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To determine the contribution of sigma B (ςB) to survival of stationary-phase Listeria monocytogenes cells following exposure to environmental stresses, we compared the viability of strain 10403S with that of an isogenic nonpolar sigB null mutant strain after exposure to heat (50°C), ethanol (16.5%), or acid (pH 2.5). Strain viabilities were also determined under the same conditions in cultures that had been previously exposed to sublethal levels of the same stresses (45°C, 5% ethanol, or pH 4.5). The ΔsigB and wild-type strains had similar viabilities following exposure to ethanol and heat, but the ΔsigB strain was almost 10,000-fold more susceptible to lethal acid stress than its parent strain. However, a 1-h preexposure to pH 4.5 yielded a 1,000-fold improvement in viability for the ΔsigB strain. These results suggest the existence in L. monocytogenes of both a ςB-dependent mechanism and a pH-dependent mechanism for acid resistance in the stationary phase. ςB contributed to resistance to both oxidative stress and carbon starvation in L. monocytogenes. The ΔsigB strain was 100-fold more sensitive to 13.8 mM cumene hydroperoxide than the wild-type strain. Following glucose depletion, the ΔsigB strain lost viability more rapidly than the parent strain. ςB contributions to viability during carbon starvation and to acid resistance and oxidative stress resistance support the hypothesis that ςB plays a role in protecting L. monocytogenes against environmental adversities.

SbrE is a ncRNA in Listeria monocytogenes, reported to be up-regulated by the alternative sigma factor σB. Initial quantitative RT-PCR (qRT-PCR) experiments on parent strains and isogenic ΔsigB strains demonstrated σB-dependent expression of SbrE across the four L. monocytogenes lineages and in L. innocua. Microarray and proteomics (MDLC/MS/MS with iTRAQ labeling) experiments with the L. monocytogenes parent strain and an isogenic ΔsbrE strain identified a single gene (lmo0636) and two proteins (Lmo0637 and Lmo2094) that showed lower expression levels in the ΔsbrE strain. qRT-PCR demonstrated an increase in SbrE transcript levels in stationary phase L. monocytogenes and in bacteria exposed to oxidative stress (mean log2 transcript levels 7.68 ± 0.57 and 1.70 ± 0.71 greater than in mid-log phase cells, respectively). However, no significant differences in growth or survival between the parent strain and ΔsbrE strain were confirmed under a variety of environmental stress conditions tested. Our data suggest that σB-dependent transcription of SbrE represents a conserved mechanism that contributes, across Listeria species, to fine-tuning of gene expression under specific environmental conditions that remain to be defined.

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.

Abstract

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.

In Listeria monocytogenes the alternative sigma factor σB plays important roles in both virulence and stress tolerance. In this study a proteomic approach was used to define components of the σB regulon in L. monocytogenes 10403S (serotype 1/2a). Using two-dimensional gel electrophoresis and the recently developed isobaric tags for relative and absolute quantitation technique, the protein expression profiles of the wild type and an isogenic ΔsigB deletion strain were compared. Overall, this study identified 38 proteins whose expression was σB dependent; 17 of these proteins were found to require the presence of σB for full expression, while 21 were expressed at a higher level in the ΔsigB mutant background. The data obtained with the two proteomic approaches showed limited overlap (four proteins were identified by both methods), a finding that highlights the complementarity of the two technologies. Overall, the proteomic data reaffirmed a role for σB in the general stress response and highlighted a probable role for σB in metabolism, especially in the utilization of alternative carbon sources. Proteomic and physiological data revealed the involvement of σB in glycerol metabolism. Five newly identified members of the σB regulon were shown to be under direct regulation of σB using reverse transcription-PCR (RT-PCR), while random amplification of cDNA ends-PCR was used to map four σB-dependent promoters upstream from lmo0796, lmo1830, lmo2391, and lmo2695. Using RT-PCR analysis of known and newly identified σB-dependent genes, as well as proteomic analyses, σB was shown to play a major role in the stationary phase of growth in complex media.

