Dataset Information

Different requirements for ? Region 4 in BvgA activation of the Bordetella pertussis promoters P(fim3) and P(fhaB).

ABSTRACT: Bordetella pertussis BvgA is a global response regulator that activates virulence genes, including adhesin-encoding fim3 and fhaB. At the fhaB promoter, P(fhaB), a BvgA binding site lies immediately upstream of the -35 promoter element recognized by Region 4 of the ? subunit of RNA polymerase (RNAP). We demonstrate that ? Region 4 is required for BvgA activation of P(fhaB), a hallmark of Class II activation. In contrast, the promoter-proximal BvgA binding site at P(fim3) includes the -35 region, which is composed of a tract of cytosines that lacks specific sequence information. We demonstrate that ? Region 4 is not required for BvgA activation at P(fim3). Nonetheless, Region 4 mutations that impair its typical interactions with core and with the -35 DNA affect P(fim3) transcription. Hydroxyl radical cleavage using RNAP with ?D581C-FeBABE positions Region 4 near the -35 region of P(fim3); cleavage using RNAP with ?276C-FeBABE or ?302C-FeBABE also positions an ? subunit C-terminal domain within the -35 region, on a different helical face from the promoter-proximal BvgA~P dimer. Our results suggest that the -35 region of P(fim3) accommodates a BvgA~P dimer, an ? subunit C-terminal domain, and ? Region 4. Molecular modeling suggests how BvgA, ? Region 4, and ? might coexist within this DNA in a conformation that suggests a novel mechanism of activation.

SUBMITTER: Decker KB

PROVIDER: S-EPMC3141349 | biostudies-literature | 2011 Jun

REPOSITORIES: biostudies-literature

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Different requirements for σ Region 4 in BvgA activation of the Bordetella pertussis promoters P(fim3) and P(fhaB).

Decker Kimberly B KB Chen Qing Q Hsieh Meng-Lun ML Boucher Philip P Stibitz Scott S Hinton Deborah M DM

Journal of molecular biology 20110422 5

Bordetella pertussis BvgA is a global response regulator that activates virulence genes, including adhesin-encoding fim3 and fhaB. At the fhaB promoter, P(fhaB), a BvgA binding site lies immediately upstream of the -35 promoter element recognized by Region 4 of the σ subunit of RNA polymerase (RNAP). We demonstrate that σ Region 4 is required for BvgA activation of P(fhaB), a hallmark of Class II activation. In contrast, the promoter-proximal BvgA binding site at P(fim3) includes the -35 region, ...[more]

PMID: 21536048

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Project description:Bordetella pertussis regulates the production of its virulence factors by the two-component system BvgAS. In the virulence phase, BvgS phosphorylates BvgA, which then activates the transcription of virulence-activated genes (vags). In the avirulence phase, such as during growth in the presence of MgSO4, BvgA is not phosphorylated and the vags are not expressed. Instead, a set of virulence-repressed genes (vrgs) is expressed. Here, we performed transcriptome sequencing (RNAseq) analyses on B. pertussis cultivated with or without MgSO4 and on a BvgA-deficient Tohama I derivative. We observed that 146 genes were less expressed under modulating conditions or in the BvgA-deficient strain than under the nonmodulating condition, while 130 genes were more expressed. Some of the genes code for proteins with regulatory functions, suggesting a BvgA/S regulation cascade. To determine which genes are directly regulated by BvgA, we performed chromatin immunoprecipitation sequencing (ChIPseq) analyses. We identified 148 BvgA-binding sites, 91 within putative promoter regions, 52 within open reading frames, and 5 in noncoding regions. Among the former, 32 are in BvgA-regulated putative promoter regions. Some vags, such as dnt and fhaL, contain no BvgA-binding site, suggesting indirect BvgA regulation. Unexpectedly, BvgA also bound to some vrg putative promoter regions. Together, these observations indicate an unrecognized complexity of BvgA/S biology.IMPORTANCEBordetella pertussis, the etiological agent of whooping cough, remains a major global health problem. Despite the global usage of whole-cell vaccines since the 1950s and of acellular vaccines in the 1990s, it still is one of the most prevalent vaccine-preventable diseases in industrialized countries. Virulence of B. pertussis is controlled by BvgA/S, a two-component system responsible for upregulation of virulence-activated genes (vags) and downregulation of virulence-repressed genes (vrgs). By transcriptome sequencing (RNAseq) analyses, we identified more than 270 vags or vrgs, and chromatin immunoprecipitation sequencing (ChIPseq) analyses revealed 148 BvgA-binding sites, 91 within putative promoter regions, 52 within open reading frames, and 5 in noncoding regions. Some vags, such as dnt and fhaL, do not contain a BvgA-binding site, suggesting indirect regulation. In contrast, several vrgs and some genes not identified by RNAseq analyses under laboratory conditions contain strong BvgA-binding sites, indicating previously unappreciated complexities of BvgA/S biology.

