Genetic and Biochemical Analyses of BvgA Interaction with the Secondary Binding Region of the fha Promoter of Bordetella pertussis

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Abstract
The BvgA-BvgS two-component signal transduction system regulates expression of virulence factors in Bordetella pertussis . The BvgA response regulator activates transcription by binding to target promoters, which include those for the genes encoding filamentous hemagglutinin ( fha ) and pertussis toxin ( ptx ). We have previously shown that at both promoters the phosphorylated form of BvgA binds multiple high- and low-affinity sites. Specifically, at the fha promoter, we proposed that there may be high- and a low-affinity binding sites for the BvgA dimer. In our present investigation, we used DNA binding analyses and in vitro and in vivo assays of promoters with substitutions and deletions to support and extend this hypothesis. Our observations indicate that (i) binding of BvgA∼P to a primary (high-affinity) site and a secondary binding region (lower affinity) is cooperative, (ii) although both the primary binding site and the secondary binding region are required for full activity of the wild-type (undeleted) promoter, deletion of two helical turns within the secondary binding region can produce a fully active or hyperactive promoter, and (iii) BvgA binding to the secondary binding region shows limited DNA sequence specificity.