The food-borne pathogen Listeria monocytogenes can acquire enhanced resistance to lethal acid conditions through multiple mechanisms. We investigated contributions of the stress-responsive alternative sigma factor, σB, which is encoded by sigB, to growth phase-dependent acid resistance (AR) and to the adaptive acid tolerance response in L. monocytogenes. At various points throughout growth, we compared the relative survival of L. monocytogenes wild-type and ΔsigB strains that had been exposed to either brain heart infusion (pH 2.5) or synthetic gastric fluid (pH 2.5) with and without prior acid adaptation. Under these conditions, survival of the ΔsigB strain was consistently lower than that of the wild-type strain throughout all phases of growth, ranging from 4 orders of magnitude less in mid-log phase to 2 orders of magnitude less in stationary phase. Survival of both ΔsigB and wild-type L. monocytogenes strains increased by 6 orders of magnitude upon entry into stationary phase, demonstrating that the L. monocytogenes growth phase-dependent AR mechanism is σB independent. σB-mediated contributions to acquired acid tolerance appear to be greatest in early logarithmic growth. Loss of a functional σB reduced the survival of L. monocytogenes at pH 2.5 to a greater extent in the presence of organic acid (100 mM acetic acid) than in the presence of inorganic acid alone (HCl), suggesting that L. monocytogenes protection against organic and inorganic acid may be mediated through different mechanisms. σB does not appear to contribute to pHi homeostasis through regulation of net proton movement across the cell membrane or by regulation of pHi buffering by the GAD system under the conditions examined in this study. In summary, a functional σB protein is necessary for full resistance of L. monocytogenes to lethal acid treatments.

To measure σB 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 σB-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 ΔsigB, ΔrsbT, and ΔrsbV 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 σB 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 σB dependent in the presence of CCCP but not under the other conditions. hly expression was not RsbT, RsbV, or σB 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 σB 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 σB regulon (e.g., clpC) appears to require networks involving multiple transcriptional regulators.

ABSTRACT

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.

IMPORTANCE

The σB transcription factor regulates expression of genes responsible for bacterial survival under changing environmental conditions and for virulence; therefore, this alternative sigma factor is important for transmission of L. monocytogenes and other Gram-positive bacteria. Regulation of σB activity is complex and tightly controlled, reflecting the key role of this factor in bacterial metabolism. We present multiple lines of evidence indicating that fluoro-phenyl-styrene-sulfonamide (FPSS) specifically inhibits activity of σB across Gram-positive bacterial genera, i.e., in both Listeria monocytogenes and Bacillus subtilis. Therefore, FPSS is an important new tool that will enable novel approaches for exploring complex regulatory networks in L. monocytogenes and other Gram-positive pathogens and for investigating small-molecule applications for controlling pathogen transmission.

Abstract

Mounting evidence suggests that σB and PrfA coregulate transcription of multiple genes in Listeria monocytogenes, therefore, the relative contributions of σB and PrfA to transcript levels of genes identified previously as differentially regulated by PrfA were measured. Group I genes are recognized virulence genes that are positively regulated by PrfA; group II genes were reported previously as negatively regulated by PrfA; and multiple group III genes were proposed to be coregulated by σB and PrfA. Transcript levels for selected genes were measured by quantitative reverse transcriptase polymerase chain reaction (RT-PCR) in L. monocytogenes 10403S as well as in otherwise isogenic ΔsigB, ΔprfA, and ΔsigBΔprfA strains grown under conditions demonstrated to induce either PrfA activity (0.2% activated charcoal) or both PrfA and σB activity (stationary phase). Although the Group I gene plcA was positively regulated by PrfA, transcript levels for the group II genes lmo0278 and lmo0178 were not affected by the prfA deletion. While the sigB deletion significantly affected transcript levels for the selected group III genes (i.e., lmo0596, lmo0654, bsh, and opuCA), with lower transcript levels in the ΔsigB strains under all conditions tested, transcript levels for these genes were not significantly affected by the prfA deletion. Our results suggest that the regulatory interactions between PrfA and σB contribute to PrfA's predominant role as a direct regulator of virulence genes critical for invasion and intracellular survival in L. monocytogenes 10403S, while σB regulates a wider range of virulence and stress response genes.

