that MgtR appears to be ideally situated in the membrane to influence the functionality of MgtC.
KeywordMeSH Terms
MD
PISEMA
distance restraints
electron spin resonance
molecular dynamics
orientational restraints
polarization inversion spin exchange at the magic angle
restrained molecular dynamics
solid-state nuclear magnetic resonance
Protein Interaction Mapping
Protein Multimerization
240. Chen  W, Ai  L, Yang  J, Ren  J, Li  Y, Guo  B,     ( 2013 )

Development of a PCR assay for rapid detection of Cronobacter spp. from food.

Canadian journal of microbiology 59 (10)
PMID : 24102218  :   DOI  :   10.1139/cjm-2013-0243    
Abstract >>
The occurrence of outbreaks of necrotizing meningitis caused by Cronobacter spp. in neonates highlights the need for rapid detection and accurate identification of this pathogenic species. The gold standard for isolation and identification of Cronobacter spp. from powdered infant formula is time consuming and labor intensive. The gyrB gene that encodes the B subunit of DNA gyrase (topoisomerase type II) was found to be suitable for the identification of Cronobacter spp. A region of the gyrB gene of 38 Cronobacter spp. strains and 5 Enterobacter spp. strains was amplified and sequenced, and a pair of primers was designed and synthesized based on the sequence of the gyrB gene. A polymerase chain reaction (PCR) system was developed and optimized to detect Cronobacter spp. The PCR assay amplified a 438 bp DNA product from all 38 Cronobacter spp. strains tested but not from 34 other bacteria. The detection limit was 1.41 pg/PCR (equivalent 282 genomic copies) when the genomic DNA of Cronobacter sakazakii ATCC 29544 was 10-fold diluted. Infant formula powders from 3 different commercial brands were inoculated with strains ATCC 29544 at a level of 56 colony-forming units, and the target fragment were produced after samples were enriched for 6 h at 37 �XC. Twenty-five food samples were evaluated by the PCR assay and the conventional method. A PCR product of the expected size was obtained from 3 samples; however, Cronobacter spp. strains were isolated from only 2 samples by the conventional method. This method is a useful tool for rapid identification of Cronobacter spp. in food and potentially environmental samples.
KeywordMeSH Terms
Food Microbiology
241. Zhang  H, You  C, Ren  J, Xu  D, Han  M, Liao  W,     ( 2014 )

A simple one-step PCR walking method and its application of bacterial rRNA for sequencing identification.

Current microbiology 68 (2)
PMID : 24126601  :   DOI  :   10.1007/s00284-013-0462-y    
Abstract >>
There are many PCR walking methods applied currently, and they all have examples of successful application in organisms which are more complex than bacteria. However, to a certain extent, it will be more convenient for researchers if the complicated operation and poor specificity for bacteria can be improved. Here, we introduced an improved one-step PCR walking method of bacteria. Using a specific primer of the known sequence together with a universal semi-random primer, the unknown sequence adjacent to a known sequence can be obtained easily by just one ordinary round PCR. The products can be gel-purified and directly sequenced. Specific primers were designed according to the gene sequence of bacterial rRNA, and the variable and adjacent gene sequences were obtained by this method. The sequence analysis of the product showed that it can improve the resolution of bacterial identification to the species level.
KeywordMeSH Terms
RNA, Bacterial
RNA, Ribosomal
242. Gong  J, Zhang  J, Xu  M, Zhu  C, Yu  Y, Liu  X, Kelly  P, Xu  B, Wang  C,     ( 2014 )

Prevalence and fimbrial genotype distribution of poultry Salmonella isolates in China (2006 to 2012).

Applied and environmental microbiology 80 (2)
PMID : 24242234  :   DOI  :   10.1128/AEM.03223-13     PMC  :   PMC3911082    
Abstract >>
In this study, a total of 323 Salmonella enterica strains were isolated from 3,566 rectal swab samples of 51 poultry farms in seven regions of 12 provinces of China between 2006 and 2012. The prevalences of Salmonella sp. carriage were 12.4% in geese (66 positive/533 samples), 10.4% in turkeys (32/309), 9.8% in chickens (167/1,706), 6.8% in ducks (41/601), and 4.1% in pigeons (17/417), respectively. These isolates belonged to 20 serovars, in which the most frequent serovars were S. enterica serovar Gallinarum biovar Pullorum (herein, S. Pullorum) (55 isolates, 17.0%), S. enterica serovar Typhimurium (50 isolates, 15.5%), and S. enterica serovar Enteritidis (39 isolates, 12.1%). Overall, S. Typhimurium was the most commonly detected serovar; among the individual species, S. Pullorum was most commonly isolated from chickens, S. Enteritidis was most common in ducks, S. Typhimurium was most common in geese and pigeons, and S. enterica serovar Saintpaul was most common in turkeys. PCR determination of 20 fimbrial genes demonstrated the presence of bcfD, csgA, fimA, stdB, and sthE genes and the absence of staA and stgA genes in these isolates, and other loci were variably distributed, with frequency values ranging from 11.8 to 99.1%. These 323 Salmonella isolates were subdivided into 41 different fimbrial genotypes, and of these isolate, 285 strains (88.2%) had 12 to 14 fimbrial genes. Our findings indicated that the Salmonella isolates from different poultry species were phenotypically and genetically diverse and that some fimbrial genes are more frequently associated with serovars or serogroups.
KeywordMeSH Terms
243. DiMarzio  M, Shariat  N, Kariyawasam  S, Barrangou  R, Dudley  EG,     ( 2013 )

Antibiotic Resistance in Salmonella enterica Serovar Typhimurium Associates with CRISPR Sequence Type.

Antimicrobial agents and chemotherapy 57 (9)
PMID : 23796925  :   DOI  :   10.1128/AAC.00913-13     PMC  :   PMC3754329    
Abstract >>
Salmonella enterica subsp. enterica serovar Typhimurium is a leading cause of food-borne salmonellosis in the United States. The number of antibiotic-resistant isolates identified in humans is steadily increasing, suggesting that the spread of antibiotic-resistant strains is a major threat to public health. S Typhimurium is commonly identified in a wide range of animal hosts, food sources, and environments, but little is known about the factors mediating the spread of antibiotic resistance in this ecologically complex serovar. Previously, we developed a subtyping method, CRISPR-multi-virulence-locus sequence typing (MVLST), which discriminates among strains of several common S. enterica serovars. Here, CRISPR-MVLST identified 22 sequence types within a collection of 76 S Typhimurium isolates from a variety of animal sources throughout central Pennsylvania. Six of the sequence types were identified in more than one isolate, and we observed statistically significant differences in resistance among these sequence types to 7 antibiotics commonly used in veterinary and human medicine, such as ceftiofur and ampicillin (P < 0.05). Importantly, five of these sequence types were subsequently identified in human clinical isolates, and a subset of these isolates had identical antibiotic resistance patterns, suggesting that these subpopulations are being transmitted through the food system. Therefore, CRISPR-MVLST is a promising subtyping method for monitoring the farm-to-fork spread of antibiotic resistance in S Typhimurium.
KeywordMeSH Terms
244. Zeinzinger  J, Pietzka  AT, Stöger  A, Kornschober  C, Kunert  R, Allerberger  F, Mach  R, Ruppitsch  W,     ( 2012 )

One-step triplex high-resolution melting analysis for rapid identification and simultaneous subtyping of frequently isolated Salmonella serovars.

Applied and environmental microbiology 78 (9)
PMID : 22344662  :   DOI  :   10.1128/AEM.07668-11     PMC  :   PMC3346493    
Abstract >>
Salmonellosis is one of the most important food-borne diseases worldwide. For outbreak investigation and infection control, accurate and fast subtyping methods are essential. A triplex gene-scanning assay was developed and evaluated for serotype-specific subtyping of Salmonella enterica isolates based on specific single-nucleotide polymorphisms in fragments of fljB, gyrB, and ycfQ. Simultaneous gene scanning of fljB, gyrB, and ycfQ by high-resolution melting-curve analysis of 417 Salmonella isolates comprising 46 different serotypes allowed the unequivocal, simple, and fast identification of 37 serotypes. Identical melting-curve profiles were obtained in some cases from Salmonella enterica serotype Enteritidis and Salmonella enterica serotype Dublin, in all cases from Salmonella enterica serotype Ohio and Salmonella enterica serotype Rissen, from Salmonella enterica serotype Mbandaka and Salmonella enterica serotype Kentucky, and from Salmonella enterica serotype Bredeney, Salmonella enterica serotype Give, and Salmonella enterica serotype Schwarzengrund. To differentiate the most frequent Salmonella serotype, Enteritidis, from some S. Dublin isolates, an additional single PCR assay was developed for specific identification of S. Enteritidis. The closed-tube triplex high-resolution melting-curve assay developed, in combination with an S. Enteritidis-specific PCR, represents an improved protocol for accurate, cost-effective, simple, and fast subtyping of 39 Salmonella serotypes. These 39 serotypes represent more than 94% of all human and more than 85% of all nonhuman Salmonella isolates (including isolates from veterinary, food, and environmental samples) obtained in the years 2008 and 2009 in Austria.
KeywordMeSH Terms
245. Kiss  J, Nagy  B, Olasz  F,     ( 2012 )

Stability, entrapment and variant formation of Salmonella genomic island 1.

PloS one 7 (2)
PMID : 22384263  :   DOI  :   10.1371/journal.pone.0032497     PMC  :   PMC3285670    
Abstract >>
The Salmonella genomic island 1 (SGI1) is a 42.4 kb integrative mobilizable element containing several antibiotic resistance determinants embedded in a complex integron segment In104. The numerous SGI1 variants identified so far, differ mainly in this segment and the explanations of their emergence were mostly based on comparative structure analyses. Here we provide experimental studies on the stability, entrapment and variant formation of this peculiar gene cluster originally found in S. Typhimurium. Segregation and conjugation tests and various molecular techniques were used to detect the emerging SGI1 variants in Salmonella populations of 17 Salmonella enterica serovar Typhimurium DT104 isolates from Hungary. The SGI1s in these isolates proved to be fully competent in excision, conjugal transfer by the IncA/C helper plasmid R55, and integration into the E. coli chromosome. A trap vector has been constructed and successfully applied to capture the island on a plasmid. Monitoring of segregation of SGI1 indicated high stability of the island. SGI1-free segregants did not accumulate during long-term propagation, but several SGI1 variants could be obtained. Most of them appeared to be identical to SGI1-B and SGI1-C, but two new variants caused by deletions via a short-homology-dependent recombination process have also been detected. We have also noticed that the presence of the conjugation helper plasmid increased the formation of these deletion variants considerably. Despite that excision of SGI1 from the chromosome was proven in SGI1(+)Salmonella populations, its complete loss could not be observed. On the other hand, we demonstrated that several variants, among them two newly identified ones, arose with detectable frequencies in these populations in a short timescale and their formation was promoted by the helper plasmid. This reflects that IncA/C helper plasmids are not only involved in the horizontal spreading of SGI1, but may also contribute to its evolution.
KeywordMeSH Terms
Drug Resistance, Bacterial
Genomic Islands
246. Gulig  PA, Chiodo  VA,     ( 1990 )

Genetic and DNA sequence analysis of the Salmonella typhimurium virulence plasmid gene encoding the 28,000-molecular-weight protein.

Infection and immunity 58 (8)
PMID : 2164511  :   PMC  :   PMC258868    
Abstract >>
We have confirmed that the 28,000-molecular-weight (28K) protein encoded by the virA gene of the 90-kilobase Salmonella typhimurium virulence plasmid is a virulence factor. It was previously shown that a Tn5 insertion, vir-22::Tn5, located in the virulence plasmid greatly attenuated virulence for mice and inhibited the production of a 28K protein (P.A. Gulig and R. Curtiss III, Infect. Immun. 56:3262-3271, 1988). Plasmid pYA426 fully complemented vir-22::Tn5 to virulence by increasing splenic infection after oral inoculation and encoded the 28K protein. To identify the virulence gene(s) of pYA426 mutated by vir-22::Tn5, we constructed nested deletions in pYA426 and examined deletion derivatives for their abilities to complement vir-22::Tn5. Only derivatives still producing the 28K protein complemented vir-22::Tn5. Furthermore, the smallest complementing derivative encoded only the 28K protein, as determined by DNA sequence analysis. Therefore, the 28K protein is sufficient for complementation of the attenuating mutation vir-22::Tn5 and must be the virulence factor inhibited by the insertion. We determined the nucleotide sequence of the 1.2-kilobase BamHI-EcoRI fragment encoding the 28K protein and identified the structural gene, virA. A 723-base-pair open reading frame which encodes a peptide with a molecular weight of 27,572 was found.
KeywordMeSH Terms
Genes, Bacterial
Virulence Factors
247. Akiyama  T, Khan  AA,     ( 2012 )

Isolation and characterization of small qnrS1-carrying plasmids from imported seafood isolates of Salmonella enterica that are highly similar to plasmids of clinical isolates.

FEMS immunology and medical microbiology 64 (3)
PMID : 22151215  :   DOI  :   10.1111/j.1574-695X.2011.00921.x    
Abstract >>
Dissemination of plasmid-mediated quinolone resistance among pathogenic bacteria is a concern for public health because of decreased sensitivity to fluoroquinolones and increased potentials to develop high fluoroquinolone resistance. Two qnrS1-positive isolates of Salmonella enterica Corvallis (468) and Typhimurium (484) from imported seafood (Thailand and Vietnam) were tested for quinolone sensitivity using disk agar diffusion and the Sensititre system. The presence of qnr genes, qnr-carrying plasmids, and mutations in the quinolone resistance determining regions were also determined. Minimal inhibitory concentrations of nalidixic acid for isolates 468 and 484 were 8 and 16 �gg mL(-1) , respectively, and those of ciprofloxacin were 1 and 2 �gg mL(-1), respectively. Disk agar diffusion indicated that isolate 468 was moderately resistant to moxifloxacin, and isolate 484 was resistant to moxifloxacin and moderately resistant to norfloxacin. Isolates 468 and 484 carried a mutation on parC, but not on gyrA, gyrB, or parE. Sequences of qnrS1-carrying plasmids from isolates 468 and 484, sized 10,039 and 10,047 bp, were nearly identical (> 99% similarity) to each other and to published sequences of plasmids from clinical isolates of Salmonella Typhimurium isolated in the United Kingdom and Taiwan, indicating a dissemination of qnrS1-carrying plasmids among different serovars of Salmonella from geographically separated sources. This is the first complete sequence of a qnrS1-carrying plasmid from imported seafood isolate of S. enterica.
KeywordMeSH Terms
248. Cain  AK, Hall  RM,     ( 2011 )

Transposon Tn5393e carrying the aphA1-containing transposon Tn6023 upstream of strAB does not confer resistance to streptomycin.

Microbial drug resistance (Larchmont, N.Y.) 17 (3)
PMID : 21702681  :   DOI  :   10.1089/mdr.2011.0037    
Abstract >>
The simplest form of transposon Tn5393 carries the strA and strB genes that confer resistance to streptomycin, in addition to its transposition determinants. Tn5393e, made up of Tn5393 and a second transposon, Tn6023, was found in an IncHI2 plasmid, pSRC125, recovered from a multiply antibiotic-resistant Salmonella enterica serovar Typhimurium isolate of bovine origin. Tn6023 is made up of the aphA1b gene flanked by two inversely oriented copies of insertion sequence (IS) IS26 and is flanked by an 8 bp duplication. It is related to several other transposons that carry aphA1b in fragments of differing length, also flanked by copies of IS26. Tn6023 is located in the tnpR gene of Tn5393, which lies upstream of the strA and strB genes, and the combined structure was designated Tn5393e. Although neither strA nor strB contain any mutations that would inactivate them, pSRC125 does not confer resistance to streptomycin, indicating that the strA and strB genes are not expressed. In Tn5393, strA and strB are transcribed from the tnpR promoter, and in Tn5393e neither this transcript nor the transcript from the aphA1b promoter in Tn6023 must reach strAB. Tn5393e was previously found in different locations in Corynebacteria, indicating that it can move and suggesting a wide distribution. The structures of several further variants of Tn5393 found in GenBank were analyzed and assigned variant designations Tn5393f-Tn5393i.
KeywordMeSH Terms
DNA Transposable Elements
249. Garbarg-Chenon  A, Godard  V, Labia  R, Nicolas  JC,     ( 1990 )

Nucleotide sequence of SHV-2 beta-lactamase gene.

Antimicrobial agents and chemotherapy 34 (7)
PMID : 2201259  :   DOI  :   10.1128/aac.34.7.1444     PMC  :   PMC175998    
Abstract >>
The nucleotide sequence of plasmid-mediated beta-lactamase SHV-2 from Salmonella typhimurium (SHV-2pHT1) was determined. The gene was very similar to chromosomally encoded beta-lactamase LEN-1 of Klebsiella pneumoniae. Compared with the sequence of the Escherichia coli SHV-2 enzyme (SHV-2E.coli) obtained by protein sequencing, the deduced amino acid sequence of SHV-2pHT1 differed by three amino acid substitutions.
KeywordMeSH Terms
250.     ( 1997 )

The flk gene of Salmonella typhimurium couples flagellar P- and L-ring assembly to flagellar morphogenesis.

Journal of bacteriology 179 (7)
PMID : 9079927  :   DOI  :   10.1128/jb.179.7.2389-2400.1997     PMC  :   PMC178978    
Abstract >>
The flagellum of Salmonella typhimurium is assembled in stages, and the negative regulatory protein, FlgM, is able to sense the completion of an intermediate stage of assembly, the basal body-hook (BBH) structure. Mutations in steps leading to the formation of the BBH structure do not express the flagellar filament structural genes, fliC and fljB, due to negative regulation by FlgM (K. L. Gillen and K. T. Hughes, J. Bacteriol. 173:6453-6459, 1991). We have discovered another novel regulatory gene, flk, which appears to sense the completion of another assembly stage in the flagellar morphogenic pathway just prior to BBH formation: the completion of the P- and L-rings. Cells that are unable to assemble the L- or P-rings do not express the flagellin structural genes. Mutations by insertional inactivation in either the flk or flgM locus allow expression of the fljB flagellin structural gene in strains defective in flagellar P- and L-ring assembly. Mutations in the flgM gene, but not mutations in the flk gene, allow expression of the fljB gene in strains defective in all of the steps leading to BBH formation. The flk gene was mapped to min 52 of the S. typhimurium linkage map between the pdxB and fabB loci. A null allele of flk was complemented in trans by a flk+ allele present in a multicopy pBR-based plasmid. DNA sequence analysis of the flk gene has revealed it to be identical to a gene of Escherichia coli of unknown function which has an overlapping, divergent promoter with the pdxB gene promoter (P. A. Schoenlein, B. B. Roa, and M. E. Winkler, J. Bacteriol. 174:6256-6263, 1992). An open reading frame of 333 amino acids corresponding to the flk gene product of S. typhimurium and 331 amino acids from the E. coli sequence was identified. The transcriptional start site of the S. typhimurium flk gene was determined and transcription of the flk gene was independent of the FlhDC and sigma28 flagellar transcription factors. The Flk protein observed in a T7 RNA polymerase-mediated expression system showed an apparent molecular mass of 35 kDa, slightly smaller than the predicted size of 37 kDa. The predicted structure of Flk is a mostly hydrophilic protein with a very C-terminal membrane-spanning segment preceded by positively charged amino acids. This finding predicts Flk to be inserted into the cytoplasmic membrane facing inside the cytoplasm.
KeywordMeSH Terms
Genes, Bacterial
251.     ( 1997 )

A secreted Salmonella protein with homology to an avirulence determinant of plant pathogenic bacteria.

Proceedings of the National Academy of Sciences of the United States of America 94 (18)
PMID : 9275221  :   DOI  :   10.1073/pnas.94.18.9887     PMC  :   PMC23287    
Abstract >>
Bacterial pathogens have evolved sophisticated mechanisms to interact with their hosts. A specialized type III protein secretion system capable of translocating bacterial proteins into host cells has emerged as a central factor in the interaction between a variety of mammalian and plant pathogenic bacteria with their hosts. Here we describe AvrA, a novel target of the centisome 63 type III protein secretion system of Salmonella enterica. AvrA shares sequence similarity with YopJ of the animal pathogen Yersinia pseudotuberculosis and AvrRxv of the plant pathogen Xanthomonas campestris pv. vesicatoria. These proteins are the first examples of putative targets of type III secretion systems in animal and plant pathogenic bacteria that share sequence similarity. They may therefore constitute a novel family of effector proteins with related functions in the cross-talk of these pathogens with their hosts.
KeywordMeSH Terms
Sequence Homology, Amino Acid
252.     ( 1997 )

Molecular analysis of the genes mediating Salmonella invasion.

FEMS immunology and medical microbiology 18 (2)
PMID : 9223615  :   DOI  :   10.1111/j.1574-695X.1997.tb01035.x    
Abstract >>
To identify invasion determinants, a genomic library of Salmonella typhimurium was cloned into a cosmid vector, pLA2917. A clone, pSI623 which was invasive for HEp-2 and Henle-407 epithelial cells, was subcloned into a plasmid vector, pGEM-7Z to define the invasion genes. The subclone, pSV6235 containing a 4.5 kbp fragment of the Salmonella genomic region, was highly invasive for HEp-2 and Henle-407 cells, compared with other subclones whose Salmonella genomic regions are 7.4, 6.3, 5.5 and 1.4 kbp, respectively. This study reflects that the invasion efficiency of the host strain, Escherichia coli to HEp-2 and Henle-407 cell lines was significantly increased by the introduction of the genomic region of the virulent S. typhimurium. Restriction enzyme analysis showed that there was a single PstI site on the 27 kbp of Salmonella genomic region of pSI623, and no PstI site was found on the 4.5 kbp of genomic region of pSV6235. This finding suggests that the invasion related genes different from the inv and spa gene clusters which were identified in S. typhimurium, may exist in genomic DNA of S. typhimurium.
KeywordMeSH Terms
Genes, Bacterial
253.     ( 1997 )

The resistance and integrase genes of pACM1, a conjugative multiple-resistance plasmid, from Klebsiella oxytoca.

