The outer membrane permeability mutation of the virulence-associated plasmid of Salmonella typhimurium is located in a traT-like gene

Abstract The SS-A mutation carried by the virulence-as-associated plasmid of Salmonella typhimurium results in increased outer membrane permeability to hydrophobic compounds. A 7.8-kilobase pair Bam HI- Sal I fragment containing the SS-A mutation was cloned from the virulence-associated plasmid into the cloning vector pACYC184. The cloned DNA segment hybridized with a radioactive probed prepared from the traT gene of R6-5. A similar DNA fragment, cloned from the wild-type virulence-associated plasmid, complemented the SS-A mutant phenotype. Both clones produced a protein that immunologically resembled the R6-5 TraT protein; however, the protein produced by the SS-A containing clone appeared truncated by approximately M _r 1000 indicating an alteration in the primary structure or processing of the protein. We conclude that the mutation producing the SS-A phenotype has occured in a traT -like gene of the Salmonella plasmid.     

The DNA supercoiling-sensitive expression of the Salmonella typhimurium his operon requires the his attenuator and is modulated by anaerobiosis and by osmolarity

Bacterial cells possess a subset of genes whose expression correlates with changes in DNA supercoiling brought about by anaerobic growth and by growth at high osmolarity. It has been shown previously that expression of the histidine biosynthetic operon of Salmonella typhimurium is derepressed by relaxation of supercoiled DNA. Here, we confirm that a his::MudJ operon fusion in S. typhimurium can be induced by treatment with the DNA gyrase inhibitor novobiocin in a dose-dependent manner, and show that the level of derepression is higher in stationary phase than in mid-exponential phase cultures. Furthermore, expression of his is repressed by anaerobiosis and by osmolarity, two environmental parameters which increase the negative supercoiling of bacterial DNA. Novobiocin induction of his is also repressed by growing the cells either at high osmolarity or anaerobically. Both environmental repression and novobiocin induction of his require the his attenuator. In addition, derepression of his expression by novobiocin and its repression by anaerobiosis or osmolarity are independent of the stringent response gene, relA.     

Virulence and mutation rates of Salmonella typhimurium strains with increased mutagenic strength in a mouse model

Two strains of Salmonella typhimurium presenting increased mutation rates, either spontaneous or mediated by DNA damage, have been constructed. One of the strains carries a null mutS mutation, while the other harbors plasmid pRW30, which contains the Escherichia coli umuDC operon. The virulence of these strains has been determined by inoculating BALB/c or Swiss mice. The 50% lethal dose of both strains is identical to that obtained for the wild-type. Likewise, the two strains and the wild-type contribute equally to animal death in mixed infections. The frequency of Nal(R) mutants recovered from animals inoculated with either wild-type or MutS(-) cells was not affected by the presence of pRW30. These results indicate that the DNA damage which S. typhimurium cells can suffer during the infectious process by host cell metabolites does not cause induction of the SOS response at levels able to trigger the error-prone DNA repair pathway.     

Genetic mapping of the chromosomal replication origin of Salmonella typhimurium.

Two hundred strains of Escherichia coli harboring Filv+ plasmids which carry a segment of the Salmonella typhimurium chromosome were isolated independently. Among them, two strains were found to harbor F' plasmids that are able to replicate in Hfr cells of E. coli; i.e., they carry a site designated poh (permissive on Hfr) of the S. typhimurium chromosome. The poh site is presumably identical with the replication origin (oriC) of the bacterial chromosome. These two plasmids carry the dnaA-uncA-rbs-ilv-cya-metE region of the chromosome of S. typhimurium. Other F' plasmids which only carried the ilv-cya-metE region were unable to be maintained in Hfr cells. The poh site (= oriC) of S. typhimurium thus is located in the uhp-ilv region of the chromosome. The two plasmids carrying the poh site of S. typhimurium can suppress the temperature-sensitive character of an E. coli mutant that carries the temperature-sensitive dnaA46 allele, when the plasmids exist in the mutant cells. This suggests that the dnaA chromosome in place of the dnaA gene product of E. coli itself. The ability of the plasmids carrying the poh site of S. typhimurium to replicate in Hfr cells of E. coli suggests that the replication system of E. coli can recognize the Salmonella replication origin.     

