Mutational analysis of the high-affinity BvgA binding site in the fha promoter of Bordetella pertussis

In order to define a consensus binding sequence for the response regulator BvgA, we have undertaken a systematic analysis of contributions made by each nucleotide within the heptad half-sites that are present in an inverted orientation at the promoter for the fha operon. Using in vitro binding assays, we examined the full complement of 21 single point mutations symmetrically arranged in this heptad repeat, Both gel shift and nitrocellulose filter-binding assays provided evidence that nucleotides at positions 3 (thymidine), 4 (cytosine) and 7 (adenine) in the binding heptad contribute substantially to sequence-specific recognition by BvgA, Furthermore, a T to A conversion at position 6 reduced binding. Selected binding site mutations were introduced into a modified fha promoter and examined for their effects on BvgA activation of promoter activity in vivo, Only those substitutions most severely affecting binding in vitro affected promoter activity in vivo, The in vivo effects of substitutions that had a significant effect on binding in vitro but did not severely affect in vivo promoter activity under standard culture conditions could be detected in vivo either in combination with additional substitutions or from their effect on the sensitivity of the mutant promoters to modulation by magnesium sulphate.