Plasmid 37 (2)
PMID : 9169202  :   DOI  :   10.1006/plas.1997.1284    
Abstract >>
pACM1 is an 85-kb conjugative plasmid from a clinical isolate of Klebsiella oxytoca that encodes resistance to beta-lactams (mediated by SHV-5 extended spectrum beta-lactamase), trimethoprim, sulfonamides, tetracycline, aminoglycosides, and mercuric chloride. The expression of the aminoglycoside resistance is difficult to detect, which could have clinical implications. A region of pACM1 containing five resistance genes and two putative integrons was characterized by restriction mapping and partial DNA sequencing. One integron appears to be class I (sull type); the second lacks a recognizable 3' conserved segment. Neither integron has the BamHI site predicted for the 5' conserved segment. Plasmids encoding SHV-5 from other bacterial strains appear to be closely related to pACM1 by restriction enzyme analysis, but have resistance/ integron regions that vary in size and content from that of pACM1. Integrase-mediated recombination might be responsible for genetic divergence in a widely distributed family of pACM1-like plasmids.
KeywordMeSH Terms
Genes, Bacterial
Genes, MDR
254.     ( 1997 )

Fluorescence-based isolation of bacterial genes expressed within host cells.

Science (New York, N.Y.) 277 (5334)
PMID : 9302299  :   DOI  :   10.1126/science.277.5334.2007    
Abstract >>
A selection strategy was devised to identify bacterial genes preferentially expressed when a bacterium associates with its host cell. Fourteen Salmonella typhimurium genes, which were under the control of at least four independent regulatory circuits, were identified to be selectively induced in host macrophages. Four genes encode virulence factors, including a component of a type III secretory apparatus. This selection methodology should be generally applicable to the identification of genes from pathogenic organisms that are induced upon association with host cells or tissues.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
255.     ( 1997 )

Propanediol utilization genes (pdu) of Salmonella typhimurium: three genes for the propanediol dehydratase.

Journal of bacteriology 179 (21)
PMID : 9352910  :   DOI  :   10.1128/jb.179.21.6633-6639.1997     PMC  :   PMC179589    
Abstract >>
The propanediol utilization (pdu) operon of Salmonella typhimurium encodes proteins required for the catabolism of propanediol, including a coenzyme B12-dependent propanediol dehydratase. A clone that expresses propanediol dehydratase activity was isolated from a Salmonella genomic library. DNA sequence analysis showed that the clone included part of the pduF gene, the pduABCDE genes, and a long partial open reading frame (ORF1). The clone included 3.9 kbp of pdu DNA which had not been previously sequenced. Complementation and expression studies with subclones constructed via PCR showed that three genes (pduCDE) are necessary and sufficient for propanediol dehydratase activity. The function of ORF1 was not determined. Analyses showed that the S. typhimurium propanediol dehydratase was related to coenzyme B12-dependent glycerol dehydratases from Citrobacter freundii and Klebsiella pneumoniae. Unexpectedly, the S. typhimurium propanediol dehydratase was found to be 98% identical in amino acid sequence to the Klebsiella oxytoca propanediol dehydratase; this is a much higher identity than expected, given the relationship between these organisms. DNA sequence analyses also supported previous studies indicating that the pdu operon was inherited along with the adjacent cobalamin biosynthesis operon by a single horizontal gene transfer.
KeywordMeSH Terms
Genes, Bacterial
256.     ( 1997 )

Sequence comparison of the genes for immunity, DNA replication, and cell lysis of the P22-related Salmonella phages ES18 and L.

Gene 195 (1)
PMID : 9300826  :   DOI  :   10.1016/s0378-1119(97)00182-0    
Abstract >>
Complementation and hybridization experiments with the generalized transducing Salmonella phages P22, ES18 and L revealed strong similarity between the phages L and P22; the genome of ES18 shows a mosaic structure. About half of its genome, including the early genes, is similar or completely homologous to P22; the other half of the morphologically different ES18 does not show any similarity to P22 nor to E. coli phage lambda. Sequence comparison of the early genes has confirmed that the C-immunity region of ES18 is identical with that of P22, whereas the same region of phage L shows poor (repressor gene) or no similarity. The 5'-terminus of the DNA replication gene 12 of ES18, however, is homologous to the same section of gene O of phage lambda. The lysis genes of ES18 again are identical to those of P22; only gene 15 is mosaic-like and has more similarity to gene Rz of phage lambda. These results will be discussed in terms of the theory of modular genome organization.
KeywordMeSH Terms
Genes, Viral
257.     ( 1997 )

The plasmid R64 thin pilus identified as a type IV pilus.

Journal of bacteriology 179 (11)
PMID : 9171405  :   DOI  :   10.1128/jb.179.11.3594-3603.1997     PMC  :   PMC179153    
Abstract >>
The entire nucleotide sequence of the pil region of the IncI1 plasmid R64 was determined. Analysis of the sequence indicated that 14 genes, designated pilI through pilV, are involved in the formation of the R64 thin pilus. Protein products of eight pil genes were identified by the maxicell procedure. The pilN product was shown to be a lipoprotein by an experiment using globomycin. A computer search revealed that several R64 pil genes have amino acid sequence homology with proteins involved in type IV pilus biogenesis, protein secretion, and transformation competence. The pilS and pilV products were suggested to be prepilins for the R64 thin pilus, and the pilU product appears to be a prepilin peptidase. These results suggest that the R64 thin pilus belongs to the type IV family, specifically group IVB, of pili. The requirement of the pilR and pilU genes for R64 liquid mating was demonstrated by constructing their frameshift mutations. Comparison of three type IVB pilus biogenesis systems, the pil system of R64, the toxin-coregulated pilus (tcp) system of Vibrio cholerae, and the bundle-forming pilus (bfp) system of enteropathogenic Escherichia coli, suggests that they have evolved from a common ancestral gene system.
KeywordMeSH Terms
258.     ( 1997 )

Requirement of a limited segment of the sog gene for plasmid R64 conjugation.

Plasmid 38 (1)
PMID : 9281491  :   DOI  :   10.1006/plas.1997.1297    
Abstract >>
The sog gene of the IncI1 plasmid R64 was sequenced and characterized. The sog gene was shown to express two acidic proteins, SogL and SogS, with 1255 and 844 amino acid residues, respectively. The SogS protein was expressed by translational reinitiation within the SogL reading frame. Analysis of dnaG-suppression activity using the Escherichia coli dnaG strain indicated that the domain for this activity was located within the N-terminal one-third segment of the SogL protein. A Deltasog mutation was constructed by replacing most of the sog coding sequence with a DNA fragment encoding a tetracycline resistance gene. Introduction of the Deltasog mutation into an R64 derivative resulted in approximately a 50-fold reduction in transfer frequency. It was observed that only a limited portion of the SogL or SogS protein corresponding to an internal 0.94-kb EcoRV-SnaBI segment of the sog gene was required for the conjugal transfer of R64.
KeywordMeSH Terms
Drosophila Proteins
Genes, Bacterial
259.     ( 1997 )

Salmonella typhimurium specifies a circular chromosome dimer resolution system which is homologous to the Xer site-specific recombination system of Escherichia coli.

Gene 198 (1��2��)
PMID : 9370270  :   DOI  :   10.1016/s0378-1119(97)00299-0    
Abstract >>
The Xer site-specific recombination system of Escherichia coli resolves both chromosome dimers and multimers of certain plasmids including those of ColE1. In this manner, Xer site-specific recombination contributes to the accurate distribution of circular chromosomes at cell division. Two related site-specific recombinases, XerC and XerD, are required for this process. The xerC and xerD genes of Salmonella typhimurium LT2 were isolated from libraries of LT2 genomic DNA by genetic complementation of E. coli Xer mutants. The putative proteins specified by the S. typhimurium genes can substitute for and are highly homologous to the corresponding proteins in E. coli. The distribution of amino acid dissimilarities differs, however, between pairs of cognate Xer proteins. The immediate genetic contexts of equivalent xer genes, i.e., in operons with genes of apparently unrelated function, are conserved between the two bacteria. This is the first description of the identification of a pair of functional homologues of the xerC and xerD genes of E. coli.
KeywordMeSH Terms
Escherichia coli Proteins
Integrases
260.     ( 1997 )

Analysis of the boundaries of Salmonella pathogenicity island 2 and the corresponding chromosomal region of Escherichia coli K-12.

Journal of bacteriology 179 (4)
PMID : 9023191  :   DOI  :   10.1128/jb.179.4.1105-1111.1997     PMC  :   PMC178805    
Abstract >>
We recently identified a pathogenicity island (SPI2) located at 30.7 centisomes on the Salmonella typhimurium chromosome. SPI2 contains genes encoding a type III secretion system whose function is distinct from that of the type III secretion system encoded by a pathogenicity island (SPI1) at 63 centisomes which is involved in epithelial cell entry. An analysis of the boundaries of SPI2 and comparison with the corresponding region of the Escherichia coli chromosome revealed that SPI2 inserted adjacent to the tRNA(Val) gene. The E. coli chromosome contains 9 kb of DNA at the region corresponding to the SPI2 insertion point which appears to be absent in S. typhimurium. The distribution of SPI1 and SPI2 was examined in various Salmonella isolates. In contrast to type III secretion system genes of SPI1, those of SPI2 are not present in Salmonella bongori, which diverged at the first branch point in the Salmonella lineage. These and other data indicate that SPI2 was acquired by a Salmonella strain already harboring SPI1 by horizontal transfer from an unknown source.
KeywordMeSH Terms
Chromosomes, Bacterial
Genes, Bacterial
261.     ( 1997 )

Conservation of the Escherichia coli dnaX programmed ribosomal frameshift signal in Salmonella typhimurium.

Journal of bacteriology 179 (13)
PMID : 9209069  :   DOI  :   10.1128/jb.179.13.4438-4442.1997     PMC  :   PMC179275    
Abstract >>
Escherichia coli DNA polymerase III subunits tau and gamma are produced from one gene, dnaX, by a programmed ribosomal frameshift which generates the C terminal of gamma within the tau reading frame. To help evaluate the role of the dispensable gamma, the distribution of tau and gamma homologs in several other species and the sequence of the Salmonella typhimurium dnaX were determined. All four enterobacteria tested produce tau and gamma homologs. S. typhimurium dnaX is 83% identical to E. coli dnaX, but all four components of the frameshift signal are 100% conserved.
KeywordMeSH Terms
Conserved Sequence
262.     ( 1997 )

A Salmonella typhimurium genetic locus which confers copper tolerance on copper-sensitive mutants of Escherichia coli.

Journal of bacteriology 179 (16)
PMID : 9260936  :   DOI  :   10.1128/jb.179.16.4977-4984.1997     PMC  :   PMC179352    
Abstract >>
Three distinct clones from a Salmonella typhimurium genomic library were identified which suppressed the copper-sensitive (Cu(s)) phenotype of cutF mutants of Escherichia coli. One of these clones, pCUTFS2, also increased the copper tolerance of cutA, -C, and -E mutants, as well as that of a lipoprotein diacylglyceryl transferase (lgt) mutant of E. coli. Characterization of pCUTFS2 revealed that the genes responsible for suppression of copper sensitivity (scs) reside on a 4.36-kb DNA fragment located near 25.4 min on the S. typhimurium genome. Sequence analysis of this fragment revealed four open reading frames (ORF120, ORF627, ORF207, and ORF168) that were organized into two operons. One operon consisted of a single gene, scsA (ORF120), whereas the other operon contained the genes scsB (ORF627), scsC (ORF207), and scsD (ORF168). Comparison of the deduced amino acid sequences of the predicted gene products showed that ScsB, ScsC, and ScsD have significant homology to thiol-disulfide interchange proteins (CutA2, DipZ, CycZ, and DsbD) from E. coli and Haemophilus influenzae, to an outer membrane protein (Com1) from Coxiella burnetii, and to thioredoxin and thioredoxin-like proteins, respectively. The two operons were subcloned on compatible plasmids, and complementation analyses indicated that all four proteins are required for the increased copper tolerance of E. coli mutants. In addition, the scs locus also restored lipoprotein modification in lgt mutants of E. coli. Sequence analyses of the S. typhimurium scs genes and adjacent DNAs revealed that the scs locus is flanked by genes with high homology to the cbpA (predicted curved DNA-binding protein) and agp (acid glucose phosphatase) genes of E. coli located at 22.90 min (1,062.07 kb) and 22.95 min (1,064.8 kb) of the E. coli chromosome, respectively. However, examination of the E. coli chromosome revealed that these genes are absent at this locus and no evidence has thus been obtained for the occurrence of the scs locus elsewhere on the genome.
KeywordMeSH Terms
Genes, Suppressor
263.     ( 1997 )

Mutational analysis of the R64 oriT region: requirement for precise location of the NikA-binding sequence.

Journal of bacteriology 179 (23)
PMID : 9393692  :   DOI  :   10.1128/jb.179.23.7291-7297.1997     PMC  :   PMC179678    
Abstract >>
Conjugative DNA transfer of IncI1 plasmid R64 is initiated by the introduction of a site- and strand-specific nick into the origin of transfer (oriT). In R64 oriT, 17-bp (repeat A and B) and 8-bp inverted-repeat sequences with mismatches are located 8 bp away from the nick site. The nicking is mediated by R64 NikA and NikB proteins. To analyze the functional organization of the R64 oriT region, various deletion, insertion, and substitution mutations were introduced into a 92-bp minimal R64 oriT sequence and their effects on oriT function were investigated. This detailed analysis confirms our previous prediction that the R64 oriT region consists of an oriT core sequence and additional sequences necessary for full oriT activity. The oriT core sequence consists of the repeat A sequence, which is recognized by R64 NikA protein, and the nick region sequence, which is conserved among various origins of transfer and is most probably recognized by NikB protein. The oriT core sequence is sufficient for NikAB-mediated oriT-specific nicking. Furthermore, it was shown that the repeat A sequence is essential for localization to a precise position relative to the nick site for oriT function. This seems to be required for the formation of a functional ternary complex consisting of NikA and NikB proteins and oriT DNA. The repeat B sequence and 8-bp inverted repeat sequences are suggested to be required for the termination of DNA transfer.
KeywordMeSH Terms
Bacterial Proteins
Conjugation, Genetic
264.     ( 1997 )

Organization of the leading region of IncN plasmid pKM101 (R46): a regulation controlled by CUP sequence elements.

Journal of molecular biology 271 (1)
PMID : 9300052  :   DOI  :   10.1006/jmbi.1997.1124    
Abstract >>
Analysis of the nucleotide sequence of the 13.8 kb leading region of the IncN plasmid pKM101 (a deletion derivative of R46) revealed eight copies of highly conserved repetitive elements, CUP (Conserved UPstream), and at least nine novel open reading frames (ORFs). Appropriate protein products were identified for eight ORFs and the analysis of their deduced amino acid sequences revealed similarities with some well-known proteins (KorA of RK2/RP4, RecX and PsiB) that may play a role in the adaptation of promiscuous plasmids to the new host. Comparison of CUP elements revealed that the CUP core is 417 nucleotides long and consists of two portions that markedly differ in GC content. The larger portion (307 nucleotides) of the core is about 74% GC and contains at least one NotI site, while the other (110 nucleotides) is only about 40% GC. The remarkable features of CUP elements is that five of them are oriented in the same direction and fused in a similar mode to the open reading frames (ORFs) that are able to encode unrelated proteins. The spacings between the right boundary of the CUP core and the potential ATG start codons of these ORFs are slightly different in length (16 to 18 bp), highly divergent in sequence but in all cases contain the conserved hexamer 5'-AGGAGT-3' at the position that is typical for the ribosome binding site of Escherichia coli. The A+T-rich portion of the CUP sequences contains the strong negatively regulated promoter and appears to function as a genetic switch that coordinately controls the expression of CUP-fused genes during the conjugal transfer. These findings suggest that seven plasmid genes fused to the CUP elements including repA and two ard genes encoding positively acting replication protein and antirestriction proteins, respectively, may be members of one regulatory network based on the CUP elements and two plasmid-encoded regulatory proteins ArdK and ArdR. At least, the ArdK protein may act as a typical repressor by binding to the promoter region of the CUP sequence. Most of the structural and functional features of organization of the CUP-controlled regulatory network are associated with the idea that the CUP elements may be involved in the natural genetic engineering process of organizing various functionally related genes in one regulon.
KeywordMeSH Terms
Escherichia coli Proteins
Plasmids
Regulon
Repetitive Sequences, Nucleic Acid
265.     ( 1997 )

Influence of genes encoding proton-translocating enzymes on suppression of Salmonella typhimurium growth and colonization.

Journal of bacteriology 179 (22)
PMID : 9371470  :   DOI  :   10.1128/jb.179.22.7186-7190.1997     PMC  :   PMC179664    
Abstract >>
Twenty-four-hour-old, aerobically grown, Luria-Bertani broth cultures of Salmonella typhimurium F98 suppressed the growth of a spectinomycin-resistant (Spcr) derivative of the same strain inoculated at 10(3) CFU ml(-1). This growth suppression is genus specific and RpoS independent, and it is not solely a result of nutrient depletion (P. A. Barrow, M. A. Lovell, and L. Zhang-Barber, J. Bacteriol. 178:3072-3076, 1996). Mutations in three genes are shown here to significantly reduce growth suppression under these conditions. The mutations were located in the nuo, cyd, and unc operons, which code for the NADH dehydrogenase I, cytochrome d oxidase, and F0F1 proton-translocating ATPase complexes, respectively. When cultures were grown under strictly anaerobic conditions, only the unc mutant did not suppress growth. Prior colonization of the alimentary tract of newly hatched chickens with the S. typhimurium F98 wild type or nuo or cyd mutants suppressed colonization by an S. typhimurium F98 Spcr derivative inoculated 24 h later. In contrast, the S. typhimurium unc mutant did not suppress colonization. The nuo and unc mutants showed poorer growth on certain carbon sources. The data support the hypothesis that growth suppression operates because of the absence of a utilizable carbon source or electron acceptor.
KeywordMeSH Terms
Electron Transport Chain Complex Proteins
Escherichia coli Proteins
266.     ( 1993 )

Molecular analysis of two ScrR repressors and of a ScrR-FruR hybrid repressor for sucrose and D-fructose specific regulons from enteric bacteria.

Molecular microbiology 9 (1)
PMID : 8412665  :   DOI  :   10.1111/j.1365-2958.1993.tb01681.x    
Abstract >>
The scr regulon of pUR400 and the chromosomally encoded scr regulon of Klebsiella pneumoniae KAY2026 are both negatively controlled by a specific repressor (ScrR). As deduced from the nucleotide sequences, both scrR genes encode polypeptides of 334 residues (85.5% identical base pairs, 91.3% identical amino acids), containing an N-terminal helix-turn-helix motif. Comparison with other regulatory proteins revealed 30.6% identical amino acids to FruR, 27.0% to Lacl and 28.1% to GalR. Six scrRs super-repressor mutations define the inducer-binding domain. The scr operator sequences were identified by in vivo titration tests of the sucrose repressor and by in vitro electrophoretic mobility shift assays. D-fructose, an intracellular product of sucrose transport and hydrolysis, and D-fructose 1-phosphate were shown to be molecular inducers of both scr regulons. An active ScrR-FruR hybrid repressor protein was constructed with the N-terminal part of the sucrose repressor of K. pneumoniae and the C-terminal part of the fructose repressor of Salmonella typhimurium LT2. Gel retardation assays showed that the hybrid protein bound to scr-specific operators, and that D-fructose 1-phosphate, the inducer for FruR, was the only inducer. In vivo, neither the operators of the fru operon nor of the pps operon, the natural targets for FruR, were recognized, but the scr operators were. These data and the data obtained from the super-repressor alleles confirm previous models on the binding of repressors of the Lacl family to their operators.
KeywordMeSH Terms
Escherichia coli Proteins
Genes, Bacterial
Genes, Synthetic
267.     ( 1996 )

Molecular characterization of the oafA locus responsible for acetylation of Salmonella typhimurium O-antigen: oafA is a member of a family of integral membrane trans-acylases.

Journal of bacteriology 178 (20)
PMID : 8830685  :   DOI  :   10.1128/jb.178.20.5904-5909.1996     PMC  :   PMC178445    
Abstract >>
Lipopolysaccharide (LPS) coats the surface of gram-negative bacteria and serves to protect the cell from its environment. The O-antigen is the outermost part of LPS and is highly variable among gram-negative bacteria. Strains of Salmonella are partly distinguished by serotypic differences in their O-antigen. In Salmonella typhimurium, the O-antigen is acetylated, conferring the 05 serotype. We have previously provided evidence that this modification significantly alters the structure of the O-antigen and creates or destroys a series of conformational epitopes. Here we report the detailed mapping, cloning, and DNA sequence of the oafA gene. The locus contains one open reading frame that is predicted to encode an inner membrane protein, consistent with its role in modification of the O-antigen subunit. The OafA protein shows homology to proteins in a number of prokaryotic and one eukaryotic species, and this defines a family of membrane proteins involved in the acylation of exported carbohydrate moieties. In many of these instances, acylation defines serotype or host range and thus has a profound effect on microbe-host interaction.
KeywordMeSH Terms
Multigene Family
268.     ( 1996 )

Sequences of beta-lactamase genes encoding CTX-M-1 (MEN-1) and CTX-M-2 and relationship of their amino acid sequences with those of other beta-lactamases.

Antimicrobial agents and chemotherapy 40 (2)
PMID : 8834913  :   PMC  :   PMC163149    
Abstract >>
Amino acid sequences determined either by protein sequencing or by DNA sequencing are identical for cefotaximases CTX-M-1 and MEN-1, whereas CTX-M-2 is 84% identical to CTX-M-1/MEN-1. Both beta-lactamases are distantly related to other plasmidic class A enzymes (homology to TEM-1 is 38.1% for CTX-M-1/MEN-1 and 36.5% for CTX-M-2); the closest relationship was with the chromosomal beta-lactamase of Klebsiella oxytoca E23004 (homologies of 74.5% for CTX-M-1/MEN-1 and 77.9% for CTX-M-2). The cefotaximases CTX-M-1/MEN-1 and CTX-M-2 represent two members of a new subgroup of plasmidic class A beta-lactamases.
KeywordMeSH Terms
Genes, Bacterial
269.     ( 1996 )

Molecular genetic relationships of the salmonellae.

Applied and environmental microbiology 62 (3)
PMID : 8975610  :   PMC  :   PMC167847    
Abstract >>
A multilocus enzyme electrophoresis analysis of 96 strains of the salmonellae distinguished 80 electrophoretic types (ETs) and placed them in eight groups, seven of which correspond precisely to the seven taxonomic groups (I, II, IIIa, IIIb, IV, V, and VI) previously defined on the basis of biotype and genomic DNA hybridization. In addition, multilocus enzyme electrophoresis identified an eighth distinctive group (designated VII) composed of five strains that had been assigned to group IV on the basis of biotype. An analysis of variation in the combined nucleotide sequences of five housekeeping genes among 16 strains representing all eight groups yielded estimates of overall genetic relationships that are fully consistent with those indicated by DNA hybridization. However, the nucleotide sequences of seven invasion genes (inv/spa) in the strains of group VII were closely similar to those of strains of group IV. These findings are interpreted as evidence that group VII represents an old, differentiated lineage to which one or more large parts of the chromosomal genome of the group IV lineage, including the 40-kb segment on which the invasion genes are located, have been horizontally transferred. All lines of molecular genetic evidence indicate that group V is very strongly differentiated from all other groups, thus supporting its current taxonomic treatment as a species, Salmonella bongori, separate from S. enterica. The Salmonella Reference Collection C, composed of the 16 strains used in DNA sequence studies, has been established for research on variation in natural populations.
KeywordMeSH Terms
Phylogeny
270.     ( 1996 )

Bacterial genetics by flow cytometry: rapid isolation of Salmonella typhimurium acid-inducible promoters by differential fluorescence induction.