The gene encoding the alternative sigma factor σB in Listeria monocytogenes is induced upon exposure of cells to several stresses. In this study, we investigated the impact of a sigB null mutation on the survival of L. monocytogenes EGD-e at low pH, during high-hydrostatic-pressure treatment, and during freezing. The survival of ΔsigB mutant exponential-phase cells at pH 2.5 was 10,000-fold lower than the survival of EGD-e wild-type cells. Moreover, the ΔsigB mutant failed to show an acid tolerance response. Upon preexposure for 1 h to pH 4.5, the survival at pH 2.5 was 100,000-fold lower for the ΔsigB mutant than for the wild type. The glutamate decarboxylase (GAD) acid resistance system is important in survival and adaptation of L. monocytogenes in acidic conditions. The σB dependence of the gad genes (gadA, gadB, gadC, gadD, and gadE) was analyzed in silico. Putative σB-dependent promoter sites were found upstream of the gadCB operon (encoding a glutamate/γ-aminobutyrate antiporter and a glutamate decarboxylase, respectively) and the lmo2434 gene (gadD, encoding a putative glutamate decarboxylase). Reverse transcriptase PCR revealed that expression of the gadCB operon and expression of gadD are indeed σB dependent. In addition, a proteomics approach was used to analyze the protein expression profiles upon acid exposure. Although the GAD proteins were not recovered, nine proteins accumulated in the wild type but not in the ΔsigB strain. These proteins included Pfk, GalE, ClpP, and Lmo1580. Exposure to pH 4.5, in order to preload cells with active σB and consequently with σ B-dependent general stress proteins, also provided considerable protection against high-hydrostatic-pressure treatment and freezing. The combined data argue that the expression of σB-dependent genes provides L. monocytogenes with nonspecific multiple-stress resistance that may be relevant for survival in the natural environment as well as during food processing.

The role of the stress response regulator σB (encoded by sigB) in directing the expression of selected putative and confirmed cold response genes was evaluated using Listeria monocytogenes 10403S and an isogenic ΔsigB mutant, which were either cold shocked at 4°C in brain heart infusion (BHI) broth for up to 30 min or grown at 4°C in BHI for 12 days. Transcript levels of the housekeeping genes rpoB and gap, the σB-dependent genes opuCA and bsh, and the cold stress genes ltrC, oppA, and fri were measured using quantitative reverse transcriptase PCR. Transcriptional start sites for ltrC, oppA, and fri were determined using rapid amplification of cDNA ends PCR. Centrifugation was found to rapidly induce σB-dependent transcription, which necessitated the use of centrifugation-independent protocols to evaluate the contributions of σB to transcription during cold shock. Our data confirmed that transcription of the cold stress genes ltrC and fri is at least partially σB dependent and experimentally identified a σB-dependent ltrC promoter. In addition, our data indicate that (i) while σB activity is induced during 30 min of cold shock, this cold shock does not induce the transcription of σB-dependent or -independent cold shock genes; (ii) σB is not required for L. monocytogenes growth at 4°C in BHI; and (iii) transcription of the putative cold stress genes opuCA, fri, and oppA is σB independent during growth at 4°C, while both bsh and ltrC show growth phase and σB-dependent transcription during growth at 4°C. We conclude that σB-dependent and σB-independent mechanisms contribute to the ability of L. monocytogenes to survive and grow at low temperatures.

