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The Salmonella typhimurium katF (rpoS) gene: cloning, nucleotide sequence, and regulation of spvR and spvABCD virulence plasmid genes.

ABSTRACT: The spv region of Salmonella virulence plasmids is essential for the development of a systemic infection in mice. Transcriptional activation of the spvABCD operon occurs during stationary growth phase and is mediated by the regulatory gene product SpvR. We have previously shown that expression of a spvRAB'-cat fusion in Escherichia coli was dependent on the katF (rpoS) locus which encodes an alternative sigma factor (sigma S). The katF gene from Salmonella typhimurium has been cloned, sequenced, and used to construct Salmonella katF mutants by allelic replacement. Using these mutants, we demonstrated by mRNA and gene fusion analyses that sigma S, in conjunction with SpvR, controls the transcription of the regulatory gene spvR. In a second series of experiments, we sought to clarify the relationship between sigma S and SpvR in the control of spvABCD transcription. It was shown that expression of a transcriptional spvAB'-lacZ fusion could be restored in E. coli and Salmonella katF mutants when spvR was expressed in trans from an exogenous promoter. Moreover, identical spvA mRNA startpoints were detected in katF+ and katF strains. These results indicate that the reduction of spvABCD transcription in katF mutants is mainly due to decreased expression of spvR. Finally, mouse inoculation studies with S. typhimurium katF mutants of both wild-type and virulence plasmid-cured strains suggest that katF contributes to Salmonella virulence via the regulation of chromosomal genes in addition to that of spv genes.

SUBMITTER: Kowarz L

PROVIDER: S-EPMC197053 | biostudies-other | 1994 Nov

REPOSITORIES: biostudies-other

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The Salmonella typhimurium katF (rpoS) gene: cloning, nucleotide sequence, and regulation of spvR and spvABCD virulence plasmid genes.

Kowarz L L Coynault C C Robbe-Saule V V Norel F F

Journal of bacteriology 19941101 22

The spv region of Salmonella virulence plasmids is essential for the development of a systemic infection in mice. Transcriptional activation of the spvABCD operon occurs during stationary growth phase and is mediated by the regulatory gene product SpvR. We have previously shown that expression of a spvRAB'-cat fusion in Escherichia coli was dependent on the katF (rpoS) locus which encodes an alternative sigma factor (sigma S). The katF gene from Salmonella typhimurium has been cloned, sequenced, ...[more]

PMID: 7961444

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Project description: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.

Project description:Salmonella typhimurium encounters a variety of acid stress situations during growth in host and nonhost environments. The organism can survive potentially lethal acid conditions (pH <4) if it is first able to adapt to mild or more moderate acid levels. The molecular events that occur during this adaptive process are collectively referred to as the acid tolerance response and vary depending on whether the cells are in log- or stationary-phase growth. The acid tolerance response of logarithmically growing cells includes the participation of an alternate sigma factor, sigmaS (RpoS), commonly associated with stationary-phase physiology. Of 51 acid shock proteins (ASPs) induced during shifts to pH 4.4, 8 are clearly dependent on sigmaS for production (I. S. Lee, J. Lin, H. K. Hall, B. Bearson, and J. W. Foster, Mol. Microbiol. 17:155-167, 1995). The acid shock induction of these proteins appears to be the result of an acid shock-induced increase in the level of sigmaS itself. We have discovered that one component of a potential signal transduction system responsible for inducing rpoS expression is the product of the mouse virulence gene mviA+. MviA exhibits extensive homology to the regulatory components of certain two-component signal transduction systems (W. H. Benjamin, Jr., and P. D. Hall, abstr. B-67, p. 38, in Abstracts of the 93rd General Meeting of the American Society for Microbiology 1993, 1993). Mutations in mviA (mviA::Km) caused the overproduction of sigmaS and sigmaS-dependent ASPs in logarithmically growing cells, as well as increases in tolerances to acid, heat, osmolarity and oxidative stresses and significant decreases in growth rate and colony size. Mutations in rpoS suppressed the mviA::Km-associated defects in growth rate, colony size, ASP production, and stress tolerance, suggesting that the effects of MviA on cell physiology occur via its control of sigmaS levels. Western blot (immunoblot) analyses of sigmaS produced from natural or arabinose-regulated promoters revealed that acid shock and MviA posttranscriptionally regulate sigmaS levels. Turnover experiments suggest that MviA regulates the stability of sigmaS protein rather than the translation of rpoS message. We propose a model in which MviA or its unknown signal transduction partner senses some consequence of acid shock, and probably other stresses, and signals the release of sigmaS from proteolysis. The increased concentration of sigmaS drives the elevated expression of the sigmaS-dependent ASPs, resulting in an increase in stress tolerance. The avirulent nature of mviA insertion mutants, therefore, appears to result from inappropriate sigmaS-dependent gene expression during pathogenesis.

Dataset Information

The Salmonella typhimurium katF (rpoS) gene: cloning, nucleotide sequence, and regulation of spvR and spvABCD virulence plasmid genes.

Publications

The Salmonella typhimurium katF (rpoS) gene: cloning, nucleotide sequence, and regulation of spvR and spvABCD virulence plasmid genes.

Similar Datasets

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