Goyard S, Ullmann A
Unité de Biochimie des Régulations Cellulaires, Institut Pasteur, Paris, France.
Mol Microbiol. 1993 Mar;7(5):693-704. doi: 10.1111/j.1365-2958.1993.tb01160.x.
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.
百日咳博德特氏菌和支气管败血博德特氏菌的cyaA基因编码一种毒素，该毒素是一种具有腺苷酸环化酶和溶血活性的双功能蛋白。在博德特氏菌中，毒力因子是在bvg调控位点的控制下，响应环境信号而合成的。在大肠杆菌中，cyaA基因不表达，也不会被bvg激活，这表明bvg对cya的激活是间接的。为了鉴定激活cyaA基因所需的顺式作用调控区域，我们构建了在启动子上游区域含有逐步缺失的cyaA-lacZY融合体，并通过化学诱变和定点诱变分离出启动子突变。缺失分析表明，转录起始位点上游从-569到-136 bp的区域是bvg反式激活所必需的，这表明多个结合位点参与了cyaA启动子的激活。在启动子上游区域未发现赋予无活性或bvg非依赖性Cya表型的单突变或双突变。相对于转录起始位点，+10和+13位的双突变使启动子在大肠杆菌中具有bvg非依赖性且功能正常。组成型突变通过提供仅由RNA聚合酶识别的新的-10和-35元件，在野生型位点下游20 bp处产生了一个新的转录起始位点。
