σB activation under environmental and energy stress conditions in Listeria monocytogenes

To measure sigma(B) 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 sigma(B)-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 Delta sigB, Delta rsbT, and Delta rsbV strains previously exhibited increased hemolytic activities compared to the hemolytic activity of the wild-type strain; therefore, transcript levels for My were also determined. RsbT, RsbV, and (TB 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 GB dependent in the presence of CCCP but not under the other conditions. hly expression was not RsbT, RsbV, or sigma(B) 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 sigma(B) 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 sigma(B) regulon (e.g., clpC) appears to require networks involving multiple transcriptional regulators.