Listeria monocytogenes σB 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 ΔctsR and ΔctsR ΔsigB strains, as well as a ΔctsR strain expressing ctsR in trans under the control of an IPTG (isopropyl-β-d-thiogalactopyranoside)-inducible promoter. These strains, along with a parent and a ΔsigB 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 ΔctsR and ΔctsR ΔsigB strains had significantly higher thermotolerances than the parent strain; however, full heat sensitivity was restored to the ΔctsR strain when ctsR was expressed in trans. Although log-phase ΔctsR was not reduced in its ability to infect human intestinal cells, the ΔctsR ΔsigB strain showed significantly lower invasion efficiency than either the parent strain or the ΔsigB strain, indicating that interactions between CtsR and σB 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 ΔctsR strain, and (iii) at least 40 genes coregulated by both CtsR and σB, including genes encoding proteins with confirmed or plausible roles in virulence and stress response. Our data demonstrate that interactions between CtsR and σB play an important role in L. monocytogenes stress resistance and virulence.

While the stress-responsive alternative sigma factor σB has been identified in different species of Bacillus, Listeria, and Staphylococcus, the σB regulon has been extensively characterized only in B. subtilis. We combined biocomputing and microarray-based strategies to identify σB-dependent genes in the facultative intracellular pathogen Listeria monocytogenes. Hidden Markov model (HMM)-based searches identified 170 candidate σB-dependent promoter sequences in the strain EGD-e genome sequence. These data were used to develop a specialized, 208-gene microarray, which included 166 genes downstream of HMM-predicted σB-dependent promoters as well as selected virulence and stress response genes. RNA for the microarray experiments was isolated from both wild-type and ΔsigB null mutant L. monocytogenes cells grown to stationary phase or exposed to osmotic stress (0.5 M KCl). Microarray analyses identified a total of 55 genes with statistically significant σB-dependent expression under the conditions used in these experiments, with at least 1.5-fold-higher expression in the wild type over the sigB mutant under either stress condition (51 genes showed at least 2.0-fold-higher expression in the wild type). Of the 55 genes exhibiting σB-dependent expression, 54 were preceded by a sequence resembling the σB promoter consensus sequence. Rapid amplification of cDNA ends-PCR was used to confirm the σB-dependent nature of a subset of eight selected promoter regions. Notably, the σB-dependent L. monocytogenes genes identified through this HMM/microarray strategy included both stress response genes (e.g., gadB, ctc, and the glutathione reductase gene lmo1433) and virulence genes (e.g., inlA, inlB, and bsh). Our data demonstrate that, in addition to regulating expression of genes important for survival under environmental stress conditions, σB also contributes to regulation of virulence gene expression in L. monocytogenes. These findings strongly suggest that σB contributes to L. monocytogenes gene expression during infection.

The alternative sigma factor σB 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 σB 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 σB; 145 of these genes were preceded by a putative σB consensus promoter. In L. innocua, 64 genes were positively regulated by σB. σB 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 σB regulon includes >140 genes that are both directly and positively regulated by σB, including genes encoding proteins with importance in stress response, virulence, transcriptional regulation, carbohydrate metabolism, and transport; (ii) a number of L. monocytogenes genes encoding flagellar proteins show higher transcript levels in the ΔsigB mutant, and both L. monocytogenes and L. innocua ΔsigB null mutants have increased motility compared to the respective isogenic parent strains, suggesting that σB affects motility and chemotaxis; and (iii) although L. monocytogenes and L. innocua differ in σB-dependent acid stress resistance and have species-specific σB-dependent genes, the L. monocytogenes and L. innocua σB regulons show considerable conservation, with a common set of at least 49 genes that are σB dependent in both species.

