The alternative sigma factor B contributes to transcription of stress response and virulence genes in diverse gram-positive bacterial species. The composition and functions of the Listeria monocytogenes and Listeria innocua B regulons were hypothesized to differ due to virulence differences between these closely related species. Transcript levels in stationary-phase cells and in cells exposed to salt stress were characterized by microarray analyses for both species. In L. monocytogenes, 168 genes were positively regulated by B ; 145 of these genes were preceded by a putative B consensus promoter. In L. innocua, 64 genes were positively regulated by B . B contributed to acid stress survival in log-phase cells for both species but to survival in stationary-phase cells only for L. monocytogenes. In summary, (i) the L. monocytogenes A sigma factor is a dissociable protein subunit that directs bacterial RNA polymerase holoenzyme to recognize a promoter sequence upstream of a gene prior to transcription initiation. New associations between alternative sigma factors and core RNA polymerase essentially reprogram promoter recognition specificities of the enzyme in response to changing environmental conditions, thus allowing expression of new sets of target genes appropriate for the conditions. The alternative sigma factor B , encoded by sigB, has been identified as contributing to the general stress response in several grampositive bacteria, including Bacillus subtilis (31), Bacillus licheniformis (7), Bacillus anthracis (24), Bacillus cereus (68), Listeria monocytogenes (2, 70), Staphylococcus aureus (75), and Corynebacterium glutamicum (47).L. monocytogenes is a non-spore-forming facultative intracellular pathogen that causes listeriosis, a serious invasive disease in both animals and humans. To establish a food-borne bacterial infection, L. monocytogenes must have the ability to survive under a variety of stress conditions, including those encountered in a wide range of nonhost environments and food matrices, as well as under rapidly changing conditions encountered during gastrointestinal passage (exposure to organic acids, bile salts, and osmotic gradients) and subsequent stages of infection (e.g., in the intracellular environment). L. monocytogenesB is activated following exposure to a number of environmental stress conditions (2) and contributes to bacterial survival under acid and oxidative stresses and during carbon starvation (13,21,22,70). In addition, transcription of several virulence genes, including prfA, bsh, inlA, and inlB, is at least partially B dependent (41-43, 53, 58, 59, 65, 66). Further, a ⌬sigB null mutant has reduced invasiveness in human intestinal epithelial cells (42) and reduced virulence in intragastrically inoculated guinea pigs (27). A previous study using a subgenomic microarray comprised of 208 L. monocytogenesspecific probes (41) identified 55 B -dependent L. monocytogenes genes with Ն1.5-fold-higher transcript levels in the parent strain than in an isogenic sigB null mutant. However, a...

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 can respond rapidly to changing environmental conditions, as illustrated by its ability to transition from a saprophyte to an orally transmitted facultative intracellular pathogen. Differential associations between various alternative σ factors and a core RNA polymerase provide a transcriptional mechanism for regulating bacterial gene expression that is crucial for survival in rapidly changing conditions. Alternative σ factors are key components of complex L. monocytogenes regulatory networks that include multiple transcriptional regulators of stress-response and virulence genes, regulation of genes encoding other regulators, and regulation of small RNAs. In this article, the contributions of various σ factors to L. monocytogenes stress response and virulence are described.

Whole-genome microarray experiments were performed to define the Listeria monocytogenes cold growth regulon and to identify genes differentially expressed during growth at 4 and 37°C. Microarray analysis using a stringent cutoff (adjusted P < 0.001; >2.0-fold change) revealed 105 and 170 genes that showed higher transcript levels in logarithmic-and stationary-phase cells, respectively, at 4°C than in cells grown at 37°C. A total of 74 and 102 genes showed lower transcript levels in logarithmic-and stationary-phase cells, respectively, grown at 4°C. Genes with higher transcript levels at 4°C in both stationary-and log-phase cells included genes encoding a two-component response regulator (lmo0287), a cold shock protein (cspL), and two RNA helicases (lmo0866 and lmo1722), whereas a number of genes encoding virulence factors and heat shock proteins showed lower transcript levels at 4°C. Selected genes that showed higher transcript levels at 4°C during both stationary and log phases were confirmed by quantitative reverse transcriptase PCR. Our data show that (i) a large number of L. monocytogenes genes are differentially expressed at 4 and 37°C, with more genes showing higher transcript levels than lower transcript levels at 4°C, (ii) L. monocytogenes genes with higher transcript levels at 4°C include a number of genes and operons with previously reported or plausible roles in cold adaptation, and (iii) L. monocytogenes genes with lower transcript levels at 4°C include a number of virulence and virulenceassociated genes as well as some heat shock genes.

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 Listeria monocytogenes genome contains more than 20 genes that encode cell surface–associated internalins. To determine the contributions of the alternative sigma factor σB and the virulence gene regulator PrfA to internalin gene expression, a subgenomic microarray was designed to contain two probes for each of 24 internalin-like genes identified in the L. monocytogenes 10403S genome. Competitive microarray hybridization was performed on RNA extracted from (i) the 10403S parent strain and an isogenic ΔsigB strain; (ii) 10403S and an isogenic ΔprfA strain; (iii) a (G155S) 10403S derivative that expresses the constitutively active PrfA (PrfA*) and the ΔprfA strain; and (iv) 10403S and an isogenic ΔsigBΔprfA strain. σB- and PrfA-dependent transcription of selected genes was further confirmed by quantitative reverse-transcriptase polymerase chain reaction. For the 24 internalin-like genes examined, (i) both σB and PrfA contributed to transcription of inlA and inlB, (ii) only σB contributed to transcription of inlC2, inlD, lmo0331, and lmo0610; (iii) only PrfA contributed to transcription of inlC and lmo2445; and (iv) neither σB nor PrfA contributed to transcription of the remaining 16 internalin-like genes under the conditions tested.

Summary We propose an empirical Bayes method based on the extreme value theory (EVT) (BE) for the analysis of data from spotted microarrays where the interest of the investigator (e.g. to identify up-regulated gene markers of a disease) or the design of the experiment (e.g. in certain ‘wild-type versus mutant’ experiments) limits identification of differentially expressed genes to those regulated in a single direction (either up or down). In such experiments, unlike in genome-wide microarrays, analysis is restricted to the tail of the distribution (extremes) of all the genes in the genome. The EVT provides a platform to account for this extreme behaviour, and is therefore a natural candidate for inference about differential expression. We compared the performance of the developed BE method with two other empirical Bayes methods on two real ‘wild-type versus mutant’ datasets where a single direction of regulation was expected due to experimental design, and in a simulation study. The BE method appears to have a better fit to the real data. In the analysis of simulated data, the BE method showed better accuracy and precision while being robust to different characteristics of microarray experiments. The BE method, therefore, seems promising and useful for inference about differential expression in microarrays where either only up- or down-regulated genes are relevant or expected.