苏云金芽胞杆菌dxr1基因的转录受SigH控制

Transcription of dxr1 is controlled by SigH in Bacillus thuringiensis

摘要:

[目的] 萜类化合物广泛分布在生物界，是重要的生命物质。目前发现有两条萜类化合物的生物合成途径，即甲羟戊酸（MVA）途径和2-甲基-D-赤藓糖醇-4-磷酸（MEP）途径。MEP代谢途径中的关键酶1-脱氧-D-木酮糖-5-磷酸还原异构化酶（DXR，EC1.1.1.267）催化1-脱氧-D-木酮糖-5-磷酸生成MEP。枯草芽胞杆菌中dxr基因编码DXR酶，而在苏云金芽胞杆菌（Bacillus thuringiensis，Bt）中有2个基因（dxr1和dxr2）编码DXR酶。通过分析Bt HD73菌株的dxr1基因的转录活性和dxr1突变体表型，明确dxr1基因的转录调控机制和功能。[方法] 通过5'RACE分析dxr1的转录起始位点；β-半乳糖苷酶活性测定分析dxr1基因启动子（Pdxr1）的转录活性；采用同源重组技术分别敲除Bt HD73菌株的dxr1和dxr2基因；利用总蛋白定量确定Cry1Ac蛋白产量；利用DXR检测试剂盒检测Bt菌株的DXR活性。[结果] dxr1基因的转录起始位点位于起始密码子上游39 bp处的G碱基；与出发菌株HD73相比，Pdxr1在sigH突变体中的转录活性明显降低；dxr1或dxr2基因的缺失对菌体生长、芽胞形成率和Cry1Ac蛋白产量无显著影响，但使DXR活性下降。[结论] Bt中dxr1基因的转录受SigH控制，dxr1基因的缺失影响DXR的活性。

关键词:苏云金芽胞杆菌;MEP代谢途径;DXR;dxr基因;转录调控

Abstract:

[Objective] Terpenoids are widely present in various organisms and play key roles in all forms of life. Two pathways have been found for the biosynthesis of terpenoids:the mevalonate (MVA) pathway and 2-methyl-D-erythritol-4-phosphate (MEP) pathway. 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) is a key enzyme in the MEP pathway that catalyzes 1-deoxy-D-xylulose-5-phosphate to generate MEP. DXR is encoded by a dxr gene in Bacillus subtilis, while two dxr genes (dxr1 and dxr2) encode DXR in Bacillus thuringiensis (Bt). To determine the mechanism of transcriptional regulation and the function of dxr gene, we analyzed the transcriptional regulation of dxr1 gene and phenotype of dxr1 and dxr2 mutants of Bt HD73. [Methods] Transcriptional start site was analyzed by 5'RACE. Transcriptional activity of dxr1 promoter (Pdxr1) was analyzed by promoter fusions with lacZ gene. dxr1 and dxr2 mutants of Bt HD73 strain were constructed by homologous recombination. Comparison of the Cry1Ac protein production was determined by protein quantitation. DXR activity was determined by DXR detection kit. [Results] A G residue located 39 bp upstream from the dxr1 start codon was identified. The transcriptional activity of Pdxr1 was sharply decreased in sigH mutant compared with that in HD73 wild-type. Mutation of dxr1 or dxr2 has no significant differences on growth, sporulation efficiency and Cry1Ac protein production. However, the DXR activity was decreased in the mutants. [Conclusion] Transcription of dxr1 is controlled by SigH. Deletion of dxr genes influenced the activity of DXR.

Key words:Bacillus thuringiensis;MEP pathway;DXR;dxr gene;transcriptional regulation