华癸根瘤菌7653R中一个RND型药物外排泵的功能表型及调控鉴定

Characterization of phenotype and expression regulation of an RND type multidrug efflux pump in Mesorhizobium huakuii 7653R

【目的】研究华癸根瘤菌7653R中MCHK_0866和MCHK_0867编码的RND家族外排泵的功能表型。【方法】对外排泵编码基因及候选调控基因在基因组上的结构进行分析。采用测定OD_(600)观察菌株生长曲线的变化。通过测定最低抑菌浓度检测菌株的药物敏感性,RT-PCR检测目的基因经特定物质处理后表达量的变化。通过细菌单杂交系统初步检测外排泵的转录调控。【结果】MCHK_0866和MCHK_0867所编码蛋白共同组成一个RND家族射流泵。缺失该外排泵后,细菌生长曲线在稳定期OD_(600)数值降低,对萘啶酸、四环素和SDS的敏感性发生变化,萘啶酸处理细菌后2个基因的表达量增加。同时,下游属于Tet R转录因子家族的基因MCHK_0869表达产物作用于MCHK_0867的启动子区域。【结论】该外排泵与萘啶酸的运输有关,缺失后自身生长受到影响,表达受到下游转录因子的调控。

[Objective] To study the function of an RND family efflux pump encoded by MCHK_0866 and MCHK_0867 in Mesorhizobium huakuii 7653 R. [Methods] Genetic organization of target genes was analyzed in genome. The change of growth was observed by measuring OD_（600）. Drug sensitivity was detected by minimal inhibitory concentrations; relative transcription level of target genes was measured by RT-PCR. Transcript regulation of the efflux pump was validated by bacterial one-hybrid system. [Results] Proteins encoded by MCHK_0866 and MCHK_0867 formed an RND family efflux pump. The OD_（600） of growth curve reduced and it showed more sensitivity to nalidixic acid, tetracycline and SDS after disrupting the efflux pump. Genes relative transcription level increased in response to nalidixic acid treatment. Meanwhile, the downstream gene MCHK_0869 belongs to Tet R family transcription factor and its expression product can interact with the promoter region of MCHK_0867. [Conclusion]The efflux pump is possibly associated with the transportation of nalidixic acid and affects rhizobial free-living growth. The pump is putatively regulated by a downstream local transcription factor.