Project description:Noncoding small RNAs (sRNAs) are crucial for the posttranscriptional regulation of gene expression in all organisms and are known to be involved in the regulation of bacterial virulence. In the human pathogen Bordetella pertussis, which causes whooping cough, virulence is controlled primarily by the master two-component system BvgA (response regulator)/BvgS (sensor kinase). In this system, BvgA is phosphorylated (Bvg+ mode) or nonphosphorylated (Bvg- mode), with global transcriptional differences between the two. B. pertussis also carries the bacterial sRNA chaperone Hfq, which has previously been shown to be required for virulence. Here, we conducted transcriptomic analyses to identify possible B. pertussis sRNAs and to determine their BvgAS dependence using transcriptome sequencing (RNA-seq) and the prokaryotic sRNA prediction program ANNOgesic. We identified 143 possible candidates (25 Bvg+ mode specific and 53 Bvg- mode specific), of which 90 were previously unreported. Northern blot analyses confirmed all of the 10 ANNOgesic candidates that we tested. Homology searches demonstrated that 9 of the confirmed sRNAs are highly conserved among B. pertussis, Bordetella parapertussis, and Bordetella bronchiseptica, with one that also has homologues in other species of the Alcaligenaceae family. Using coimmunoprecipitation with a B. pertussis FLAG-tagged Hfq, we demonstrated that 3 of the sRNAs interact directly with Hfq, which is the first identification of sRNA binding to B. pertussis Hfq. Our study demonstrates that ANNOgesic is a highly useful tool for the identification of sRNAs in this system and that its combination with molecular techniques is a successful way to identify various BvgAS-dependent and Hfq-binding sRNAs. IMPORTANCE Noncoding small RNAs (sRNAs) are crucial for posttranscriptional regulation of gene expression in all organisms and are known to be involved in the regulation of bacterial virulence. We have investigated the presence of sRNAs in the obligate human pathogen B. pertussis, using transcriptome sequencing (RNA-seq) and the recently developed prokaryotic sRNA search program ANNOgesic. This analysis has identified 143 sRNA candidates (90 previously unreported). We have classified their dependence on the B. pertussis two-component system required for virulence, namely, BvgAS, based on their expression in the presence/absence of the phosphorylated response regulator BvgA, confirmed several by Northern analyses, and demonstrated that 3 bind directly to B. pertussis Hfq, the RNA chaperone involved in mediating sRNA effects. Our study demonstrates the utility of combining RNA-seq, ANNOgesic, and molecular techniques to identify various BvgAS-dependent and Hfq-binding sRNAs, which may unveil the roles of sRNAs in pertussis pathogenesis.

Dataset Information

Different requirements for ? Region 4 in BvgA activation of the Bordetella pertussis promoters P(fim3) and P(fhaB).

Publications

Different requirements for σ Region 4 in BvgA activation of the Bordetella pertussis promoters P(fim3) and P(fhaB).

Similar Datasets

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