SummaryEntry into intestinal epithelial cells is an essential feature in the pathogenicity of Salmonella typhi, which causes typhoid fever in humans. This process requires intact motility and secretion of the invasion-promoting Sip proteins, which are targets of the type III secretion machinery encoded by the inv, spa and prg loci. During our investigations into the entry of S. typhi into cultured epithelial cells, we observed that the secretion of Sip proteins and flagellin was impaired in Vi-expressing strains. We report here that the production of Sip proteins, flagellin and Vi antigen is differentially modulated by the RcsB-RcsC regulatory system and osmolarity. This regulation occurs at both transcriptional and post-translational levels. Under low-osmolarity conditions, the transcription of iagA, invF and sipB genes is negatively controlled by the RcsB regulator, which probably acts in association with the viaB locusencoded TviA protein. The cell surface-associated Vi polysaccharide, which was maximally produced under these growth conditions, prevented the secretion of Sip proteins and flagellin. As the NaCl concentration in the growth medium was increased, transcription of iagA, invF and sipB was found to be markedly increased, whereas transcription of genes involved in Vi antigen biosynthesis was greatly reduced. The expression of iagA, whose product is involved in invF and sipB transcription, occurred selectively during the exponential growth phase and was maximal in the presence of 300 mM NaCl. At this osmolarity, large amounts of Sips and flagellin were secreted in culture supernatants. As expected from these results, and given the essential role of Sip proteins and motility in entry, RcsB and osmolarity modulated the invasive capacity of S. typhi. Together, these findings might reflect the adaptive response of S. typhi to the environments encountered during the different stages of pathogenesis.

The rpoS gene encodes the alternative sigma factor 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.

A Salmonella typhi chromosomal locus composed of five adjacent genes, designated sipEBCDA, was identified by transposon mutagenesis as being essential for cell invasion. Products of the sip genes exhibit extensive sequence similarities to the effectors of Shigella entry into epithelial cells encoded by the virulence plasmid-borne ipa operon. Expression of sipE and sipB in a Shigella non-invasive ipaB mutant restored the ability to invade epithelial cells. The structural and functional conservation of the Sip and Ipa proteins suggests that Salmonella and Shigella entry processes are promoted by similar effectors.

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.