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Microarray-based analysis of the Staphylococcus aureus sigmaB regulon.

ABSTRACT: Microarray-based analysis of the transcriptional profiles of the genetically distinct Staphylococcus aureus strains COL, GP268, and Newman indicate that a total of 251 open reading frames (ORFs) are influenced by sigmaB activity. While sigmaB was found to positively control 198 genes by a factor of > or =2 in at least two of the three genetic lineages analyzed, 53 ORFs were repressed in the presence of sigmaB. Gene products that were found to be influenced by sigmaB are putatively involved in all manner of cellular processes, including cell envelope biosynthesis and turnover, intermediary metabolism, and signaling pathways. Most of the genes and/or operons identified as upregulated by sigmaB were preceded by a nucleotide sequence that resembled the sigmaB consensus promoter sequence of Bacillus subtilis. A conspicuous number of virulence-associated genes were identified as regulated by sigmaB activity, with many adhesins upregulated and prominently represented in this group, while transcription of various exoproteins and toxins were repressed. The data presented here suggest that the sigmaB of S. aureus controls a large regulon and is an important modulator of virulence gene expression that is likely to act conversely to RNAIII, the effector molecule of the agr locus. We propose that this alternative transcription factor may be of importance for the invading pathogen to fine-tune its virulence factor production in response to changing host environments.

SUBMITTER: Bischoff M

PROVIDER: S-EPMC421609 | biostudies-literature | 2004 Jul

REPOSITORIES: biostudies-literature

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Microarray-based analysis of the Staphylococcus aureus sigmaB regulon.

Bischoff Markus M Dunman Paul P Kormanec Jan J Macapagal Daphne D Murphy Ellen E Mounts William W Berger-Bächi Brigitte B Projan Steven S

Journal of bacteriology 20040701 13

Microarray-based analysis of the transcriptional profiles of the genetically distinct Staphylococcus aureus strains COL, GP268, and Newman indicate that a total of 251 open reading frames (ORFs) are influenced by sigmaB activity. While sigmaB was found to positively control 198 genes by a factor of > or =2 in at least two of the three genetic lineages analyzed, 53 ORFs were repressed in the presence of sigmaB. Gene products that were found to be influenced by sigmaB are putatively involved in al ...[more]

PMID: 15205410

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Project description:The alternative sigma factor sigma(B) of Staphylococcus aureus controls the expression of a variety of genes, including virulence determinants and global regulators. Genetic manipulations and transcriptional start point (TSP) analyses showed that the sigB operon is transcribed from at least two differentially controlled promoters: a putative sigma(A)-dependent promoter, termed sigB(p1), giving rise to a 3.6-kb transcript covering sa2059-sa2058-rsbU-rsbV-rsbW-sigB, and a sigma(B)-dependent promoter, sigB(p3), initiating a 1.6-kb transcript covering rsbV-rsbW-sigB. TSP and promoter-reporter gene fusion experiments indicated that a third promoter, tentatively termed sigB(p2) and proposed to lead to a 2.5-kb transcript, including rsbU-rsbV-rsbW-sigB, might govern the expression of the sigB operon. Environmental stresses, such as heat shock and salt stress, induced a rapid response within minutes from promoters sigB(p1) and sigB(p3). In vitro, the sigB(p1) promoter was active in the early growth stages, while the sigB(p2) and sigB(p3) promoters produced transcripts throughout the growth cycle, with sigB(p3) peaking around the transition state between exponential growth and stationary phase. The amount of sigB transcripts, however, did not reflect the concentration of sigma(B) measured in cell extracts, which remained constant over the entire growth cycle. In a guinea pig cage model of infection, sigB transcripts were as abundant 2 and 8 days postinoculation as values found in vitro, demonstrating that sigB is indeed transcribed during the course of infection. Physical interactions between staphylococcal RsbU-RsbV, RsbV-RsbW, and RsbW-sigma(B) were inferred from a yeast (Saccharomyces cerevisiae) two-hybrid approach, indicating the presence of a partner-switching mechanism in the sigma(B) activation cascade similar to that of Bacillus subtilis. The finding that overexpression of RsbU was sufficient to trigger an immediate and strong activation of sigma(B), however, signals a relevant difference in the regulation of sigma(B) activation between B. subtilis and S. aureus in the cascade upstream of RsbU.

Project description:SarA, a transcriptional regulator of Staphylococcus aureus, is a major global regulatory system that coordinates the expression of target genes involved in its pathogenicity. Various studies have identified a large number of SarA target genes, but an in-depth characterization of the sarA regulon, including small regulatory RNAs (sRNAs), has not yet been done. In this study, we utilized transcriptome sequencing (RNA-Seq) and chromatin immunoprecipitation sequencing (ChIP-Seq) to determine a comprehensive list of SarA-regulated targets, including both mRNAs and sRNAs. RNA-Seq analysis indicated 390 mRNAs and 51 sRNAs differentially expressed in a ΔsarA mutant, while ChIP-Seq revealed 354 mRNAs and 55 sRNA targets in the S. aureus genome. We confirmed the authenticity of several novel SarA targets by Northern blotting and electrophoretic mobility shift assays. Among them, we characterized repression of sprG2, a gene that encodes the toxin of a type I toxin-antitoxin system, indicating a multilayer lockdown of toxin expression by both SarA and its cognate antitoxin, SprF2. Finally, a novel SarA consensus DNA binding sequence was generated using the upstream promoter sequences of 15 novel SarA-regulated sRNA targets. A genome-wide scan with a deduced SarA motif enabled the discovery of new potential SarA target genes which were not identified in our RNA-Seq and ChIP-Seq analyses. The strength of this new consensus was confirmed with one predicted sRNA target. The RNA-Seq and ChIP-Seq combinatory analysis gives a snapshot of the regulation, whereas bioinformatic analysis reveals a permanent view of targets based on sequence. Altogether these experimental and in silico methodologies are effective to characterize transcriptional factor (TF) regulons and functions. IMPORTANCE Staphylococcus aureus, a commensal and opportunist pathogen, is responsible for a large number of human and animal infections, from benign to severe. Gene expression adaptation during infection requires a complex network of regulators, including transcriptional factors (TF) and sRNAs. TF SarA influences virulence, metabolism, biofilm formation, and resistance to some antibiotics. SarA directly regulates expression of around 20 mRNAs and a few sRNAs. Here, we combined high-throughput expression screening methods combined with binding assays and bioinformatics for an in-depth investigation of the SarA regulon. This combinatory approach allowed the identification of 85 unprecedented mRNAs and sRNAs targets, with at least 14 being primary. Among novel SarA direct targets, we characterized repression of sprG2, a gene that encodes the toxin of a toxin-antitoxin system, indicating a multilayer lockdown of toxin expression by both SarA and its cognate antitoxin, SprF2.