Sigma B (σB) is an alternative sigma factor that controls the transcriptional response to stress in Listeria monocytogenes and is also known to play a role in the virulence of this human pathogen. In the present study we investigated the impact of a sigB deletion on the proteome of L. monocytogenes grown in a chemically defined medium both in the presence and in the absence of osmotic stress (0.5 M NaCl). Two new phenotypes associated with the sigB deletion were identified using this medium. (i) Unexpectedly, the strain with the ΔsigB deletion was found to grow faster than the parent strain in the growth medium, but only when 0.5 M NaCl was present. This phenomenon was independent of the carbon source provided in the medium. (ii) The ΔsigB mutant was found to have unusual Gram staining properties compared to the parent, suggesting that σB contributes to the maintenance of an intact cell wall. A proteomic analysis was performed by two-dimensional gel electrophoresis, using cells growing in the exponential and stationary phases. Overall, 11 proteins were found to be differentially expressed in the wild type and the ΔsigB mutant; 10 of these proteins were expressed at lower levels in the mutant, and 1 was overexpressed in the mutant. All 11 proteins were identified by tandem mass spectrometry, and putative functions were assigned based on homology to proteins from other bacteria. Five proteins had putative functions related to carbon utilization (Lmo0539, Lmo0783, Lmo0913, Lmo1830, and Lmo2696), while three proteins were similar to proteins whose functions are unknown but that are known to be stress inducible (Lmo0796, Lmo2391, and Lmo2748). To gain further insight into the role of σB in L. monocytogenes, we deleted the genes encoding four of the proteins, lmo0796, lmo0913, lmo2391, and lmo2748. Phenotypic characterization of the mutants revealed that Lmo2748 plays a role in osmotolerance, while Lmo0796, Lmo0913, and Lmo2391 were all implicated in acid stress tolerance to various degrees. Invasion assays performed with Caco-2 cells indicated that none of the four genes was required for mammalian cell invasion. Microscopic analysis suggested that loss of Lmo2748 might contribute to the cell wall defect observed in the ΔsigB mutant. Overall, this study highlighted two new phenotypes associated with the loss of σB. It also demonstrated clear roles for σB in both osmotic and low-pH stress tolerance and identified specific components of the σB regulon that contribute to the responses observed.

Some Listeria monocytogenes internalins are recognized as contributing to invasion of mammalian tissue culture cells. While PrfA is well established as a positive regulator of L. monocytogenes virulence gene expression, the stress-responsive σB has been recognized only recently as contributing to expression of virulence genes, including some that encode internalins. To measure the relative contributions of PrfA and σB to internalin gene transcription, we used reverse transcription-PCR to quantify transcript levels for eight internalin genes (inlA, inlB, inlC, inlC2, inlD, inlE, inlF, and inlG) in L. monocytogenes 10403S and in isogenic ΔprfA, ΔsigB, and ΔsigB ΔprfA strains. Strains were grown under defined conditions to produce (i) active PrfA, (ii) active σB and active PrfA, (iii) inactive PrfA, and (iv) active σB and inactive PrfA. Under the conditions tested, σB and PrfA contributed differentially to the expression of the various internalins such that (i) both σB and PrfA contributed to inlA and inlB transcription, (ii) only PrfA contributed to inlC transcription, (iii) only σB contributed to inlC2 and inlD transcription, and (iv) neither σB nor PrfA contributed to inlF and inlG transcription. inlE transcript levels were undetectable. The important role for σB in regulating expression of L. monocytogenes internalins suggests that exposure of this organism to environmental stress conditions, such as those encountered in the gastrointestinal tract, may activate internalin transcription. Interplay between σB and PrfA also appears to be critical for regulating transcription of some virulence genes, including inlA, inlB, and prfA.

A remarkable feature of many small non-coding RNAs (sRNAs) of Escherichia coli and Salmonella is their accumulation in the stationary phase of bacterial growth. Several stress response regulators and sigma factors have been reported to direct the transcription of stationary phase-specific sRNAs, but a widely conserved sRNA gene that is controlled by the major stationary phase and stress sigma factor, σS (RpoS), has remained elusive. We have studied in Salmonella the conserved SdsR sRNA, previously known as RyeB, one of the most abundant stationary phase-specific sRNAs in E. coli. Alignments of the sdsR promoter region and genetic analysis strongly suggest that this sRNA gene is selectively transcribed by σS. We show that SdsR down-regulates the synthesis of the major Salmonella porin OmpD by Hfq-dependent base pairing; SdsR thus represents the fourth sRNA to regulate this major outer membrane porin. Similar to the InvR, MicC and RybB sRNAs, SdsR recognizes the ompD mRNA in the coding sequence, suggesting that this mRNA may be primarily targeted downstream of the start codon. The SdsR-binding site in ompD was localized by 3′-RACE, an experimental approach that promises to be of use in predicting other sRNA–target interactions in bacteria.

