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

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.