SigmaB activation under environmental and energy stress conditions in Listeria monocytogenes.

To measure sigmaB activation in Listeria monocytogenes under environmental or energy stress conditions, quantitative reverse transcriptase PCR (TaqMan) was used to determine the levels of transcripts for the sigmaB -dependent opuCA and clpC genes in strains having null mutations in genes encoding regulator of sigma B proteins (rsbT and rsbV) and sigma B (sigB) and in the L. monocytogenes wild-type 10403S strain under different stress conditions. The DeltasigB, DeltarsbT, and DeltarsbV strains previously exhibited increased hemolytic activities compared to the hemolytic activity of the wild-type strain; therefore, transcript levels for hly were also determined. RsbT, RsbV, and sigmaB were all required for opuCA expression during growth under carbon-limiting conditions or following exposure to pH 4.5, salt, ethanol, or the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP). Expression of clpC was RsbT, RsbV, and sigmaB dependent in the presence of CCCP but not under the other conditions. hly expression was not RsbT, RsbV, or sigmaB dependent in the presence of either CCCP or salt. opuCA transcript levels did not increase in the presence of rapidly lethal stresses (i.e., pH 2.5 or 13 mM cumene hydroperoxide) despite the enhanced survival of the wild type compared with the survival of the mutant strains under these conditions. These findings highlight the importance of complementing phenotypic characterizations with gene expression studies to identify direct and indirect effects of null mutations in regulatory genes, such as sigB. Overall, our data show that while sigmaB activation occurs through a single pathway under both environmental and energy stress conditions, regulation of expression of some stress response and virulence genes in the sigmaB regulon (e.g., clpC) appears to require networks involving multiple transcriptional regulators.