PHOSPHORYLATION-DEPENDENT BINDING OF BVGA TO THE UPSTREAM REGION OF THE CYAA GENE OF BORDETELLA-PERTUSSIS

In Bordetella pertussis, transcription of virulence-associated genes i s regulated by the BvgS and BvgA proteins, members of the bacterial tw o-component signal-transduction family. BvgS is the transmembrane sens or and BvgA, in its phosphorylated form, is believed to be the key tra nscriptional activator in B. pertussis. However, the BvgA recognition sites in most virulence promoters have not yet been identified. To inv estigate the interaction of BvgA with the upstream region of cyaA, the gene encoding adenylate cyclase haemolysin, we have produced large am ounts of BvgA in Escherichia coli. The protein was purified from inclu sion bodies and then phosphorylated by acetyl phosphate. Using electro phoretic mobility-shift and footprinting assays, we provide evidence t hat BvgA cannot bind to the cyaA promoter unless it is phosphorylated. The phosphorylated form of BvgA (BvgA-P) is able to bind specifically to the upstream region of cyaA. Analysis of this region revealed that an unexpectedly large sequence, from -137 to -51, appears to be the t arget for BvgA-P binding, and probably contains multiple binding sites .