Molecular microbiology 22 (2)
PMID : 8930920  :   DOI  :   10.1046/j.1365-2958.1996.00120.x    
Abstract >>
The ability of Salmonella typhimurium to survive and replicate within murine macrophages is dependent on a low phagosomal pH. This requirement for an acidic vacuole suggests that low pH is an important environmental stimulus for the transcription of genes necessary for intracellular survival. To study the behaviour of acid-inducible genes in response to the phagosomal environment, we have applied a novel enrichment strategy, termed differential fluorescence induction (DFI), to screen an S. typhimurium library for promoters that are upregulated at pH 4.5. DFI utilizes a fluorescence-enhanced green fluorescent protein (GFP) and a fluorescence-activated cell sorter (FACS) to perform genetic selection. In the presence of an inducing stimulus, such as low pH, a FACS is used to sort highly fluorescent bacterial clones bearing random promoters fused to the mutant GFP protein (GFPmut). This population is then amplified at neutral pH and the least fluorescent population is sorted. Sequential sorts for fluorescent and non-fluorescent bacteria in the presence or absence of inducing conditions rapidly enriches for promoter fusions that are regulated by the inducing stimulus. We have identified eight acid-inducible promoters and quantified their expression in response to pH 4.5 and to the phagosome milieu. These acid-inducible promoters exhibited extensive homology to promoter regions of genes encoding for cell-surface-maintenance enzymes, stress proteins, and generalized efflux pumps. Only a subset of these promoters was induced in macrophages with kinetics and levels of expression that do not necessarily correlate with in vitro pH-shock induction. This suggests that while low pH is a relevant inducer of intracellular gene expression, additional stimuli in the macrophage can modulate the expression of acid-inducible genes.
KeywordMeSH Terms
Flow Cytometry
Promoter Regions, Genetic
271.     ( 1997 )

Simultaneous prevention of glutamine synthesis and high-affinity transport attenuates Salmonella typhimurium virulence.

Infection and immunity 65 (2)
PMID : 9009317  :   PMC  :   PMC176100    
Abstract >>
In Salmonella typhimurium, transcription of the glnA gene (encoding glutamine synthetase) is under the control of the nitrogen-regulatory (ntr) system comprising the alternate sigma factor sigma54 (NtrA) and the two-component sensor-transcriptional activator pair NtrB and NtrC. The glnA, ntrB, and ntrC genes form an operon. We measured the virulence of S. typhimurium strains with nitrogen-regulatory mutations after intraperitoneal (i.p.) or oral inoculations of BALB/c mice. Strains with single mutations in glnA, ntrA, ntrB, or ntrC had i.p. 50% lethal doses (LD50s) of <10 bacteria, similar to the wild-type strain. However, a strain with a delta(glnA-ntrC) operon deletion had an i.p. LD50 of >10(5) bacteria, as did delta glnA ntrA and delta glnA ntrC strains, suggesting that glnA strains require an ntr-transcribed gene for full virulence. High-level transcription of the glutamine transport operon (glnHPQ) is dependent upon both ntrA and ntrC, as determined by glnHp-lacZ fusion measurements. Moreover, delta glnA glnH and delta glnA glnQ strains are attenuated, similar to delta glnA ntrA and delta glnA ntrC strains. These results reveal that access of S. typhimurium to host glutamine depends on the ntr system, which apparently is required for the transcription of the glutamine transport genes. The delta(glnA-ntrC) strain exhibited a reduced ability to survive within the macrophage cell line J774, identifying a potential host environment with low levels of glutamine. Finally, the delta(glnA-ntrC) strain, when inoculated at doses as low as 10 organisms, provided mice with protective immunity against challenge by the wild-type strain, demonstrating its potential use as a live vaccine.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
272.     ( 1996 )

Characterization of beta-lactamase gene blaPER-2, which encodes an extended-spectrum class A beta-lactamase.

Antimicrobial agents and chemotherapy 40 (3)
PMID : 8851581  :   PMC  :   PMC163168    
Abstract >>
Plasmidic extended-spectrum beta-lactamases of Ambler class A are mostly inactive against ceftibuten. Salmonella typhimurium JMC isolated in Argentina harbors a bla gene located on a plasmid (pMVP-5) which confers transferable resistance to oxyiminocephalosporins, aztreonam, and ceftibuten. The beta-lactamase PER-2 (formerly ceftibutenase-1; CTI-1) is highly susceptible to inhibition by clavulanate and is located at a pI of 5.4 after isoelectric focusing. The blaPER-2 gene was cloned and sequenced. The nucleotide sequence of a 2.2-kb insert in vector pBluescript includes an open reading frame of 927 bp. Comparison of the deduced amino acid sequence of PER-2 with those of other beta-lactamases indicates that PER-2 is not closely related to TEM or SHV enzymes (25 to 26% homology). PER-2 is most closely related to PER-1 (86.4% homology), an Ambler class A enzyme first detected in Pseudomonas aeruginosa. An enzyme with an amino acid sequence identical to that of PER-1, meanwhile, was found in various members of the family Enterobacteriaceae isolated from patients in Turkey. Our data indicate that PER-2 and PER-1 represent a new group of Ambler class A extended-spectrum beta-lactamases. PER-2 so far has been detected only in pathogens (S. typhimurium, Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis) isolated from patients in South America, while the incidence of PER-1-producing strains so far has been restricted to Turkey, where it occurs both in members of the family Enterobacteriaceae and in P. aeruginosa.
KeywordMeSH Terms
273.     ( 1996 )

Transcriptional activation of Salmonella typhimurium invasion genes by a member of the phosphorylated response-regulator superfamily.

Molecular microbiology 22 (4)
PMID : 8951818  :   DOI  :   10.1046/j.1365-2958.1996.d01-1719.x    
Abstract >>
The Salmonella typhimurium PhoP-repressed locus prgHIJK encodes components of a sec-independent type III secretion apparatus. This apparatus is composed of at least 17 proteins encoded on a 40 kb pathogenicity Island located at centisome 63 on the S. typhimurium chromosome. The secretion apparatus and some of its targets, SapB, SapC and SspD, are necessary for epithelial cell invasion. The transcription of many invasion genes, including prgHIJK, is coordinately activated by HilA, a transcription factor encoded within the pathogenicity island. In this report we identify sirA, a gene located outside the pathogenicity island that is essential for induction of prgHIJK and hilA transcription. sirA encodes a 234-amino-acid protein that is essential for S. typhimurium Ssp (Salmonella secreted protein) secretion and invasion and is similar to response regulators of two-component regulatory systems. sirA-mutant phenotypes could be suppressed by two DNA clones from unlinked loci, designated sirB and sirC. These data suggest that SirA may be phosphorylated in response to S. typhimurium sensing a mammalian microenvironment. Furthermore, SirA phosphorylation is predicted to initiate a cascade of transcription-factor synthesis which results in invasion-gene transcription, Ssp secretion, and bacterial invasion of epithelia.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
Transcriptional Activation
274.     ( 1996 )

Nucleotide sequence and characterization of the trbABC region of the IncI1 Plasmid R64: existence of the pnd gene for plasmid maintenance within the transfer region.

Journal of bacteriology 178 (6)
PMID : 8626273  :   DOI  :   10.1128/jb.178.6.1491-1497.1996     PMC  :   PMC177830    
Abstract >>
A 6.72-kb DNA sequence between the exc gene and the oriT operon within the transfer region of IncI1 plasmid R64 was sequenced and characterized. Three novel transfer genes, trbA, trbB, and trbC, were found in this region, along with the pnd gene responsible for plasmid maintenance. The trbABC genes appear to be organized into an operon located adjacent to the oriT operon in the opposite orientation. The trbA and trbC genes were shown to be indispensable for R64 plasmid transfer, while residual transfer activity was detected in the case of R64 derivatives carrying the trbB++ deletion mutation. The T7 RNA polymerase-promoter system revealed that the trbB gene produced a 43-kDa protein and the trbC gene produced an 85-kDa protein. The nucleotide sequence of the pnd gene is nearly identical to that of plasmid R483, indicating a function in plasmid maintenance. The plasmid stability test indicated that the mini-R64 derivatives with the pnd gene are more stably maintained in Escherichia coli cells under nonselective conditions than the mini-R64 derivatives without the pnd gene. It was also shown that the R64 transfer system itself is involved in plasmid stability to a certain degree. Deletion of the pnd gene from the tra+ mini-R64 derivative did not affect transfer frequency. DNA segments between the exc and trbA genes for IncI1 plasmids R64, Colb-P9, and R144 were compared in terms of their physical and genetic organization.
KeywordMeSH Terms
Conjugation, Genetic
Escherichia coli Proteins
Genes, Bacterial
Periplasmic Proteins
275.     ( 1993 )

Plasmid pKM101 encodes two nonhomologous antirestriction proteins (ArdA and ArdB) whose expression is controlled by homologous regulatory sequences.

Journal of bacteriology 175 (15)
PMID : 8393008  :   DOI  :   10.1128/jb.175.15.4843-4850.1993     PMC  :   PMC204937    
Abstract >>
The IncN plasmid pKM101 (a derivative of R46) encodes the antirestriction protein ArdB (alleviation of restriction of DNA) in addition to another antirestriction protein, ArdA, described previously. The relevant gene, ardB, was located in the leading region of pKM101, about 7 kb from oriT. The nucleotide sequence of ardB was determined, and an appropriate polypeptide was identified in maxicells of Escherichia coli. Like ArdA, ArdB efficiently inhibits restriction by members of the three known families of type I systems of E. coli and only slightly affects the type II enzyme, EcoRI. However, in contrast to ArdA, ArdB is ineffective against the modification activity of the type I (EcoK) system. Comparison of deduced amino acid sequences of ArdA and ArdB revealed only one small region of similarity (nine residues), suggesting that this region may be somehow involved in the interaction with the type I restriction systems. We also found that the expression of both ardA and ardB genes is controlled jointly by two pKM101-encoded proteins, ArdK and ArdR, with molecular weights of about 15,000 and 20,000, respectively. The finding that the sequences immediately upstream of ardA and ardB share about 94% identity over 218 bp suggests that their expression may be controlled by ArdK and ArdR at the transcriptional level. Deletion studies and promoter probe analysis of these sequences revealed the regions responsible for the action of ArdK and ArdR as regulatory proteins. We propose that both types of antirestriction proteins may play a pivotal role in overcoming the host restriction barrier by self-transmissible broad-host-range plasmids. It seems likely that the ardKR-dependent regulatory system serves in this case as a genetic switch that controls the expression of plasmid-encoded antirestriction functions during mating.
KeywordMeSH Terms
276.     ( 1993 )

Substrate-induced inactivation of the OXA2 beta-lactamase.

The Biochemical journal 295 (Pt 3) (N/A)
PMID : 8240304  :   DOI  :   10.1042/bj2950871     PMC  :   PMC1134642    
Abstract >>
The hydrolysis time courses of 22 beta-lactam antibiotics by the class D OXA2 beta-lactamase were studied. Among these, only three appeared to correspond to the integrated Henri-Michaelis equation. 'Burst' kinetics, implying branched pathways, were observed with most penicillins, cephalosporins and with flomoxef and imipenem. Kinetic parameters characteristic of the different phases of the hydrolysis were determined for some substrates. Mechanisms generally accepted to explain such reversible partial inactivations involving branches at either the free enzyme or the acyl-enzyme were inadequate to explain the enzyme behaviour. The hydrolysis of imipenem was characterized by the occurrence of two 'bursts', and that of nitrocefin by a partial substrate-induced inactivation complicated by a competitive inhibition by the hydrolysis product.
KeywordMeSH Terms
277.     ( 1996 )

Identification of a pathogenicity island required for Salmonella survival in host cells.

Proceedings of the National Academy of Sciences of the United States of America 93 (15)
PMID : 8755556  :   DOI  :   10.1073/pnas.93.15.7800     PMC  :   PMC38828    
Abstract >>
We have identified a region unique to the Salmonella typhimurium chromosome that is essential for virulence in mice. This region harbors at least three genes: two (spiA and spiB) encode products that are similar to proteins found in type III secretion systems, and a third (spiR) encodes a putative regulator. A strain with a mutation in spiA was unable to survive within macrophages but displayed wild-type levels of epithelial cell invasion. The culture supernatants of the spi mutants lacked a modified form of flagellin, which was present in the supernatant of the wild-type strain. This suggests that the Spi secretory apparatus exports a protease, or a protein that can alter the activity of a secreted protease. The "pathogenicity island" harboring the spi genes may encode the virulence determinants that set Salmonella apart from other enteric pathogens.
KeywordMeSH Terms
Genes, Bacterial
278.     ( 1993 )

Nucleotide sequence and characterization of the traABCD region of IncI1 plasmid R64.

Journal of bacteriology 175 (16)
PMID : 8349545  :   DOI  :   10.1128/jb.175.16.5035-5042.1993     PMC  :   PMC204969    
Abstract >>
A 3.6-kb BglII-SmaI segment of the transfer region of IncI1 plasmid R64drd-11 was sequenced and characterized. Analysis of the DNA sequence indicated the presence of four genes, traA, traB, traC, and traD, in this region. The expression of the traB, traC, and traD genes was examined by maxicell experiments and that of the traA gene was examined by constructing the traA-lacZ fusion gene. The introduction of frameshift mutations into the four genes indicated that the traB and traC genes are essential for conjugal transfer in liquid medium and on a solid surface. Both were also required for the formation of the thin pilus, which is the receptor for phages I alpha and PR64FS. Upstream of the traA gene, a promoter sequence for sigma 70 of E. coli RNA polymerase was identified by S1 nuclease mapping and primer extension experiments.
KeywordMeSH Terms
Bacterial Proteins
Escherichia coli Proteins
Membrane Proteins
279.     ( 1996 )

Molecular analysis of the scrA and scrB genes from Klebsiella pneumoniae and plasmid pUR400, which encode the sucrose transport protein Enzyme II Scr of the phosphotransferase system and a sucrose-6-phosphate invertase.

Molecular & general genetics : MGG 250 (2)
PMID : 8628219  :   DOI  :   10.1007/bf02174179    
Abstract >>
The Klebsiella pneumoniae genes scrA and scrB are indispensable for sucrose (Scr) utilisation. Gene scrA codes for an Enzyme IIScr (IIScr) transport protein of the phosphoenolpyruvate-dependent carbohydrate: phosphotransferase system (PTS), while scrB encodes a sucrose 6-phosphate specific invertase. A 3.7 kbscr AB DNA fragment has been cloned from K. pneumoniae and expressed in Escherichia coli. Its nucleotide sequence was determined and the coding regions for scrA (1371 bp) and scrB (1401 bp) were identified by genetic complementation, enzyme activity test and radiolabelling of the gene products. In addition, the nucleotide sequence of the scrB gene from conjugative plasmid pUR400 isolated from Salmonella typhimurium was also determined and errors in the previously published sequence of the scrA gene of pUR400 were corrected. Extensive similarity was found between the sequences of ScrA and other Enzymes II, as well as between the two invertases and other sucrose hydrolysing enzymes. Based on the analysis of seven IIScr proteins, a hypothetical model of the secondary structure of IIScr is proposed.
KeywordMeSH Terms
280.     ( 1996 )

The predicted amino acid sequence of the Salmonella typhimurium virulence gene mviAA(+) strongly indicates that MviA is a regulator protein of a previously unknown S. typhimurium response regulator family.

Infection and immunity 64 (6)
PMID : 8675354  :   PMC  :   PMC174083    
Abstract >>
The Salmonella typhimurium virulence gene mviA+ has a predicted amino acid sequence with homology to the N-terminal 112-amino-acid sequence of response regulator proteins. A previously described mutant allele (mviA), which restores virulence to avirulent LT2 strains, was shown to contain a point mutation which would be predicted to cause a single amino acid change, V-102-->G (W. H. Benjamin, Jr., J. Yother, P. Hall, and D. E. Briles, J. Exp. Med. 1,74:1073-1083, 1991). A comparison of the nucleotide sequence of mviA+ with that of the Escherichia coli and Salmonella typhi genes revealed a high degree of conservation.
KeywordMeSH Terms
Genes, Bacterial
281.     ( 1996 )

Salmonella typhimurium acrB-like gene: identification and role in resistance to biliary salts and detergents and in murine infection.

FEMS microbiology letters 135 (2��3��)
PMID : 8595853  :   DOI  :   10.1111/j.1574-6968.1996.tb07983.x    
Abstract >>
Salmonella serotype typhimurium transpositional mutants altered in resistance to biliary salts and detergents were isolated previously. We have characterized further the LX1054 mutant strain, the most sensitive of them. The chromosomal DNA segment flanking transposon insertion was cloned and sequenced. The highest level of identity was found for the acrB (formerly acrE) gene of Escherichia coli, a gene encoding a drug efflux pump of the Acr family. LX1054 exhibited a reduced capacity to colonize the intestinal tract. After passages in mice, the mutant strain lost the sensitive phenotype. In vitro, a resumption of growth appeared after 17 h of culture in medium with cholate or other tested biological or chemical detergents. Then, the acquired resistant phenotype seemed stable. The data suggested a role of S. typhimurium acrB-like gene in resistance to biliary salts and detergents and in mice intestinal colonization. However, the local and transient sensitivity observed in vivo, and the in vitro adaptations suggest that several detergent-resistance mechanisms operate in S. typhimurium.
KeywordMeSH Terms
Carrier Proteins
Escherichia coli Proteins
Genes, Bacterial
282.     ( 1993 )

Nucleotide sequence of a 13.9 kb segment of the 90 kb virulence plasmid of Salmonella typhimurium: the presence of fimbrial biosynthetic genes.

Molecular microbiology 8 (3)
PMID : 8100983  :   DOI  :   10.1111/j.1365-2958.1993.tb01599.x    
Abstract >>
The 90kb plasmid resident in Salmonella typhimurium confers increased virulence in mice by promoting the spread of infection after invasion of the intestinal epithelium. The nucleotide sequence of a 13.9kb segment of this plasmid known to encode an outer membrane protein related in sequence to components of fimbrial biosynthesis in enteric bacteria was determined. This cloned segment between the repB and repC replicon regions programmed expression of abundant surface fimbriae in Escherichia coli and S. typhimurium cells. A 7kb region contained seven open reading frames, the protein products of five of which were related in sequence to regulatory, structural, and assembly proteins of adherence fimbriae/pili, such as the P and K88 pili. These five genes and two adjacent ones which were not markedly related to proteins in the data bases comprise the pef (plasmid-encoded fimbriae) locus. Transposon TnphoA insertions in four genes in the pef locus (pefA, pefC, orf5 and orf6) resulted in active PhoA fusions and blocked or reduced the surface presentation of fimbriae, indicating that the proteins encoded by these four genes are translocated at least across the cytoplasmic membrane and contribute to formation of the fimbrial structure. The differences in genetic organization and protein sequence relatedness from other fimbrial gene clusters suggest that the pef locus might encode a novel type of fimbria. Between the pef and the repB loci, there were five open reading frames, one of which (orf8) gave rise to active PhoA fusions but was not necessary for fimbrial expression. Two of the other proteins were homologous to transcription regulatory proteins and a third was the rck gene, which encodes an outer membrane protein that confers complement resistance to serum-sensitive hosts.
KeywordMeSH Terms
Genes, Bacterial
283.     ( 1994 )

Surface exclusion gene of IncI1 plasmid R64: nucleotide sequence and analysis of deletion mutants.

Plasmid 32 (1)
PMID : 7991676  :   DOI  :   10.1006/plas.1994.1047    
Abstract >>
We have cloned and sequenced the exc gene determining surface exclusion of IncI1 plasmid R64. R64 exc gene is highly homologous to the R144 exc gene. The R64 exc gene may encode two proteins with 220 and 147 amino acid residues by inframe reinitiation of translation, as the R144 exc gene does, while the larger protein with 220 amino acids was shown to be essential for surface exclusion function. R64 derivatives carrying deletion or insertion mutations in the exc gene transferred at a frequency similar to that of wild-type R64, indicating that the exc gene is not essential for the transfer of R64.
KeywordMeSH Terms
Genes, Bacterial
284.     ( 1994 )

Intermediate filament-like network formed in vitro by a bacterial coiled coil protein.

The Journal of biological chemistry 269 (14)
PMID : 8144657  :  
Abstract >>
The TlpA protein encoded by the virulence plasmid of Salmonella enterica is an alpha-helical 371-amino acid protein possessing characteristics similar to eukaryotic coiled coil proteins (Koski, P., Saarilahti, H., Sukupolvi, S., Taira, S., Rikkonen, P., Osterlund, K., Hurme, R., and Rhen, M. (1992) J. Biol. Chem. 267, 12258-12265). In this paper we have investigated inter- and intramolecular associations and the morphology of structures formed by TlpA. Dynamics and temperature stability of TlpA dimers were studied by examining the feasibility and conditions in which TlpA would form an artificial heterodimer with its truncated derivative. Formation of heterodimers, bridged by Cu(2+)-catalyzed air oxidation of adjacent Cys residues, showed that TlpA dimers are dynamic chain exchanging structures at 37 degrees C, whereas they were nonexchanging at room temperature or on ice. Chemical cross-linking suggested higher order interaction between TlpA dimers. Electron microscopy studies revealed two levels of TlpA organization in vitro: thin filaments and rods, 2-5 nm in diameter, and a higher ordered filament network consisting of tonofilament-like formations with a diameter of 8-15 nm. Electron microscopy of thin-sectioned Escherichia coli over-producing TlpA showed an extraordinary intracellular assembly of proteinacious lamellae with a striated appearance and a 38-nm periodicity. This study describes for the first time a bacterial protein capable of organizing itself into an ordered and suspectedly dynamic intermediate filament-like architecture.
KeywordMeSH Terms
285.     ( 1994 )

Escherichia coli-Salmonella typhimurium hybrid nusA genes: identification of a short motif required for action of the lambda N transcription antitermination protein.

