Project description:A screening process was used to determine the aciduric capacities of a diverse set of Listeria monocytogenes strains and was based on the capacity to grow at pH 5.0 in the presence 4 different organic acids. Food and clinical isolates tended to be more resistant but strain genetic groupings were found to be only weakly linked to sodium diacetate (SDA)-resistance. Representative and comparatively aciduric food isolates FW04/0023 and FW04/0025 were found to accumulate reduced levels of acetate anion and K+ ion during growth in the presence of SDA, compared to more acid sensitive reference strains EGD (ATCC BAA-739) and ATCC 19111. The aciduric nature of FW04/0023 and FW04/0025 was also reflected by their comparatively high tolerance to pH 2.4 acid challenge; a property boosted by the presence of SDA, and growth-phase dependent acid tolerance. Transcriptomic analyses revealed increased levels of SDA did not activate or promulgate the response of any of the known acid protective mechanisms, except for acetoin biosynthesis. Elevated levels of unprotonated SDA (20 mM SDA at pH 5.0) was found to have broad effects on gene expression that could be differentiated from effects caused by mildly acidic conditions (pH 5.0) and substantial strain variation was also found. Collated SDA mainly effect genes associated with virulence, cell wall processes, DNA repair, and cofactor and lipid biosynthesis. Strain FW04/0025 was more responsive to elevated unprotonated SDA increasing the expression of 222 genes (>2-fold change, p<0.05), compared to 112 genes for strain EGD. Key differences between the strains in relation to SDA-enhanced transcript abundance in FW04/0023 were primarily associated with the cell wall, oxidative stress management, intermediary metabolism, and several hypothetical proteins. This complement of different responses could potentially alter phenotypes resulting in different cell envelope properties and metabolic properties that effect accumulation of acetate anion. The data demonstrates that amongst different L. monocytogenes strains acetate has differing effects that may ultimately influence growth efficiency under stressful conditions relevant to acidic foods and the gastrointestinal environment. The microarray component of the experiments had the aim of determining: i) To examine the affect of elevated levels of unprotonated sodium diacetate (21 mM sodium diacetate added to cultures at pH 5.0, resulting in ~7.7 mM unprotonated acetate) compared to mild acid stress at pH 5.0 in which lower levels of acetate accumulates through natural end-product formation. ii) To examine the gene expression responses of two L. monocytogenes strains that had different intrinsic acid resistances, including an organic acid resistant strain FW04/0025 and a more sensitive reference strain EGD on which genome the microarray oligonucleotide set is based.

Project description:A screening process was used to determine the aciduric capacities of a diverse set of Listeria monocytogenes strains and was based on the capacity to grow at pH 5.0 in the presence 4 different organic acids. Food and clinical isolates tended to be more resistant but strain genetic groupings were found to be only weakly linked to sodium diacetate (SDA)-resistance. Representative and comparatively aciduric food isolates FW04/0023 and FW04/0025 were found to accumulate reduced levels of acetate anion and K+ ion during growth in the presence of SDA, compared to more acid sensitive reference strains EGD (ATCC BAA-739) and ATCC 19111. The aciduric nature of FW04/0023 and FW04/0025 was also reflected by their comparatively high tolerance to pH 2.4 acid challenge; a property boosted by the presence of SDA, and growth-phase dependent acid tolerance. Transcriptomic analyses revealed increased levels of SDA did not activate or promulgate the response of any of the known acid protective mechanisms, except for acetoin biosynthesis. Elevated levels of unprotonated SDA (20 mM SDA at pH 5.0) was found to have broad effects on gene expression that could be differentiated from effects caused by mildly acidic conditions (pH 5.0) and substantial strain variation was also found. Collated SDA mainly effect genes associated with virulence, cell wall processes, DNA repair, and cofactor and lipid biosynthesis. Strain FW04/0025 was more responsive to elevated unprotonated SDA increasing the expression of 222 genes (>2-fold change, p<0.05), compared to 112 genes for strain EGD. Key differences between the strains in relation to SDA-enhanced transcript abundance in FW04/0023 were primarily associated with the cell wall, oxidative stress management, intermediary metabolism, and several hypothetical proteins. This complement of different responses could potentially alter phenotypes resulting in different cell envelope properties and metabolic properties that effect accumulation of acetate anion. The data demonstrates that amongst different L. monocytogenes strains acetate has differing effects that may ultimately influence growth efficiency under stressful conditions relevant to acidic foods and the gastrointestinal environment. The microarray component of the experiments had the aim of determining: i) To examine the affect of elevated levels of unprotonated sodium diacetate (21 mM sodium diacetate added to cultures at pH 5.0, resulting in ~7.7 mM unprotonated acetate) compared to mild acid stress at pH 5.0 in which lower levels of acetate accumulates through natural end-product formation. ii) To examine the gene expression responses of two L. monocytogenes strains that had different intrinsic acid resistances, including an organic acid resistant strain FW04/0025 and a more sensitive reference strain EGD on which genome the microarray oligonucleotide set is based. Two to four biologically replicated control cultures were labelled with Cy3 for each treatment sample with two sets of experimental conditions investigated for two different L. monocytogenes strains . Condition 1) â Mildly acidic stress: Control culture (Cy3-labelled RNA) - strains were grown to early- mid exponential growth phase at 25Â°C (in a shaking water bath) in brain-heart infusion broth at pH 7.3. Test cultures (Cy5-labelled RNA) â strains grown to early- mid exponential growth at 25Â°C phase in BHI broth adjusted to pH 5.0 (in a shaking water bath). Condition 2) â Organic acid (sodium diacetate) stress: Control culture (Cy3-labelled RNA) - strains were grown to exponential growth phase at 25Â°C (in a shaking water bath) in BHI broth at pH 5.0. Test cultures (Cy5-labelled RNA) â strains grown to exponential growth at 25Â°C phase in BHI broth adjusted to pH 5.0 and amended with 21 mM (0.3% w/v)sodium diacetate (in a shaking water bath).

