Project description:To characterize regulons of alternative sigma factor SigH, SigL, and SigC in Listeria monocytogenes, in-frame mutant strains were created in the 10403S background. Regulons controlled by these 3 alternative sigma factors were characterized by whole-genome microarrays. The L. monocytogenes 10403S wild type and sigma factor null mutation strains were grown at 37 °C to stationary phase (defined in this study as growth to OD600 = 1.0, followed by incubation for an additional 3 h) prior to RNA isolation. Transcriptional profiles of 10403S wild type were compared to those of null mutation strain. In addition to stationary phase condition, SigC regulon was further characterized using heat stress (cultures grown to log phase at OD600 = 0.4, 37 °C and then exposed to heat at 55 °C for 10 min) and a condition with IPTG-inducible expression of sigC (sigC gene is placed under Pspac promoter using pLIV2 vector in wild type 10403S background). Under these conditions, expression profiles were compared between (i) wild type and sigC null mutant for heat stress and (ii) IPTG-inducible sigC strain and sigC null mutant, respectively. Using adjusted P < 0.05 and ≥ 1.5 fold change as cutoff values, microarray analyses identified 169 SigH-dependent, 51 SigL-dependent, and 3 SigC-dependent genes. Keywords: Listeria monocytogenes, alternative sigma factor, SigH, SigL, SigC
Project description:These studies were designed to examine the transcription of Listeria monocytogenes strains 10403S and LO28 during intracellular replication in mammalian macrophages. Duplicate WT Listeria monocytogenes (strains 10403S and LO28) were used to infect mouse bone marrow-derived macrophages (BMMs). Bacterial RNA was harvested at 4 hours post-infection.
Project description:These studies were designed to examine the transcription of Listeria monocytogenes strains 10403S and LO28 during intracellular replication in mammalian macrophages.
Project description:In several gram-positive bacterial genera including Bacillus, Staphylococcus, and Listeria, sigma B (σB) has been identified as a stress-responsive alternative sigma factor responsible for initiating transcription of genes (the σB regulon) involved in response to stress-inducing environmental conditions. In L. monocytogenes, a foodborne pathogen of considerable threat to public health and the food industry, σB is involved in regulation of stress response and virulence gene expression. We have defined the σB regulon in L. monocytogenes during early stationary phase and under salt stress (0.3M NaCl) conditions using whole-genome microarrays, identifying 168 genes that generated ≥2.0-fold higher signals in the parental strain 10403S than in an isogenic sigB null mutant (ΔsigB), categorized into nine functional groups including stress-response genes (12), virulence genes (5), and genes related to transport (26) and metabolism (45). To gain a broader biological perspective of the σB stress response system, we applied these microarrays to Listeria innocua under the same environmental conditions. Our studies revealed 64 candidates in the L. innocua σB regulon with ≥2.0-fold higher signals in the parent than in a ΔsigB mutant; 49 of the 64 genes overlap with the L. monocytogenes σB regulon, indicating extensive overlap in σB-controlled genes between the two species. Further transcriptional analysis using TaqMan quantitative real time RT-PCR was performed for selected genes that displayed contrasting fold changes among the four microarray data sets (two stress conditions per species). We report novel members of the L. monocytogenes σB regulon, as well as the initial definition of the L. innocua σB regulon. Our comparative studies of the σB stress response systems in L. monocytogenes and L. innocua revealed features of the σB regulon that are conserved and unique to the two species. Keywords: Listeria monocytogenes, Listeria innocua, SigB regulon, salt stress, 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:These studies were designed to examine the acute Listeria monocytogenes transcriptional response to mammalian (porcine) bile. Triplicate WT Listeria monocytogenes (strain 10403S) were grown to mid-log in BHI at 37 °C. Samples were divided, and either treated or not treated by addition of porcine bile (Sigma, to 1% final) for 30 minutes.
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. Independent RNA isolations were performed for strain 10403S with and without exposure to ClO2 from cells grown to early log phase. Four biological replicates were used in competitive whole-genome microarray experiments. For each set of hybridizations, RNA from a control sample of Listeria monocytogenes was hybridized with RNA from a culture of L. monocytogenes following exposure to ClO2. Dye swapping was performed for the four replicates to mitigate any concerns of dye bias.
Project description:In this study, RNA-seq was used to compare the transcriptomes of Listeria monocytogenes 10403S::ÎBCHL Prha-sigH and ÎBCHL Prha. RNA-seq was performed on ÎBCHL Prha-sigH and ÎBCHL Prha RNA samples representing three independent biological replicates at log phase in Brain Heart Infusion (BHI) broth under rhamnose induction. Indexed and purified cDNA libraries (6 libraries including 3 replicates for 2 strains) were loaded together onto an independent flow cell without any other samples; sequencing was carried out by running Hiseq 2500 (single-end, 150-bp per read). Reads alignment was carried out using the Burrows-Wheeler Aligner (BWA). Differential expression of genes in different strains was statistically assessed using the BaySeq method. To identify sigH-dependent promoters, a new method of moving sliding windows of 50 nt along the whole genome was used to compare the normalized RNA-seq coverage (NRC) between the two strains. Using the standard whole gene differential expression analysis, significant upregulation of 5 genes in 4 operons was found in the sigH overexpressing strain. While with the sliding windiow analysis, 2 additional ÏH-dependent promoters were identified. Our results show that three ÏH-dependent transcritption units that encode competence proteins, including the comEABC , comGABCDEFG and coiA. Transcriptome profiles of L. monocytogenes 10403S::ÎBCHL Prha-sigH and ÎBCHL Prha were generated by deep sequencing, in triplicate, using Illumina Hiseq 2500.
Project description:The stationary phase stress response transcriptome of the human bacterial pathogen Listeria monocytogenes was defined using RNA sequencing (RNA-Seq) with the Illumina Genome Analyzer. Specifically, bacterial transcriptomes were compared between stationary phase cells of L. monocytogenes 10403S and an otherwise isogenic DsigB mutant, which does not express the alternative sigma factor σB, a major regulator of genes contributing to stress response. Keywords: Transcriptome and differential expression analyses
Project description:The stationary phase stress response transcriptome of the human bacterial pathogen Listeria monocytogenes was defined using RNA sequencing (RNA-Seq) with the Illumina Genome Analyzer. Specifically, bacterial transcriptomes were compared between stationary phase cells of L. monocytogenes 10403S and an otherwise isogenic DsigB mutant, which does not express the alternative sigma factor M-OM-^CB, a major regulator of genes contributing to stress response. Keywords: Transcriptome and differential expression analyses a laboratory strain, 10403S and its otherwise isogenic mutant lacking sigB were analyzed. Two replicates of each strain were analyzed for a total of 4 runs