Transcriptomic and Phenotypic Analyses Suggest a Network between the Transcriptional Regulators HrcA and σ^BinListeria monocytogenes

Abstract

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...

Comparative Analysis of the σB-Dependent Stress Responses inListeria monocytogenesandListeria innocuaStrains Exposed to Selected Stress Conditions

“…For example, microarray data did not show effects of the sigB deletion on transcript levels for mogR, which encodes a transcriptional repressor of flagellar and motility genes (30). While hrcA, which encodes a negative regulator, is positively regulated by B , microarray experiments showed no evidence that HrcA regulates flagellar genes (37). Previous studies have shown that an L. monocytogenes ⌬degU strain as well as a ⌬mogR strain has attenuated virulence in mouse models of infection (30,44,71).…”

Population Diversity of Listeria monocytogenes LO28: Phenotypic and Genotypic Characterization of Variants Resistant to High Hydrostatic Pressure

“…The first cluster contains five immotile variants showing extreme HHP and heat resistance. Resistance to various stresses, as seen for most variants, might be explained by simultaneous activation through regulatory networks of the different stress mechanisms, as previously described for HrcA, CtsR, and B in L. monocytogenes (10,11). The second cluster contains two stressresistant variants with anaerobic growth and aerobic biofilm formation similar to the wild type (Fig.…”

Identification of a σ B -Dependent Small Noncoding RNA in Listeria monocytogenes

“…In addition, a large number of genes were found to be negatively influenced by B , in particular genes encoding flagellar and ribosomal proteins. Intriguingly, many of the B -dependent effects observed in these studies appear to be indirect, i.e., through secondary gene regulatory systems, such as the stress-related transcriptional regulators HrcA and CtsR, the key virulence regulator PrfA, and the RNA chaperone Hfq (15,16).…”