Novel architectural features of Bordetella pertussis fimbrial subunit promoters and their activation by the global virulence regulator BvgA. Mol Microbiol

Division of Bacterial, Parasitic, and Allergenic Products, Center for Biologics Evaluation and Research, FDA, Bethesda, MD 20892, USA.
Molecular Microbiology (Impact Factor: 4.42). 09/2010; 77(5):1326-40. DOI: 10.1111/j.1365-2958.2010.07293.x
Source: PubMed


A prominent feature of the promoters of Bordetella pertussis fimbrial subunit genes fim2, fim3 and fimX is the presence of a 'C-stretch', a monotonic run of C residues. The C-stretch renders these genes capable of phase variation, through spontaneous variations in its length. For each of these we determined the length of the C-stretch that gave maximal transcriptional activity, and found that the three optimized promoters align perfectly, with identical distances between conserved upstream sequences and the downstream -10 elements and transcriptional start sites. We also demonstrated, for Pfim3, that the conserved sequence corresponds to BvgA binding sites. The more upstream of the two binding sites is predicted to be high affinity, by comparison to a functionally derived consensus BvgA-binding sequence. The other binding site is a fairly poor match to this consensus, with 10 of 14 bp belonging to the C-stretch. Interestingly, the centre of this downstream site of BvgA binding coincides exactly with the centre of the expected typical location of a -35 sequence. However, the lack of a recognizable -35 element (CCCCCC versus TTGACA), and the occupation of this site by BvgA∼P suggest that activation of the fim promoters involves unusual interactions among BvgA, RNA polymerase and promoter DNA.

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Available from: Qing Chen
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    • "IHF to the nadA promoter and the repressive activity of a transcriptional regulator called NadR (Martin et al. 2005; Metruccio et al. 2009). A similar situation can be found in the promoter of fim genes encoding fimbrial subunits in B. pertussis (Chen et al. 2010). In this case, the length of a poly-C tract acts as a spacer that optimizes interaction between the BvgA response regulator protein of the BvgSA twocomponent system and RNA polymerase and results in changes in the transcriptional activity of these genes. "

    Full-text · Chapter · Jan 2014
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    • "The resulting plasmid produced His 6 -tagged σ ΔR4 . pETσ BpΔR4 was constructed by PCR amplification of the region encoding residues 1–674 from pETsigmaBp (Chen et al., 2010), using primers, which included an NdeI site (upstream) and EcoRI site (downstream). "
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    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 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, which is composed of a tract of cytosines that lacks specific sequence information. We demonstrate that σ 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 σD581C-FeBABE positions Region 4 near the -35 region of P(fim3); cleavage using RNAP with α276C-FeBABE or α302C-FeBABE also positions an α 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 α subunit C-terminal domain, and σ Region 4. Molecular modeling suggests how BvgA, σ Region 4, and α might coexist within this DNA in a conformation that suggests a novel mechanism of activation.
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