Symbiotic Function Characterization Of RND-type Efflux Pump Genes In Mesorhizobium Huakuii 7653R

Although the bacteria efflux pumps had been studied a long history, but it’s function about translate drugs mainly focus on patheogens. There are very less research about efflux pumps in rhizobium, efflux pump system genes in the functional phenotype and mechanism in symbiosis is worth in-depth study. This work investigate the RND efflux family genes in Mesorhizobium huakuii 7653 R.The main results obtained are as follows.Firstly, we analyzed the transcriptome data, MCHK₀866 and MCHK₀867up-regulated expression in symbiosis condition and the products of this two genes forms a RND-type efflux pumps. Genetic organization of target genes was analyzed in genome.The change of growth curve was observed by measuring OD600. 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 preliminarily validated by bacterial one-hybrid system. Proteins encoded by MCHK₀866 and MCHK₀867 forms a RND family efflux pump. The OD600 of growth curve reduced and it showed more sensitivity to nalidixic acid, tetracycline and SDS after disruptive efflux pump. Genes relative transcription level increased in response to nalidixic acid treatment.Meanwhile, the downstream gene MCHK₀869 expression product which belongs to TetR family transcription factor can bound on the promoter region of MCHK₀867. 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.Secondly, we have chosen three genes which no significant expression change in nodule, constructing insert mutant respectively. Inoculated these muatnts on Astragalus sinicus for identify symbiosis phenotype. It was found disrupted MCHK₀811 and MCHK₃948 were defective on nodulation and nitrogenease activity. Disrupted MCHK₆517 did not influence on symbiosis.The above results provide experimental material and work foundation for study rhizobium efflux pumps involved in symbiosis.