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Identification and characterization of a Salmonella typhimurium oxygen-regulated gene required for bacterial internalization.

ABSTRACT: Growth of Salmonella typhimurium in a low-oxygen environment induces the ability of these bacteria to enter mammalian cells. We have carried out a search for invasion genes that are expressed under low-oxygen conditions by using Tn5lacZY transcriptional fusions. Several noninvasive oxygen-regulated lacZY insertion strains have been identified. The invasion defect in one of these noninvasive S. typhimurium strains, BJ66, has been complemented by introduction of a cosmid (pBDJ125) from an S. typhimurium SL1344 gene bank. A 1.9-kb EcoRV DNA fragment subcloned from this cosmid, containing a single open reading frame (orgA), restores the ability of BJ66 to invade mammalian cells. Comparative searches of the GenBank and EMBL sequence data banks with the nucleotide sequence of the gene and deduced amino acid sequence of the protein reveal no significant similarities. Interestingly, hybridization of an orgA gene probe with a P22 chromosomal mapping library demonstrated that the orgA gene maps to a region on the chromosome between 57.5 and 60 min where other Salmonella invasion genes have been mapped. Other enteroinvasive bacteria (Shigella flexneri, Escherichia coli, Yersinia spp., and Listeria monocytogenes) lack sequences which cross hybridize to the probe. We have compared the virulence of S. typhimurium SL1344 and an isogenic orgA mutant in a mouse model of typhoid fever. The orgA mutant was as virulent as the wild-type strain was when inoculated intraperitoneally but is significantly reduced (> 60-fold) in its ability to cause disease by an oral route of infection.

SUBMITTER: Jones BD

PROVIDER: S-EPMC303026 | biostudies-other | 1994 Sep

REPOSITORIES: biostudies-other

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Identification and characterization of a Salmonella typhimurium oxygen-regulated gene required for bacterial internalization.

Jones B D BD Falkow S S

Infection and immunity 19940901 9

Growth of Salmonella typhimurium in a low-oxygen environment induces the ability of these bacteria to enter mammalian cells. We have carried out a search for invasion genes that are expressed under low-oxygen conditions by using Tn5lacZY transcriptional fusions. Several noninvasive oxygen-regulated lacZY insertion strains have been identified. The invasion defect in one of these noninvasive S. typhimurium strains, BJ66, has been complemented by introduction of a cosmid (pBDJ125) from an S. typhi ...[more]

PMID: 8063389

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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.

Dataset Information

Identification and characterization of a Salmonella typhimurium oxygen-regulated gene required for bacterial internalization.

Publications

Identification and characterization of a Salmonella typhimurium oxygen-regulated gene required for bacterial internalization.

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