Multiple weak interactions between BvgA~P and ptx promoter DNA strongly activate transcription of pertussis toxin genes in Bordetella pertussis

Pertussis toxin is the preeminent virulence factor and major protective antigen produced by Bordetella pertussis , the human respiratory pathogen and etiologic agent of whooping cough. Genes for its synthesis and export are encoded by the 12 kb ptx-ptl operon, which is under the control of the pertussis promoter, P ptx . Expression of this operon, like that of all other known protein virulence factors, is regulated by the BvgAS two-component global regulatory system. Although P ptx has been studied for years, characterization of its promoter architecture vis-à-vis BvgA-binding has lagged behind that of other promoters, mainly due to its lower affinity for BvgA~P. Here we take advantage of a mutant BvgA protein (Δ127–129), which enhances ptx transcription in B . pertussis and also demonstrates enhanced binding affinity to P ptx . By using this mutant protein labeled with FeBABE, binding of six head-to-head dimers of BvgA~P was observed, with a spacing of 22 bp, revealing a binding geometry similar to that of other BvgA-activated promoters carrying at least one strong binding site. All of these six BvgA-binding sites lack sequence features associated with strong binding. A genetic analysis indicated the degree to which each contributes to P ptx activity. Thus the weak/medium binding affinity of P ptx revealed in this study explains its lower responsiveness to phosphorylated BvgA, relative to other promoters containing a high affinity binding site, such as that of the fha operon.