Effects Of Mutation In The BjaI-bjaR₁ Quorum Sensing System Of Bradyrhizobium Diazoefficiens On Bacterial Nitrogen Metabolism

Bacterial quorum sensing（QS）refers to the regulation of bacterial gene expression dependent on bacterial population density.The QS system is a common way of bacterial information exchange.As an important part of gene expression regulatory networks,bacteria can sense population density,coordinate population behavior,adapt to changing environments,or participate in host-host interactions.Chinahas a long history of soybean planting.Bradyrhizobium diazoefficiens is a endosymotic bacteria of soybean and has excellent nodulation nitrogen fixation efficiency.B.d iazoefficiens USDA110（B.d 110）is the model bacterium of this species.The literature shows that Bja I-Bja R₁on chromosome B.d 110 is homologous to the classical Lux I-Lux R of the bacterial QS system.In this study,B.d 110 was used as the research object to explore the effect of the mutation of its quorum sensing system Lux R-type Bja R₁transcription factor on bacterial nitrogen metabolism.The research will deepen the theoretical understanding of Bradyrhizobium diazoefficiens-soybean symbiotic nitrogen fixation system,and develop high-efficiency nitrogen-fixing strains,consequently improving the efficiency of nodulation nitrogen fixation,reducing environmental soil pollution,and be important for the development of green agriculture.The results of the study are as follows:1.Under the bacgorund of wild strain B.d 110 and the mutant strain B.dΔbja R₁,the promoter of genes,bja I encoding homoserine lactones signal synthase gene,nif H nitrogen fixation reductase gene were transctiptionally fused with reporter gene gus A.Besides,transcriptional fusions of the key genes involved in the process of nitrogen assimilation and metabolism,gln II（encoding glutamine synthase）,gln B（encoding nitrogen metabolism regulators）and amt B（encoding ammonia transporter）with reporter gene lac Z were also constructed.2.Studies have shown that the deletion of bja R₁leads to a significant increase in the nitrogenase activity（ARA,acetylene reduction activity）and the promoter activity of nif H gene of in-nodule bacteria indicating that that the lack of bja R₁could significantly improve the nitrogen fixation ability of bacteria on soybean;gln II-lac Z,gln B-lac Z and amt B-lac Z had the highest activity within the root nodules of soybean inoculated with B.diazoefficiens at 20 days,while nif H-gus A activity reached the highest at 30 days,implying that nitrogen assimilation metabolism process in bacteroids gradually depressed before the onset of the nitrogen fixation.In addition,the transcriptional fusions activities of the above genes related to nitrogen assimilation metabolism in thebackground of B.dΔbja R₁were significantly lower than those in the wild strain,no matter under free-living or inoculation conditions,so the more severe repression of nitrogen assimilation metabolism process in the B.dΔbja R₁mutant strain may be one of the reasons to contribute its significantly enhanced nitrogen fixation ability.3.In the free-living state,compared with the wild strain B.d 110,the addition of exogenous inorganic nitrogen compounds（NH₄Cl,Na NO₃and Na NO₂）significantly promoted the growth of the B.dΔbja R₁,indicating that bja R₁is involved in the bacterial response to environmental nitrogen nutrients.In addition,soybean inoculation experiments showed that the projected area,surface area,total root length,total root volume and nitrogenase activity of roots of soybean with inoculation of B.dΔbja R₁were higher than those of plants with wild type strain when high nitrogen solution with the supplementation of 20 m M NH₄⁺-N and NO₂^--N was applied,indicating that the B.dΔbja R₁mutant can alleviate the inhibitory effect of high concentrations of inorganic nitrogen compounds on soybean symbiotic nitrogen fixation,which is most likely a strain that is insensitive to nitrogen assimilation and metabolism.In conclusion,the quorum sensing system in Bradyrhizobium diazoefficiens may be involved in regulating the nitrogen metabolism of rhizobia,thereby affecting the nitrogen fixation of rhizobia.