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An advanced bioinformatics approach for analyzing RNA-seq data reveals sigma H-dependent regulation of competence genes in Listeria monocytogenes.

ABSTRACT: Alternative ? factors are important transcriptional regulators in bacteria. While ?(B) has been shown to control a large regulon and play important roles in stress response and virulence in the pathogen Listeria monocytogenes, the function of ?(H) has not yet been well defined in Listeria, even though ?(H) controls a large regulon in the closely related non-pathogenic Bacillus subtilis.Using RNA-seq characterization of a L. monocytogenes strain with deletions of all 4 genes encoding alternative ? factors (?BCHL), which was further modified to overexpress sigH (?BCHL::P rha -sigH), we identified 6 transcription units (TUs) that are transcribed from ?(H)-dependent promoters. Five of these TUs had not been previously identified. Identification of these promoters was facilitated by use of a bio-informatics approach that compared normalized RNA-seq coverage (NRC), between ?BCHL::P rha -sigH and a ?BCHL control, using sliding windows of 51 nt along the whole genome rather than comparing NRC calculated only for whole genes. Interestingly, we found that three operons that encode competence genes (comGABCDEFG, comEABC, coiA) are transcribed from ?(H)-dependent promoters. While these promoters were highly conserved in L. monocytogenes, none of them were found in all Listeria spp. and coiA and its ?(H)-dependent promoter were only found in L. monocytogenes.Our data indicate that a number of L. monocytogenes competence genes are regulated by ?(H). This ?(H)-dependent regulation of competence related genes is conserved in the pathogen L. monocytogenes, but not in other non-pathogenic Listeria strains. Combined with prior data that indicated a role of ?(H) in virulence in a mouse model, this suggests a possible novel role of ?(H)-dependent competence genes in L. monocytogenes virulence. Development and implementation of a sliding window approach to identify differential transcription using RNA-seq data, not only allowed for identification of ?(H)-dependent promoters, but also provides a general approach for sensitive identification of differentially transcribed promoters and genes, particularly for genes that are transcribed from multiple promoter elements only some of which show differential transcription.

SUBMITTER: Liu Y

PROVIDER: S-EPMC4754846 | biostudies-literature | 2016 Feb

REPOSITORIES: biostudies-literature

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An advanced bioinformatics approach for analyzing RNA-seq data reveals sigma H-dependent regulation of competence genes in Listeria monocytogenes.

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

BMC genomics 20160216

<h4>Background</h4>Alternative σ factors are important transcriptional regulators in bacteria. While σ(B) has been shown to control a large regulon and play important roles in stress response and virulence in the pathogen Listeria monocytogenes, the function of σ(H) has not yet been well defined in Listeria, even though σ(H) controls a large regulon in the closely related non-pathogenic Bacillus subtilis.<h4>Results</h4>Using RNA-seq characterization of a L. monocytogenes strain with deletions o ...[more]

PMID: 26880300

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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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An advanced bioinformatics approach for analyzing RNA-seq data reveals sigma H-dependent regulation of competence genes in Listeria monocytogenes.

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An advanced bioinformatics approach for analyzing RNA-seq data reveals sigma H-dependent regulation of competence genes in Listeria monocytogenes.

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