Effect Of C-di-GMP Signal In Rhizobium Etli CFN42 On Symbiotic Phenotype

In nature,biological nitrogen fixation plays an important role in maintaining nitrogen balance.Rhizobia forms nodules through symbiosis with legumes,and differentiate into bacteroids in the nodules to convert atmospheric nitrogen into ammonia to meet the nitrogen supply required by legumes.This symbiotic nitrogen fixation method is biological nitrogen fixation the most efficient one,accounting for nearly 70%of the amount of biological nitrogen fixation,so it has important theoretical and applied research value.Cyclic diguanylic acid(c-di-GMP)is a common second messenger in bacteria.It plays an important role in the life activities of bacteria and participates in regulating a series of physiological phenotypes of bacteria,including the formation of biofilms.The movement of bacteria and the formation of extracellular polysaccharides.In some bacteria,the c-di-GMP signal and the quorum sensing system will have a molecular dialogue(Cross-talk),but in rhizobia,the relationship between c-di-GMP signal and symbiotic nitrogen fixation is less researched.This study takes Rhizobium etli CFN42 as the research object,using molecular biology,physiology and other methods to research the influence of c-di-GMP signal on the symbiotic nitrogen fixation ability of this strain and its relationship with the quorum sensing system(Cin/R).The main results are as follows:1.By constructing c-di-GMP signal enhancement strains CFN42(wspR),(overexpressing diguanylate cyclase-DGC),CFN42ΔcinR(wspR)and silent CFN42(yhjH),(overexpressing phosphodiesterase-PDE),CFN42ΔcinR(yhjH)strains,to detect the relevant physiological phenotypes of these strains.The results show that the enhanced c-di-GMP signal can promote the formation of biofilms and the production of extracellular polysaccharides of the strains(WT and AcinR),and promote the motility of the bacteria.After testing the autoinducer of these strains,it was found that the β-galactase activity of all WT strains was around 2500 Miller Units,and all AcinR strains were below 20 Miller Units(no AHLs signal molecules were produced).The above results indicate that c-di-GMP signal is involved in regulating a series of physiological phenotypes such as Rhizobium etli CFN42 biofilm formation and motility,but there is no molecular dialogue with the quorum sensing CinI/R system.2.Perform inoculation experiments on these strains.The results show that after the c-di-GMP signal is enhanced,the nodulation time is about the 4th day after inoculation(seeding age 7 days),and the nodulation rate is faster than the control group.Summary at 42 days the number of nodules was about 130/plant,while the number of nodules in the control group was only about 90/plant;the c-di-GMP signal silenced strains began to nodulate at about 10 days of seedling age,and their nodulation rate was slower than that of the control group,and the final conclusion is about 70 nodules/plant.The nitrogenase activity of different plants was measured for 2-4 weeks,and the results showed that the nitrogenase activity of the treatment group with enhanced c-di-GMP signal was about 500,300,200 μM C2H4/g/m/plant,and the nitrogenase activity of the c-di-GMP signal silence treatment group was about 300,700,600 μM C2H4/g/m/plant,and the nitrogenase activity of the control group was about 380,500,400 μM C2H4/g/m/plant.From the overall view of 2-4 weeks,the nitrogenase activity of the treatment group with enhanced c-di-GMP signal showed a gradual decline trend,and the other two groups showed an increase and stable trend.By counting the number of bacteria in the root nodule in 2-4 weeks,the effect of c-di-GMP signal on the colonization ability of the strain was detected.The results showed that the number of viable bacteria that can be cultured in the root nodule has no relationship with the strength of c-di-GMP signal in 2-4 weeks,that is,the c-di-GMP signal does not affect the colonization and survival of the strain in the root nodule.The above results indicate that the c-di-GMP signal has a promoting effect on the nodulation ability of CFN42,but has a certain inhibition on the nitrogenase activity as a whole,and has no effect on the survival ability of the rhizobia.3.Through bioinformatics analysis,it is found that there are 34 c-di-GMP signal-related domains in the CFN42 genome,26 of which have c-di-GMP signal synthesis and degradation-related domains.The 26 genes were tested by qPCR in the biofilm state and suspension culture state of the strain,and it was found that the expression of 17 genes increased in the biofilm.These 26 genes were overexpressed in CFN42.Through biofilm experiments and combined with previous data analysis,it was found that the expression products of genes RHE_CH01586,RHE_CH01761,RHE_CH02005,and RHE_CH02526 were DGC,while the expression products of gene RHE_CH03639 were PDE.The overexpression strains of the DGC gene RHE_CH02526 and the PDE gene RHE CH03639 were selected for inoculation experiments.The results showed that when the gene RHE_CH02526 was overexpressed,the expression of c-di-GMP signal was enhanced,and nitrogenase was in symbiotic state the activity detection is consistent with the phenotype of the strain CFN42(wspR),and after the gene RHE_CH03639 is overexpressed,the nitrogenase activity detection in the symbiotic state is consistent with the phenotype of the strain CFN42(yhjH)(signal weakened type).In general,there is a complex c-di-GMP regulatory network in Rhizobium etli CFN42.This study found that the DGC-related gene RHE CH02526 and the PDE-related gene RHE_CH03639 are involved in R.etli physiological processes such as symbiotic nitrogen fixation and biofilm formation provide a theoretical basis for subsequent studies on the receptor and gene expression regulation of c-di-GMP signal in CFN42.