Identification of RpoS (ς ^S )-Regulated Genes in Salmonella enterica Serovar Typhimurium

Abstract

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.

Escherichia coli transcription factor YncC (McbR) regulates colanic acid and biofilm formation by repressing expression of periplasmic protein YbiM (McbA)

“…Moreover, MG1655 yncC and MG1655 have the same specific growth rate during the early exponential phase, but obtain different cell densities at the late exponential stage and stationary stage (the yncC mutant enters stationary stage with a significantly lower cell density, turbidity of 600 nm of 2.7 ± 0.2 vs 5.3 ± 0.1 for the wild-type strain). This suggests YncC functions at the late exponential stage and stationary stage, which are consistent with the finding that the homolog of E. coli yncC in the Gram-negative bacterium Salmonella enterica serovar typhimurium is under the regulation of the stationary-phase sigma factor s S (RpoS) (Ibanez-Ruiz et al, 2000). This mimics bacterial AI-2 internalization behavior (Xavier and Bassler, 2005) and also agrees with the concept that RpoS is intimately associated with E. coli quorum sensing (DeLisa et al, 2001a), which suggests that the quorum-sensing process, stress response process and biofilm development are tightly linked.…”

Salmonella spp. survival strategies within the host gastrointestinal tract

“…Various studies have shown rpoS null mutants to display increased susceptibility to acid pH and attenuated virulence in BALB/c mice after the peroral route of infection (Fang et al, 1992;Lee et al, 1995;Coynault et al, 1996;Bearson et al, 2006;Domínguez-Bernal et al, 2008;Karasova et al, 2009). Although many genes with known functions in stress responses have been identified within the rpoS regulon of S. Typhimurium (Ibanez-Ruiz et al, 2000), further work is required in order to determine the nature of the full regulon and to elucidate the environmental factors that influence its expression within specific microenvironments in the host.…”

Virulence‐Defective Strains of Salmonella enterica Serovar Typhi as Candidates for Education at Level 2 Facilities

“…At least 70 genes are known to be regulated by RpoS, with many more probably still to be found (6,11). Moreover, the functions of RpoS and the known RpoS-regulated genes are not completely understood (11).…”

“…At least 70 genes are known to be regulated by RpoS, with many more probably still to be found (6,11). Moreover, the functions of RpoS and the known RpoS-regulated genes are not completely understood (11). Though the rpoS-deleted strain of S. enterica serovar Typhi exhibited low cytotoxicity in our previous work (12), the safety of rpoS-deleted virulencedefective strains GTC 3P460 and GTC 3P461 was checked in this work to ensure their suitability for use in a teaching laboratory.…”

“…A number of virulence factors are included in the large regulatory network of more than 70 genes controlled by RpoS (11).…”