Project description:Full title: Probing the pan genome of a foodborne bacterial pathogen Listeria monocytogenes: Implications for its niche adaptation, pathogenesis, and evolution Listeria monocytogenes is a foodborne bacterial pathogen well known for adaptability to diverse environmental and host niches, and a high fatality rate among infected, immuno-compromised individuals. Three genetic lineages have been identified within this species. Strains of genetic lineages I and II account for more than ninety percent of foodborne disease outbreaks worldwide, whereas strains from genetic lineage III are rarely implicated in human infectious for unknown, yet intriguing, reasons. Here we have probed the genomic diversity of 26 L. monocytogenes strains using both whole-genome sequences and a novel 385,000 probe pan-genome microarray, fully tiling the genomes of 20 representative strains. Using these methods to identify genes highly conserved in lineages I and II but rare in lineage III, we have identified 86 genes and 8 small RNAs that play roles in bacterial stress resistance, pathogenicity, and niche, potentially explaining the predominance of L. monocytogenes lineages I and II in foodborne disease outbreaks. Extending gene content analysis to all lineages revealed a L. monocytogenes core genome of approximately 2,350 genes (80% of each individual genome) and a pan-genomic reservoir of >4,000 unique genes. Combined gene content data from both sequences and arrays was used to reconstruct an informative phylogeny for the L. monocytogenes species that confirms three distinct lineages and describes the relationship of 9 new lineage III genomes. Comparative analysis of 18 fully sequenced L. monocytogenes lineage I and II genomes shows a high level of genomic conservation and synteny, indicative of a closed pan-genome, with moderate domain shuffling and sequence drift associated with bacteriophages is present in all lineages. In contrast with lineages I and II, notable genomic diversity and characteristics of an open pan-genome were observed in the lineage III genomes, including many strain-specific genes and a more complex conservation pattern. This indicates that the L. monocytogenes pan-genome has not yet been fully sampled by genome sequencing, and additional sequencing of lineage III genomes is necessary to survey the full diversity of this intriguing species and reveal its mechanisms for adaptability and virulence. This is a Listeria monocytogenes pan-genome tilling array designed using PanArray algorithm. 9 experimental strains (F2-569, M1-002, F2-208, J2-071, J1-208, W1-111, W1-110, F2-524, F2-501) vs reference (EGD-e) strain.
Project description:Full title: Probing the pan genome of a foodborne bacterial pathogen Listeria monocytogenes: Implications for its niche adaptation, pathogenesis, and evolution Listeria monocytogenes is a foodborne bacterial pathogen well known for adaptability to diverse environmental and host niches, and a high fatality rate among infected, immuno-compromised individuals. Three genetic lineages have been identified within this species. Strains of genetic lineages I and II account for more than ninety percent of foodborne disease outbreaks worldwide, whereas strains from genetic lineage III are rarely implicated in human infectious for unknown, yet intriguing, reasons. Here we have probed the genomic diversity of 26 L. monocytogenes strains using both whole-genome sequences and a novel 385,000 probe pan-genome microarray, fully tiling the genomes of 20 representative strains. Using these methods to identify genes highly conserved in lineages I and II but rare in lineage III, we have identified 86 genes and 8 small RNAs that play roles in bacterial stress resistance, pathogenicity, and niche, potentially explaining the predominance of L. monocytogenes lineages I and II in foodborne disease outbreaks. Extending gene content analysis to all lineages revealed a L. monocytogenes core genome of approximately 2,350 genes (80% of each individual genome) and a pan-genomic reservoir of >4,000 unique genes. Combined gene content data from both sequences and arrays was used to reconstruct an informative phylogeny for the L. monocytogenes species that confirms three distinct lineages and describes the relationship of 9 new lineage III genomes. Comparative analysis of 18 fully sequenced L. monocytogenes lineage I and II genomes shows a high level of genomic conservation and synteny, indicative of a closed pan-genome, with moderate domain shuffling and sequence drift associated with bacteriophages is present in all lineages. In contrast with lineages I and II, notable genomic diversity and characteristics of an open pan-genome were observed in the lineage III genomes, including many strain-specific genes and a more complex conservation pattern. This indicates that the L. monocytogenes pan-genome has not yet been fully sampled by genome sequencing, and additional sequencing of lineage III genomes is necessary to survey the full diversity of this intriguing species and reveal its mechanisms for adaptability and virulence.
