单核细胞增生李斯特菌LadR蛋白对外排泵MdrL的调控机制研究

Regulation of Efflux Pump MdrL by LadR in Listeria monocytogenes

研究单核细胞增生李斯特菌(Listeria monocytogenes,Lm)调控子LadR对外排泵MdrL的调控作用及方式。采用同源重组技术构建ladR基因缺失菌株ΔladR;通过实时荧光定量PCR检测mdrL基因在野生株EGD-e和突变株ΔladR中的转录水平;构建重组蛋白表达菌株BL21(DE3)-ladR并纯化重组蛋白His6-LadR;通过凝胶阻滞试验检测LadR蛋白与mdrL基因启动子区域的结合活性。成功构建ladR基因缺失菌株ΔladR。与野生株EGD-e相比,突变株ΔladR中mdrL基因的转录水平提高约51倍;成功构建重组蛋白表达菌株BL21(DE3)-ladR,纯化得到浓度为1 mg/mL的重组蛋白His6-LadR;凝胶阻滞实验结果显示,重组蛋白His6-LadR可与mdrL基因的启动子区域特异性结合。Lm的调控子LadR通过与mdrL基因启动子区域特异性结合实现对外排泵MdrL的负调控。

The aim of this study is to investigate the role of LadR in the regulation of efflux pump MdrL in Listeria monocytogenes.The mutant strainΔladR was constructed from the wild type strain EGD-e by homologous recombination.The transcriptional levels of mdrL in the wild type strain EGD-e and the mutant strainΔladR were tested by quantitative reverse transcriptase PCR.The expression strain BL21(DE3)-ladR was constructed,and then the recombination protein His6-LadR was purified.Electrophoretic mobility shift assay(EMSA)was employed to assess the binding activity of LadR to the mdrL promoter.As results,the mutant strainΔladR was constructed successfully.Compared to the wild type strain EGD-e,the transcriptional level of mdrL inΔladR showed about51-fold up-regulation.The expression strain BL21(DE3)-ladR was also constructed successfully,and the concentration of the purified recombinant protein His6-LadR was1mg/mL.Results of EMSA showed that His6-LadR protein was able to bind to the promoter of gene mdrL specifically.Conclusively,the regulator LadR negatively regulates the efflux pump MdrL by specifically binding to the promoter of mdrL.