Journal of bacteriology 176 (5)
PMID : 8113180  :   DOI  :   10.1128/jb.176.5.1394-1404.1994     PMC  :   PMC205205    
Abstract >>
The Escherichia coli nusA gene, nusAEc, encodes an essential protein that influences transcription elongation. Derivatives of E. coli in which the Salmonella typhimurium nusA gene, nusASt, has replaced nusAEc are viable. Thus, NusASt can substitute for NusAEc in supporting essential bacterial activities. However, hybrid E. coli strains with the nusASt substitution do not effectively support transcription antitermination mediated by the N gene product of phage lambda. We report the DNA sequence of nusASt, showing that the derived amino acid sequence is 95% identical to the derived amino acid sequence of nusAEc. The alignment of the amino acid sequences reveals scattered single amino acid differences and one region of significant heterogeneity. In this region, called 449, NusAEc has four amino acids and NusASt has nine amino acids. Functional studies of hybrid nusA genes, constructed from nusAEc and nusASt, show that the 449 region of the NusAEc protein is important for lambda N-mediated transcription antitermination. A hybrid that has a substitution of the four E. coli codons for the nine S. typhimurium codons, but is otherwise nusASt, supports the action of the N antitermination protein. The 449 region and, presumably, adjacent sequences appear to compose a functional domain of NusAEc important for the action of the N transcription antitermination protein of phage lambda.
KeywordMeSH Terms
Peptide Elongation Factors
Recombination, Genetic
286.     ( 1994 )

The control region of the pdu/cob regulon in Salmonella typhimurium.

Journal of bacteriology 176 (17)
PMID : 8071226  :   DOI  :   10.1128/jb.176.17.5474-5482.1994     PMC  :   PMC196736    
Abstract >>
The pdu operon encodes proteins for the catabolism of 1,2-propanediol; the nearby cob operon encodes enzymes for the biosynthesis of adenosyl-cobalamin (vitamin B12), a cofactor required for the use of propanediol. These operons are transcribed divergently from distinct promoters separated by several kilobases. The regulation of the two operons is tightly integrated in that both require the positive activator protein PocR and both are subject to global control by the Crp and ArcA proteins. We have determined the DNA nucleotide sequences of the promoter-proximal portion of the pdu operon and the region between the pdu and cob operons. Four open reading frames have been identified, pduB, pduA, pduF, and pocR. The pduA and pduB genes are the first two genes of the pdu operon (transcribed clockwise). The pduA gene encodes a hydrophobic protein with 56% amino acid identity to a 10.9-kDa protein which serves as a component of the carboxysomes of several photosynthetic bacteria. The pduF gene encodes a hydrophobic protein with a strong similarity to the GlpF protein of Escherichia coli, which facilitates the diffusion of glycerol. The N-terminal end of the PduF protein includes a motif for a membrane lipoprotein-lipid attachment site as well as a motif characteristic of the MIP (major intrinsic protein) family of transmembrane channel proteins. We presume that the PduF protein facilitates the diffusion of propanediol. The pocR gene encodes the positive regulatory protein of the cob and pdu operons and shares the helix-turn-helix DNA binding motif of the AraC family of regulatory proteins. The mutations cobR4 and cobR58 cause constitutive, pocR-independent expression of the cob operon under both aerobic and anaerobic conditions. Evidence that each mutation is a deletion creating a new promoter near the normal promoter site of the cob operon is presented.
KeywordMeSH Terms
Aquaporins
Escherichia coli Proteins
Gene Expression Regulation, Bacterial
Operon
Regulatory Sequences, Nucleic Acid
Regulon
287.     ( 1994 )

Entry exclusion of the IncN plasmid pKM101 is mediated by a single hydrophilic protein containing a lipid attachment motif.

Plasmid 31 (2)
PMID : 8029323  :   DOI  :   10.1006/plas.1994.1017    
Abstract >>
The eex gene(s) of pKM101, which mediates that plasmid's entry exclusion phenotype, is located within a cluster of tra genes that are thought to encode a mating bridge. We have determined the DNA sequence of the region containing eex, including the points of insertion of two flanking tra::Tn5 insertions. Sequence analysis of this interval strongly suggests that it contains only one gene. This gene is transcribed in a clockwise direction on the circular map of pKM101 and appears to be translationally coupled to neighboring tra genes. A Tn5 insertion that causes a null eex mutation disrupts this ORF. Computer analysis of the predicted eex protein suggests that it may be exported and covalently modified by the addition of lipid moieties. eex was amplified by polymerase chain reaction, subcloned, and fused to the Escherichia coli lac promoter. The resulting plasmid was sufficient to mediate entry exclusion. The degree of exclusion was greatly enhanced by overexpression of this gene.
KeywordMeSH Terms
Escherichia coli Proteins
Fimbriae Proteins
Lipid Metabolism
Membrane Proteins
Plasmids
288. Post  DA, Hove-Jensen  B, Switzer  RL,     ( 1993 )

Characterization of the hemA-prs region of the Escherichia coli and Salmonella typhimurium chromosomes: identification of two open reading frames and implications for prs expression.

Journal of general microbiology 139 (2)
PMID : 7679718  :   DOI  :   10.1099/00221287-139-2-259    
Abstract >>
The prs gene, encoding phosphoribosylpyrophosphate synthetase, is preceded by a leader, which is 302 bp long in Escherichia coli and 417 bp in Salmonella typhimurium. A potential open reading frame (ORF) extends across the prs promoter and into the leader. The region between the prs coding region and an upstream gene (hemA) in E. coli and S. typhimurium was cloned, sequenced and shown to encode two ORFs of unknown function. ORF 1 encodes a 23 kDa protein and ORF 2 a 31 kDa protein, as observed by denaturing PAGE of extracts of cells bearing plasmids encoding the ORFs. Both ORFs are transcribed in the same direction as the prs gene with ORF 2 extending into the prs leader. Northern blot analysis showed that the prs message in E. coli was on 1.3 and 2.7 kb transcripts. The shorter transcript encoded the prs gene only, while the longer transcript also encoded the two ORFs. Thus, the prs gene is transcribed from two promoters, the first promoter (P1) originating upstream of ORF 1, and expressing the prs gene in a tricistronic operon and a second promoter (P2), located within the ORF 2 coding frame, which transcribes the prs gene only. The transcripts encoding prs only were 20 times as abundant as the tricistronic transcripts under all conditions examined. This was the case whether cells containing plasmid-encoded or only chromosomally encoded copies of the hemA-prs region were probed for these transcripts. Derepression of the prs gene upon pyrimidine starvation was shown to be due to an increase in the amount of message originating from the promoter P2.
KeywordMeSH Terms
Open Reading Frames
289.     ( 1994 )

Molecular characterization of an enterotoxin from Salmonella typhimurium.

Microbial pathogenesis 16 (2)
PMID : 8047004  :   DOI  :   10.1006/mpat.1994.1010    
Abstract >>
In this study, we describe the molecular and antigenic characteristics of a cloned enterotoxin from Salmonella typhimurium strain Q1. The full length Salmonella enterotoxin gene (stn), localized on a 2.8 kb ClaI/PstI DNA fragment, was cloned from a genomic library of Salmonella. Based on nucleotide sequence analysis, the stn gene contained 749 bp that would encode a protein having a molecular size of 29,073. The most unusual feature of the stn gene was the presence of a rare initiation codon (TTG) in lieu of the typical ATG codon, which required site-directed mutagenesis to confirm the precise initiation site. The expression of the stn gene in a bacteriophage T7 RNA polymerase/promoter system was enhanced by introducing a typical ATG start codon and an optimal Shine-Dalgarno sequence upstream of the stn gene by site-directed mutagenesis. The stn gene was located opposite the hydHG operon that regulates labile hydrogenase activity in Salmonella species and Escherichia coli. The overall amino acid sequence of the enterotoxin was quite dissimilar to any other published sequence, including cholera toxin or other adenylate cyclase-activating proteins. However, an intriguing similarity in a small region of the amino acid sequence of Stn was observed with portions of the amino acid sequences from several other protein toxins known to ADP-ribosylate host cell proteins. This region of homology may indicate a conserved motif, within the active site, that is involved in the stimulation of adenylate cyclase activity.
KeywordMeSH Terms
Genes, Bacterial
290. Bunny  KL, Hall  RM, Stokes  HW,     ( 1995 )

New mobile gene cassettes containing an aminoglycoside resistance gene, aacA7, and a chloramphenicol resistance gene, catB3, in an integron in pBWH301.

Antimicrobial agents and chemotherapy 39 (3)
PMID : 7793874  :   DOI  :   10.1128/aac.39.3.686     PMC  :   PMC162606    
Abstract >>
The multidrug resistance plasmid pBWH301 was shown to contain a sull-associated integron with five inserted gene cassettes, aacA7-catB3-aadB-oxa2-orfD, all of which can be mobilized by the integron-encoded DNA integrase. The aadB, oxa2, and orfD cassettes are identical to known cassettes. The aacA7 gene encodes a protein that is a member of one of the three known families of aminoglycoside acetyltransferases classified as AAC(6')-I. The chloramphenicol acetyltransferase encoded by the catB3 gene is closely related to members of a recently identified family of chloramphenicol acetyltransferases. The catB3 gene displays a relatively high degree of sequence identity to a chromosomally located open reading frame in Pseudomonas aeruginosa, and this may represent evidence for the acquisition by a cassette of a chromosomal gene.
KeywordMeSH Terms
291. Pohlman  RF, Genetti  HD, Winans  SC,     ( 1994 )

Common ancestry between IncN conjugal transfer genes and macromolecular export systems of plant and animal pathogens.

Molecular microbiology 14 (4)
PMID : 7891554  :   DOI  :   10.1111/j.1365-2958.1994.tb01304.x    
Abstract >>
The DNA sequence of a cluster of pKM101 conjugal transfer genes was determined and aligned with the genetic map of the plasmid. Eighteen genes were identified, at least eight and probably 11 of which are required for efficient conjugation. These tra genes are homologous to and colinear with genes found in the virB operon of Agrobacterium tumefaciens Ti plasmids. Seven pKM101 tra genes are also homologous to ptl genes of Bordetella pertussis, which direct the export of pertussis toxin. We used TnphoA to construct translational fusions between pKM101 genes and the Escherichia coli phoA gene, which encodes alkaline phosphatase, and provide evidence that at least 11 of the 18 genes are either fully or partially exported from the cytoplasm.
KeywordMeSH Terms
Biological Evolution
Conjugation, Genetic
Genes, Bacterial
292. Bäumler  AJ, Heffron  F,     ( 1995 )

Identification and sequence analysis of lpfABCDE, a putative fimbrial operon of Salmonella typhimurium.

Journal of bacteriology 177 (8)
PMID : 7721701  :   DOI  :   10.1128/jb.177.8.2087-2097.1995     PMC  :   PMC176853    
Abstract >>
A chromosomal region present in Salmonella typhimurium but absent from related species was identified by hybridization. A DNA probe originating from 78 min on the S. typhimurium chromosome hybridized with DNA from Salmonella enteritidis, Salmonella heidelberg, and Salmonella dublin but not with DNA from Salmonella typhi, Salmonella arizonae, Escherichia coli, and Shigella serotypes. Cloning and sequence analysis revealed that the corresponding region of the S. typhimurium chromosome encodes a fimbrial operon. Long fimbriae inserted at the poles of the bacterium were observed by electron microscopy when this fimbrial operon was introduced into a nonpiliated E. coli strain. The genes encoding these fimbriae were therefore termed lpfABCDE, for long polar fimbriae. Genetically, the lpf operon was found to be most closely related to the fim operon of S. typhimurium, both in gene order and in conservation of the deduced amino acid sequences.
KeywordMeSH Terms
Genes, Bacterial
Operon
293.     ( 1994 )

Integrons found in different locations have identical 5' ends but variable 3' ends.

Journal of bacteriology 176 (20)
PMID : 7929000  :   DOI  :   10.1128/jb.176.20.6286-6294.1994     PMC  :   PMC196970    
Abstract >>
The positions of the outer boundaries of the 5'- and 3'-conserved segment sequences of integrons found at several different locations have been determined. The position of the 5' end of the 5'-conserved segment is the same for six independently located integrons, In1 (R46), In2 (Tn21), In3 (R388), In4 (Tn1696), In5 (pSCH884), and In0 (pVS1). However, the extent of the 3'-conserved segment differs in each integron. The sequences of In2 and In0 diverge first from the conserved sequence, and their divergence point corresponds to the 3'-conserved segment endpoint defined previously (H.W. Stokes and R.M. Hall, Mol. Microbiol. 3:1669-1683, 1989), which now represents the endpoint of a 359-base deletion in In0 and In2. The sequence identity in In3, In1, In4, and In5 extends beyond this point, but each sequence diverges from the conserved sequence at a different point within a short region. Insertions of IS6100 were identified adjacent to the end of the conserved region in In1 and 123 bases beyond the divergence point of In4. These 123 bases are identical to the sequence found at the mer end of the 11.2-kb insertion in Tn21 but are inverted. In5 and In0 are bounded by the same 25-base inverted repeat that bounds the 11.2-kb insert in Tn21, and this insert now corresponds to In2. However, while In0, In2, and In5 have features characteristic of transposable elements, differences in the structures of these three integrons and the absence of evidence of mobility currently preclude the identification of all of the sequences associated with a functional transposon of this type.
KeywordMeSH Terms
294. Yoshioka  K, Aizawa  S, Yamaguchi  S,     ( 1995 )

Flagellar filament structure and cell motility of Salmonella typhimurium mutants lacking part of the outer domain of flagellin.

Journal of bacteriology 177 (4)
PMID : 7860589  :   DOI  :   10.1128/jb.177.4.1090-1093.1995     PMC  :   PMC176707    
Abstract >>
We have isolated spontaneous mutants of Salmonella typhimurium which can swim in the presence of antifilament antibodies. The molecular masses of flagellins isolated from these mutants were smaller than that (52 kDa) of wild-type flagellin. Two mutants which produced the smallest flagellins (42 and 41 kDa) were selected, and the domain structures of the flagellins were analyzed by trypsin digestion and then subjected to amino acid sequencing. The two flagellins have deletions at Ala-204 to Lys-292 and Thr-183 to Lys-279, respectively. These deleted parts belong to the outer domain (D3) of flagellin, which is believed to be at the surface of the filament. These mutant filaments aggregated side by side in the presence of salt, resulting in disordered motility.
KeywordMeSH Terms
295. Li  J, Ochman  H, Groisman  EA, Boyd  EF, Solomon  F, Nelson  K, Selander  RK,     ( 1995 )

Relationship between evolutionary rate and cellular location among the Inv/Spa invasion proteins of Salmonella enterica.

Proceedings of the National Academy of Sciences of the United States of America 92 (16)
PMID : 7638176  :   DOI  :   10.1073/pnas.92.16.7252     PMC  :   PMC41317    
Abstract >>
For 21 strains of Salmonella enterica, nucleotide sequences were obtained for three invasion genes, spaO, spaP, and spaQ, of the chromosomal inv/spa complex, the products of which form a protein export system required for entry of the bacteria into nonphagocytic host cells. These genes are present in all eight subspecies of the salmonellae, and homologues occur in a variety of other bacteria, including the enteric pathogens Shigella and Yersinia, in which they are plasmid borne. Evolutionary diversification of the invasion genes among the subspecies of S. enterica has been generally similar in pattern and average rate to that of housekeeping genes. However, the range of variation in evolutionary rate among the invasion genes is unusually large, and there is a relationship between the evolutionary rate and cellular location of the invasion proteins, possibly reflecting diversifying selection on exported proteins in adaptation to variable host factors in extracellular environments. The SpaO protein, which is hypervariable in S. enterica and exhibits only 24% sequence identity with its homologues in Shigella and Yersinia, is secreted. In contrast, the membrane-associated proteins SpaP, SpaQ, and InvA are weakly polymorphic and have > 60% sequence identity with the corresponding proteins of other enteric bacteria. Acquisition of the inv/spa genes may have been a key event in the evolution of the salmonellae as pathogens, following which the invention of flagellar phase shifting facilitated niche expansion to include warm-blooded vertebrates.
KeywordMeSH Terms
Adhesins, Bacterial
Antigens, Bacterial
Biological Evolution
Membrane Glycoproteins
Membrane Proteins
296. Sacerdot  C, Dessen  P, Hershey  JW, Plumbridge  JA, Grunberg-Manago  M,     ( 1984 )

Sequence of the initiation factor IF2 gene: unusual protein features and homologies with elongation factors.

Proceedings of the National Academy of Sciences of the United States of America 81 (24)
PMID : 6096856  :   DOI  :   10.1073/pnas.81.24.7787     PMC  :   PMC392237    
Abstract >>
The gene for protein synthesis initiation factor IF2 in Escherichia coli, infB, is located downstream from nusA on the same operon. We sequenced about 3 kilobases of DNA beginning within nusA and including the entire infB structural gene plus another 392 bases downstream. This region contains no obvious strong promoter signals, but a possible transcriptional termination or pausing site occurs downstream from infB. The putative initiator codon for IF2 alpha (97,300 daltons) is AUG; that for IF2 beta (79,700 daltons) is GUG, located 471 bases downstream in the same reading frame. The codon usage for IF2 is typical of other highly expressed proteins in E. coli and suggests that IF2 mRNA is efficiently translated. IF2 alpha contains two adjacent regions (residues 104-155 and 167-214) that are rich in alanine and charged amino acids and that show striking periodicities in their sequences. These regions may alternate between flexible and helical conformations, thereby drawing together the NH2-terminal and COOH-terminal globular domains of the factor as IF2 interacts with ribosomes or tRNA. Certain regions of the DNA and protein sequences of IF2 share strong homologies with elongation factor EF-Tu and lesser homology with EF-G. In particular, a region of EF-Tu implicated in GTP binding contains sequences and secondary structure that are conserved in IF2. The homologies indicate that the genes for IF2 and the elongation factors are derived at least in part from a common ancestor.
KeywordMeSH Terms
Genes
Genes, Bacterial
297. Gemmill  RM, Tripp  M, Friedman  SB, Calvo  JM,     ( 1984 )

Promoter mutation causing catabolite repression of the Salmonella typhimurium leucine operon.

Journal of bacteriology 158 (3)
PMID : 6327652  :   PMC  :   PMC215533    
Abstract >>
Two mutations that affect expression of the Salmonella typhimurium leu operon were investigated. leu operon DNA from these mutant strains was cloned, and nucleotide sequences of the leu control regions were determined. leu-500, which eliminates expression of all four leu genes simultaneously, is a point mutation in the -10 region of the leu promoter. leu-2012 is a point mutation within the -35 region of the leu promoter. leu-2012 suppressed leucine auxotrophy caused by leu-500 only when the medium contained a carbon source that does not cause catabolite repression. A cya mutation (adenylate cyclase deficiency) introduced into the leu-500 leu-2012 strain caused leu enzymes to be made only if cAMP was supplied exogenously. A leu-500 leu-2012 strain containing a crp mutation (cAMP receptor protein deficiency), on the other hand, could not make leu enzymes even in the presence of cAMP. In vitro transcription experiments demonstrated that the leu-2012 mutation created a new transcription initiation site. RNA polymerase utilized this site in vitro in the absence of added cAMP receptor protein and cAMP.
KeywordMeSH Terms
Genes
Genes, Bacterial
Mutation
Operon
Suppression, Genetic
298. Dammel  CS, Noller  HF,     ( 1995 )

Suppression of a cold-sensitive mutation in 16S rRNA by overexpression of a novel ribosome-binding factor, RbfA.

Genes & development 9 (5)
PMID : 7535280  :   DOI  :   10.1101/gad.9.5.626    
Abstract >>
A novel 15-kDa protein, RbfA, has been identified by virtue of its ability to act as a high copy suppressor of a previously characterized dominant cold-sensitive mutation (C23U) in 16S rRNA. RbfA is found associated with free 30S ribosomal subunits, but not with 70S ribosomes or polysomes, and is essential for maximal cell growth, particularly at low temperatures. Cells lacking RbfA in a wild-type rRNA background exhibit a cold-sensitive phenotype that is strikingly similar to that of the cold-sensitive C23U rRNA mutant. The observed patterns of allele specificity of suppression and synthetic lethality in cells containing an RbfA knockout in combination with various 16S rRNA mutations suggests that RbfA interacts with the 5'-terminal helix region of 16S rRNA, possibly during a late step of 30S maturation.
KeywordMeSH Terms
Escherichia coli Proteins
Ribosomal Proteins
Suppression, Genetic
299. Joyner  SS, Fling  ME, Stone  D, Baccanari  DP,     ( 1984 )

Characterization of an R-plasmid dihydrofolate reductase with a monomeric structure.

The Journal of biological chemistry 259 (9)
PMID : 6371010  :  
Abstract >>
A plasmid-encoded dihydrofolate reductase that originated in a clinical isolate of Salmonella typhimurium (phage type 179) moderately resistant to trimethoprim has been isolated and characterized. The dihydrofolate reductase (called type III) was purified to homogeneity using a combination of gel filtration, hydrophobic chromatography, and methotrexate affinity chromatography. Polyacrylamide gel electrophoresis under denaturing and nondenaturing conditions indicated that the enzyme is a 16,900 molecular weight monomeric protein. Kinetic analyses showed that trimethoprim is a relatively tight binding inhibitor (Ki = 19 nM) competitive with dihydrofolate. The enzyme is also extremely sensitive to methotrexate inhibition (Ki = 9 pM) and has a high affinity for dihydrofolate (Km = 0.4 microM). The sequence of the first 20 NH2-terminal residues of the protein shows 50% homology with the trimethoprim-sensitive chromosomal Escherichia coli dihydrofolate reductase and suggests that the two enzymes may be closely related. This is the first example of a plasmid encoding for a monomeric dihydrofolate reductase only moderately resistant to trimethoprim, and a resistance mechanism, dependent in part on the high dihydrofolate affinity of the type III enzyme, is proposed.
KeywordMeSH Terms
R Factors
300. Holland  S, Dale  JW,     ( 1984 )

Improved purification and characterization of the OXA-2 beta-lactamase.

The Biochemical journal 224 (3)
PMID : 6335398  :   DOI  :   10.1042/bj2241009     PMC  :   PMC1144540    
Abstract >>
An improved and scaled-up procedure has been developed for purifying the OXA-2 plasmid-mediated beta-lactamase. This has enabled us to improve the characterization of this enzyme, including a revised determination of its amino acid composition and the sequence of the N-terminal region of the protein.
KeywordMeSH Terms
301. Simon  M, Zieg  J, Silverman  M, Mandel  G, Doolittle  R,     ( 1980 )

Phase variation: evolution of a controlling element.