Project description:Background The catabolite control protein A (CcpA) is a member of the LacI/GalR family of transcriptional regulators controlling carbon-metabolism pathways in low-GC Gram positive bacteria. It functions as a catabolite repressor or activator, allowing the bacteria to utilize the preferred carbon source over secondary carbon sources. This study is the first CcpA-dependent transcriptome and proteome analysis in S. aureus wild type and ccpA-deleted mutant, focussing on short-time effects of glucose under stable pH conditions. Results The addition of glucose to exponentially growing S. aureus increased enzymes of glycolytic pathway, indicating a higher glycolytic activity, while proteins required for the complete oxidation in the TCA cycle were repressed via CcpA. Phosphotransacetylase and acetate kinase, converting acetylCoA to acetate with a concomitant substrate-level phosphorylation were neither regulated by glucose nor by CcpA. Most CcpA directly repressed genes were involved in utilization of amino acids as secondary carbon sources. More genes were found to be differentially expressed by CcpA in a glucose-independent manner than in the classical, glucose dependent way, suggesting that glucose-independent regulation by CcpA may be of particular importance in S. aureus. In the presence of glucose, CcpA was found to regulate expression of genes involved in metabolism, but that of genes coding for virulence determinants. Conclusions This study identified the CcpA regulon of exponentially growing S. aureus, for the first time. As in other bacteria, the CcpA-regulon of S. aureus comprised a large amount of metabolic genes but also some 50 genes associated with virulence. CcpA seemed to work in a glucose- as well as glucose-independent way. The transcriptomes of strain Newman and its isogenic ccpA-deleted mutant were determined in early exponential growth and 30 min after the addition of 10 mM glucose, under controlled pH conditions. In the absence of glucose, the wild type grew slightly faster than the mutant, reaching an OD600 of 1 approximately 20 min earlier than the mutant. Adding 10 mM glucose at OD600 1 increased the growth rate of the wild type but had only a minor effect on that of the mutant. 60 min after glucose addition, glucose was depleted down to 0.3 mM by the wild type, while still 3 mM glucose was left in the culture of the mutant. Despite increased glucose consumption rates in the wild type, acetate production was only slightly enhanced compared to the mutant. No lactate was excreted at any time point sampled. Acidification of the medium upon glucose metabolism was prevented by buffering, maintaining a pH of 7.5 for both strains and under both growth conditions for at least 2 h after glucose addition, allowing to rule out any pH effects.

Project description:BackgroundStaphylococcus aureus (S. aureus), is responsible for many infectious diseases, ranging from benign skin infections to life-threatening endocarditis and toxic shock syndrome. Ortho-phenylphenol (OPP) is an antimicrobial agent and an active ingredient of EPA-registered disinfectants with wide human exposure in various agricultural, hospital and veterinary disinfectant products. Despite many uses, an understanding of a cellular response to OPP and it's mechanism of action, targeted genes, and the connectivity between targeted genes and the rest of cell metabolism remains obscure.ResultsHerein, we performed a genome-wide transcriptome analysis of the cellular responses of S. aureus when exposed to 0.82 mM of OPP for 20 and 60 min. Our data indicated that OPP downregulated the biosynthesis of many amino acids, which are required for protein synthesis. In particular, the genes encoding the enzymes of the diaminopimelate (DAP) pathway which results in lysine biosynthesis were significantly downregualted. Intriguingly, we revealed that the transcription of genes encoding ribosomal proteins was upregulated by OPP and at the same time, the genes encoding iron acquisition and transport were downregulated. The genes encoding virulence factors were upregulated and genes encoding phospholipids were downregulated upon 20 min exposure to OPP.ConclusionBy using microarray analysis that enables us to simultaneously and globally examine the complete transcriptome during cellular responses, we have revealed novel information regarding the mode of action of OPP on Staphylococcus: OPP inhibits anabolism of many amino acids and highly downregulates the genes that encode the enzymes involved in the DAP pathway. Lysine and DAP are essential for building up the peptidoglycan cell wall. It was concluded that the mode of action of OPP is similar to the mechanism of action of some antibiotics. The discovery of this phenomenon provides useful information that will benefit further antimicrobial research on S. aureus.

Dataset Information

Microarray-based analysis of the Staphylococcus aureus sigmaB regulon.

Publications

Microarray-based analysis of the Staphylococcus aureus sigmaB regulon.

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