Proteomic Analysis Mediated By The Quorum Sensing System BjaR₁-bjaI Of Bradyrhizobium Diazoefficiens

Rhizobium-soybean nitrogen-fixing symbiosis is not only a key step in the global nitrogen ecological cycle,but also a nitrogen input link in the production of legume crops.Symbiotic nitrogen fixation is environmentally friendly and can reduce the overuse of inorganic nitrogenous fertilizer,which is conducive to the sustainable development of agriculture.The biological role of quorum sensing（QS）system in other legume rhizobium has been well studied,but its mechanism in Bradyrhizobium diazoefficiens is understood.In this study,the lux R-lux I type bja R₁-bja I QS system of Bradyrhizobium diazoefficiens USDA110（B.d 110）,a type strain of Bradyrhizobium diazoefficiens,was studied as follows:（1）the effects of QS system mutant strain on soybean growth,nodulation ability and nitrogenase activity were investigated;（2）the regulatory network mediated by Bja R₁ regulatory protein,an important component of the system,under different living conditions of rhizobium（aerobic free-living,bacteroid and anaerobic nitrate respiration）was analyzed by proteomics;（3）the influence of QS system on anaerobic nitrate respiration process was further explored through nitrate respiration physiological and biochemical experiments;（4）the expression of key transcription factor genes fix K₂ and nnr R in the denitrification signaling pathway was analyzed by using lac Z（encodingβ-galactosidase）reporter gene promoter fusion technique.The key findings were as follows:1.Under low nitrogen cultivation condition,compared with wild strain,inoculation of bja I mutant strains and bja R₁-bja I mutant strains was more conducive to soybean growth,especially the aboveground biomass（dry weight）increased. Meanwhile,nodule numbers,nodules dry weight and nitrogenase activity were significantly increased;compared with low nitrogen condition,under high nitrogen （NH₄⁺-N）cultivation,the number of nodules in soybean inoculated by the two mutants decreased,while the dry weight of nodules and the activity of nitrogenase did not change,but were significantly higher than those inoculated by wild strains. These results indicate that the quorum-sensing system mutation is beneficial to symbiotic nitrogen fixation in soybean and insensitive to ammonia repression.2.Proteomics results under aerobic free-living state showed that the deletion mutation of bja R₁ inhibited（or delayed）the expression of a large number of proteins in the nitrogen metabolism process of rhizobium.In addition,under the condition of high cell density,it significantly affects the expression of transmembrane transporters,heme binding,and electron transfer proteins in the periplasmic space,which may affect the transport process of nitrogen compounds, amino acid catabolism,and electron transport chain.3.Proteomic analysis under symbiotic living state（bacteroid）showed that the deletion of bja R₁ increased the expression of nitrogenase complex（Nif DKH）; similar to the results of aerobic free-living state,the expression of other proteins in the nitrogen metabolic pathway was decreased.In contrast,the deletion of bja R₁ significantly up-regulated the expression of ribosomal pathway-related proteins in the early symbionts,thereby affecting important processes such as ribosomal assembly,peptide biosynthesis,and translation.4.Since nitrogen metabolism（including denitrification process）in bja R₁ mutant strain was inhibited under the above two living conditions,this study also analyzed the proteome of rhizobium under anaerobic nitrate respiration conditions.The results showed that the deletion of bja R₁ gene significantly inhibited the expression of key enzymes in the denitrification process,which was confirmed by further denitrification physiological and biochemical experiments.Moreover,the promoter activity of key transcription factors in the denitrification process significantly decreased.In summary,this study constructed a regulatory network of Lux R-type transcription factors at the translation level of the QS system of B.d 110.It was found that the QS system mainly affects the nitrogen metabolism process of the bacteria,thereby tightly controlling the symbiotic nitrogen fixation process of B.d 110.