Study Of The Function Of Glutathione Synthase Gene GshB In Mesorhizobium Huakuii

Glutathione?GSH?is active sulfhydryl peptides,which widely exists in cells.It acts as an antioxidant plays an important physiological role in maintainin nomal redox environment ofthe cell.Biosynthesis of GSH involves two sequential reactions catalyzed by?-glutamylcysteine synthetase?encoded by gshA?and glutathione synthetase?encoded by gshB?.Mesorhizobium huakuii 7653R is a gram-negative bacterium which can coexist with leguminous plant Astragalus sinicu.It can fix N₂ in the atmosphere by forming root nodules with host plants and convert it into ammonia as source of nitrogen for plant growth.In turn,the host plant must provide some energy for nitrogen fixation.By bioinformatics analysis,M.huaguii 7653R gshB?MCHK₅809?was found to display significant sequence similarity to glutathione synthetase.In this study,the roles of M.huaguii gshB in free-living bacteria,resistance to oxidative stress and during N-fixing symbiosis on Astragalus sinicus by analyzing the phenotype of the gshB mutant strain.To investigate the contribution of the GSH to growth of M.huakuii,the free-living growth and oxidative stress inhibition zone experiments were conducted.M.huakuii gshB mutant cannot efficiently utilize various compounds,such as fructose,maltose,glucose,and mannitol,as the sole carbon source.It also cannot efficiently utilize various amino acids,including glutamic acid,cereal amino acids,reduced glutathione,cysteine,aspartic acid,alanine,as a sole carbon and nitrogen source.Compared to that of strain 7653R,the inhibition zone of the gshB mutant was significantly larger in inorganic oxide hydrogen peroxide?H2O2?and organic oxide tert-butyl hydro peroxide?t-BHP?,but not in Cumene hydroperoxide?CuOOH?.In order to study the symbiotic phenotype of gshB mutant,Astragalus sinicu was inoculated with M.huakuii widetype 7653R and gshB mutant MHgshB.The experiment result showed plants inoculated with the gshB mutant shows a 70%reduction in nitrogen fixation,although nodules are pink and its nodulation number is not significantly differen from that of the wild type 7653R.Parrafin section and ultra-structural microscope studies revealed that cells infected by MHgshB mutant contained a few normal bacteroids and numerous senescences.These results indicated that the gshB is important for symbiotic nitrogen fixation and premature aging of root nodules.By quantitative real-time PCR?qRT-PCR?,the expression of gshB relative genes under H2O2 stress and symbiotic conditions was measured.The results showed that the lack of GSH increases,but not deduces,the transcription of the branched amino acid transporter genes braE?braG,and the amino acid permease genes aapJ?aapQ,suggesting GSH does not regulate the activity of transporters via Bra and Aap.The catalase-peroxidase gene katG was significantly up regulated.However,there was no significant difference in expression of electron transport genes Hmus?RhtA,suggesting the absence of GSH results in an increase in the performance of other antioxidant pathways.In symbiosis,the expression of nitrogen-fixing genes nifH,nifD in the mutant nodules are down-regulated significantly.The decline of gene performance associated with nitrogen fixation has important significance for the reduction of root nodule nitrogen fixation and premature senility of root nodules.