A set of seven Listeria monocytogenes 10403S mutant strains, each bearing an in-frame null mutation in a gene encoding a key regulatory protein, was used to characterize transcriptional networks in L. , and at least one additional regulator). Comparative phenotypic characterization measuring acid resistance, heat resistance, intracellular growth in J774 cells, invasion into Caco-2 epithelial cells, and virulence in the guinea pig model indicated contributions of (i)B to acid resistance, (ii) CtsR to heat resistance, and (iii) PrfA, B , and CtsR to virulence-associated characteristics. Loss of the remaining transcriptional regulators (i.e., sigH, sigL, or sigC) resulted in limited phenotypic consequences associated with stress survival and virulence. Identification of overlaps among the regulons provides strong evidence supporting the existence of complex regulatory networks that appear to provide the cell with regulatory redundancies, along with the ability to fine-tune gene expression in response to rapidly changing environmental conditions.

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Listeria monocytogenes HrcA and CtsR negatively regulate class I and III stress response genes, respectively, while B positively regulates the transcription of class II stress response genes. To define the HrcA regulon and identify interactions between HrcA, CtsR, and B , we characterized newly generated L. monocytogenes ⌬hrcA, ⌬ctsR ⌬hrcA, and ⌬hrcA ⌬sigB strains, along with previously described ⌬sigB, ⌬ctsR, and ⌬ctsR ⌬sigB strains, using phenotypic assays (i.e., heat resistance, acid resistance, and invasion of human intestinal epithelial cells) and performed whole-genome transcriptome analysis of the ⌬hrcA strain. The hrcA and sigB deletions had significant effects on heat resistance. While the hrcA deletion had no significant effect on acid resistance or invasion efficiency in Caco-2 cells, a linear regression model revealed a significant (P ‫؍‬ 0.0493) effect of interactions between the hrcA deletion and the ctsR deletion on invasiveness. Microarray-based transcriptome analyses and promoter searches identified (i) 25 HrcA-repressed genes, including two operons (the groESL and dnaK operons, both confirmed as HrcA regulated by quantitative real-time PCR) and one gene directly repressed by HrcA, and (ii) 36 genes that showed lower transcript levels in the ⌬hrcA strain and thus appear to be indirectly upregulated by HrcA. A number of genes were found to be coregulated by either HrcA and CtsR (2 genes), HrcA and B (31 genes), or all three regulators (5 genes, e.g., gadCB). Combined with previous evidence that B appears to directly regulate hrcA transcription, our data suggest that HrcA and B , as well as CtsR, form a regulatory network that contributes to the transcription of a number of L. monocytogenes genes.Listeria monocytogenes is a gram-positive food-borne pathogen that can cause severe invasive disease in humans, as well as in a number of different animal species (31). The capacity of L. monocytogenes to survive and multiply under a wide range of environmental stress conditions appears to be critical for the food-borne transmission of this pathogen (10). A number of transcriptional regulators (e.g., PrfA, B , HrcA, and CtsR) that are important for the transcription of stress response and virulence genes have been identified in this organism (20,25,32,37). While clear evidence for interactions between PrfA and B has been reported (26,40,45), our understanding of interactions among other L. monocytogenes transcriptional regulators is limited. As no L. monocytogenes hrcA null mutant appears to have previously been reported, our understanding of the contributions of the negative regulator HrcA to stress response, transcriptional regulation, and regulatory networks is limited. In a number of gram-positive bacteria, including Bacillus subtilis, HrcA (heat regulation at CIRCE) has been found to repress the dnaK and groESL operons by binding to a region designated as the controlling inverted-repeat chaperone expression (CIRCE) element (38). Sequence analyses in L. monocytogenes also identified putative CIRCE ele...

The stress-responsive alternative sigma factor σB is conserved across diverse Gram-positive bacterial genera. In Listeria monocytogenes, σB regulates transcription of >150 genes, including genes contributing to virulence and to bacterial survival under host-associated stress conditions, such as those encountered in the human gastrointestinal lumen. An inhibitor of L. monocytogenes σB activity was identified by screening ~57,000 natural and synthesized small molecules using a high-throughput cell-based assay. The compound fluoro-phenyl-styrene-sulfonamide (FPSS) (IC50 = 3.5 µM) downregulated the majority of genes previously identified as members of the σB regulon in L. monocytogenes 10403S, thus generating a transcriptional profile comparable to that of a 10403S ΔsigB strain. Specifically, of the 208 genes downregulated by FPSS, 75% had been identified previously as positively regulated by σB. Downregulated genes included key virulence and stress response genes, such as inlA, inlB, bsh, hfq, opuC, and bilE. From a functional perspective, FPSS also inhibited L. monocytogenes invasion of human intestinal epithelial cells and bile salt hydrolase activity. The ability of FPSS to inhibit σB activity in both L. monocytogenes and Bacillus subtilis indicates its utility as a specific inhibitor of σB across multiple Gram-positive genera.