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:Transcriptional profile of wild type L. monocytogenes (EGDe) and a pycA mutant strain was compared on growth in BHI. The human pathogen L. monocytogenes is a facultatively intracellular bacterium that survives and replicates in the cytosol of many mammalian cells. The listerial metabolism, especially under intracellular conditions , is still poorly understood. Recent studies analyzed the carbon metabolism of L. monocytogenes by the 13C-isotopologue perturbation method in a defined minimal medium containing [U-13C6]glucose. It was shown that these bacteria produce oxaloacetate mainly by carboxylation of pyruvate due to an incomplete tricarboxylic acid cycle. Here we report that a pycA insertion mutant defective in pyruvate carboxylase (PYC) still grows, albeit at a reduced rate, in BHI medium, but is unable to multiply in a defined minimal medium with glucose or glycerol 36 as carbon source. Transcriptional profiling was performed on the pycA mutant and the wild type strain grown in BHI to get a closer insight into the effect of the pycA mutation in Listeria monocytogenes.
Project description:Listeria monocytogenes cells (strain LI0521) were digested with trypsin for the identification of surface proteins. The supernatant was filter-sterilized and subjected to identification by LC-MS/MS. Concurrently secreted or shed proteins were identified by isolating filter-sterilized supernatants following incubation of L. monocytogenes cells in buffer without trypsin. This was followed by trypsin digest of the sterilized supernatant and identification by LC-MS/MS.
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:The SOS response is a conserved pathway that is activated under certain stress conditions and is regulated by the repressor LexA and the activator RecA. The food-borne pathogen Listeria monocytogenes contains RecA and LexA homologs, but their roles in Listeria have not been established. In this study, we identified the SOS regulon in L. monocytogenes by comparing the transcription profiles of the wild-type strain and the ΔrecA mutant strain after exposure to the DNA damaging agent mitomycinC (MMC). The SOS response is an inducible pathway involved in DNA repair, restart of stalled replication forks, and in induction of genetic variation in stressed and stationary phase cells. It is regulated by LexA and RecA. LexA is an autoregulatory repressor which binds to a consensus sequence in the promoter region of the SOS response genes, thereby repressing transcription. A consensus LexA binding motif for L. monocytogenes has not been identified thus far. Generally, the SOS response is induced under circumstances in which single stranded DNA accumulates in the cell. This results in activation of RecA, which in turn stimulates cleavage of LexA, and ultimately in the induction of the SOS response. Keywords: stress response, loop design, SOS response, mitomycin c, listeria monocytogenes, RecA, LexA
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:Transcriptional profile of wild type L. monocytogenes (EGDe) and a pycA mutant strain was compared on growth in BHI. The human pathogen L. monocytogenes is a facultatively intracellular bacterium that survives and replicates in the cytosol of many mammalian cells. The listerial metabolism, especially under intracellular conditions , is still poorly understood. Recent studies analyzed the carbon metabolism of L. monocytogenes by the 13C-isotopologue perturbation method in a defined minimal medium containing [U-13C6]glucose. It was shown that these bacteria produce oxaloacetate mainly by carboxylation of pyruvate due to an incomplete tricarboxylic acid cycle. Here we report that a pycA insertion mutant defective in pyruvate carboxylase (PYC) still grows, albeit at a reduced rate, in BHI medium, but is unable to multiply in a defined minimal medium with glucose or glycerol 36 as carbon source. Transcriptional profiling was performed on the pycA mutant and the wild type strain grown in BHI to get a closer insight into the effect of the pycA mutation in Listeria monocytogenes. RNA from the two strains were isolated after growth in BHI and and compared using whole genome oligonucleotide microarrays