Molecular Response Mechanism Of Sinorhizobium Meliloti LsrB Protein Sensing Oxidation

Legumes can form symbiotic relationship with rhizobia.Plants provide carbohydrates and an environment suitable for their survival and metabolism for the ammonia nitrogen transformed by rhizobia from atmospheric nitrogen.Nearly three quarters of the nitrogen sources in the biological community come from this symbiotic system.Rhizobia-legumes symbiotic nitrogen fixation system is also the highest-efficiency nitrogen fixation.The process of symbiotic nitrogen fixation is mediated by the recognition,transmission and transformation of molecular signals between a series of rhizobia and host plants.Lys R family protein of Sinorhizobium meliloti plays a very important role in symbiotic nitrogen fixation.In our earlier research,a symbiotic essential protein LsrB was found in this family.The lsr B deletion mutant is more sensitive to oxidant,and the nodule induced by lsr B has abnormal development and a large amount of hydrogen peroxide accumulation in vivo.The phenotypes of three cysteine-sites lsr B mutant were similar.We predicted that LsrB is a new redox protein that depends on the active catalytic Cys site.In order to further explore the molecular mechanism of its sensing oxidation signal,we did the research on two different domain and 3catalytic Cys sites.With the LsrB DNA binding domain deletion mutant?lsr B₁₁₆ and LsrB substrate domain deletion mutant?lsr B₁₈₇constructed and stored in our lab,the expression of catalase encoding gene kat A was detected by q-RTPCR.The expression of kat A in?lsr B₁₁₆ was significantly increased,but the rate of increase was much less than that in?lsr B₁₈₇.We detected the protein level of Kat A by native-PAGE staining,and found that the activity of Kat A in?lsr B₁₁₆ was slightly higher than wild type,but lower than?lsr B₁₈₇ and?lsr B.According to the result that?lsr B₁₈₇ mutant was more sensitive to GSSG,the above results showed that the oxidation of?lsr B₁₈₇,a mutant with substrate binding domain deletion of lsr B,suffer more intense oxidative stress.The ratio of GSH/GSSG in?lsr B₁₁₆is closer to that in wild type,while the ratio of GSH/GSSG in?lsr B₁₈₇ is similar to that in?lsr B.Our results show that the substrate binding domain of LsrB balance the redox state of meliloti.We transformed the constructed expression vector of lsr B substrate binding domain fragment with each Cys site mutation into lsr B deletion mutant.We found that each mutation of Cys site lead to a significant decrease in the number of symbiotic effective nitrogen fixation nodules and biological nitrogen fixation,while the mutation of Cys146 site lead to a seriously effect on the symbiotic nitrogen fixation efficiency of Bacteroides,indicating that Cys146 may be an antioxidant-functional site.We cloned the lsr B homologous genes of three rhizobia:Bradyrhizobium japonicum USDA110with Cys172&Cys238 sites,Mesorhizobium loti MAFF303099 with Cys146 site only and Azorhizobium caulinodans ORS571 with no Cys residue.The lsr B homologous genes were transferred into lsr B deletion mutants by expression vector.The results of oxidant sensitivity analysis showed that Plsr B_bj and Plsr B_ml with Cys residues could resume the antioxidant defects of lsr B deletion mutants,but Plsr B_ac could not.The symbiotic phenotype analysis showed that the strains expressing LsrB_ac^V146C and LsrB_ac^V172C completely recovered the symbiotic defect of lsr B deletion mutant,but LsrB_ac^V231C and LsrB_ac^T269C did not.The results showed that Cys146 or Cys172 in the substrate binding domain was the key sites for ROS detoxification.The results of transcriptome sequencing and CHIP-Sequencing in our group's previous work suggest that LsrB may directly regulate the expression of its own gene.To verify this hypothesis,we purified lsr B promoter fragment with biotin-Tag and LsrB protein in vitro,and confirmed their direct binding by electrophoretic mobility assay?EMSA?.The competitive experiment shows that the binding is specific.Cys238 residue of LsrB is essential for the expression of lsr B downstream genes.The electrophoretic mobility assay showed that the formation of LsrB-DNA complex depends on Cys238,and Cys146 is not required under any condition.In order to confirm the binding site on the lsr B gene promoter,we synthesized the lsr B upstream promoter fragment labeled by FAM fluorescence,and collected the promoter sequence fragment binding to LsrB protein through DNase I footprinting assay.The results showed that the expression of lsr B gene was self-regulated by its coding protein directly binding with the conserved motif on the promoter.With NBT staining and rhizobia-attachment experiments,we found that:compared with the aquatic conditions,the ROS level of the main root near the root tip and root hair area of Alfalfa growing in the land soil conditions was higher,while the bacterial colonies on the roots growing in the aquatic environment were more.These results support our conjecture that the Cys146and Cys172 residues of LsrB are the products of selection and evolution under different oxidative stress in legume nodules.We detected the LsrB protein level in the product by anti-Flag monoclonal antibody immunoprecipitation.The experimental results show that LsrB protein can interact with Grx and Trx in vivo.The Cys residue in LsrB is necessary for the interaction between LsrB and Grx protein.The decrease of Cys sites lead to a weaker interaction,and the LsrB protein with only one Cys residue can't interact with Trx A.These results suggest that Grx and Trx may be the reducers of oxidized LsrB protein,and the reductive reaction depends on the Cys residues on the substrate binding domain of LsrB protein.In conclusion,LsrB is a new type of oxidoreductase protein evolved from Lys R transcription factor family in Sinorhizobium meliloti.The expression of lsr B gene is self-regulated by its coding protein directly interacting with the conserved motif on the promoter.The detoxification function of LsrB against ROS under oxidative stress depends on the Cys146 residue on the substrate binding domain,which is the product of selection and evolution under different oxidative stress in legume nodules.The oxidized LsrB protein may be reduced by Thioredoxin or Glutaredoxin.