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

Bordetella pertussis BvgA is a global response regulator that activates virulence genes, including adhesin-encoding fim3 and fhaB. At the fhaB promoter, PfₕₐB, 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 PfₕₐB, a hallmark of Class II activation. In contrast, the promoter-proximal BvgA binding site at Pfᵢₘ₃ 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 Pfᵢₘ₃. Nonetheless, Region 4 mutations that impair its typical interactions with core and with the −35 DNA affect Pfᵢₘ₃ transcription. Hydroxyl radical cleavage using RNAP with σD581C–FeBABE positions Region 4 near the −35 region of Pfᵢₘ₃; 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 Pfᵢₘ₃ 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.