Functional analysis of the cya promoter of Bordetella pertussis

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Abstract
The cyaA gene of Bordetella pertussis and of Bordetella bronchiseptica encodes a toxin which is a bifunctional protein exhibiting adenylate cyclase and haemolytic activities. In Bordetella, virulence factors are synthesized under the control of the bvg regulatory locus, in response to environmental signals. In Escherichia coli the cyaA gene is not expressed, nor is it activated by bvg indicating that the activation of cya by bvg is indirect. To characterize cis-acting regulatory regions required for the activation of the cyaA gene we constructed cyaA-lacZY fusions containing progressive deletions in the promoter upstream region and isolated promoter mutations by chemical and site-directed mutagenesis. Deletion analysis shows that a region extending from -569 to -136 bp upstream from the start site of transcription is required for transactivation by bvg, suggesting that multiple binding sites are involved in the activation of the cyaA promoter. No single or double mutations in the promoter upstream region were found which conferred inactive or bvg-independent Cya phenotype. A double mutation in positions +10 and +13, relative to the transcription start site, rendered the promoter bvg-independent and functional in E. coli. The constitutive mutations create a new transcription start site, 20 bp downstream from the wild-type site, by providing new -10 and -35 elements recognized by RNA polymerase alone.