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

摘要

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. 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 RNA polymerase with σD581C-FeBABE positions Region 4 near the −35 region of Pfim3; cleavage using RNA polymerase with α276C–FeBABE or α302C–FeBABE also positions an αCTD 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 αCTD, 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.