The alternate RNA polymerase sigma factor gene, sigF, which is expressed in stationary phase and under stress conditions in vitro, has been deleted in the virulent CDC1551 strain of Mycobacterium tuberculosis. The growth rate of the ΔsigF mutant was identical to that of the isogenic wild-type strain in exponential phase, although in stationary phase the mutant achieved a higher density than the wild type. The mutant showed increased susceptibility to rifampin and rifapentine. Additionally, the ΔsigF mutant displayed diminished uptake of chenodeoxycholate, and this effect was reversed by complementation with a wild-type sigF gene. No differences in short-term intracellular growth between mutant and wild-type organisms within human monocytes were observed. Similarly, the organisms did not differ in their susceptibilities to lymphocyte-mediated inhibition of intracellular growth. However, mice infected with the ΔsigF mutant showed a median time to death of 246 days compared with 161 days for wild-type strain-infected animals (P < 0.001). These data indicate that M. tuberculosis sigF is a nonessential alternate sigma factor both in axenic culture and for survival in macrophages in vitro. While the ΔsigF mutant produces a lethal infection of mice, it is less virulent than its wild-type counterpart by time-to-death analysis.

Listeria monocytogenes HrcA and CtsR negatively regulate class I and III stress response genes, respectively, while σB positively regulates the transcription of class II stress response genes. To define the HrcA regulon and identify interactions between HrcA, CtsR, and σB, we characterized newly generated L. monocytogenes ΔhrcA, ΔctsR ΔhrcA, and ΔhrcA ΔsigB strains, along with previously described ΔsigB, ΔctsR, and ΔctsR ΔsigB 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 ΔhrcA 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 ΔhrcA 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 σB (31 genes), or all three regulators (5 genes, e.g., gadCB). Combined with previous evidence that σB appears to directly regulate hrcA transcription, our data suggest that HrcA and σB, as well as CtsR, form a regulatory network that contributes to the transcription of a number of L. monocytogenes genes.

In gram-negative bacteria, the RNA-binding protein Hfq has emerged as an important regulatory factor in a variety of physiological processes, including stress resistance and virulence. In Escherichia coli, Hfq modulates the stability or the translation of mRNAs and interacts with numerous small regulatory RNAs. Here, we studied the role of Hfq in the stress tolerance and virulence of the gram-positive food-borne human pathogen Listeria monocytogenes. We present evidence that Hfq is involved in the ability of L. monocytogenes to tolerate osmotic and ethanol stress and contributes to long-term survival under amino acid-limiting conditions. However, Hfq is not required for resistance to acid and oxidative stress. Transcription of hfq is induced under various stress conditions, including osmotic and ethanol stress and at the entry into the stationary growth phase, thus supporting the view that Hfq is important for the growth and survival of L. monocytogenes in harsh environments. The stress-inducible transcription of hfq depends on the alternative sigma factor σB, which controls the expression of numerous stress- and virulence-associated genes in L. monocytogenes. Infection studies showed that Hfq contributes to pathogenesis in mice, yet plays no role in the infection of cultured cell lines. This study provides, for the first time, information on the role of Hfq in the stress tolerance and virulence of a gram-positive pathogen.

Stationary-phase cells of Listeria monocytogenes grown in glucose-free or glucose-containing media were exposed for 90 min to various stresses, including acid stress (pH 4.0 to 7.0), osmotic stress (10.5 to 20.5% NaCl), and various temperatures (−5 to 50°C), and were further exposed to pH 3.5. Exposure to a mildly acidic (pH 5.0 to 6.0) environment provided protection of the pathogen against acid upon subsequent exposure. This adaptive response, however, was found to be strongly dependent on other environmental conditions during the shock, such as temperature or the simultaneous presence of a second stress factor (NaCl). Growth of L. monocytogenes in the presence of glucose resulted in enhanced survival of the pathogen at pH 3.5. Sublethal stresses other than acidic stresses, i.e., osmotic, heat, and low-temperature stresses, did not affect the acid resistance of L. monocytogenes (P > 0.5). More-severe levels of these stresses, however, resulted in sensitization of the pathogen to acid.

