Functional Role Of Quorum Sensing Of Bradyrhizobium Diazofeficiens In Symbiotic Interaction With Soybean

The quorum sensing(QS)system is a kind of information exchange between bacteria depending on population density.It can not only regulate the biological behavior of free-living bacteria,but also participate during the interaction between bacteria and associated host.Bradyrhizobium diazoefficiens USDA110,as an endosymbiont of soybean,is known for its superior nodulation formation and nitrogen fixation capability.The present study focused on biofunction of the QS of a model strain Bradyrhizobium diazoefficiens USDA110(B.d 110).The obtained research fruits can deepen the understanding of the soybean-Bradyrhizobium symbiosis,providing not only theoretical basis for the agricultural application of rhizobium,but also novel knowledges about this bacterium in global nitrogen ecological cycle.In this study,the biological role of blr1062(bjaR1)-blr1063(bjaI),which are QS genes of LuxI-LuxR type,in B.d 110 was characterized.The results are as follows:1.Regulatory gene blr1062 and structural gene blr1063 function as an operon in a cyclic regulation pattern.blr1063 gene was strongly induced by soybean isoflavonoid genistein,but greatly inhibited by soybean seed extract(SSE).The motility of blr1062 mutant was 48.3% higher than that of wild strain in semi-solid YMB medium,indicating that blr1062 mutation is beneficial to rapid colonization of soybean root at the early stage of symbiosis.2.Compared with the wild type,blr1062 mutant strain enhanced slightly the total number of root nodules and their dry weight,accompanied by a more occupation on the lateral roots.However,the activity of nitrogenase and the surface area of soybean roots inoculated with this mutant strain were significantly increased by 82.6% and9.9%,respectively.Here,the deletion of blr1062 was beneficial to nodulation and nitrogen fixation,further suggesting that Blr1062 is probably a negative factor controlling these symbiotic processes.3.Based on the literature,the chromosomes-integrated lacZ transcriptional fusions were constructed with key genes that have been reported to be involved in theregulation of nodulation genes in both blr1062 mutant and wild type strains.Results showed that in blr1062 mutant strain,the induction of nwsA,nolA and nodD2 genes that are involved in negative regulation of nodule number decreased significantly,while that of nodY which is participated in the synthesis of nodulation factor,increased significantly;However,the induction of nodD1 and nodW were not substantially changed by the deletion of blr1062.In addition,the expression of genes(ttsI and rhcN)related to type III secretion system were decreased in blr1062 mutant,but the difference was not obvious.These results suggest that the QS system may be involved in the negative regulation of nodule formation through NolA and NodD2.These data can explain from the level of molecular for the slightly increased nodule number of soybeans inoculated with blr1062 mutant as described above.4.Transcriptome data displayed that the genes above showed expression pattern similar to their lacZ activity.Moreover,the mutation of blr1062 gene significantly reduced the expression of a great number of genes involved in the nitrogen metabolism.Further,blr1062 mutant strain was grown more quickly than wild strain in the minimum medium added with different nitrogen sources.It suggests that QS system may inhibit the assimilation process of nitrogen metabolism of rhizobium in bacteroid,resulting in the enhancement of activity of nitrogenase.However,the mechanism was not clear.Although differentially expressed proteins were not correlated to those differential genes obtained by transcriptome,a large number of proteins involved in the pathway of the QS system are dramatically down-regulated in proteomic analysis.Furthermore,the expression of glutamine synthetase and glutamate dehydrogenase were significantly decreased in blr1062 mutant strain,and it was validated by the assay of their enzyme activities.Taken together,the quorum sensing system of Bradyrhizobium diazoefficiens may participate in the negative regulation of nodule formation through NodD2 and the process of nitrogen metabolism,consequently affecting the nitrogen fixation capability.