Science (New York, N.Y.) 209 (4463)
PMID : 6251543  :   DOI  :   10.1126/science.6251543    
Abstract >>
Phase variation in bacteria is regulated by homologous recombination at a specific DNA site. This recombinational event causes the inversion of a 970-base-pair DNA sequence that includes the promoter necessary for transcription of a flagellar gene. The invertible segment is flanked by two sites that are necessary for the inversion and contains a gene (hin) whose product mediates the inversion event. The hin gene shows extensive homology with the TnpR gene carried on the Tn3 transposon. It is also homologous with the gin gene carried on bacteriophage mu. These relationships suggest that the phase variation system may have evolved by the association of a transposon with a resident gene and the subsequent specialization of these elements to regulate flagellar antigen expression.
KeywordMeSH Terms
DNA Transposable Elements
302. Bernardi  A, Bernardi  F,     ( 1984 )

Complete sequence of pSC101.

Nucleic acids research 12 (24)
PMID : 6096829  :   DOI  :   10.1093/nar/12.24.9415     PMC  :   PMC320470    
Abstract >>
We have completed the pSC101 sequence. The coding capacities of the newly sequenced regions show the presence of two large open reading frames close to the oriT region. Their size and localization suggest that these polypeptide chains could be involved in the transfer process of pSC101.
KeywordMeSH Terms
Plasmids
303. Sweet  GD, Kay  CM, Kay  WW,     ( 1984 )

Tricarboxylate-binding proteins of Salmonella typhimurium. Purification, crystallization, and physical properties.

The Journal of biological chemistry 259 (3)
PMID : 6141166  :  
Abstract >>
Citrate transport in Salmonella typhimurium involves inducible periplasmic components. Two forms of a tricarboxylate-binding protein, C1 and C2, were isolated, in high yield, from the periplasm of a cyclic AMP phosphodiesterase mutant. These immunologically cross-reactive Mr = 29,000 proteins were crystallized using ammonium sulfate. CD measurements indicated considerable secondary structure: 24% a helix, and 12% beta structure. The amino acid compositions of C1 and C2 were identical. The NH2-terminal sequence of C1 was determined; C2 was found to have a blocked NH2 terminus (pyroglutamate). C1 and C2 are products of the same gene (Somers, J. M., and Kay, W. W. (1983) Mol. Gen. Genet. 190, 20-26). C1 and C2 bound a variety of citrate analogues and organic acids, with a predominant specificity for tricarboxylates (citrate KD 1.4 X 10(-7) M), and both required a deprotonated central carboxyl group for binding. Citrate was not bound to C protein as either a salt or metal ion complex.
KeywordMeSH Terms
304. Burns  DM, Burger  MJ, Beacham  IR,     ( 1995 )

Silent genes in bacteria: the previously designated 'cryptic' ilvHI locus of 'Salmonella typhimurium LT2' is active in natural isolates.

FEMS microbiology letters 131 (2)
PMID : 7557326  :   DOI  :   10.1111/j.1574-6968.1995.tb07772.x    
Abstract >>
Gene ilvG in Escherichia coli K-12 and ilvI in 'Salmonella typhimurium LT2' (S. enterica serotype Typhimurium, strain LT2) are inactive due to frameshift or nonsense mutations, respectively. These inactive genes have been suggested to be part of 'cryptic' genetic systems which are defined as being of long-term regulatory and evolutionary significance. We have shown that the nonsense mutation in ilvI is present only in derivatives of the laboratory strain 'S. typhimurium LT2'. All natural isolates of Salmonella examined have an arginine codon at the corresponding location of their ilvI sequences. Further, two randomly selected natural isolates of serotype Typhimurium are shown to each have an active ALS III isozyme. Our findings strongly suggest that the only Salmonella strains which lack a functional ilvHI locus are LT2 isolates. We suggest that the mutations leading to inactivation of both ilvI in 'S. typhimurium LT2' and ilvG in E. coli K-12 are more likely to have been acquired during laboratory storage and/or cultivation, rather than representing cryptic systems of gene regulation.
KeywordMeSH Terms
Genes, Bacterial
305. Gensberg  K, Jin  YF, Piddock  LJ,     ( 1995 )

A novel gyrB mutation in a fluoroquinolone-resistant clinical isolate of Salmonella typhimurium.

FEMS microbiology letters 132 (1��2��)
PMID : 7590165  :   DOI  :   10.1111/j.1574-6968.1995.tb07810.x    
Abstract >>
In order to study the role of gyrB in antibiotic resistance in post-ciprofloxacin therapy fluoroquinolone-resistant clinical isolates of Salmonella typhimurium, plasmid pBP548, which contains the Escherichia coli gyrB gene, was used in complementation studies. In a heterodiploid strain, the wild-type (quinolone sensitive) allele is dominant over the resistant allele therefore, eleven clinical isolates were complemented with gyrB encoded on pBP548. Only one transformant, L18pBP548, exhibited increased susceptibility to the quinolones nalidixic acid, ciprofloxacin and sparfloxacin. The amino acid sequence of the gyrase B protein from a wild-type and the pre-therapy S. typhimurium (deduced from the nucleotide sequence) was identical to that of E. coli from codons 436 to 470; however, a point mutation was identified in codon 463 of gyrB of the quinolone-resistant post-therapy isolate L18, giving rise to an amino acid substitution of serine to tyrosine.
KeywordMeSH Terms
Fluoroquinolones
Mutation
306. Bastin  DA, Stevenson  G, Brown  PK, Haase  A, Reeves  PR,     ( 1993 )

Repeat unit polysaccharides of bacteria: a model for polymerization resembling that of ribosomes and fatty acid synthetase, with a novel mechanism for determining chain length.

Molecular microbiology 7 (5)
PMID : 7682279  :   DOI  :   10.1111/j.1365-2958.1993.tb01163.x    
Abstract >>
We report the identification and sequence from Escherichia coli and Salmonella enterica strains of the cld gene, encoding the chain-length determinant (CLD) which confers a modal distribution of chain length on the O-antigen component of lipopolysaccharide (LPS). The distribution of chain lengths in the absence of this gene fits a model in which as the chain is extended there is a constant probability of 0.165 of transfer of growing chain to LPS core, with termination of chain extension. The data for E. coli O111 fit a model in which the CLD reduces this probability for short chains and increases it to 0.4 for longer chains, leading to a reduced number of short chain molecules but an increase in numbers of longer molecules and transfer of essentially all molecules by chain length 21. We put forward a model for O-antigen polymerase which resembles the ribosome and fatty acid synthetase in having two sites, with the growing chain being transferred from a D site onto the new unit at the R site to extend the chain and then back to the D site to repeat the process. It is proposed that the CLD protein and polymerase form a complex which has two states: 'E' facilitating extension and 'T' facilitating transfer to core. The complex is postulated to enter the E state as O-antigen polymerization starts, and to shift to the T state after a predetermined time, the CLD acting as a molecular clock. The CLD is not O-antigen or species-specific but the modal value does depend on the source of the cld gene.
KeywordMeSH Terms
Escherichia coli Proteins
Models, Biological
307. Lisitsyn  NA, Monastyrskaya  GS, Sverdlov  ED,     ( 1988 )

Genes coding for RNA polymerase beta subunit in bacteria. Structure/function analysis.

European journal of biochemistry 177 (2)
PMID : 3056723  :   DOI  :   10.1111/j.1432-1033.1988.tb14385.x    
Abstract >>
The nucleotide sequence of the rpoB gene of Salmonella typhimurium has been determined in this work. It was compared with known sequences of the gene from other sources and the conservative regions were detected. This allowed some interesting conclusions to be made about the distribution of the functional domains in bacterial RNA polymerase and about the three-dimensional structure of its beta subunit.
KeywordMeSH Terms
Genes, Bacterial
308. Tay  MYF, Pathirage  S, Chandrasekaran  L, Wickramasuriya  U, Sadeepanie  N, Waidyarathna  KDK, Liyanage  LDC, Seow  KLG, Hendriksen  RS, Takeuchi  MT, Schlundt  J,     ( 2019 )

Whole-Genome Sequencing Analysis of Nontyphoidal Salmonella enterica of Chicken Meat and Human Origin Under Surveillance in Sri Lanka.

Foodborne pathogens and disease 16 (7)
PMID : 31099590  :   DOI  :   10.1089/fpd.2018.2604     PMC  :   PMC6653781    
Abstract >>
A total of 73 nontyphoidal Salmonella enterica isolates, 33 from raw chicken meat and 40 from routine clinical specimens, were collected between 2015 and 2017 from eight cities in Sri Lanka for a pilot study of whole-genome sequencing for Salmonella surveillance. The isolates were characterized by conventional serotyping and whole-genome sequencing. The raw sequenced data were assembled and analyzed to predict Salmonella serotypes, determine sequence type (ST) profiles of genome and plasmid, and identify plasmid replicon sequences and antimicrobial resistance (AMR) genes. The most common serovar isolated from chicken meat was Salmonella enterica serovar Agona of ST13 (n = 16), in contrast to Salmonella enterica serovar Enteritidis of ST11 (n = 21) in human. Salmonella enterica serovar Corvallis is the only serovar that was overlapping between human and chicken meat. The level of agreement between serotyping and serotype prediction results was 100%. Among the 33 chicken isolates, multidrug resistance (MDR) was observed in five isolates, including two Salmonella enterica serovar Kentucky ST314, which harbored six different classes of AMR determinants. Among the 40 human isolates, MDR was detected in two Salmonella enterica serovar Chester (ST2063) isolates containing five different antibiotic classes of AMR determinants. Out of 73 isolates, the only human Salmonella enterica serovar Typhimurium strain of ST36 was found to possess extended-spectrum beta-lactamase (ESBL) gene, blaCTX-M-15, and it was positive for ESBL production. In summary, this study identified S. enterica serovars that were dominating in chicken meat and human and showed the genomic differences among the chicken meat and human strains. It should be noted that the limited number of isolates and sampling at a different time period means that thorough source attribution is not possible. To the best of our knowledge, this is the first report on the use of whole-genome sequencing analysis of nontyphoidal S. enterica isolated from chicken meat and human in Sri Lanka.
KeywordMeSH Terms
CTX-M-15
Salmonella enterica
Sri Lanka
chicken meat
human
surveillance
whole genome sequencing
CTX-M-15
Salmonella enterica
Sri Lanka
chicken meat
human
surveillance
whole genome sequencing
309. Selker  E, Yanofsky  C,     ( 1979 )

Nucleotide sequence of the trpC-trpB intercistronic region from Salmonella typhimurium.

Journal of molecular biology 130 (2)
PMID : 381671  :   DOI  :   10.1016/0022-2836(79)90422-4    
Abstract >>
N/A
KeywordMeSH Terms
DNA, Bacterial
310. Haughn  GW, Wessler  SR, Gemmill  RM, Calvo  JM,     ( 1986 )

High A + T content conserved in DNA sequences upstream of leuABCD in Escherichia coli and Salmonella typhimurium.

Journal of bacteriology 166 (3)
PMID : 3519576  :   DOI  :   10.1128/jb.166.3.1113-1117.1986     PMC  :   PMC215239    
Abstract >>
The nucleotide sequence of over 800 base pairs of DNA upstream of leuP was determined for Escherichia coli and Salmonella typhimurium. In both of these enteric bacteria, approximately 500 base pairs of A + T-rich sequences separates leuP from an upstream open reading frame. Although these A + T-rich sequences share little homology, the distribution of A + T base pairs within the region is strikingly conserved. Deletion of the A + T-rich sequences upstream of the E. coli leu operon does not markedly affect the strength of the leu promoter in vivo.
KeywordMeSH Terms
311. Liljeström  P, Luokkamäki  M, Palva  ET,     ( 1987 )

Isolation and characterization of a substitution mutation in the ompR gene of Salmonella typhimurium LT2.

Journal of bacteriology 169 (1)
PMID : 3539926  :   DOI  :   10.1128/jb.169.1.438-441.1987     PMC  :   PMC211790    
Abstract >>
The expression of the genes ompC and ompF encoding major outer membrane proteins is dependent on the ompR-envZ operon. Here we describe the isolation and characterization of an ompR mutation, a single-base-pair change, that results in an Arg-to-Cys substitution. When present in multiple copies, the mutant allele conferred a dominant OmpC- OmpF+ phenotype. Furthermore, the mutant allele exhibited allele-specific negative complementation with other ompR mutations. This ability, together with its dominant character, suggested that the OmpR protein is capable of multimerization.
KeywordMeSH Terms
Genes, Bacterial
Mutation
312. Kubo  A, Kusukawa  A, Komano  T,     ( 1988 )

Nucleotide sequence of the rci gene encoding shufflon-specific DNA recombinase in the IncI1 plasmid R64: homology to the site-specific recombinases of integrase family.

Molecular & general genetics : MGG 213 (1)
PMID : 3065610  :   DOI  :   10.1007/bf00333394    
Abstract >>
Shufflon is a novel type of DNA rearrangement in which four DNA segments are flanked by seven 19-bp repeat sequences. The site-specific recombination between any inverted repeats results in an inversion of the DNA segment(s) either independently or in groups. The recombination is mediated by a gene designated rci. We have determined the nucleotide sequence of the rci gene and found that it encodes a basic protein with 384 amino acid residues. The rci gene was fused with lacZ and its gene product was identified by Western blot analysis. The Rci protein shows regional homologies to the site-specific recombinases encoded by the bacteriophage genomes, including those of lambda, phi 80, P22, P2, 186, P4 and P1.
KeywordMeSH Terms
Genes
Genes, Bacterial
Plasmids
313. Dale  JW, Godwin  D, Mossakowska  D, Stephenson  P, Wall  S,     ( 1985 )

Sequence of the OXA2 beta-lactamase: comparison with other penicillin-reactive enzymes.

FEBS letters 191 (1)
PMID : 3876949  :   DOI  :   10.1016/0014-5793(85)80989-3    
Abstract >>
The nucleotide sequence of the unusual plasmid-mediated OXA2 beta-lactamase is presented, and compared with other beta-lactamases. The OXA2 enzyme has similar features at the presumed active site, but no other significant regions of homology with other penicillin-reactive enzymes. The active site homology may therefore represent convergent evolution of otherwise dissimilar genes.
KeywordMeSH Terms
314. Lee  HS, Lee  S, Kim  JS, Lee  HR, Shin  HC, Lee  MS, Jin  KS, Kim  CH, Ku  B, Ryu  CM, Kim  SJ,     ( 2018 )

Structural and Physiological Exploration of Salmonella Typhi YfdX Uncovers Its Dual Function in Bacterial Antibiotic Stress and Virulence.

Frontiers in microbiology 9 (N/A)
PMID : 30692978  :   DOI  :   10.3389/fmicb.2018.03329     PMC  :   PMC6339873    
Abstract >>
YfdX is a prokaryotic protein encoded by several pathogenic bacteria including Salmonella enterica serovar Typhi, which causes one of the most fatal infectious diseases, typhoid fever. YfdX is a product of the yfdXWUVE operon and is known to be under the control of EvgA, a regulator protein controlling the expression of several proteins involved in response to environmental stress, in Escherichia coli. Nevertheless, unlike other proteins encoded by the same operon, the structural and physiological aspects of YfdX have been poorly characterized. Here, we identified a previously unknown pH-dependent stoichiometric conversion of S. Typhi YfdX between dimeric and tetrameric states; this conversion was further analyzed via determining its structure by X-ray crystallography at high resolution and by small-angle X-ray scattering in a solution state and via structure-based mutant studies. Biologically, YfdX was proven to be critically involved in Salmonella susceptibility to two �]-lactam antibiotics, penicillin G and carbenicillin, as bacterial growth significantly impaired by its deficiency upon treatment with each of the two antibiotics was recovered by chromosomal complementation. Furthermore, by using Galleria mellonella larvae as an in vivo model of Salmonella infection, we demonstrated that Salmonella virulence was remarkably enhanced by YfdX deficiency, which was complemented by a transient expression of the wild-type or dimeric mutant but not by that of the monomeric mutant. The present study work provides direct evidence regarding the participation of YfdX in Salmonella antibiotic susceptibility and in the modulation of bacterial virulence, providing a new insight into this pathogen's strategies for survival and growth.
KeywordMeSH Terms
STY3178
Salmonella Typhi
YfdX
antibiotics susceptibility
virulence
315. Barcak  GJ, Wolf  RE,     ( 1988 )

Comparative nucleotide sequence analysis of growth-rate-regulated gnd alleles from natural isolates of Escherichia coli and from Salmonella typhimurium LT-2.

Journal of bacteriology 170 (1)
PMID : 3275621  :   DOI  :   10.1128/jb.170.1.372-379.1988     PMC  :   PMC210652    
Abstract >>
A comparative study of gnd genes from Escherichia coli strains isolated from natural populations and laboratory strains and from Salmonella typhimurium was undertaken. In the accompanying paper (G. J. Barcak and R. E. Wolf, Jr., J. Bacteriol. 170:365-371, 1988), we showed that the growth-rate-dependent regulation of gnd expression was conserved among four natural E. coli isolates and E. coli B/r in a manner qualitatively similar to that of the gene from E. coli K-12. Here, we report the DNA sequence of the 5' regulatory region and the first 125 codons of the structural gene for the five E. coli gnd genes and the gnd gene from S. typhimurium LT-2. The sequences differed from one another by 5% on the average. All sequences defined putative secondary structures of the mRNA leader, which were previously proposed to be important in the regulation of the K-12 gene. In addition, a sequence between codons 69 and 74, which is highly complementary to the ribosome-binding site of the mRNA, was conserved in all the genes. The sequence data are discussed with respect to potential regulatory consequences.
KeywordMeSH Terms
Alleles
316. Gibson  MM, Ellis  EM, Graeme-Cook  KA, Higgins  CF,     ( 1987 )

OmpR and EnvZ are pleiotropic regulatory proteins: positive regulation of the tripeptide permease (tppB) of Salmonella typhimurium.

Molecular & general genetics : MGG 207 (1)
PMID : 3037276  :   DOI  :   10.1007/bf00331499    
Abstract >>
The tppB locus of Salmonella typhimurium encodes the anaerobically-induced tripeptide permease. We have demonstrated that expression of tppB requires the function of the ompR and envZ gene products, originally identified as positive regulatory proteins required for the osmotic regulation of porin expression. Significantly, tppB expression is not osmotically regulated. We have also identified three additional genes whose expression depends on OmpR. Thus OmpR and EnvZ serve a more general regulatory role than has previously been supposed. This study provides the first detailed genetic analysis of the ompB locus of S. typhimurium.
KeywordMeSH Terms
Gene Expression Regulation
317. Komano  T, Toyoshima  A, Morita  K, Nisioka  T,     ( 1988 )

Cloning and nucleotide sequence of the oriT region of the IncI1 plasmid R64.

Journal of bacteriology 170 (9)
PMID : 3045094  :   DOI  :   10.1128/jb.170.9.4385-4387.1988     PMC  :   PMC211456    
Abstract >>
The nucleotide sequence at the oriT region of the IncI1 plasmid R64 was determined. A recombinant plasmid carrying a 141-base-pair R64 sequence was mobilized with a normal frequency, while a plasmid carrying only 44 base pairs of this R64 sequence was mobilized with a frequency 1/10 that of the original plasmid. The oriT region of the R64 plasmid contains two inverted-repeat sequences.
KeywordMeSH Terms
Conjugation, Genetic
R Factors
318. Hall  RM,     ( 1987 )

pKM101 is an IS46-promoted deletion of R46.

Nucleic acids research 15 (13)
PMID : 3037494  :   DOI  :   10.1093/nar/15.13.5479     PMC  :   PMC305975    
Abstract >>
N/A
KeywordMeSH Terms
Chromosome Deletion
DNA Transposable Elements
R Factors
319. Lengeler  JW, Ebner  R,     ( 1988 )

DNA sequence of the gene scrA encoding the sucrose transport protein EnzymeII(Scr) of the phosphotransferase system from enteric bacteria: homology of the EnzymeII(Scr) and EnzymeII(Bgl) proteins.

Molecular microbiology 2 (1)
PMID : 3285123  :  
Abstract >>
The nucleotide sequence of the structural gene, scrA, which codes for sucrose-specific EnzymeII(Scr) (EII(Scr)) of the phosphoenolpyruvate-dependent carbohydrate:phosphotransferase system (PTS), was determined. EllScr requires an EnzymeIII, the product of the gene crr, for full activity. The gene scrA is preceded immediately by a classical Shine-Dalgarno sequence (AAGAGGGTA). It contains 1368 nucleotides with an increased GC-content (58%) corresponding to a polypeptide of 455 amino acid residues (Mr 47,500). The protein has the hydropathic profile (average hydropathy +0.82) of an integral membrane protein lacking extended alpha-helical structures and a signal peptide. Comparison with the sequence of the beta-glucoside-specific EnzymeII (EII(Bgl), 625 amino acids, Mr 66,480; Bramley and Kornberg, 1987a; Schnetz et al., 1987) revealed strong homologies between EiI(Scr) and the first 458 residues of EII(Bgl). The 162 carboxyterminal residues of EII(Bgl), however, showed a high homology with the sequence of EnzymeIII (Nelson et al., 1984), a homology also described recently by Bramley and Kornberg (1987b). The evolutionary and functional significance of the similarities with four other EnzymesII is discussed.
KeywordMeSH Terms
Genes
Genes, Bacterial
320. Faccone  D, Lucero  C, Albornoz  E, Petroni  A, Ceriana  P, Campos  J, Viñas  MR, Francis  G, N/A  N/A, Melano  RG, Corso  A,     ( 2018 )

Emergence of azithromycin resistance mediated by the mph(A) gene in Salmonella Typhimurium clinical isolates in Latin America.

Journal of global antimicrobial resistance 13 (N/A)
PMID : 29705223  :   DOI  :   10.1016/j.jgar.2018.04.011    
Abstract >>
N/A
KeywordMeSH Terms
321. M?ka  ?, Ma?kiw  E, Stasiak  M, Wo?kowicz  T, Kowalska  J, Postupolski  J, Popowska  M,     ( 2018 )

Ciprofloxacin and nalidixic acid resistance of Salmonella spp. isolated from retail food in Poland.