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

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.     

Characterization of the Salmonella typhimurium pagC/pagD chromosomal region.

The PhoP/PhoQ two-component system regulates Salmonella typhimurium genes that are essential to bacterial virulence and survival within macrophages. The best characterized of these PhoP-activated genes (pag) is pagC, which encodes a 188-amino-acid envelope protein (W. S. Pulkkinen and S. I. Miller, J. Bacteriol. 173:86-93, 1991). We here report the identification of four genes (pagD, envE, msgA, and envF) located 5' to pagC. Each gene is transcribed from its own promoter, two of which (msgA and pagD) were defined by primer extension analysis. Three of these genes (pagD, envE, and envF) are predicted to encode envelope proteins. The pagD gene is transcribed in a direction opposite from that of and adjacent to pagC and is positively regulated by PhoP/PhoQ. Transposon insertions within pagD and msgA attenuate bacterial virulence and survival within macrophages; however, deletion of pagD has no effect on virulence. The product of the envF gene is predicted to be a lipoprotein on the basis of the presence of a consensus lipid attachment site. The low G + C content of these genes and the homology of msgA to Shigella plasmid DNA suggest that this region may have been acquired by horizontal transmission.     

RNA polymerase involvement in the regulation of expression of Salmonella typhimurium pyr genes. Isolation and characterization of a fluorouracil-resistant mutant with high, constitutive expression of the pyrB and pyrE genes due to a mutation in rpoBC

A mutant of Salmonella typhimurium with a defect in the regulation of pyr-gene expression was obtained during a selection for mutants resistant to a combination of the two pyrimidine analogs, 5-fluorouracil and 5-fluorouridine. The mutant possesses 4-fold elevated pools of the pyrimidine nucleoside triphosphatases, UTP and CTP. The specific activities of aspartate transcarbamylase and orotate phosphoribosyltransferase are 40-fold and 7-fold higher in the mutant than in the parent strain when grown in minimal media. Furthermore, the synthesis of the two enzymes in the mutant is not repressed following addition of exogenous pyrimidines. The levels of carbamoylphosphate synthase and orotidine 5'-monophosphate decarboxylase are approximately 3-fold enhanced, while the activities of dihydroorotase and dihydroorotate oxidase appear largely unaffected by the mutation. The mutation responsible for these effects was shown to map between two known point mutations in the rpoBC gene cluster encoding the beta and beta' subunits of RNA polymerase. These observations indicate a regulatory function of RNA polymerase in the control of pyr-gene expression in S. typhimurium.     

Reverse mutation tests in Salmonella typhimurium and chromosomal aberration tests in mammalian cells in culture on fluorinated pyrimidine derivatives

