Influence of the sigB gene on the cold stress survival and subsequent recovery of two Listeria monocytogenes serotypes

108

117

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.

Section: Discussionmentioning

confidence: 92%

Microarray-Based Characterization of theListeria monocytogenesCold Regulon in Log- and Stationary-Phase Cells

Chan

Raengpradub

Boor

et al. 2007

108

117

“…The characterization of sigB null mutants has also shown that L. monocytogenes B contributes to survival under a variety of stress conditions, including acid and oxidative stresses and during carbon starvation (3,10,35). CtsR (class three stress gene repressor) is a transcriptional repressor which regulates at least 64 genes in L. monocytogenes, including directly repressing at least 10 genes (e.g., clpC, clpB, and clpP) (21, 37).…”

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confidence: 99%

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

Oliver

Raengpradub

et al. 2007

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...

“…In L. monocytogenes, B contributes to cell survival under stress conditions, including exposure to low pH, oxidative stress, carbon starvation, and growth at low temperatures (14,15,35,54,55 plays a major role in mouse septic arthritis (24), although it is not essential for infection in the mouse abscess model, the mouse hematogenous pyelonephritis model, or the rat osteomyelitis model (7,38).…”

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confidence: 99%

“…In L. monocytogenes, B contributes to cell survival under stress conditions, including exposure to low pH, oxidative stress, carbon starvation, and growth at low temperatures (14,15,35,54,55). Loss of B also reduces the ability of L. monocytogenes to invade human intestinal epithelial cells (20,29,30,31), as well as its virulence in the murine (36,55) and guinea pig models (20).…”

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confidence: 99%

σ^BActivation under Environmental and Energy Stress Conditions inListeria monocytogenes

Chaturongakul

Boor

2006

To measure 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 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 ⌬sigB, ⌬rsbT, and ⌬rsbV strains previously exhibited increased hemolytic activities compared to the hemolytic activity of the wild-type strain; therefore, transcript levels for hly were also determined. RsbT, RsbV, and B 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 B dependent in the presence of CCCP but not under the other conditions. hly expression was not RsbT, RsbV, or 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 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 B regulon (e.g., clpC) appears to require networks involving multiple transcriptional regulators.Listeria monocytogenes is a non-spore-forming, gram-positive, facultative intracellular pathogen. The emergence of this organism as a difficult-to-control food-borne pathogen is at least in part due to its ability to survive in a broad range of ecological niches (13) and in many different hosts, including both animals and humans (11,50). Contamination of foods with L. monocytogenes raises both public health and economic concerns (33, 57). Although rare, listeriosis is a severe disease that results in death in 20 to 30% of reported cases (33). Infection in humans occurs predominantly among pregnant women, newborns, the elderly, and immunocompromised adults.B is a stress-responsive alternative sigma factor that has been identified in various low-GϩC-content gram-positive bacteria, including the genera Bacillus, Staphylococcus, and Listeria. In L. monocytogenes, B contributes to cell survival under stress conditions, including exposure to low pH, oxidative stress, carbon starvation, and growth at low temperatures (14,15,35,54,55 plays a major role in mouse septic arthritis (24), although it is not essential for infection in the mouse abscess model, the mouse hematogenous pyelonephritis model, or the rat osteomyelitis ...