International journal of food microbiology 276 (N/A)
PMID : 29649749  :   DOI  :   10.1016/j.ijfoodmicro.2018.03.012    
Abstract >>
Distribution of amino acid substitutions in the quinolone resistance-determining region (QRDR) of gyrA, gyrB, parC, parE and determinants of plasmid-mediated quinolone resistance (PMQR) were investigated among quinolone-resistant Salmonella spp. strains isolated from retail food in Poland in the years 2008-2013. Ten different amino acid substitutions were identified in QRDRs. Five different amino acid substitutions were identified in gyrA: Ser83Tyr, Ser83Phe, Asp87Tyr, Asp87Asn, Asp87Gly, two amino acid substitutions in parC: Thr57Ser, Ser80Ile and in parE: Leu445Phe, Arg511Ser. One substitution - Ser464Phe - was detected within gyrB. In gyrA a single substitution (Ser83Tyr) was identified the most frequently - 34.8% (63/181). Second most frequently identified variant (21.0%-38/181) was a co-existence of two single substitutions in gyrA: Ser83Tyr and parC: Thr57Ser. In four isolates co-existed three substitutions in three different genes: gyrA: Ser83Tyr + parC: Thr57Ser + parE: Leu445Phe (two isolates), gyrA: Ser83Phe + parC: Thr57Ser + parE: Leu445Phe, and gyrA: Ser83Tyr + parC: Thr57Ser + parE: Arg511Ser. In the two isolates four substitutions were identified - in gyrA: Ser83Phe + Asp87Tyr and in parC: Thr57Ser + Ser80Ile. Among resistant isolates, MIC values varied between 32 and 2048 mg/L (nalidixic acid) and between 0.125 and 16 mg/L (ciprofloxacin). MIC values of two isolates harboring qnrS1without any substitutions were 32 mg/L (NA) and 0.5-1.0 mg/L (CIP). The highest MIC values for NA and CIP were observed in two isolates of Salmonella spp. carrying double substitutions in gyrA: Ser83Phe + Asp87Tyr and parC: Thr57Ser + Ser80Ile. MIC value for NA was 2048 mg/L while for CIP - 16 mg/L.
KeywordMeSH Terms
Antimicrobial resistance
Food
PMQR
QRDR mutations
Quinolones
Salmonella spp.
Food Microbiology
322. Elkenany  RM, Eladl  AH, El-Shafei  RA,     ( 2018 )

Genetic characterisation of class 1 integrons among multidrug-resistant Salmonella serotypes in broiler chicken farms.

Journal of global antimicrobial resistance 14 (N/A)
PMID : 29684574  :   DOI  :   10.1016/j.jgar.2018.04.009    
Abstract >>
Antimicrobial resistance in Salmonella serotypes has been reported. Integrons play an important role in the dissemination of antimicrobial resistance genes in bacteria. Scarce literature is available on the identification of integrons in Salmonella isolated from broiler chickens. In this study, antimicrobial susceptibility testing and characterisation of class 1 integrons among multidrug-resistant (MDR) Salmonella enterica serotypes in broiler chicken farms in Egypt were performed. Antimicrobial susceptibility was determined by the disk diffusion method. PCR was performed to detect antimicrobial resistance genes and class 1 integrons in the tested Salmonella serotypes. Gene sequencing of the variable region of a class 1 integron was performed. Salmonella spp. were detected in 26 (13.5%) of 192 broiler samples, with Salmonella Enteritidis being the most frequently detected serotype, followed by Salmonella Kentucky and Salmonella Typhimurium and other serotypes. A very high resistance rate was observed to trimethoprim/sulfamethoxazole (100%), whilst a low resistance rate was observed to cefuroxime (57.7%). MDR S. enterica isolates displayed resistance to ciprofloxacin and azithromycin. Class 1 integrons were detected in 20 (76.9%) of the 26 Salmonella isolates. A high prevalence of class 1 integrons, as the first recorded percentage in the literature, associated with MDR Salmonella isolates was observed. Antimicrobial resistance rates in Salmonella serotypes from broiler chicken farms were alarming, especially for ciprofloxacin and azithromycin. Thus, another therapeutic strategy other than antimicrobials is recommended to prevent outbreaks of MDR Salmonella.
KeywordMeSH Terms
Antimicrobial resistance
Broilers
Integrons
Salmonella
323. Chiariotti  L, Alifano  P, Carlomagno  MS, Bruni  CB,     ( 1986 )

Nucleotide sequence of the Escherichia coli hisD gene and of the Escherichia coli and Salmonella typhimurium hisIE region.

Molecular & general genetics : MGG 203 (3)
PMID : 3018428  :   DOI  :   10.1007/bf00422061    
Abstract >>
In this paper we report the nucleotide sequence of the hisD gene of Escherichia coli and of the his IE region of both E. coli and Salmonella typhimurium. The hisD gene codes for a bifunctional enzyme, L-histidinol:NAD+ oxidoreductase, of 434 amino acids with a molecular mass of 46,199 daltons. We established that the hisIE region of both S. typhimurium and E. coli is composed of a single gene and not, as previously believed, of two separate genes. The derived amino acid sequence indicates that the hisIE gene codes for a bifunctional protein of 203 amino acids with an approximate molecular mass of 22,700 daltons. We also determined the nucleotide sequence of a deletion mutant in S. typhimurium which abolishes the hisF and hisI functions but retains the hisE function. We deduced that the mutant produces a chimeric protein fusing the aminoterminal region of the upstream hisF gene to the carboxyl-terminal domain of the hisIE gene which encodes for the hisE function. In view of these results the structural and functional organization of the histidine operon in enteric bacteria needs to be revised. The operon is composed of only 8 genes and the pathway leading to the biosynthesis of the amino acid requires 11 enzymatic steps.
KeywordMeSH Terms
Genes
Genes, Bacterial
324. Wajid  M, Awan  AB, Saleemi  MK, Weinreich  J, Schierack  P, Sarwar  Y, Ali  A,     ( N/A )

Multiple Drug Resistance and Virulence Profiling of Salmonella enterica Serovars Typhimurium and Enteritidis from Poultry Farms of Faisalabad, Pakistan.

Microbial drug resistance (Larchmont, N.Y.) 25 (1)
PMID : 30113248  :   DOI  :   10.1089/mdr.2018.0121    
Abstract >>
The zoonotic serovars of Salmonella enterica particularly Typhimurium and Enteritidis pose a continuous global threat to poultry industry and public health. We report the prevalence of Salmonella Typhimurium and Salmonella Enteritidis serovars in local poultry, phenotypic antimicrobial resistance profiling, and molecular detection of antimicrobial resistance and virulence genes. A total of 340 clinical samples were collected and 239 carried Salmonella, which were identified by genus-specific PCR (invA gene) and by matrix-assisted laser desorption/ionization time-of-flight. The 68 and 22 isolates were confirmed as Salmonella Typhimurium (stm gene) and Salmonella Enteritidis (sdfI gene) respectively. Pulsed-field gel electrophoresis (PFGE) revealed 27 and 9 PFGE types of Salmonella Typhimurium and Salmonella Enteritidis respectively. Among 24 antimicrobials tested, highest resistance was observed against pefloxacin while highest susceptibility was found for ertapenem in Salmonella Typhimurium and aztreonam in Salmonella Enteritidis. All isolates were found multiple drug resistant, 98.8% as motile and 8.8% as extended spectrum beta lactamase producers. Most prevalent resistance gene in Salmonella Typhimurium was parE (69.1%) while in Salmonella Enteritidis blaTEM-1 (72.7%). High prevalence of SopE gene in Salmonella Typhimurium (91.1%) and Salmonella Enteritidis (81.8%) indicated their zoonotic potential. The study is first of its kind from this region and highlights the emerging trends of antimicrobial resistance of global concern.
KeywordMeSH Terms
Enteritidis
Typhimurium
antimicrobial resistance
poultry
virulence profiling
325. Komano  T, Kubo  A, Nisioka  T,     ( 1987 )

Shufflon: multi-inversion of four contiguous DNA segments of plasmid R64 creates seven different open reading frames.

Nucleic acids research 15 (3)
PMID : 3029698  :   DOI  :   10.1093/nar/15.3.1165     PMC  :   PMC340515    
Abstract >>
The IncI alpha plasmid R64 was found to bear a highly mobile DNA segment which was designated as a clustered inversion region (J. Bacteriol. 165, 94-100, 1986). The clustered inversion region consists of four DNA segments designated respectively as A, B, C and D which differ in molecular size and restriction sites. The four DNA segments invert independently or in groups resulting in a complex DNA rearrangement. We now show the nucleotide sequence of the clustered inversion region of R64. The present results suggest that the clustered inversion region is a biological switch to select one of seven open reading frames whose primary structures at the region proximal to N-termini are constant while those at the C-terminal region are variable. A name, "Shufflon" was proposed to call this kind of the clustered inversion region.
KeywordMeSH Terms
Chromosome Inversion
Plasmids
326. Tran-Dien  A, Le Hello  S, Bouchier  C, Weill  FX,     ( 2018 )

Early transmissible ampicillin resistance in zoonotic Salmonella enterica serotype Typhimurium in the late 1950s: a retrospective, whole-genome sequencing study.

The Lancet. Infectious diseases 18 (2)
PMID : 29198740  :   DOI  :   10.1016/S1473-3099(17)30705-3    
Abstract >>
Ampicillin, the first semi-synthetic penicillin active against Enterobacteriaceae, was released onto the market in 1961. The first outbreaks of disease caused by ampicillin-resistant strains of Salmonella enterica serotype Typhimurium were identified in the UK in 1962 and 1964. We aimed to date the emergence of this resistance in historical isolates of S enterica serotype Typhimurium. In this retrospective, whole-genome sequencing study, we analysed 288 S enterica serotype Typhimurium isolates collected between 1911 and 1969 from 31 countries on four continents and from various sources including human beings, animals, feed, and food. All isolates were tested for antimicrobial drug susceptibility with the disc diffusion method, and isolates shown to be resistant to ampicillin underwent resistance-transfer experiments. To provide insights into population structure and mechanisms of ampicillin resistance, we did whole-genome sequencing on a subset of 225 isolates, selected to maximise source, spatiotemporal, and genetic diversity. 11 (4%) of 288 isolates were resistant to ampicillin because of acquisition of various �] lactamase genes, including blaTEM-1, carried by various plasmids, including the virulence plasmid of S enterica serotype Typhimurium. These 11 isolates were from three phylogenomic groups. One isolate producing TEM-1 �] lactamase was isolated in France in 1959 and two isolates producing TEM-1 �] lactamase were isolated in Tunisia in 1960, before ampicillin went on sale. The vectors for ampicillin resistance were different from those reported in the strains responsible for the outbreaks in the UK in the 1960s. The association between antibiotic use and selection of resistance determinants is not as direct as often presumed. Our results suggest that the non-clinical use of narrow-spectrum penicillins (eg, benzylpenicillin) might have favoured the diffusion of plasmids carrying the blaTEM-1 gene in S enterica serotype Typhimurium in the late 1950s. Institut Pasteur, Sant? publique France, the French Government's Investissement d'Avenir programme, the Fondation Le Roch-Les Mousquetaires.
KeywordMeSH Terms
Ampicillin Resistance
Food Microbiology
Gene Transfer, Horizontal
Plasmids
327. Lin  QP, Gao  ZQ, Geng  Z, Zhang  H, Dong  YH,     ( 2017 )

Crystal structure of the putative cytoplasmic protein STM0279 (Hcp2) from Salmonella typhimurium.

Acta crystallographica. Section F, Structural biology communications 73 (Pt 8)
PMID : 28777089  :   DOI  :   10.1107/S2053230X17010512     PMC  :   PMC5544003    
Abstract >>
STM0279 is a putative cytoplasmic protein from Salmonella typhimurium and was recently renamed haemolysin co-regulated protein 2 (Hcp2), with the neighbouring STM0276 being Hcp1. Both of them are encoded by the type VI secretion system (T6SS) of the Salmonella pathogenicity island 6 (SPI-6) locus and have high sequence identity. The Hcp proteins may function as a vital component of the T6SS nanotube and as a transporter and chaperone of diverse effectors from the bacterial T6SS. In this study, the crystal structure and the oligomeric state in solution of Hcp2 from S. typhimurium (StHcp2) were investigated. The crystal structure refined to 3.0 ? resolution showed that the protein is composed of a �]-barrel domain with extended loops and can form hexameric rings as observed in known Hcp homologues. Mutation of the extended loop was found to partly destabilize the hexameric conformation into monomers or cause the production of inclusion bodies, suggesting it has an important role in hexameric ring formation.
KeywordMeSH Terms
T6SS assembly
crystal structure
haemolysin co-regulated protein
hexameric rings
type VI secretion system
328. Purcell  BK, Pruckler  J, Clegg  S,     ( 1987 )

Nucleotide sequences of the genes encoding type 1 fimbrial subunits of Klebsiella pneumoniae and Salmonella typhimurium.

Journal of bacteriology 169 (12)
PMID : 2890624  :   DOI  :   10.1128/jb.169.12.5831-5834.1987     PMC  :   PMC214169    
Abstract >>
The nucleotide sequences of the genes encoding the subunits of Klebsiella pneumoniae and Salmonella typhimurium type 1 fimbriae were determined. Comparison of the predicted amino acid sequences of the two subunits revealed domains in which the sequences were highly conserved. Both gene products possessed signal peptides, a fact consistent with the transport of the fimbrial subunit across the membrane, but these regions showed no amino acid homology between the two proteins. The predicted N-terminal amino acid sequences of the processed fimbrial subunits were in good agreement with those obtained by purification of the fimbrial subunits.
KeywordMeSH Terms
Fimbriae, Bacterial
Genes, Bacterial
329. Zhang  YN, Peng  J, Wang  Q, Pei  ZF, Zhang  WJ, Niu  ZX,     ( 2008 )

Appearance of blaCMY-2)gene-positive Salmonella isolates of pig origin in China.

International journal of antimicrobial agents 31 (3)
PMID : 18178065  :   DOI  :   10.1016/j.ijantimicag.2007.10.023    
Abstract >>
KeywordMeSH Terms
330.     ( 1999 )

The SPI-3 pathogenicity island of Salmonella enterica.

Journal of bacteriology 181 (3)
PMID : 9922266  :   PMC  :   PMC93469    
Abstract >>
Pathogenicity islands are chromosomal clusters of pathogen-specific virulence genes often found at tRNA loci. We have determined the molecular genetic structure of SPI-3, a 17-kb pathogenicity island located at the selC tRNA locus of Salmonella enterica serovar Typhimurium. The G+C content of SPI-3 (47.5%) differs from that of the Salmonella genome (52%), consistent with the notion that these sequences have been horizontally acquired. SPI-3 harbors 10 open reading frames organized in six transcriptional units, which include the previously described mgtCB operon encoding the macrophage survival protein MgtC and the Mg2+ transporter MgtB. Among the newly identified open reading frames, one exhibits sequence similarity to the ToxR regulatory protein of Vibrio cholerae and one is similar to the AIDA-I adhesin of enteropathogenic Escherichia coli. The distribution of SPI-3 sequences varies among the salmonellae: the right end of the island, which harbors the virulence gene mgtC, is present in all eight subspecies of Salmonella; however, a four-gene cluster at the center of SPI-3 is found in only some of the subspecies and is bracketed by remnants of insertion sequences, suggesting a multistep process in the evolution of SPI-3 sequences.
KeywordMeSH Terms
Cation Transport Proteins
Multigene Family
Operon
331.     ( 1999 )

Molecular basis for resistance to silver cations in Salmonella.

Nature medicine 5 (2)
PMID : 9930866  :   DOI  :   10.1038/5545    
Abstract >>
Here we report the genetic and proposed molecular basis for silver resistance in pathogenic microorganisms. The silver resistance determinant from a hospital burn ward Salmonella plasmid contains nine open reading frames, arranged in three measured and divergently transcribed RNAs. The resistance determinant encodes a periplasmic silver-specific binding protein (SilE) plus apparently two parallel efflux pumps: one, a P-type ATPase (SilP); the other, a membrane potential-dependent three-polypeptide cation/proton antiporter (SilCBA). The sil determinant is governed by a two-component membrane sensor and transcriptional responder comprising silS and silR, which are co-transcribed. The availability of the sil silver-resistance determinant will be the basis for mechanistic molecular and biochemical studies as well as molecular epidemiology of silver resistance in clinical settings in which silver is used as a biocide.
KeywordMeSH Terms
ATP Binding Cassette Transporter, Subfamily B
Bacterial Proteins
332.     ( 2012 )

Molecular serotyping of Salmonella enterica by complete rpoB gene sequencing.

Journal of microbiology (Seoul, Korea) 50 (6)
PMID : 23274983  :   DOI  :   10.1007/s12275-012-2547-x    
Abstract >>
Serotyping has been the gold standard for identifying Salmonella, but it requires large amounts of standard antisera. Multilocus sequence typing (MLST) has been applied to identify Salmonella serovars, but the recombination of 4-7 housekeeping genes and multiple analytic steps diminish its applicability. In the present study, we determined the complete sequences of the RNA polymerase beta subunit gene (rpoB) and 7 housekeeping genes (aroC, dnaN, hemD, hisD, purE, sucA, and thrA) for 76 strains of 33 Salmonella enterica serovars and conducted phylogenetic analyses together with the corresponding gene sequences of 24 reference strains registered in the GenBank database. Based on the phylogenetic analyses, 100 strains from 40 serovars and 91 strains from 37 serovars were classified into 60 rpoB (RST) and 49 multilocus sequence types (ST), respectively. The nucleotide similarities were 98.8-100% and 96.9-100% for the complete rpoB gene and the seven concatenated housekeeping genes, respectively. The strains of 35 and 30 serovars formed serovar-specific branches or clusters in the rpoB and housekeeping gene phylogenetic trees, respectively. Therefore, complete rpoB gene sequencing and phylogenetic analysis may be a useful method for identifying Salmonella serovars that is a simpler, more cost-effective, and less time-consuming alternative or complementary method to MLST and conventional serotyping.
KeywordMeSH Terms
Multilocus Sequence Typing
333.     ( 2012 )

Evolution of a multiple antibiotic resistance region in IncHI1 plasmids: reshaping resistance regions in situ.

The Journal of antimicrobial chemotherapy 67 (12)
PMID : 22888274  :   DOI  :   10.1093/jac/dks317    
Abstract >>
To determine the structure of the resistance region in an IncHI1 plasmid conferring resistance to multiple antibiotics, including gentamicin, recovered from a Salmonella enterica serovar Typhimurium isolate from a horse. Plasmids were recovered by conjugation. The plasmid type, resistance genes and their context were identified by PCR, cloning, hybridization and DNA sequencing. The sequence was compared using bioinformatic tools with available resistance region sequences. In isolate SRC27, an IncI1 plasmid, pSRC27-I, conferred streptomycin resistance via the strA and strB genes contained within Tn5393a. An IncHI1 plasmid, pSRC27-H, was found to carry the aacC2 gentamicin resistance gene within a 34.6 kb multiple antibiotic resistance region that included nine further antibiotic resistance genes, aadA2, aphA1, bla(TEM), catA1, dfrA12, strA and strB, sul2 and tetA(B), conferring resistance to streptomycin and spectinomycin, kanamycin and neomycin, ampicillin, chloramphenicol, trimethoprim, streptomycin, sulfamethoxazole and tetracycline, respectively. This complex resistance region has evolved from Tn2670 and Tn10 via loss and gain of DNA segments. It includes Tn6029 and Tn4352, and a new transposon carrying the aacC2 gene. It also contains five copies of IS26, two of IS1 and one each of IS10 and ISCfr1. This region of pSRC27-H is related to ones present at the same position in three sequenced IncHI1 plasmids, pHCM1, pO111_1 and pMAK1, but has acquired new segments carrying antibiotic resistance genes. Evolution via loss and gain of resistance genes has occurred within the large resistance region of pHCM1-type IncHI1 plasmids leading to different resistance phenotypes.
KeywordMeSH Terms
Drug Resistance, Multiple, Bacterial
Evolution, Molecular
Plasmids
334.     ( 2013 )

Development of ceftriaxone resistance affects the virulence properties of Salmonella enterica serotype Typhimurium strains.

Foodborne pathogens and disease 10 (1)
PMID : 23320420  :   DOI  :   10.1089/fpd.2012.1216    
Abstract >>
Development of antibiotic resistance may alter the virulence properties of bacterial organisms. In this study, nine clinical ceftriaxone-susceptible Salmonella enterica serotype Typhimurium strains were subjected to stepwise selection with increasing concentrations of ceftriaxone in culture media. Mutations in virulence-associated genes and antibiotic efflux genes were analyzed by polymerase chain reaction (PCR) and DNA sequencing. The expression levels of virulence genes invA and stn as well as efflux pump genes tolC, arcA, and arcB before and after the selection were measured by real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The stepwise selection resulted in the development of Salmonella strains that were highly resistant to ceftriaxone. Sequence analysis did not reveal any mutations or deletions in the examined virulence genes and regulatory gene, but a silent mutation (T423C) in acrR (encoding a repressor for the efflux pump) was detected in most of the ceftriaxone-resistant strains. The qRT-PCR revealed increased expression of the AcrAB-TolC efflux pump and decreased expression of invA and stn in the ceftriaxone-resistant strains. Moreover, decreased invasion into cultured epithelial cells and reduced growth rates were observed with the resistant strains. These results suggest that acquisition of ceftriaxone resistance is associated with the overexpression of the AcrAB-TolC efflux pump and leads to reduced virulence in Salmonella Typhimurium.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
335.     ( 2012 )

High clonality and diversity of virulence determinants among bla(PSE)-positive Salmonella Typhimurim isolates recovered in three geographically distant Spanish hospitals.

Diagnostic microbiology and infectious disease 74 (4)
PMID : 23000287  :   DOI  :   10.1016/j.diagmicrobio.2012.08.011    
Abstract >>
Molecular typing, the presence of Salmonella genomic island 1 (SGI1), and virulence factors were studied in 45 bla(PSE)-positive S. Typhimurium isolates from 3 hospitals. All isolates belonged to sequence type ST19, presented low clonal diversity, and harbored SGI1. The wide diversity of virulence factors was classified into 3 major virulotype groups.
KeywordMeSH Terms
Genetic Variation
336.     ( 1999 )

Integration of minitransposons for expression of the Escherichia coli elt genes at a preferred site in Salmonella typhimurium identifies a novel putative fimbrial locus.

Archives of microbiology 171 (2)
PMID : 9914309  :  
Abstract >>
An asd-complementing mini-Tn5 transposon was constructed for random insertion of the Escherichia coli LT enterotoxin genes (elt) into the genome of Deltaasd attenuated strains of Salmonella typhimurium. Transfer of the minitransposon to different S. typhimurium strains resulted in random integration only in strain chi4072, while in strain chi3987, which harbours the virulence plasmid, over 20% of the insertions occurred at the same site. Expression of elt was found to be highest in Salmonella isolates carrying the mini-Tn5 integrated at the preferred site, which was mapped to an uncharacterised region of the virulence plasmid. Sequence analysis of the integration site showed that it lies within an open reading frame with sequence similarity to E. coli leuO and contiguous to a novel fimbrial locus.
KeywordMeSH Terms
DNA Transposable Elements
Escherichia coli Proteins
337.     ( 2013 )

pMdT1, a small ColE1-like plasmid mobilizing a new variant of the aac(6')-Ib-cr gene in Salmonella enterica serovar Typhimurium.

