title: Synergistic binding of RNA polymerase and BvgA phosphate to the pertussis toxin promoter of Bordetella pertussis.
abstract: Regulation of virulence factor expression in Bordetella pertussis is mediated by the BvgAS two-component regulatory system. Although previous studies have demonstrated that the transcriptional regulation of the filamentous hemagglutinin gene (fhaB) involves binding of the BvgA activator directly to the fhaB promoter region, the mechanism of pertussis toxin operon (ptx) regulation by BvgA has remained unclear. We demonstrate in vitro the specific binding of BvgA to a region upstream of the ptx promoter that encompasses a 20-bp directly repeated sequence (positions -157 to -117) previously shown to be critical for BvgA-dependent activation. This binding is strictly dependent on the phosphorylation of BvgA, which can be obtained by incubation of BvgA with acetyl phosphate. By DNase I protection studies, we demonstrate the synergistic binding of BvgA-phosphate and purified Escherichia coli RNA polymerase to the ptx promoter. In the presence of the polymerase holoenzyme, a greatly extended footprint encompassing the region between -163 and the putative polymerase binding site was observed. The implications of these observations for pertussis toxin expression and regulation are discussed.
authors.affiliation: Department of Bacterial Products, Food and Drug Administration, Bethesda, Maryland 20892, USA.
authors.forename: P E
authors.initials: PE
authors.lastname: Boucher
authors.forename: S
authors.initials: S
authors.lastname: Stibitz
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D001886:Bordetella pertussis / Q000235:genetics*; D004269:DNA, Bacterial / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics; D008969:Molecular Sequence Data; D010755:Organophosphates; D037342:Pertussis Toxin; D010710:Phosphates / Q000378:metabolism; D010766:Phosphorylation; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics; D014157:Transcription Factors / Q000378:metabolism*; D010566:Virulence Factors, Bordetella / Q000235:genetics*
chemical_list: D001426:Bacterial Proteins; C065601:BvgA protein, Bacteria; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; C489314:EvgA protein, E coli; D010755:Organophosphates; D010710:Phosphates; D014157:Transcription Factors; D010566:Virulence Factors, Bordetella; C011632:acetyl phosphate; D037342:Pertussis Toxin; D012321:DNA-Directed RNA Polymerases