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.

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...

Listeria monocytogenesB positively regulates the transcription of class II stress response genes; CtsR negatively regulates class III stress response genes. To identify interactions between these two stress response systems, we constructed L. monocytogenes ⌬ctsR and ⌬ctsR ⌬sigB strains, as well as a ⌬ctsR strain expressing ctsR in trans under the control of an IPTG (isopropyl-␤-D-thiogalactopyranoside)-inducible promoter. These strains, along with a parent and a ⌬sigB strain, were assayed for motility, heat resistance, and invasion of human intestinal epithelial cells, as well as by whole-genome transcriptomic and quantitative real-time PCR analyses. Both ⌬ctsR and ⌬ctsR ⌬sigB strains had significantly higher thermotolerances than the parent strain; however, full heat sensitivity was restored to the ⌬ctsR strain when ctsR was expressed in trans. Although log-phase ⌬ctsR was not reduced in its ability to infect human intestinal cells, the ⌬ctsR ⌬sigB strain showed significantly lower invasion efficiency than either the parent strain or the ⌬sigB strain, indicating that interactions between CtsR and B contribute to invasiveness. Statistical analyses also confirmed interactions between the ctsR and the sigB null mutations in both heat resistance and invasion phenotypes. Microarray transcriptomic analyses and promoter searches identified (i) 42 CtsR-repressed genes, (ii) 22 genes with lower transcript levels in the ⌬ctsR strain, and (iii) at least 40 genes coregulated by both CtsR and B , including genes encoding proteins with confirmed or plausible roles in virulence and stress response. Our data demonstrate that interactions between CtsR and B play an important role in L. monocytogenes stress resistance and virulence.

The ability of Listeria monocytogenes to grow at refrigeration temperatures is critical for transmission of this foodborne pathogen. We evaluated the contributions of different transcriptional regulators and two-component regulatory systems to L. monocytogenes cold adaptation and cold growth. L. monocytogenes parent strain 10403S and selected isogenic null mutants in genes encoding four alternative sigma factors (sigB, sigH, sigC, and sigL), two regulators of sigmaB (rsbT and rsbV), two negative regulators (ctsR and hrcA), and 15 two-component response regulators were grown in brain heart infusion broth at 4 degrees C with (i) a high-concentration starting inoculum (10(8) CFU/ml), (ii) a low-concentration starting inoculum (102 CFU/ml), and (iii) a high-concentration starting inoculum of cold-adapted cells. With a starting inoculum of 10(8) CFU/ml, null mutants in genes encoding selected alternative sigma factors (DeltasigH, DeltasigC, and DeltasigL), a negative regulator (DeltactsR), regulators of sigmaB (DeltarsbT and DeltarsbV), and selected two-component response regulators (DeltalisR, Deltalmo1172, and Deltalmo1060) had significantly reduced growth (P < 0.05) compared with the parent strain after 12 days at 4 degrees C. The growth defect for DeltasigL was limited and was not confirmed by optical density (OD600) measurement data. With a starting inoculum of 102 CFU/ml and after monitoring growth at 4 degrees C over 84 days, only the DeltactsR strain had a consistent but limited growth defect; the other mutant strains had either no growth defects or limited growth defects apparent at only one or two of the nine sampling points evaluated during the 84-day growth period (DeltasigB, DeltasigC, and Deltalmo1172). With a 10(8) CFU/ml starting inoculum of cold-adapted cells, none of the mutant strains that had a growth defect when inoculation was performed with cells pregrown at 37 degrees C had reduced growth as compared with the parent strain after 12 days at 4 degrees C, suggesting a specific defect in the ability of these mutant strains to adapt to 4 degrees C after growth at 37 degrees C. Our data indicate (i) selected sigma factors and two-component regulators may contribute to cold adaptation even though two-component regulatory systems, alternative sigma factors, and the negative regulators CtsR and HrcA appear to have limited contributions to L. monocytogenes growth at 4 degrees C in rich media, and (ii) inoculum concentration and pregrowth conditions affect the L. monocytogenes cold-growth phenotype.

An understanding of Listeria transmission and contamination patterns in processing environments of ready-to-eat foods is critical for improving control of Listeria monocytogenes. A cold-smoked fish processing operation was the site used to study variability in Listeria contamination in a processing environment associated with a ready-to-eat food product throughout one production week (five consecutive days). Intensive testing was conducted on finished products and environmental samples collected at the beginning, middle, and end of each working day. A total of 20 finished products and 22 to 36 environmental samples were collected at each sampling time, and an additional 12 environmental samples were collected on days 4 and 5. Overall, a total of 782 samples, 300 finished products and 482 environmental samples, were tested. All samples were collected from processing steps after smoking, including skinning, trimming, slicing, staging, and packing. A total of 28 finished and 57 environmental samples (9.3 and 11.8%, respectively) were positive for Listeria spp. (including 1 and 5 samples positive for L. monocytogenes, respectively). DNA sequencing of the sigB gene allowed differentiation of eight Listeria subtypes. Listeria prevalence varied significantly between days, and a high prevalence in both environmental samples and finished products on day 3 was likely associated with a point source contamination event by a single Listeria welshimeri subtype. There were no consistent differences in Listeria prevalence among samples collected from the beginning, middle, and end of the production day, but subtype data often revealed unique contamination patterns for samples collected at different times of a given day. Listeria contamination patterns and prevalences were highly variable between days and within a given day. These findings indicate that chance events play an important role in the contamination of finished products, thus complicating efforts to define Listeria transmission patterns in processing environments associated with ready-to-eat foods.