The Journal of antimicrobial chemotherapy 68 (6)
PMID : 23361643  :   DOI  :   10.1093/jac/dkt001    
Abstract >>
To characterize a 5.9 kb aac(6')-Ib-cr-harbouring plasmid that was detected in a clinical Salmonella Typhimurium DT104B strain. Extraction and purification of plasmid DNA and electrotransformation assays were carried out in order to obtain kanamycin-resistant transformants. MICs of several fluoroquinolones and aminoglycosides were determined. DNA sequencing was performed by primer walking on purified plasmid preparations. The new plasmid nucleotide sequence was analysed and compared with available sequences using bioinformatic tools. pMdT1 is a 5.9 kb mobilizable ColE1-like plasmid that harbours aac(6')-Ib-cr4, a gene encoding a new variant of the AAC(6')-Ib-cr protein (225 amino acids). This active protein conferred resistance to tobramycin and kanamycin, and also decreased susceptibility to ciprofloxacin and norfloxacin in the transformant strain, as MICs demonstrated. The mobilization region, necessary for horizontal transfer and composed of the mobA, mobB, mobC and mobD genes, displayed a high degree of identity with those from representative ColE1-like plasmids. The basis of mobility (bom), oriT and origin of replication regions were also detected. Apart from the acetylase-encoding gene, three other open reading frames (ORFs) were determined. No similarities were found when the ORF1 sequence was compared with the sequences included in GenBank. The deduced ORF2 protein predicted a CopG-like structure characteristic of transcriptional regulators, and the deduced ORF3 protein was identical to macrophage stimulating factors. The pMdT1 is the smallest mobilizable ColE1-like plasmid containing an aac(6')-Ib-cr gene that has been described so far.
KeywordMeSH Terms
PMQR
Salmonella Typhimurium
mob genes
plasmid-mediated quinolone resistance
338.     ( 1998 )

Class 1 integron-borne multiple-antibiotic resistance carried by IncFI and IncL/M plasmids in Salmonella enterica serotype typhimurium.

Antimicrobial agents and chemotherapy 42 (12)
PMID : 9835490  :   PMC  :   PMC105998    
Abstract >>
The presence and genetic content of integrons were investigated for 37 epidemiologically unrelated multiple-drug-resistant strains of Salmonella enterica serotype Typhimurium from humans. All isolates were resistant to ampicillin, chloramphenicol, kanamycin, streptomycin, sulfonamides, and trimethoprim, as well as to tetracycline and/or nalidixic acid; 20% of them were also resistant to gentamicin and amikacin. Three different class 1 integrons (In-t1, In-t2, and In-t3) were identified by Southern blot hybridization, PCR, and DNA sequencing, and these integrons were found to carry the aadB, catB3, oxa1, aadA1a, aacA4, and aacC1 gene cassettes. Integrons In-t1 (aadB and catB3) and In-t2 (oxa1 and aadA1a) were both located on a conjugative IncFI plasmid of 140 kb. In-t3 (aacA4, aacC1, and aadAIa) was located on an IncL/M plasmid of 100 kb which was present, in association with the IncFI plasmid, in gentamicin- and amikacin-resistant isolates. Despite the extensive similarity at the level of the antibiotic resistance phenotype, integrons were not found on the prototypic IncFI plasmids carried by epidemic Salmonella strains isolated during the late 1970s. The recent appearance and the coexistence of multiple integrons on two conjugative plasmids in the same Salmonella isolate are examples of how mobile gene cassettes may contribute to the acquisition and dissemination of antibiotic resistance.
KeywordMeSH Terms
339.     ( 1998 )

S. typhimurium encodes an activator of Rho GTPases that induces membrane ruffling and nuclear responses in host cells.

Cell 93 (5)
PMID : 9630225  :   DOI  :   10.1016/s0092-8674(00)81442-7    
Abstract >>
S. typhimurium stimulates signaling pathways leading to membrane ruffling, actin cytoskeleton rearrangements, and nuclear responses. The stimulation requires a protein secretion system (type III) that translocates bacterial proteins into the host cell. We show that SopE, a substrate of this secretion system, stimulates cytoskeletal reorganization and JNK activation in a CDC42- and Rac-1-dependent manner. A lambda gt11 cDNA library screen for proteins that interact with SopE identified Rac-1 and CDC42. Furthermore, purified SopE was shown to stimulate GDP/GTP nucleotide exchange in several Rho GTPases in vitro, including Rac-1 and CDC42. These findings establish a paradigm for microbial stimulation of cellular responses in which the pathogen induces signaling events by directly engaging the signaling machinery within the host cell.
KeywordMeSH Terms
Mitogen-Activated Protein Kinases
340.     ( 1999 )

Lipid A mutant Salmonella with suppressed virulence and TNFalpha induction retain tumor-targeting in vivo.

Nature biotechnology 17 (1)
PMID : 9920266  :   DOI  :   10.1038/5205    
Abstract >>
Systemically administered tumor-targeted Salmonella has been developed as an anticancer agent, although its use could be limited by the potential induction of tumor necrosis factor alpha (TNFalpha)-mediated septic shock stimulated by lipid A. Genetic modifications of tumor-targeting Salmonella that alter lipid A and increase safety must, however, retain the useful properties of this bacteria. We report here that disruption of the Salmonella msbB gene reduces TNFalpha induction and increases the LD50 of this pathogenic bacteria by 10,000-fold. Notwithstanding this enormous difference, Salmonella retains its tumor-targeting properties, exhibiting tumor accumulation ratios in excess of 1000:1 compared with normal tissues. Administration of this bacteria to mice bearing melanoma results in tumors that are less than 6% the size of tumors in untreated controls at day 18. Thus, the antitumor activity previously demonstrated using tumor-targeting Salmonella with normal lipid A is retained. Lipid modification of tumor-specific bacterial vectors provides a means for reducing septic shock and further suggests that the antitumor activity of these bacteria may be independent of TNFalpha.
KeywordMeSH Terms
Acyltransferases
Escherichia coli Proteins
341.     ( 1998 )

Two novel plasmid-mediated cefotaxime-hydrolyzing beta-lactamases (CTX-M-5 and CTX-M-6) from Salmonella typhimurium.

FEMS microbiology letters 165 (2)
PMID : 9742701  :   DOI  :   10.1111/j.1574-6968.1998.tb13159.x    
Abstract >>
Two novel plasmid-mediated beta-lactamases (CTX-M-5 and CTX-M-6) produced by Salmonella typhimurium clinical strains were characterized. The enzymes exhibited a pI of 8.4, hydrolyzed oxyimino-beta-lactams and were susceptible to mechanism-based beta-lactamase inhibitors. The respective bla genes were cloned and sequenced. The deduced amino acid sequences showed a high degree of homology with those of the previously described plasmid class A CTX-M-type enzymes and appeared related to the chromosomal beta-lactamases of Klebsiella oxytoca.
KeywordMeSH Terms
342.     ( 1998 )

The sfiX, rfe and metN genes of Salmonella typhimurium and their involvement in the His(c) pleiotropic response.

Molecular & general genetics : MGG 259 (1)
PMID : 9738879  :   DOI  :   10.1007/s004380050787    
Abstract >>
Two loci involved in the pleiotropic response of His(c) strains of Salmonella typhimurium (sfiX and sfiY) have been characterized at the molecular level. The sfiX gene (CS 44) has been identified as a homolog of the E. coli gene sanA, located downstream of the cytidine deaminase gene (cdd). The cdd-sanA (or cdd-sfiX) operon shows a highly conserved structure in E. coli and Salmonella. Like its E. coli homolog, the sfiX gene of S. typhimurium is required for vancomycin resistance at high temperature. The dual effect of sfiX mutations (induction of vancomycin sensitivity and suppression of cell division inhibition) suggests a link between SfiX function and murein synthesis. The sfiY locus (CS 85), contains two genes arranged in a single transcriptional unit. The upstream gene is a homolog of the E. coli gene rfe; mutations in this gene suppress the cell division defect of His(c) strains. The suppressor effect of rfe mutations can be reproduced by tunicamycin, suggesting that suppression of filamentation results from an increase in the intracellular concentration of UDP-N-acetyl-D-glucosamine. The gene located downstream of rfe is also found in E. coli but its function is unknown. Insertions in rfe suppress the methionine requirement of His(c) strains of S. typhimurium by a polar effect on the downstream gene, tentatively designated metN. Complementation with a rfe+ clone indicates that the rfe gene is not involved in the methionine requirement of His(c) strains. Thus metN expression appears to cause methionine auxotrophy in a His(c) background.
KeywordMeSH Terms
Escherichia coli Proteins
343.     ( 1998 )

Identification of a new site for ferrichrome transport by comparison of the FhuA proteins of Escherichia coli, Salmonella paratyphi B, Salmonella typhimurium, and Pantoea agglomerans.

Journal of bacteriology 180 (15)
PMID : 9683481  :   PMC  :   PMC107368    
Abstract >>
The fhuA genes of Salmonella paratyphi B, Salmonella typhimurium, and Pantoea agglomerans were sequenced and compared with the known fhuA sequence of Escherichia coli. The highly similar FhuA proteins displayed the largest difference in the predicted gating loop, which in E. coli controls the permeability of the FhuA channel and serves as the principal binding site for the phages T1, T5, and phi80. All the FhuA proteins contained the region in the gating loops required in E. coli for ferrichrome and albomycin transport. The three subdomains required for phage binding were contained in the gating loop of S. paratyphi B which is infected by the E. coli phages, whereas two of the subdomains were deleted in S. typhimurium and P. agglomerans which are resistant to the E. coli phages. Small deletions in a surface loop adjacent to the gating loop, residues 236 to 243 and 236 to 248, inactivated E. coli FhuA with regard to transport of ferrichrome and albomycin, but sensitivity to T1 and T5 was fully retained and sensitivity to phi80 and colicin M was reduced 10-fold. Full-size FhuA hybrid proteins of S. paratyphi B and S. typhimurium displayed S. paratyphi B FhuA activity when the hybrids contained two-thirds of either the N- or the C-terminal portions of S. paratyphi B and displayed S. typhimurium FhuA activity to phage ES18 when the hybrid contained two-thirds of the N-terminal region of the S. typhimurium FhuA. The central segment of the S. paratyphi B FhuA flanked on both sides by S. typhimurium FhuA regions conferred full sensitivity only to phage T5. The data support the essential role of the gating loop for the transport of ferrichrome and albomycin, identified an additional loop for ferrichrome and albomycin uptake, and suggest that several segments and their proper conformation, determined by the entire FhuA protein, contribute to the multiple FhuA activities.
KeywordMeSH Terms
Escherichia coli Proteins
344.     ( 1998 )

Macrophage-dependent induction of the Salmonella pathogenicity island 2 type III secretion system and its role in intracellular survival.

Molecular microbiology 30 (1)
PMID : 9786194  :   DOI  :   10.1046/j.1365-2958.1998.01048.x    
Abstract >>
Salmonella pathogenicity island 2 (SPI-2) encodes a putative type III secretion system necessary for systemic infection in animals. We have investigated the transcriptional organization and regulation of SPI-2 by creating gfp fusions throughout the entire gene cluster. These gfp fusions demonstrated that SPI-2 genes encoding structural, regulatory and previously uncharacterized putative secreted proteins are preferentially expressed in the intracellular environment of the host macrophage. Furthermore, the transcription of these genes within host cells was dependent on the two-component regulatory system SsrA/SsrB and an acidic phagosomal environment. Most SPI-2 mutants failed to replicate to the same level as wild-type strains in murine macrophages and human epithelial cells. In orally infected mice, SPI-2 mutants colonized the Peyer's patches but did not progress to the mesenteric lymph nodes. We conclude that SPI-2 genes are specifically expressed upon entry into mammalian cells and are required for intracellular growth in host cells in vivo and in vitro.
KeywordMeSH Terms
Membrane Glycoproteins
345.     ( 1998 )

The apeE gene of Salmonella typhimurium encodes an outer membrane esterase not present in Escherichia coli.

Journal of bacteriology 180 (14)
PMID : 9657991  :   PMC  :   PMC107316    
Abstract >>
Salmonella typhimurium apeR mutations lead to overproduction of an outer membrane-associated N-acetyl phenylalanine beta-naphthyl ester-cleaving esterase that is encoded by the apeE gene (P. Collin-Osdoby and C. G. Miller, Mol. Gen. Genet. 243:674-680, 1994). This paper reports the cloning and nucleotide sequencing of the S. typhimurium apeE gene as well as some properties of the esterase that it encodes. The predicted product of apeE is a 69.9-kDa protein which is processed to a 67-kDa species by removal of a signal peptide. The predicted amino acid sequence of ApeE indicates that it is a member of the GDSL family of serine esterases/lipases. It is most similar to a lipase excreted by the entomopathogenic bacterium Photorhabdus luminescens. The Salmonella esterase catalyzes the hydrolysis of a variety of fatty acid naphthyl esters and of C6 to C16 fatty acid p-nitrophenyl esters but will not hydrolyze peptide bonds. A rapid diagnostic test reported to be useful in distinguishing Salmonella spp. from related organisms makes use of the ability of Salmonella to hydrolyze the chromogenic ester substrate methyl umbelliferyl caprylate. We report that the apeE gene product is the enzyme in Salmonella uniquely responsible for the hydrolysis of this substrate. Southern blot analysis indicates that Escherichia coli K-12 does not contain a close analog of apeE, and it appears that the apeE gene is contained in a region of DNA present in Salmonella but not in E. coli.
KeywordMeSH Terms
346.     ( 1998 )

Complex metabolic phenotypes caused by a mutation in yjgF, encoding a member of the highly conserved YER057c/YjgF family of proteins.

Journal of bacteriology 180 (24)
PMID : 9851994  :   PMC  :   PMC107753    
Abstract >>
The oxidative pentose phosphate pathway is required for function of the alternative pyrimidine biosynthetic pathway, a pathway that allows thiamine synthesis in the absence of the PurF enzyme in Salmonella typhimurium. Mutants that no longer required function of the oxidative pentose phosphate pathway for thiamine synthesis were isolated. Further phenotypic analyses of these mutants demonstrated that they were also sensitive to the presence of serine in the medium, suggesting a partial defect in isoleucine biosynthesis. Genetic characterization showed that these pleiotropic phenotypes were caused by null mutations in yjgF, a previously uncharacterized open reading frame encoding a hypothetical 13.5-kDa protein. The YjgF protein belongs to a class of proteins of unknown function that exhibit striking conservation across a wide range of organisms, from bacteria to humans. This work represents the first detailed phenotypic characterization of yjgF mutants in any organism and provides important clues as to the function of this highly conserved class of proteins. Results also suggest a connection between function of the isoleucine biosynthetic pathway and the requirement for the pentose phosphate pathway in thiamine synthesis.
KeywordMeSH Terms
Mutation
347.     ( 1998 )

Identification and sequence analysis of a 27-kilobase chromosomal fragment containing a Salmonella pathogenicity island located at 92 minutes on the chromosome map of Salmonella enterica serovar typhimurium LT2.

Infection and immunity 66 (7)
PMID : 9632606  :   PMC  :   PMC108353    
Abstract >>
Using a genomic approach, we have identified a new Salmonella pathogenicity island, SPI-4, which is the fourth Salmonella pathogenicity island to be identified. SPI-4 was located at 92 min on the chromosome map and was flanked by the ssb and soxSR loci. The DNA sequence covering the entire SPI-4 and both boundaries was determined. The size of SPI-4 was about 25 kb and it contains 18 putative open reading frames (ORFs). Three of these ORFs encode proteins that have significant homology with proteins involved in toxin secretion. Another five ORFs encode proteins that have significant homology with hypothetical proteins from Synechocystis sp. strain PCC6803 or Acinetobacter calcoaceticus. The rest of the ORFs encode novel proteins, one of which has five membrane-spanning domains. SPI-4 is likely to carry a type I secretion system involved in toxin secretion. Furthermore, a previously identified locus (ims98), which is required for intramacrophage survival, was also mapped within the SPI-4 region. These findings suggested that SPI-4 is needed for intramacrophage survival.
KeywordMeSH Terms
Chromosome Mapping
Genes, Bacterial
Multigene Family
348.     ( 1999 )

Analysis of the type 1 pilin gene cluster fim in Salmonella: its distinct evolutionary histories in the 5' and 3' regions.

Journal of bacteriology 181 (4)
PMID : 9973358  :   PMC  :   PMC93509    
Abstract >>
The type 1 pilin encoded by fim is present in both Escherichia coli and Salmonella natural isolates, but several lines of evidence indicate that similarities at the fim locus may be an example of independent acquisition rather than common ancestry. For example, the fim gene cluster is found at different chromosomal locations and with distinct gene orders in these closely related species. In this work we examined the fim gene cluster of Salmonella, the genes of which show high nucleotide sequence divergence from their E. coli counterparts, as well as a different G+C content and codon usage. DNA hybridization analysis revealed that, among the salmonellae, the fim gene cluster is present in all isolates of S. enterica but is absent from S. bongori. Molecular phylogenetic analyses of the fimA and fimI genes yield an estimate of phylogeny that is in satisfactory congruence with housekeeping and other virulence genes examined in this species. In contrast, phylogenetic analyses of the fimZ, fimY, and fimW genes indicate that horizontal transfer of this region has occurred more than once. There is also size variation in the fimZ, fimY, and fimW intergenic regions in the 3' region, and these genes are absent in isolate S2983 of subspecies IIIa. Interestingly, the G+C contents of the fimZ, fimY, and fimW genes are less than 46%, which is considerably lower than those of the other six genes of the fim cluster. This study demonstrates that horizontal transmission of all or part of the same gene cluster can occur repeatedly, with the result that different regions of a single gene cluster may have different evolutionary histories.
KeywordMeSH Terms
Evolution, Molecular
Genes, Bacterial
Multigene Family
349.     ( 1998 )

CTX-M-5, a novel cefotaxime-hydrolyzing beta-lactamase from an outbreak of Salmonella typhimurium in Latvia.

Antimicrobial agents and chemotherapy 42 (8)
PMID : 9687393  :   PMC  :   PMC105719    
Abstract >>
At a children's hospital in Riga, Latvia, isolates identified as Salmonella typhimurium were found to be resistant to expanded-spectrum cephalosporins. Two of the resistant strains were analyzed for the mechanism of cephalosporin resistance. Isoelectric focusing revealed a common beta-lactamase with a pI of 8.8. In addition, one of the strains produced a pI 7.6 beta-lactamase. A transconjugant producing only the pI 7.6 enzyme was susceptible to expanded-spectrum cephalosporins; therefore, this enzyme was most likely SHV-1. Transformants producing only the pI 8.8 beta-lactamase were resistant to cefotaxime and aztreonam but were susceptible or intermediate to ceftazidime. A substrate profile determined spectrophotometrically with purified enzyme revealed potent activity against cefotaxime, with a relative kcat value of 95 (benzylpenicillin equal to 100). The enzyme showed lower relative kcat values for ceftazidime (3.3) and aztreonam (9.3). In addition, the enzyme was inhibited by clavulanate, sulbactam and tazobactam, with 50% inhibitory concentrations of 19, 100, and 3.4 nM, respectively. These results indicated the presence of an unusual extended-spectrum beta-lactamase. The gene expressing the pI 8.8 beta-lactamase was cloned. Nucleotide sequencing revealed a beta-lactamase gene that differs from the gene encoding CTX-M-2, which also originated from S. typhimurium, by 11 nucleotides, 4 of which result in amino acid substitutions: Ala27Thr, Val230Gly, Glu254Ala, and Ile278Val. These results indicated the presence of a novel extended-spectrum beta-lactamase, designated CTX-M-5, that specifically confers resistance to cefotaxime.
KeywordMeSH Terms
Disease Outbreaks
350.     ( 1998 )

Effect of substitution of Asn for Arg-276 in the cefotaxime-hydrolyzing class A beta-lactamase CTX-M-4.

FEMS microbiology letters 169 (2)
PMID : 9868772  :   DOI  :   10.1111/j.1574-6968.1998.tb13331.x    
Abstract >>
The effect of substitution of asparagine for arginine at position 276 (Ambler's numbering) on the properties of the extended-spectrum beta-lactamase CTX-M-4 was studied. Compared with CTX-M-4, the mutant beta-lactamase CTX-M-4(R276N) conferred lower levels of resistance to cefotaxime, ceftriaxone and aztreonam while the levels of resistance to penicillins and penicillin-inhibitor combinations were similar. Arg-276-->Asn substitution rendered CTX-M-4 slightly less susceptible to inhibition by clavulanate and tazobactam. It also caused a three-fold reduction in the relative rate of hydrolysis of cefotaxime. These results indicate that Arg-276 in CTX-M-type beta-lactamases may be implicated in hydrolysis of oxyimino-beta-lactams; they do not, however, support the hypothesis that Arg-276 is the functional equivalent of Arg-244 found in other class A beta-lactamases.
KeywordMeSH Terms
Bacterial Proteins
351.     ( 1998 )

Production of monoclonal antibodies specific for the i and 1,2 flagellar antigens of Salmonella typhimurium and characterization of their respective epitopes.

Applied and environmental microbiology 64 (12)
PMID : 9835604  :   PMC  :   PMC90964    
Abstract >>
Salmonella typhimurium expresses two antigenically distinct flagellins, each containing a different H antigen (i and 1,2), the combination of which is highly specific for this serotype. In this study, overlapping recombinant flagellin fragments were constructed from the fliC (H:i) and fljB (H:1,2) flagellin genes, and the expression products were tested for binding to H antigen-specific monoclonal and polyclonal antibodies. A minimal area, 86 amino acids for H:i and 102 amino acids for H:1,2, located in the central variable domain of each flagellin was required for the binding of serotype-specific antibodies, providing further evidence for the presence of a discontinuous H epitope. Two peptides comprising these areas were shown to be highly suitable for application as antigens in an enzyme-linked immunosorbent assay detecting S. typhimurium-specific antibody.
KeywordMeSH Terms
Antibodies, Monoclonal
Bacterial Proteins
352.     ( 1998 )

Genes encoding putative effector proteins of the type III secretion system of Salmonella pathogenicity island 2 are required for bacterial virulence and proliferation in macrophages.

