RNA-sequencing of L. monocytogenes under PrfA-inducing conditions refines the PrfA regulon and reveals a putative novel PrfA-dependent gene and its associated PrfA box
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PROVIDER: PRJNA610791 | ENA |
REPOSITORIES: ENA
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Project description:Purpose: The objective of the present study was to refine the PrfA regulatory network using RNA-seq to characterize the L. monocytogenes transcriptome in a wt, ∆prfA, and prfA* under PrfA-inducing conditions. Methods: Sequence reads were aligned to the L. monocytogenes 10403S genome using the BWA-MEM algorithm, version 0.7.3a. The data for coverage per base on the sense and antisense strands were analyzed separately using SAMtools. Differential expression of genes in each strain comparison (wt/∆prfA, wt/prfA*, and prfA*/∆prfA) were analyzed using the baySeq package for R version 2.2.0. Transcripts were considered differentially expressed if the FDR (False Discovery Rate) was ≤ 0.05, the FC (Fold Change) was ≥ 2.0 for up-regulated transcripts and ≤ 0.5 for down-regulated transcripts. Results: Our data indicate that we (i) confirmed transcription of known PrfA-dependent genes and (ii) identified a novel PrfA box and its putative ������A-dependent promoter region. Conclusions: Combined with many studies that have investigated the PrfA regulon and virulence in L. monocytogenes, we have shown tha PrfA directly regulates a small subset of genes; however, using RNA-seq and bioinformatics we were able to identify a novel PrfA-dependent gene and its putative PrfA box. This finding highlights the importance of using advanced approaches to analyze RNA-seq data, which can potentially reveal previously unidentified genes regulated under specific conditions.
2020-10-06 | GSE146507 | GEO
Project description:Listeria monocytogenes SigB and PrfA are pleiotropic regulators of stress response and virulence gene expression, which have been shown to co-regulate genes in L. monocytogenes. We performed whole genome transcriptional profiling in the presence of PrfA* and active SigB, to identify the overlaps between the PrfA virulence regulon and the SigB stress response regulon. In L. monocytogenes, the PrfA* allele contributes to the activation of virulence genes to a level comparable to that of intracellular growing L. monocytogenes. Our results showed that the core PrfA regulon consists of 12 genes previously described as PrfA regulated. Furthermore, we found that the role of SigB during virulence gene regulation changes, dependent on the presence or absence of PrfA*. In the absence of PrfA*, SigB activated the transcription of virulence genes such as inlA and inlB. In the presence of PrfA*, SigB negatively influenced the transcription of genes in the PrfA core regulon. The observed effect of SigB on the transcript level of PrfA regulated genes was shown to reduce the cytotoxic effect of the PrfA* allele in HepG-2 cells. Our results indicate that the SigB-PrfA regulatory network is important for the adjustment of virulence gene transcription to ensure L. monocytogenes success as an intracellular pathogen. Keywords: comparison of gene expression of regulatory mutants
2008-10-01 | GSE11347 | GEO
Project description:Listeria monocytogenes SigB and PrfA are pleiotropic regulators of stress response and virulence gene expression, which have been shown to co-regulate genes in L. monocytogenes. We performed whole genome transcriptional profiling in the presence of PrfA* and active SigB, to identify the overlaps between the PrfA virulence regulon and the SigB stress response regulon. In L. monocytogenes, the PrfA* allele contributes to the activation of virulence genes to a level comparable to that of intracellular growing L. monocytogenes. Our results showed that the core PrfA regulon consists of 12 genes previously described as PrfA regulated. Furthermore, we found that the role of SigB during virulence gene regulation changes, dependent on the presence or absence of PrfA*. In the absence of PrfA*, SigB activated the transcription of virulence genes such as inlA and inlB. In the presence of PrfA*, SigB negatively influenced the transcription of genes in the PrfA core regulon. The observed effect of SigB on the transcript level of PrfA regulated genes was shown to reduce the cytotoxic effect of the PrfA* allele in HepG-2 cells. Our results indicate that the SigB-PrfA regulatory network is important for the adjustment of virulence gene transcription to ensure L. monocytogenes success as an intracellular pathogen. Keywords: comparison of gene expression of regulatory mutants The experimental design included 4 mutant strains of L. monocytogenes 10403S (PrfA*, delta prfA, delta prfA delta sigB, and PrfA* delta sigB), of which cDNA generated from 4 biological replicates were hybridized in all possible pairwise comparisons. Data were analyzed using a one way ANOVA in R/MAANOVA to determine significant differences in gene expression among the different strains. A two way ANOVA implemented in R/MAANOVA determined significant differences in gene expression due to the presence or absence of SigB and PrfA.
2010-05-19 | E-GEOD-11347 | biostudies-arrayexpress
Project description:The Alphaproteobacterium Caulobacter crescentus inhabits low-nutrient environments and can tolerate certain levels of heavy metal in these sites. It has been reported that C. crescentus responds to chromium exposure by altering expression of a large number of genes. In this work, we showed that the ECF sigma factor SigF is one of the regulatory proteins involved in the control of this transcriptional response. Microarray experiments from cells under dichromate stress showed that SigF controls a small regulon consisting of seven genes. Promoter analyses revealed that a conserved SigF-dependent sequence is located upstream of all but one gene of this regulon. Surprisingly, sigF itself was not found to be strongly auto-regulated under dichromate exposure. Furthermore, we showed that the second gene (CC3252) in the sigF operon acts as a negative regulator of SigF function, as overexpression of this putative membrane protein abolishes the transcriptional activation of sigF and its regulon under dichromate stress. Additionally, we found that substitution of one or both the conserved cysteines in the protein encoded by CC3252 (C131 and C181) affects the ability of this protein in maintaining expression of SigF-dependent genes at basal levels. Interestingly, we showed that the activation of SigF is not caused by reactive oxygen species generated as a consequence of dichromate exposure. Therefore, dichromate most probably may lead to conformational changes in the putative anti-sigma factor CC3252, releasing SigF to bind RNA polymerase core and drive transcription of its regulon.
