苏云金芽胞杆菌BkdR和CcpA对bkd基因簇的转录调控

bkd gene cluster is regulated by BkdR and CcpA in Bacillus thuringiensis

摘要:

[目的] 通过分析苏云金芽胞杆菌（Bacillus thuringiensis，Bt）转录调控因子BkdR和多效调控因子CcpA对亮氨酸、异亮氨酸、缬氨酸代谢基因簇bkd的转录调控，明确bkd基因簇的转录调控机制。[方法] 通过β-半乳糖苷酶活性测定分析bkd基因簇启动子的诱导转录活性，采用同源重组技术敲除Bt HD73菌株的ccpA基因，通过融合His标签的方法在大肠杆菌中表达纯化BkdR和CcpA蛋白，通过凝胶阻滞实验明确BkdR和CcpA蛋白与bkd基因簇启动子的结合作用。[结果] 亮氨酸、异亮氨酸、缬氨酸可诱导bkd基因簇启动子Pptb的转录活性。Pptb的诱导活性在bkdR突变体中明显降低，而在ccpA突变体中明显上升。BkdR和CcpA蛋白与Pptb均有结合作用。[结论] bkd基因簇的转录活性受BkdR正调控，而受CcpA负调控。

Abstract:

[Objective] We analyzed the transcriptional regulation of bkd gene cluster by BkdR and CcpA in Bacillus thuringiensis (Bt), which is involved in leucine, isoleucine and valine metabolism. To determine the mechanism of transcriptional regulation of bkd gene cluster.[Methods] Induced transcriptional activity of bkd promoter (Pptb) was analyzed by promoter fusions with lacZ gene. ccpA insertion mutant was constructed by homologous recombination. Purification of BkdR and CcpA was using HiTrap chelating column. The binding of bkd promoter with CcpA protein was verified by electrophoresis mobility shift assays.[Results] Transcriptional activity of Pptb was induced by leucine, isoleucine, and valine. The induced transcriptional activity of Pptb was sharply decreased in bkdR mutant, but increased in ccpA mutant. Pptb could bind to the BkdR and CcpA protein, respectively.[Conclusion] The induced transcription of bkd gene cluster is positively regulated by BkdR and negatively regulated by CcpA.