Molecular microbiology 30 (1)
PMID : 9786193  :   DOI  :   10.1046/j.1365-2958.1998.01047.x    
Abstract >>
The type III secretion system of Salmonella pathogenicity island 2 (SPI-2) is required for systemic infection of this pathogen in mice. Cloning and sequencing of a central region of SPI-2 revealed the presence of genes encoding putative chaperones and effector proteins of the secretion system. The predicted products of the sseB, sseC and sseD genes display weak but significant similarity to amino acid sequences of EspA, EspD and EspB, which are secreted by the type III secretion system encoded by the locus of enterocyte effacement of enteropathogenic Escherichia coli. The transcriptional activity of an sseA::luc fusion gene was shown to be dependent on ssrA, which is required for the expression of genes encoding components of the secretion system apparatus. Strains carrying nonpolar mutations in sseA, sseB or sseC were severely attenuated in virulence, strains carrying mutations in sseF or sseG were weakly attenuated, and a strain with a mutation in sseE had no detectable virulence defect. These phenotypes were reflected in the ability of mutant strains to grow within a variety of macrophage cell types: strains carrying mutations in sseA, sseB or sseC failed to accumulate, whereas the growth rates of strains carrying mutations in sseE, sseF or sseG were only modestly reduced. These data suggest that, in vivo, one of the functions of the SPI-2 secretion system is to enable intracellular bacterial proliferation.
KeywordMeSH Terms
Acetyltransferases
Genes, Bacterial
353.     ( 1998 )

Identification of PhoP-PhoQ activated genes within a duplicated region of the Salmonella typhimurium chromosome.

Microbial pathogenesis 25 (2)
PMID : 9712687  :   DOI  :   10.1006/mpat.1998.0217    
Abstract >>
Salmonellae virulence requires the PhoP-PhoQ two-component regulatory system. PhoP-PhoQ activate the transcription of genes following phagocytosis by macrophages which are necessary for survival within the phagosome environment. Thirteen previously undefined PhoP-activated gene fusions generated by MudJ and TnphoA (pag A, and E-P, respectively) were cloned and sequenced. Most pag products show no similarity to proteins in the database, while others are predicted to encode: a UDP-glucose dehydrogenase (pagA); a protein with similarity to the product of an E. coli aluminium-induced gene (pagH); a protein encoded within a Salmonella-unique region adjacent to the sinR gene (pagN); a protein similar to a product of the Yersinia virulence plasmid (pagO); and a protein with similarity to CrcA which is necessary for resistance of E. coli to camphor (pagP). Of the pag characterized, only pagK, M and O were closely linked. pagJ and pagK were shown to be unlinked but nearly identical in DNA sequence, as each was located within a 1.6 kb DNA duplication. The translations of sequences surrounding pagJ and pagK show similarity to proteins from extrachromosomal elements as well as those involved in DNA transposition and rearrangement, suggesting that this region may have been or is a mobile element. The transcriptional start sites of pagK, M, and J were determined; however, comparison to other known pag gene promoters failed to reveal a consensus sequence for PhoP-regulated activation. DNA sequences hybridizing to a Salmonella typhimurium pagK specific probe were found in S. enteritidis but absent in other Salmonella serotypes and Enterobacteriaceae tested, suggesting that these genes are specific for broad host range Salmonellae that cause diarrhoea in humans. Cumulatively, these data further demonstrate: (1) that PhoP-PhoQ is a global regulator of the production of diverse envelope or secreted proteins; (2) that PhoP-PhoQ regulate the production of proteins of redundant function; and (3) that pag are often located in regions of horizontally acquired DNA that are absent in other Enterobacteriaceae.
KeywordMeSH Terms
Chromosome Mapping
Chromosomes, Bacterial
Gene Expression Regulation, Bacterial
354.     ( 1998 )

Salmonella typhimurium encodes an SdiA homolog, a putative quorum sensor of the LuxR family, that regulates genes on the virulence plasmid.

Journal of bacteriology 180 (5)
PMID : 9495757  :   PMC  :   PMC107006    
Abstract >>
Quorum sensing is a phenomenon in which bacteria sense and respond to their own population density by releasing and sensing pheromones. In gram-negative bacteria, quorum sensing is often performed by the LuxR family of transcriptional regulators, which affect phenotypes as diverse as conjugation, bioluminescence, and virulence gene expression. The gene encoding one LuxR family member, named sdiA (suppressor of cell division inhibition), is present in the Escherichia coli genome. In this report, we have cloned the Salmonella typhimurium homolog of SdiA and performed a systematic screen for sdiA-regulated genes. A 4.4-kb fragment encoding the S. typhimurium sdiA gene was sequenced and found to encode the 3' end of YecC (homologous to amino acid transporters of the ABC family), all of SdiA and SirA (Salmonella invasion regulator), and the 5' end of UvrC. This gene organization is conserved between E. coli and S. typhimurium. We determined that the S. typhimurium sdiA gene was able to weakly complement the E. coli sdiA gene for activation of ftsQAZ at promoter 2 and for suppression of filamentation caused by an ftsZ(Ts) allele. To better understand the function of sdiA in S. typhimurium, we screened 10,000 random lacZY transcriptional fusions (MudJ transposon mutations) for regulation by sdiA. Ten positively regulated fusions were isolated. Seven of the fusions were within an apparent operon containing ORF8, ORF9, rck (resistance to complement killing), and ORF11 of the S. typhimurium virulence plasmid. The three ORFs have now been named srgA, srgB, and srgC (for sdiA-regulated gene), respectively. The DNA sequence adjacent to the remaining three fusions shared no similarity with previously described genes.
KeywordMeSH Terms
Bacterial Proteins
Escherichia coli Proteins
Gene Expression Regulation, Bacterial
355.     ( 1998 )

A promoter relay mechanism for sequential gene activation.

Journal of bacteriology 180 (3)
PMID : 9457867  :   PMC  :   PMC106931    
Abstract >>
The effect of DNA supercoiling on gene expression is dependent not only on specific genes but also on the sequence context of the genes. This position-dependent supercoiling effect on gene activation is best illustrated in the study of the suppression of the leu-500 mutation of the leuABCD operon in a Salmonella typhimurium topA mutant. In this communication, we report a novel promoter relay mechanism whereby several genes are sequentially expressed in a position-dependent manner: the ilvIH promoter (pilvIH) activates a cryptic leuO promoter (pleuO) located between the two divergently arrayed ilvIH and leu-500 promoters. Both the cis-acting pleuO activity and the trans-acting LeuO protein are necessary for subsequent activation of the leu-500 promoter (pleu-500). Furthermore, pleuO can be functionally replaced with the inducible tac promoter (ptac) for leu-500 activation, suggesting that transcription-driven DNA supercoiling underlies the relay mechanism. This is the first example of several related genes communicating via a promoter relay mechanism for their coordinated expression.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
Promoter Regions, Genetic
Transcriptional Activation
356.     ( 1997 )

Periplasmic superoxide dismutase protects Salmonella from products of phagocyte NADPH-oxidase and nitric oxide synthase.

Proceedings of the National Academy of Sciences of the United States of America 94 (25)
PMID : 9391141  :   DOI  :   10.1073/pnas.94.25.13997     PMC  :   PMC28421    
Abstract >>
Superoxide dismutase (SOD) catalyzes the conversion of superoxide radical to hydrogen peroxide. Periplasmic localization of bacterial Cu,Zn-SOD has suggested a role of this enzyme in defense against extracellular phagocyte-derived reactive oxygen species. Sequence analysis of regions flanking the Salmonella typhimurium sodC gene encoding Cu,Zn-SOD demonstrates significant homology to lambda phage proteins, reflecting possible bacteriophage-mediated horizontal gene transfer of this determinant among pathogenic bacteria. Salmonella deficient in Cu,Zn-SOD has reduced survival in macrophages and attenuated virulence in mice, which can be restored by abrogation of either the phagocyte respiratory burst or inducible nitric oxide synthase. Moreover, a sodC mutant is extremely susceptible to the combination of superoxide and nitric oxide. These observations suggest that SOD protects periplasmic or inner membrane targets by diverting superoxide and limiting peroxynitrite formation, and they demonstrate the ability of the respiratory burst and nitric oxide synthase to synergistically kill microbial pathogens in vivo.
KeywordMeSH Terms
357.     ( 1998 )

A temperature-sensitive DNA adenine methyltransferase mutant of Salmonella typhimurium.

Archives of microbiology 169 (6)
PMID : 9575240  :  
Abstract >>
A temperature-sensitive mutant of Salmonella typhimurium was isolated earlier after transposon mutagenesis with Tn10d Tet. The mutant D220 grows well at 28 degreesC but has a lower growth rate and forms filaments at 37 degreesC. Transposon-flanking fragments of mutant D220 DNA were cloned and sequenced. The transposon was inserted in the dam gene between positions 803 and 804 (assigned allele number: dam-231 : : Tn10d Tet) and resulted in a predicted ten-amino-acid-shorter Dam protein. The insertion created a stop codon that led to a truncated Dam protein with a temperature-sensitive phenotype. The insertion dam-231 : : Tn10d Tet resulted in a dam "leaky" phenotype since methylated and unmethylated adenines in GATC sequences were present. In addition, the dam-231 : : Tn10d Tet insertion rendered dam mutants temperature-sensitive for growth depending upon the genetic background of the S. typhimurium strain. The wild-type dam gene of S. typhimurium exhibited 82% identity with the Escherichia coli dam gene.
KeywordMeSH Terms
358.     ( 1998 )

Purification and characterization of thin pili of IncI1 plasmids ColIb-P9 and R64: formation of PilV-specific cell aggregates by type IV pili.

Journal of bacteriology 180 (11)
PMID : 9603870  :   PMC  :   PMC107247    
Abstract >>
Thin pili of the closely related IncI1 plasmids ColIb-P9 and R64 are required only for liquid mating and belong to the type IV family of pili. They were sedimented by ultracentrifugation from culture medium in which Escherichia coli cells harboring ColIb-P9- or R64-derived plasmids had been grown, and then the pili were purified by CsCl density gradient centrifugation. In negatively stained thin pilus samples, long rods with a diameter of 6 nm, characteristic of type IV pili, were observed under an electron microscope. Gel electrophoretic analysis of purified ColIb-P9 thin pili indicated that thin pili consist of two kinds of proteins, pilin and the PilV protein. Pilin was demonstrated to be the product of the pilS gene. Pilin was first synthesized as a 22-kDa prepilin from the pilS gene and subsequently processed to a 19-kDa protein by the function of the pilU product. The N-terminal amino group of the processed protein was shown to be modified. The C-terminal segments of the pilV products vary among six or seven different types, as a result of shufflon DNA rearrangements of the pilV gene. These PilV proteins were revealed to comprise a minor component of thin pili. Formation of PilV-specific cell aggregates by ColIb-P9 and R64 thin pili was demonstrated and may play an important role in liquid mating.
KeywordMeSH Terms
359.     ( 1997 )

E. coli translation initiation factor IF2--an extremely conserved protein. Comparative sequence analysis of the infB gene in clinical isolates of E. coli.

FEBS letters 419 (2��3��)
PMID : 9428651  :   DOI  :   10.1016/s0014-5793(97)01472-5    
Abstract >>
The functionally uncharacterised N-terminal of translation initiation factor IF2 has been found to be extremely variable when comparing different bacterial species. In order to study the intraspecies variability of IF2 the 2670 basepairs nucleotide sequence of the infB gene (encoding IF2) was determined in 10 clinical isolates of E. coli. The N-terminal domains (I, II and III) were completely conserved indicating a specific function of this region of IF2. Only one polymorphic position was found in the deduced 890 amino acid sequence. This Gln/Gly490 is located within the central GTP/GDP-binding domain IV of IF2. The results are further evidence that IF2 from E. coli has reached a highly defined level of structural and functional development.
KeywordMeSH Terms
Genes, Bacterial
Polymorphism, Genetic
360.     ( 1998 )

Sequence of the gene encoding a plasmid-mediated cefotaxime-hydrolyzing class A beta-lactamase (CTX-M-4): involvement of serine 237 in cephalosporin hydrolysis.

Antimicrobial agents and chemotherapy 42 (5)
PMID : 9593162  :   PMC  :   PMC105797    
Abstract >>
The sequence of the gene encoding a novel cefotaxime-hydrolyzing beta-lactamase (CTX-M-4) was determined. It was located in a plasmid harbored by a Salmonella typhimurium strain. CTX-M-4 was similar to the plasmidic cefotaxime-hydrolyzing beta-lactamases CTX-M-2 and Toho-1 and related to the chromosomal beta-lactamase of Klebsiella oxytoca. A Ser-237-->Ala substitution, introduced by site-directed mutagenesis, caused minor alterations in the interaction of CTX-M-4 with beta-lactams, reducing slightly the relative hydrolytic activity against cefotaxime and the susceptibility to inhibition by clavulanate.
KeywordMeSH Terms
Bacterial Proteins
361.     ( 1998 )

Role for the Salmonella flavohemoglobin in protection from nitric oxide.

The Journal of biological chemistry 273 (20)
PMID : 9575213  :   DOI  :   10.1074/jbc.273.20.12543    
Abstract >>
Hemoglobin homologs are being identified in an expanding number of unicellular prokaryotic and eukaryotic organisms. Many of these hemoglobins are twodomain proteins that possess a flavin-containing reductase in their C terminus. Determination of a function for these flavohemoglobins has been elusive. A Salmonella typhimurium strain harboring a deletion in the flavohemoglobin gene shows no difference in growth under oxidative stress conditions but displays an increased sensitivity to acidified nitrite and S-nitrosothiols, both of which produce nitric oxide. The effect is seen aerobically or anaerobically, indicating that oxygen is not required for flavohemoglobin function. These results suggest a role for the bacterial flavohemoglobins that is independent of oxygen metabolism and provide evidence for a bacterial route of protection from nitric oxide that is distinct from oxidative stress responses.
KeywordMeSH Terms
362.     ( 1998 )

The assembly system for the lipopolysaccharide R2 core-type of Escherichia coli is a hybrid of those found in Escherichia coli K-12 and Salmonella enterica. Structure and function of the R2 WaaK and WaaL homologs.

The Journal of biological chemistry 273 (15)
PMID : 9535865  :   DOI  :   10.1074/jbc.273.15.8849    
Abstract >>
In Escherichia coli F632, the 14-kilobase pair chromosomal region located between waaC (formerly rfaC) and waaA (kdtA) contains genes encoding enzymes required for the synthesis of the type R2 core oligosaccharide portion of lipopolysaccharide. Ten of the 13 open reading frames encode predicted products sharing greater than 90% total similarity with homologs in E. coli K-12. However, the products of waaK (rfaK) and waaL (rfaL) each resemble homologs in Salmonella enterica serovar Typhimurium but share little similarity with E. coli K-12. The F632 WaaK and WaaL proteins therefore define differences between the type R2 and K-12 outer core oligosaccharides of E. coli lipopolysaccharides. Based on the chemical structure of the core oligosaccharide of an E. coli F632 waaK::aacC1 mutant and in vitro glycosyltransferase analyses, waaK encodes UDP-N-acetylglucosamine:(glucose) lipopolysaccharide alpha1, 2-N-acetylglucosaminyltransferase. The WaaK enzyme adds a terminal GlcNAc side branch substituent that is crucial for the recognition of core oligosaccharide acceptor by the O-polysaccharide ligase, WaaL. Results of complementation analyses of E. coli K-12 and F632 waaL mutants suggest that structural differences between the WaaL proteins play a role in recognition of, and interaction with, terminal lipopolysaccharide core moieties.
KeywordMeSH Terms
Escherichia coli Proteins
Membrane Proteins
363.     ( 1997 )

Expression of thin aggregative fimbriae promotes interaction of Salmonella typhimurium SR-11 with mouse small intestinal epithelial cells.

Infection and immunity 65 (12)
PMID : 9393832  :   PMC  :   PMC175765    
Abstract >>
The factors that mediate binding of Salmonella typhimurium to small intestinal epithelial cells have not been fully characterized. In this paper we demonstrate that elimination of production of thin aggregative fiber by a transposon insertion within the gene encoding the subunit protein of the fiber reduced binding of S. typhimurium SR-11 to a conditionally immortalized proximal small intestinal epithelial cell line established from transgenic mice. This binding defect could be overcome by transcomplementation with a wild-type allele. The conditionally immortalized cell line should prove useful in identifying the epithelial cell receptor for bacterial attachment since expression of its bacterial binding activity can be induced by manipulating the line's proliferative status.
KeywordMeSH Terms
364.     ( 1998 )

A substrate of the centisome 63 type III protein secretion system of Salmonella typhimurium is encoded by a cryptic bacteriophage.

Proceedings of the National Academy of Sciences of the United States of America 95 (5)
PMID : 9482928  :   DOI  :   10.1073/pnas.95.5.2574     PMC  :   PMC19418    
Abstract >>
Salmonella enterica has evolved a type III protein secretion system that allows these enteropathogens to translocate effector molecules directly into the host cell cytoplasm. These effectors mediate a variety of responses, including cytoskeletal rearrangements, cytokine production, and in certain cells, the induction of apoptosis. We report here the characterization of a substrate of this secretion system in S. enterica serovar typhimurium (Salmonella typhimurium) that is homologous to the SopE protein of Salmonella dublin implicated in bacterial entry into cultured epithelial cells. The sopE locus is located within a cluster of genes that encode tail and tail fiber proteins of a cryptic P2-like prophage, outside of the centisome 63 pathogenicity island that encodes the invasion-associated type III secretion system. Southern hybridization analysis revealed that sopE is present in only a subset of S. enterica serovars and that the flanking bacteriophage genes are also highly polymorphic. Encoding effector proteins that are delivered through type III secretion systems in highly mobile genetic elements may allow pathogens to adapt rapidly by facilitating the assembly of an appropriate set of effector proteins required for successful replication in a new environment.
KeywordMeSH Terms
365.     ( 1998 )

Virulence of antibiotic-resistant Salmonella typhimurium.

Proceedings of the National Academy of Sciences of the United States of America 95 (7)
PMID : 9520473  :   DOI  :   10.1073/pnas.95.7.3949     PMC  :   PMC19943    
Abstract >>
We show that most Salmonella typhimurium mutants resistant to streptomycin, rifampicin, and nalidixic acid are avirulent in mice. Of seven resistant mutants examined, six were avirulent and one was similar to the wild type in competition experiments in mice. The avirulent-resistant mutants rapidly accumulated various types of compensatory mutations that restored virulence without concomitant loss of resistance. Such second-site compensatory mutations were more common then reversion to the sensitive wild type. We infer from these results that a reduction in the use of antibiotics might not result in the disappearance of the resistant bacteria already present in human and environmental reservoirs. Thus, second-site compensatory mutations could increase the fitness of resistant bacteria and allow them to persist and compete successfully with sensitive strains even in an antibiotic-free environment.
KeywordMeSH Terms
366.     ( 1998 )

CobD, a novel enzyme with L-threonine-O-3-phosphate decarboxylase activity, is responsible for the synthesis of (R)-1-amino-2-propanol O-2-phosphate, a proposed new intermediate in cobalamin biosynthesis in Salmonella typhimurium LT2.

The Journal of biological chemistry 273 (5)
PMID : 9446573  :   DOI  :   10.1074/jbc.273.5.2684    
Abstract >>
The cobD gene of Salmonella typhimurium LT2 has been cloned, sequenced, and overexpressed. The overexpressed protein had a molecular mass of approximately 40 kDa, in agreement with the mass predicted by the deduced amino acid sequence (40.8 kDa). Computer analysis of the deduced amino acid sequence of CobD identified a consensus pyridoxal phosphate-binding motif. The role of CobD in cobalamin biosynthesis in this bacterium has been established. CobD was shown to decarboxylate L-threonine O-3-phosphate to yield (R)-1-amino-2-propanol O-2-phosphate. We propose that the latter is a substrate in the reaction catalyzed by the CbiB enzyme proposed to be responsible for the conversion of adenosylcobyric acid to adenosylcobinamide and that the product of the reaction is adenosylcobinamide phosphate, not adenosylcobinamide as previously thought. The implications of these findings are discussed in light of the demonstrated kinase activity of the CobU enzyme (O'Toole, G. A., and Escalante-Semerena, J. C. (1995) J. Biol. Chem. 270, 23560-23569) responsible for the conversion of adenosylcobinamide to adenosylcobinamide phosphate. These findings shed light on the strategy used by this bacterium for the assimilation of exogenous unphosphorylated cobinamide from its environment. To our knowledge, CobD is the first enzyme reported to have L-threonine-O-3-phosphate decarboxylase activity, and computer analysis of its amino acid sequence suggests that it may be a member of a new class of pyridoxal phosphate-dependent decarboxylases.
KeywordMeSH Terms
Transaminases
367.     ( 1998 )

Identification of Salmonella typhimurium genes required for colonization of the chicken alimentary tract and for virulence in newly hatched chicks.

Infection and immunity 66 (5)
PMID : 9573095  :   PMC  :   PMC108169    
Abstract >>
From a collection of 2,800 Tn5-TC1 transposon mutants of Salmonella typhimurium F98, 18 that showed reduced intestinal colonization of 3-week-old chicks were identified. The sites of transposon insertion were determined for most of the mutants and included insertions in the lipopolysaccharide biosynthesis genes rfaK, rfaY, rfbK, and rfbB and the genes dksA, clpB, hupA, and sipC. In addition, identification was made of an insertion into a novel gene that encodes a protein showing similarity to the IIC component of the mannose class of phosphoenolpyruvate-carbohydrate phosphotransferase systems, which we putatively called ptsC. Transduction of most of the transposon mutations to a fresh S. typhimurium F98 genetic background and construction of defined mutations in the rfbK, dksA, hupA, sipC, and ptsC genes of S. typhimurium F98 supported the role in colonization of all but the pts locus. The virulence of the rfbK, dksA, hupA, sipC, and ptsC defined mutants and clpB and rfaY transductants in 1-day-old chicks was tested. All but the ptsC and rfaY mutants were attenuated for virulence. A number of other phenotypes associated with some of the mutations are described.
KeywordMeSH Terms
Genes, Bacterial
368.     ( 1998 )

Structure of the sucrose-specific porin ScrY from Salmonella typhimurium and its complex with sucrose.

Nature structural biology 5 (1)
PMID : 9437428  :  
Abstract >>
The X-ray structure of a sucrose-specific porin (ScrY) from Salmonella typhimurium has been determined by multiple isomorphous replacement at 2.4 A resolution both in its uncomplexed form and with bound sucrose. ScrY is a noncrystallographic trimer of identical subunits, each with 413 structurally well-defined amino acids. A monomer is built up of 18 anti-parallel beta-strands surrounding a hydrophilic pore, with a topology closely similar to that of maltoporin. Two non-overlapping sucrose-binding sites were identified in difference Fourier maps. The higher permeability for sucrose of ScrY as compared to maltoporin is mainly accounted for by differences in their pore-lining residues.
KeywordMeSH Terms
Bacterial Proteins

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