Project description:Listeria monocytogenes strains classify into at least three distinct phylogenetic lineages. Correlations exist between lineage classification and source of bacterial isolation, e.g., human clinical and food isolates usually classify into either lineage I or II, however, human clinical isolates are over-represented in lineage I while food isolates are over-represented in lineage II. σB, a transcriptional regulator previously demonstrated to contribute to environmental stress response and virulence in L. monocytogenes lineage II strains, was hypothesized to provide differential capabilities for L. monocytogenes survival in various niches (e.g., food vs. human clinical). To determine if σB contributions to stress response and virulence differ across diverse L. monocytogenes strains, ΔsigB mutations were created in strains from lineages I, II, IIIA, and IIIB. Paired parent and ΔsigB mutant strains were tested for acid and oxidative stress survival, 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 acid and oxidative stress survival and Caco-2 cell invasion in lineage I, II, and IIIB strains, σB contributions 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 each strain. Our results suggest that σB universally contributes to L. monocytogenes virulence, but specific σB-regulated stress response phenotypes vary among strains.
Project description:Listeria monocytogenes strains classify into at least three distinct phylogenetic lineages. Correlations exist between lineage classification and source of bacterial isolation, e.g., human clinical and food isolates usually classify into either lineage I or II, however, human clinical isolates are over-represented in lineage I while food isolates are over-represented in lineage II. σB, a transcriptional regulator previously demonstrated to contribute to environmental stress response and virulence in L. monocytogenes lineage II strains, was hypothesized to provide differential capabilities for L. monocytogenes survival in various niches (e.g., food vs. human clinical). To determine if σB contributions to stress response and virulence differ across diverse L. monocytogenes strains, ΔsigB mutations were created in strains from lineages I, II, IIIA, and IIIB. Paired parent and ΔsigB mutant strains were tested for acid and oxidative stress survival, 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 acid and oxidative stress survival and Caco-2 cell invasion in lineage I, II, and IIIB strains, σB contributions 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 each strain. Our results suggest that σB universally contributes to L. monocytogenes virulence, but specific σB-regulated stress response phenotypes vary among strains.
Project description:The foodborne pathogen Listeria monocytogenes uses a number of transcriptional regulators, including the negative regulator CtsR, to control gene expression under different environmental conditions and in response to stress. Gene expression patterns of DctsR log phase cells were compared to both wt and ictsR-mcsA log phase cells grown with 0.5mM IPTG to identify CtsR-dependent genes.We identified 62 CtsR-dependent genes that showed significant expression ratios (adj. P < 0.05), with ≥ 1.5-fold differential expression either between ΔctsR and wt or between ΔctsR and ictsR-mcsA. Keywords: Listeria monocytogenes, CtsR regulon, log phase
Project description:The foodborne pathogen Listeria monocytogenes uses a number of transcriptional regulators, including the negative regulator HrcA, to control gene expression under different environmental conditions and in response to stress. Gene expression patterns of DhrcA stationary phase cells were compared to wt to identify hrcA-dependent genes. We identified 61 HrcA-dependent genes that showed significant expression ratios (adj. P < 0.05), with ≥ 1.5-fold differential expression between ΔhrcA and wt. Combined with microarray analysis, Hidden Markov Model searches show HrcA directly repress at least 8 genes. Keywords: Listeria monocytogenes, HrcA regulon, stationary phase
Project description:Listeria monocytogenes strain 10403S has been studied extensively for stress response activity toward multiple stressors (acid, osmotic, cold, high temperature, etc.) as well as multiple stress regulons (SigB, CtsR, HrcA, etc.). Here we aimed to determine the transcriptional response of Listeria monocytogenes in early log phase towards the strong oxidative stress imposed by ClO2. The elucidation of such a response allows for further a more completel understanding of the mechanism of inactivation by sanitizers, specifically ClO2.
Project description:As the foodborne pathogen Listeria monocytogenes has the ability to grow at refrigeration temperatures, whole-genome microarray experiments were performed using L. monocytogenes strain 10403S to define the cold stress regulon and to identify genes differentially expressed during growth at 4°C and 37°C. Microarray analysis using a stringent cutoff (adjusted p<0.001; fold-change >2.0) revealed 105 and 170 genes that showed higher transcript levels in logarithmic- and stationary-phase cells, respectively, at 4°C (compared to cells at 37°C). A total of 74 and 102 genes showed lower transcript levels in logarithmic- and stationary-phase cells grown at 4°C, respectively. Genes upregulated at 4°C during both stationary- and log-phase included those encoding a two-component response regulator (lmo0287), a cold shock protein (cspL), and two RNA helicases (lmo0866 and lmo1722), whereas genes encoding selected virulence factors and heat shock proteins were downregulated at 4°C. Selected genes that were upregulated at 4°C during both stationary- and log-phase were confirmed by quantitative reverse transcriptase PCR. Our data show (i) a large number of L. monocytogenes genes are differentially expressed at 4 and 37°C with a larger number of genes showing higher transcript level at 4°C than genes showing lower transcript levels at 4°C; (ii) L. monocytogenes genes upregulated at 4°C include a number of genes and operons with previously reported or plausible roles in cold adaptation; and (iii) L. monocytogenes genes downregulated at 4°C include a number of virulence and virulence-associated genes as well as some heat shock genes. Keywords: Listeria monocytogenes, cold regulon, temperature