Reverse mutation (Ames) tests with Salmonella typhimurium TA98, TA100 and TA1537, and chromosomal aberration tests in vitro with a Chinese hamster fibroblast cell line (CHL), were carried out on fluorinated pyrimidine derivatives, such as 5-fluorouracil (5-FU), 1-(2-tetrahydrofuryl)-5-fluorouracil (FT), 5-fluorodeoxyuridine (FUdR), 1,3-bis(2-tetrahydrofuryl)-5-fluorouracil (FD-1) and a mixture of uracil and FT in the molar ratio 4 : 1 (UFT) (Fujii et al., 1978). For comparison, similar tests were also carried out on 4 anti-metabolic agents, a metabolite of FD-1 and a component of UFT, such as cytosine-1-beta-D-arabinofuranoside (AraC), 6-mercaptopurine (6-MP), 6-thioguanine (6-TG), 8-azaguanine (8-AG), 3-(2-tetrahydrofuryl)-5-fluorouracil (3-FT) and uracil. The anti-bacterial action of 4 fluorinated pyrimidine derivatives such as 5-FU, FT, FD-1 and UFT to TA100 was tested under the condition that buffer, S9 mix, S9 and albumin were present. 6-MP was only positive in the Ames test with TA100 in the system without S9 mix, while all others failed to show mutagenic activity. On the other hand, all compounds tested, except uracil, induced chromosomal aberrations on CHL cells in the system without metabolic activation. FT was degraded by S9, but there was no significant difference in the killing activity of FT among with buffer, S9 mix and albumin. The killing activity of 5-FU was the strongest with buffer, and it was slightly binding to albumin. The killing activity of 5-FU was mostly decreased by S9 mix. FD-1 showed the strongest anti-bacterial action when S9 mix was present but it was degraded by S9. UFT showed no anti-bacterial action in any conditions.     

Effect of mutation of lysine-120, located at the entry to the active site of O-acetylserine sulfhydrylase-A from Salmonella typhimurium

O-Acetylserine sulfhydrylase catalyzes the final step of the biosynthesis of L-cysteine, the replacement of the beta-acetoxy group of O-acetyl-L-serine (OAS) by a thiol. The enzyme undergoes a conformational change to close the site upon formation of the external Schiff base (ESB) with OAS. Mutation of K120 to Q was predicted to destabilize the closed form of the ESB and decrease the rate. The K120Q mutant enzyme was prepared and characterized by UV-visible absorbance, fluorescence, visible CD, and 31P NMR spectral studies, as well as steady state and pre-steady state kinetic studies. Spectra suggest a shift in the tautomeric equilibrium toward the neutral enolimine and an increase in the rate of interconversion of the open and closed forms of the enzyme. A decrease in the rate of both half reactions likely reflects the stabilization of the ESB as a result of the increased rate of equilibration of the open and closed forms of the enzyme along the reaction pathway. Data suggest a role of K120 in helping to stabilize the closed conformation by participating in a new hydrogen bond to the backbone carbonyl of A231.     

The EnvZ protein of Salmonella typhimurium LT-2 and Escherichia coli K-12 is located in the cytoplasmic membrane

Abstract The osmoregulated expression of the porin proteins OmpC and OmpF in S. typhimurium and E. coli is dependent on the regulatory proteins OmpR and EnvZ. The function of the EnvZ protein is not clear. In order to establish the cellular location of EnvZ two different methods of buoyant sucrose density centrifugation was employed. The presence of EnvZ in the different fractions was visualised by immunoblotting. It was conclusively shown that the EnvZ protein is located in the cytoplasmic membrane fraction. The result is in agreement with the available sequence data which shows that the EnvZ polypeptide contains two long hydrophobic stretches.     

A recombinant Salmonella typhimurium vaccine strain is taken up and survives within murine Peyer's patch dendritic cells

The attenuated Salmonella typhimurium PhoP^c strain is avirulent but immunogenic via the oral route in mice and is attenuated in survival in macrophage cell lines. In this study, the fate of PhoP^c bacteria expressing green fluorescent protein was investigated in murine Peyer's patches. The survival of PhoP^c was monitored after orogastric inoculation of BALB/c mice. Bacteria persisted for several weeks in the Peyer's patches and were also recovered from the mesenteric lymph nodes and spleen. Confocal microscopy analysis identified dendritic cells as the Peyer's patch cell type that internalized PhoP^c expressing green fluorescent protein at early time points. In addition, live PhoP^c were found in Peyer's patch dendritic cells and not in B cells 3 days after orogastric inoculation. Taken together, these results provide strong evidence that PhoP^c is internalized and survives within Peyer's patch dendritic cells. As these cells are potent antigen-presenting cells, these data could explain the immunogenicity of S. typhimurium vaccine strains in vivo .