Listeria monocytogenes strains are classified in at least three distinct phylogenetic lineages. There are correlations between lineage classification and source of bacterial isolation; e.g., human clinical and food isolates usually are classified in either lineage I or II. However, human clinical isolates are overrepresented in lineage I, while food isolates are overrepresented in lineage II. σB, a transcriptional regulator previously demonstrated to contribute to environmental stress responses and virulence in L. monocytogenes lineage II strains, was hypothesized to provide differential abilities for L. monocytogenes survival in various niches (e.g., food and human clinical niches). To determine if the contributions of σB to stress response and virulence differ across diverse L. monocytogenes strains, ΔsigB mutations were created in strains belonging to lineages I, II, IIIA, and IIIB. Paired parent and ΔsigB mutant strains were tested for survival under acid and oxidative stress conditions, Caco-2 cell invasion efficiency, and virulence using the guinea pig listeriosis infection model. Parent and ΔsigB mutant strain transcriptomes were compared using whole-genome expression microarrays. σB contributed to virulence in each strain. However, while σB contributed significantly to survival under acid and oxidative stress conditions and Caco-2 cell invasion in lineage I, II, and IIIB strains, the contributions of σB were not significant for these phenotypes in the lineage IIIA strain. A core set of 63 genes was positively regulated by σB in all four strains; different total numbers of genes were positively regulated by σB in the strains. Our results suggest that σB universally contributes to L. monocytogenes virulence but specific σB-regulated stress response phenotypes vary among strains.

Abstract

Alkali stress is an important means of inactivating undesirable pathogens in a wide range of situations. Unfortunately, Listeria monocytogenes can launch an alkaline tolerance response, significantly increasing persistence of the pathogen in such environments. This study compared transcriptome patterns of alkali and nonalkali-stressed L. monocytogenes 10403S cells, to elucidate the mechanisms by which Listeria adapts and/or grows during short- or long-term alkali stress. Transcription profiles associated with alkali shock (AS) were obtained by DNA microarray analysis of midexponential cells suspended in pH 9 media for 15, 30, or 60 min. Transcription profiles associated with alkali adaptation (AA) were obtained similarly from cells grown to midexponential phase at pH 9. Comparison of AS and AA transcription profiles with control cell profiles identified a high number of differentially regulated open-reading frames in all tested conditions. Rapid (15 min) changes in expression included upregulation of genes encoding for multiple metabolic pathways (including those associated with Na+/H+ antiporters), ATP-binding cassette transporters of functional compatible solutes, motility, and virulence-associated genes as well as the σB controlled stress resistance network. Slower (30 min and more) responses to AS and adaptation during growth in alkaline conditions (AA) involved a different pattern of changes in mRNA concentrations, and genes involved in proton export.

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 virulence-associated genes as well as some heat shock genes.

Bacteria withstand starvation during long-term stationary phase through the acquisition of mutations that increase bacterial fitness. The evolution of the Growth Advantage in Stationary Phase (GASP) phenotype results in the ability of bacteria from an aged culture to outcompete bacteria from a younger culture when the two are mixed together. The GASP phenotype was first described for Escherichia coli but has not been examined for an environmental bacterial pathogen which must balance long-term survival strategies that promote fitness in the outside environment with those that promote fitness within the host. Listeria monocytogenes is an environmental bacterium that lives as a saprophyte in soil but is capable of replicating within the cytosol of mammalian cells. Here we demonstrate the ability of L. monocytogenes to express GASP via the acquisition of mutations during long-term stationary growth. L. monocytogenes GASP occurred through mechanisms that were both dependent and independent of the stress responsive alternative sigma factor SigB. Constitutive activation of the central virulence transcriptional regulator PrfA interfered with the development of GASP, however L. monocytogenes GASP cultures retained full virulence in mice. These results indicate that L. monocytogenes can accrue mutations that optimize fitness during long-term stationary growth without negatively impacting virulence.