肺炎克雷伯菌CRP对kfuABC操纵子的转录调控机制研究

Study of transcriptional regulation mechanism of kfuABC operon by cAMP receptor protein in Klebsiella pneumoniae

摘要:

目的：利用分子生物学实验研究肺炎克雷伯菌环磷酸腺苷受体蛋白（cAMP receptor protein，CRP）对kfuABC操纵子的转录调控机制。方法：设计相关跨基因引物，用Real-time PCR方法验证kfuABC的转录结构。以肺炎克雷伯菌野生株NTUH-K2044（wild type，WT）的总RNA为模板，利用引物延伸的方法寻找kfuA的转录起始位点，确定其核心启动子区。构建含kfuA启动子区DNA序列的LacZ重组质粒，并将其转入突变株?驻crp（肺炎克雷伯菌野生株基因组中crp基因敲除）和WT中，通过测定比较两株菌中β-半乳糖苷酶活性差异来判定CRP调控子对kfuA的调控关系；分别提取?驻crp和WT的总RNA，进一步采用实时定量Real-time PCR的方法验证CRP对kfuA的调控关系。通过凝胶阻滞实验验证His-CRP是否对kfuA启动子区具有直接的结合作用，进一步采用 DNaseⅠ足迹实验确定具体的结合位点。结果：肺炎克雷伯菌kfuA、kfuB和kfuC位于同一个操纵子kfuABC；引物延伸实验结果显示kfuABC只有一个转录起始位点T；CRP能够结合到kfuABC启动子区-204到-171之间的碱基序列上（转录起始位点为+1），抑制其转录表达。结论：CRP能直接结合到kfuABC启动子区抑制其转录表达。

Abstract:

Objective：To investigate the transcriptional regulation mechanism of kfuABC operon by cAMP receptor protein（CRP） in Klebsiella pneumoniae. Methods：Real-time PCR was firstly used to validate the operon structure of kfuABC by using the intergenic re－gion spanning primers. Primer extension assay was employed to detect the transcriptional start site of kfuA by using the total RNAs of wild type（WT） as template. The entire promoter-proximal region of kfuA was amplified and cloned into the plasmid pHRP309 con－taining a promoterless lacZ reporter gene. The recombinant LacZ reporter plasmid kfuA∷pHRP309 was transformed into the crp null mutant（?驻crp） and WT，respectively，to measure the promoter activity（the β-Galactosidase activity） of kfuA by using the β-Galac－tosidase enzyme assay system. Then，total RNAs were extracted from ?驻crp and WT strains，and the quantitative Real-time PCR was carried out to calculate the transcriptional variation of kfuA in the ?驻crp and WT. The over-expressed His-CRP was purified under native conditions with nickel loaded HiTrap Chelating Sepharose columns（Amersham），and the entire promoter-proximal region of the kfuA gene was amplified by PCR from WT strain. Then，the electrophoretic mobility shift assay and DNaseⅠ footprinting experiment were applied to analyze the DNA-binding site of His-CRP to kfuA promoter region in vitro. Results：The transcription of kfuABC operon was regulated by CRP negatively. Primer extension assay detected only one transcriptional start site. The DNaseⅠ footprinting assays showed that the binding site was located from 204 bp to 171 bp upstream of kfuABC. Conclusion：The transcription of kfuABC oper－on is repressed by CRP via directly combination with promoter region of kfuABC operon in Klebsiella pneumoniae.