Studying Function Of Sinorhizobium Meliloti LsrB Protein Domains

Rhizobia-legume symbiotic nitrogen fixation is one of the most important biochemical reactions in nature.About three-quarters of the nitrogen sources in biosphere are derived from biological nitrogen fixation.This process is that nitrogen-fixing microorganisms convert nitrogen to the ammonia that can be absorbed by plant.Symbiotic nitrogen fixation between rhizobia and legumes is the most efficient biological nitrogen fixation.Symbiotic nitrogen fixation is a very complex biological process,including the molecular recognition,transmission and exchange between rhizobia and host legumes.Sinorhizobium meliloti Lys R family proteins are very important.The Lys R family proteins are a class of transcriptional regulators consisting of a DNA binding domain and a substrate binding domain.The Lys R family protein Lsr B,which is necessary for rhizobia-legume symbiotic nitrogen fixation,was screened out.The results showed that the oxidant can induce lsr B gene expression,and the lsr B mutant was sensitive to oxidant and we found the accumulation of hydrogen peroxide in alfalfa nodules induced by lsr B mutant.Therefore,we speculate that Lsr B may be a redox protein.To verify this hypothesis,we conducted the following studies: 1.Analyzing function of Lsr B protein domains by constructing deletion mutantsFirstly,we knocked out DNA fragments on the genome of Sinorhizobium meliloti that encode the Lsr B protein DNA binding domain and the substrate binding domain,and then analyze the oxidant sensitivity of the mutants.We found that the mutant(?lsr B187)lacking the Lsr B protein substrate binding domain was more sensitive to the oxidant than the mutant(?lsr B116)lacking the Lsr B protein DNA binding domain,which indicated that Lsr B substrate binding domain has antioxidant capacity.We inoculated alfalfa with the mutants and observed symbiotic phenotype of alfalfa.We found that the Lsr B DNA binding domain mutant(?lsr B116)can induce alfalfa to form more red nodules and less white nodules.In addition,the aboveground dry weight of alfalfa inoculated with ?lsr B116 is also significantly higher than alfalfa inoculated with ?lsr B187.Our study shows that the substrate binding domain of Lsr B has independent antioxidant activity.2.Analyzing function of Lsr B protein domains by overexpression techniquesTo further confirm the above results,we expressed the DNA binding domain and the substrate binding domain of Lsr B protein in the background of lsr B deletion mutant(?lsr B),respectively.Oxidative susceptibility experiments showed that the strains expressing Lsr B protein substrate binding domain had better resistance to oxidants.We observed the symbiotic phenotype of the alfalfa inoculated with the strains and found that the alfalfa inoculated with the strains expressing Lsr B protein substrate binding domain grew better.In addition,the strains expressing the Lsr B protein substrate binding domain could induce alfalfa to form more red nodules,and the aboveground dry weight of the inoculated alfalfa was also higher than that of the alfalfa noculated with the strain expressing the Lsr B protein DNA binding domain.Our results demonstrate that the Lsr B substrate binding domain has an independent antioxidant function.3.Analyzing expression of Lsr B substrate binding domain in Sinorhizobium melilotiWe detected that the DNA fragment encoding the Lsr B substrate binding domain was transcribable in deletion mutants and expression strains by q RT-PCR.We also demonstrated that the substrate binding domain of Lsr B was expressed in overexpression strains by Western Blot experiments.The experimental results further confirm that the phenotype of Lsr B domain deletion mutants and overexpressing strains is indeed due to the substrate binding domain of Lsr B.4.Expressing Lsr B domains in a strain with abnormal redox status and analyzing their functionWe overexpressed the Lsr B protein substrate binding domain in gsh A mutant with abnormal redox status.Oxidative susceptibility experiments showed that the growth of the strain expressing the Lsr B protein substrate binding domain(gsh A-/ plsr B187)was well recovered and the antioxidant capacity was enhanced.After inoculating the alfalfa,the growth and nitrogen fixation of plant swere also recovered.The results of this study show that the substrate binding domain of Lsr B has an independent redox function.5.Analyzing cysteine sites on Lsr B substrate binding domainLsr B substrate binding domain has three cysteine residues(Cys),through the Lsr B protein amino acid sequences analysis,Cys146 is the most conservative cysteine residue.Since the Lsr B protein retains a Cys,it still has complete function and the redox function of the Lsr B substrate binding domain is weak.Therefore,we try to add a cysteine residue to the 146 site of Azorhizobium caulinodans Lsr B protein because there is no any cysteine residue on this protein.Then we expressed this reformed protein in the background of Sinorhizobium meliloti lsr B deletion mutant.The results showed that due to the mutation of Cys146,the antioxidant capacity of the strain was significantly improved.The growth of the alfalfa inoculated with the strain expressing A.caulinodans Lsr BV146 C was also recovered,and it could induce alfalfa to form more red nodules and less white nodules.The dry weight of the plant was almost equal to that of wild-type Sinorhizobium meliloti.Thus,our results suggest that Cys146 may be the predominant redox site on Lsr B.In summary,Lsr B is a novel Lys R family redox protein in Sinorhizobium meliloti.The Lsr B protein plays a major role through its substrate binding domain,and the Cys146 may be the most important redox catalytic site.