Project description:Listeria monocytogenes is able to colonize human and animal intestinal tracts and to subsequently cross the intestinal barrier, causing systemic infection. For successful establishment of infection, L. monocytogenes must survive the low pH environment of the stomach. L. monocytogenes encodes a functional ArgR, a transcriptional regulator belonging to the ArgR/AhrC arginine repressor family. We aimed at clarifying the specific functions of ArgR in arginine metabolism regulation, and more importantly, in acid tolerance of L. monocytogenes. We showed that ArgR in the presence of 10 mM arginine represses transcription and expression of the argGH and argCJBDF operons, indicating that L. monocytogenes ArgR plays the classical role of ArgR/AhrC family proteins in feedback inhibition of the arginine biosynthetic pathway. Notably, transcription and expression of arcA (encoding arginine deiminase) and sigB (encoding an alternative sigma factor B) were also markedly repressed by ArgR when bacteria were exposed to pH 5.5 in the absence of arginine. However, addition of arginine enabled ArgR to derepress the transcription and expression of these two genes. Electrophoretic mobility shift assays showed that ArgR binds to the putative ARG boxes in the promoter regions of argC, argG, arcA, and sigB. Reporter gene analysis with gfp under control of the argG promoter demonstrated that ArgR was able to activate the argG promoter. Unexpectedly, deletion of argR significantly increased bacterial survival in BHI medium adjusted to pH 3.5 with lactic acid. We conclude that this phenomenon is due to activation of arcA and sigB. Collectively, our results show that L. monocytogenes ArgR finely tunes arginine metabolism through negative transcriptional regulation of the arginine biosynthetic operons and of the catabolic arcA gene in an arginine-independent manner during lactic acid-induced acid stress. ArgR also appears to activate catabolism as well as sigB transcription by anti-repression in an arginine-dependent way.

Project description:Listeria monocytogenes is well known to survive and grow under several stress conditions, including salt stress, which is important for growth in certain foods as well as for host infection. To characterize the contributions, to salt stress response, of transcriptional regulators important for stress response and virulence (i.e., σ(B) and PrfA), we analyzed three L. monocytogenes parent strains and isogenic mutants (ΔsigB, ΔprfA, and ΔsigBΔprfA), representing different serotypes and lineages, for their ability to grow, at 25°C, in BHI with 1.9 M NaCl. With regard to growth rate, only the lineage IV strain presented a significant difference between the parent strain and both of its respective mutants lacking prfA (ΔprfA and ΔsigBΔprfA). Conversely, the lineage I and II parent strains showed significantly shorter lag phase in comparison to their respective ΔsigB mutant strains. Intestinal epithelial cell invasion assay and hemolytic activity assays showed a significant role for σ(B) in the former and for PrfA in the latter. To explore the mechanism that may contribute to the extended lag phase in the ΔsigB mutant strain and survival and growth of the parent strain upon salt shock, whole genome transcription profiling was performed to compare transcript levels between the lineage I, serotype 1/2b, parent strain and its isogenic ΔsigB mutant after 30 min of lag phase growth at 25°C in the presence of 1.9M NaCl (salt shock) without aeration. Microarray data showed significantly higher transcript levels for 173 genes in the parent strain as compared to the ΔsigB strain. Overall, 102 of the 173 σ(B) up-regulated genes had been identified in previous studies, indicating that 71 genes were newly identified as being up-regulated by σ(B) in this study. We hypothesize that, among these genes newly identified as σ(B) up-regulated, four genes (lmo2174, lmo0530, lmo0527 and lmo0529) may play a major role in response to salt stress. Lmo2174 contains domains that facilitate sensing and producing a transduction signal in the form of cyclic di-GMP, which may activate the enzymes Lmo0527, Lmo0529 and Lmo0530, which encode proteins similar to those responsible for synthesis of exopolysaccharides that may protect the cell by changing the cell wall structure during salt stress. Overall, our data showed that σ(B), but not PrfA, contributes to growth under salt stress. Moreover, we show that the σ(B) regulon of a L. monocytogenes lineage I strain challenged with salt shock includes salt stress-specific as well as previously unidentified σ(B) up-regulated genes.

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Transcriptomic and phenotypic responses of Listeria monocytogenes strains possessing different growth efficiencies under acidic conditions.

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Transcriptomic and phenotypic responses of Listeria monocytogenes strains possessing different growth efficiencies under acidic conditions.

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