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Home Alone: Elimination of All but One Alternative Sigma Factor in Listeria monocytogenes Allows Prediction of New Roles for ?B.

ABSTRACT: Among Listeria monocytogenes' four alternative ? factors, ?B controls the largest regulon. As ?B-dependent transcription of some genes may be masked by overlaps among regulons, and as some ?B-dependent genes are expressed only under very specific conditions, we hypothesized that the ?B regulon is not yet fully defined. To further extend our understanding of the ?B regulon, we used RNA-seq to identify ?B-dependent genes in an L. monocytogenes strain that expresses ?B following rhamnose induction, and in which genes encoding the other alternative sigma factors have been deleted. Analysis of RNA-seq data with multiple bioinformatics approaches, including a sliding window method that detects differentially transcribed 5' untranslated regions (UTRs), identified 105 ?B-dependent transcription units (TUs) comprising 201 genes preceded by ?B-dependent promoters. Of these 105 TUs, 7 TUs comprising 15 genes had not been identified previously as ?B-dependent. An additional 23 genes not reported previously as ?B-dependent were identified in 9 previously recognized ?B-dependent TUs. Overall, 38 of these 201 genes had not been identified previously as members of the L. monocytogenes ?B regulon. These newly identified ?B-dependent genes encode proteins annotated as being involved in transcriptional regulation, oxidative and osmotic stress response, and in metabolism of energy, carbon and nucleotides. In total, 18 putative ?B-dependent promoters were newly identified. Interestingly, a number of genes previously identified as ?B-dependent did not show significant evidence for ?B-dependent transcription in our experiments. Based on promoter analyses, a number of these genes showed evidence for co-regulation by ?B and other transcriptional factors, suggesting that some ?B-dependent genes require additional transcriptional regulators along with ?B for transcription. Over-expression of a single alternative sigma factor in the absence of all other alternative sigma factors allowed us to: (i) identify new ?B-dependent functions in L. monocytogenes, such as regulation of genes involved in 1,2-propanediol utilization (LMRG_00594-LMRG_00611) and biosynthesis of pyrimidine nucleotides (LMRG_00978-LMRG_00985); and (ii) identify new ?B-dependent genes involved in stress response and pathogenesis functions. These data further support that ?B not only regulates stress response functions, but also plays a broad role in L. monocytogenes homeostasis and resilience.

SUBMITTER: Liu Y

PROVIDER: S-EPMC5641562 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Home Alone: Elimination of All but One Alternative Sigma Factor in Listeria monocytogenes Allows Prediction of New Roles for σB.

Liu Yichang Y Orsi Renato H RH Boor Kathryn J KJ Wiedmann Martin M Guariglia-Oropeza Veronica V

Frontiers in microbiology 20171011

Among Listeria monocytogenes' four alternative σ factors, σB controls the largest regulon. As σB-dependent transcription of some genes may be masked by overlaps among regulons, and as some σB-dependent genes are expressed only under very specific conditions, we hypothesized that the σB regulon is not yet fully defined. To further extend our understanding of the σB regulon, we used RNA-seq to identify σB-dependent genes in a ...[more]

PMID: 29075236

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Project description:BackgroundTranscriptional regulation by alternative sigma (σ) factors represents an important mechanism that allows bacteria to rapidly regulate transcript and protein levels in response to changing environmental conditions. While the role of the alternative σ factor σB has been comparatively well characterized in L. monocytogenes, our understanding of the roles of the three other L. monocytogenes alternative σ factors is still limited. In this study, we employed a quantitative proteomics approach using Isobaric Tags for Relative and Absolute Quantitation (iTRAQ) to characterize the L. monocytogenes σL, σH, and σC protein regulons. Proteomic comparisons used a quadruple alternative σ factor mutant strain (ΔBCHL) and strains expressing a single alternative σ factor (i.e., σL, σH, and σC; strains ΔBCH, ΔBCL, and ΔBHL) to eliminate potential redundancies between σ factors.ResultsAmong the three alternative σ factors studied here, σH provides positive regulation for the largest number of proteins, consistent with previous transcriptomic studies, while σL appears to contribute to negative regulation of a number of proteins. σC was found to regulate a small number of proteins in L. monocytogenes grown to stationary phase at 37°C. Proteins identified as being regulated by multiple alternative σ factors include MptA, which is a component of a PTS system with a potential role in regulation of PrfA activity.ConclusionsThis study provides initial insights into global regulation of protein production by the L. monocytogenes alternative σ factors σL, σH, and σC. While, among these σ factors, σH appears to positively regulate the largest number of proteins, we also identified PTS systems that appear to be co-regulated by multiple alternative σ factors. Future studies should not only explore potential roles of alternative σ factors in activating a "cascade" of PTS systems that potentially regulate PrfA, but also may want to explore the σL and σC regulons under different environmental conditions to identify conditions where these σ factors may regulate larger numbers of proteins or genes.

Project description:While the stress-responsive alternative sigma factor sigma(B) has been identified in different species of Bacillus, Listeria, and Staphylococcus, the sigma(B) regulon has been extensively characterized only in B. subtilis. We combined biocomputing and microarray-based strategies to identify sigma(B)-dependent genes in the facultative intracellular pathogen Listeria monocytogenes. Hidden Markov model (HMM)-based searches identified 170 candidate sigma(B)-dependent promoter sequences in the strain EGD-e genome sequence. These data were used to develop a specialized, 208-gene microarray, which included 166 genes downstream of HMM-predicted sigma(B)-dependent promoters as well as selected virulence and stress response genes. RNA for the microarray experiments was isolated from both wild-type and Delta sigB null mutant L. monocytogenes cells grown to stationary phase or exposed to osmotic stress (0.5 M KCl). Microarray analyses identified a total of 55 genes with statistically significant sigma(B)-dependent expression under the conditions used in these experiments, with at least 1.5-fold-higher expression in the wild type over the sigB mutant under either stress condition (51 genes showed at least 2.0-fold-higher expression in the wild type). Of the 55 genes exhibiting sigma(B)-dependent expression, 54 were preceded by a sequence resembling the sigma(B) promoter consensus sequence. Rapid amplification of cDNA ends-PCR was used to confirm the sigma(B)-dependent nature of a subset of eight selected promoter regions. Notably, the sigma(B)-dependent L. monocytogenes genes identified through this HMM/microarray strategy included both stress response genes (e.g., gadB, ctc, and the glutathione reductase gene lmo1433) and virulence genes (e.g., inlA, inlB, and bsh). Our data demonstrate that, in addition to regulating expression of genes important for survival under environmental stress conditions, sigma(B) also contributes to regulation of virulence gene expression in L. monocytogenes. These findings strongly suggest that sigma(B) contributes to L. monocytogenes gene expression during infection.

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Home Alone: Elimination of All but One Alternative Sigma Factor in Listeria monocytogenes Allows Prediction of New Roles for ?B.

Publications

Home Alone: Elimination of All but One Alternative Sigma Factor in <i>Listeria monocytogenes</i> Allows Prediction of New Roles for σ<sup>B</sup>.

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