2012-10-02 | GSE32406 | GEO
Project description:External signals are key for bacteria to sense their immediate environment and fine-tune gene expression accordingly. The foodborne pathogen Listeria monocytogenes senses a range of environmental cues in order to activate or deactivate the virulence-inducing transcriptional factor PrfA during transition between infectious and saprophytic lifecycles. Chitin is an abundant biopolymer formed from linked β-(1–4)-N-acetyl-D-glucosamine residues associated with fungi, the exoskeleton of insects and often incorporated into foods as a thickener or stabiliser. L. monocytogenes evolved to hydrolyse chitin, presumably, to facilitate nutrient acquisition from competitive environments such as soil where the polymer is abundant. Since mammals do not produce chitin, we reasoned that the polymer could serve as an environmental signal contributing to repression of L. monocytogenes PrfA-dependent expression outside the host. This study shows a significant downregulation of the core PrfA-regulon during virulence-inducing conditions in vitro in the presence of chitin. Our data suggest this phenomenon occurs through a mechanism that differs from PTS-transport of oligosaccharides generated from either degradation or chitinase-mediated hydrolysis of the polymer. Importantly, an indication that chitin can repress virulence expression of a constitutively active PrfA* mutant is shown, possibly mediated via a post-translational modification inhibiting PrfA* activity. To our knowledge, chitin as a molecule with anti-virulence properties against a pathogenic bacterium has not been previously reported. Thus, our findings identify chitin as a signal which may downregulate the virulence potential of the pathogen and may provide an alternative approach towards reducing disease risk.
2020-12-22 | GSE154844 | GEO
Project description:The Alphaproteobacterium Caulobacter crescentus inhabits low-nutrient environments and can tolerate certain levels of heavy metal in these sites. It has been reported that C. crescentus responds to chromium exposure by altering expression of a large number of genes. In this work, we showed that the ECF sigma factor SigF is one of the regulatory proteins involved in the control of this transcriptional response. Microarray experiments from cells under dichromate stress showed that SigF controls a small regulon consisting of seven genes. Promoter analyses revealed that a conserved SigF-dependent sequence is located upstream of all but one gene of this regulon. Surprisingly, sigF itself was not found to be strongly auto-regulated under dichromate exposure. Furthermore, we showed that the second gene (CC3252) in the sigF operon acts as a negative regulator of SigF function, as overexpression of this putative membrane protein abolishes the transcriptional activation of sigF and its regulon under dichromate stress. Additionally, we found that substitution of one or both the conserved cysteines in the protein encoded by CC3252 (C131 and C181) affects the ability of this protein in maintaining expression of SigF-dependent genes at basal levels. Interestingly, we showed that the activation of SigF is not caused by reactive oxygen species generated as a consequence of dichromate exposure. Therefore, dichromate most probably may lead to conformational changes in the putative anti-sigma factor CC3252, releasing SigF to bind RNA polymerase core and drive transcription of its regulon. Three replicates perfomed with independent biological samples
2012-10-02 | E-GEOD-32406 | biostudies-arrayexpress
Project description:Listeria monocytogenes is a food-borne pathogen which causes listeriosis. It is an intracellular parasite invading the epithelial cells where it escapes from the vacuole into the host cytoplasm to replicate, using actin-based motility to move within and between cells. The intracellular life cycle is well documented whereas the time spent in the lumen of the intestine is poorly understood. The aim of this study was to investigate the mechanism by which L. monocytogenes adapts to the environment of the small intestine prior to invasion. Specifically, to determine if the PrfA regulon, that encodes the virulence factors of L. monocytogenes, is switched on by signals within the intestinal lumen. L. monocytogenes were grown under aerobic or microaerobic conditions with glucose or glycerol as carbon source.
2023-11-24 | E-MTAB-12856 | biostudies-arrayexpress
Project description:uncultured putative methanotroph Metagenome
| PRJNA445632 | ENA
Project description:In this study, we first assess the role of the GAL regulon in enabling efficient galactose utilization for cell growth by decoupling its regulatory responses from sugar catabolism. We provide evidence that regulon-controlled galactose assimilation is more efficient than constitutive expression of the catabolic genes in supporting fast growth rates to higher cell densities. Next, we assessed whether a regulon could enable more complete and efficient utilization of a nutrient that is non-native to this yeast – xylose. We first adapt the GAL regulon to respond to xylose through directed evolution of Gal3p, enabling coupling of nutrient stimulus with sensing, computation, and regulatory actuation. Next, by using a rational, model-guided approach, we test two different positive feedback signal transduction loop designs for the regulon and demonstrate their individual merits and weaknesses. We also show that implementation of a GAL-type xylose-responsive regulon can regulate multiple genes across the yeast genome and enable more homogeneous population-wide gene expression. By integrating a minimal set of heterologous catabolic genes into the synthetic regulon we demonstrate high cellular growth rates and high final cell densities on xylose as well as better growth in non-inducing carbon sources. Finally, we compare the genome-wide expression profiles of strains grown with regulon assistance and conventionally engineered strains to identify mechanistic reasons that account for the different phenotypes observed. We posit that this study strongly supports the need to re-evaluate how nutrient assimilation systems are currently implemented and introduces a new and unexplored paradigm of adapting a native regulon for efficient non-native sugar assimilation.
2018-05-30 | GSE110818 | GEO
Project description:Elucidating the BT4338 regulon
| PRJNA553806 | ENA