Two-component systems, composed of a sensor kinase and a response regulator RR, are a major tool used by microorganisms to adapt to environmental conditions. Typically, phosphorylation of the RR by the sensor kinase results in gene activation. In the case of B. pertussis, the RR BvgA is phosphorylated by the sensor kinase BvgS to yield BvgA-P that binds to the promoter regions of the virulence genes, activating their transcription during infection. These genes include adhesins, needed to adhere to the ciliated epithelial cells within the upper respiratory tract, and toxins, which cause the major symptoms of whooping cough disease. The serologically distinct fimbriae 2 and fimbriae 3, which are composed primarily of the major subunit proteins Fim2 and Fim3, are adhesins that are common components of the acellular pertussis vaccine. Although it is known that RR phosphorylation is typically required for gene activation, few studies have addressed how and if phosphorylation affects specific steps during transcription initiation. We characterized transcription complexes made with RNA polymerase and BvgA, in its nonphosphorylated or phosphorylated (BvgA∼P) state at Pfim3, the promoter for the fim3 gene, using gel retardation, potassium permanganate and DNase I footprinting, cleavage reactions with protein conjugated with iron bromoacetamidobenzyl-EDTA, and in vitro transcription. Our previous work has shown that the level of nonphosphorylated BvgA remains high in vivo under conditions in which BvgA is phosphorylated. We have found now that surprisingly both BvgA and BvgA∼P form open and initiating complexes with RNA polymerase at Pfim3. However, phosphorylation of BvgA is needed to generate the correct conformation that can transition to competent elongation. Footprints obtained with the complexes made with nonphosphorylated BvgA are atypical; while the initiating complex with BvgA synthesizes short RNA, it does not generate full-length transcripts. Extended incubation of the BvgA/RNA polymerase initiated complex in the presence of heparin generates a stable, but defective species that depends on the initial transcribed sequence of fim3. Our results suggest that the presence of nonphosphorylated BvgA down-regulates Pfim3 activity when phosphorylated BvgA is present and may allow the bacterium to quickly adapt to the loss of inducing conditions by rapidly eliminating Pfim3 activation once the signal for BvgA phosphorylation is removed.
