SummaryThecyaAgene ofBordetella pertussisand ofBordetella bronchisepticaencodes a toxin which is a bifunctional protein exhibiting adenylate cyclase and haemolytic activities. InBordetella, virulence factors are synthesized under the control of thebvgregulatory locus, in response to environmental signals. InEscherichia colithecyaAgene is not expressed, nor is it activated bybvgindicating that the activation ofcyabybvgis indirect. To characterizecis‐acting regulatory regions required for the activation of thecyaAgene we constructedcyaA–lacZYfusions 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 bybvg, suggesting that multiple binding sites are involved in the activation of thecyaApromoter. No single or double mutations in the promoter upstream region were found which conferred inactive orbvg‐independent Cya phenotype. A double mutation in positions +10 and +13, relative to the transcription start site, rendered the promoterbvg‐independent and functional inE. coli.The constitutive mutations create a new transcription start site, 20 bp downstream from the witd‐type site, by providing new −10 and −35 elements recognized by R
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