Different Requirements for σ Region 4 in BvgA Activation of the Bordetella pertussis promoters Pfim3 and PfhaB

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.