Alkyl Hydroperoxide Reductase Subunit C(AhpC) Is Critical For Oxidative Stress Resistance,Nodulation And Nitrogen Fixation In Azorhizobium Caulinodans

The legume-rhizobial interaction results in the formation of symbiotic nodules in which,rhizobia fix nitrogen.During the process of symbiosis reactive oxygen specie（ROS）are generated.The response of rhizobia to ROS thus is important for successful nodulation and nitrogen fixation.In this study,we investigated how Azorhizobium caulinodans,a rhizobium that form both root and stem nodules on its host plant,regulates ROS resistance.The stem nodule were completely exposure to oxygen,and its cortex cells contained releasing oxygen chloroplasts,through photosynthesis.Thus stem bacteroid were more likely to be attacked by ROS.For these reasons,it is important to study the ROS resistance mechanisms of Azorhizobium caulinodans ORS571.In rhizobia,ROS detoxification comes from the enzyme catalase,which catalyzes the conversion of H2O2 into water and diatomic oxygen.In most bacteria,these processes are controlled by the transcriptional activator OxyR,a member of the LysR family of transcriptional regulators.OxyR is known to activate/repress the expression of the oxyR regulon,which consists of several genes,including ahpCF,katG,gorA,grxA.trxC,dps.OxyR activates the expression of the socalled oxyR regulon.We found the in-fram deletions of a gene encoding alkyhydroperoxide reductase ahpC or a gene encoding bcp shows hypersusceptable to killing by ROS such as H2O2 and t-BOOH,CuOOH.In this study,We first reported azc₁050 and azc₄162 encode respectively Alkyl Hydroperoxide Reductase Subunit C（AhpC）and its regulator OxyR,azc₁350 encodes AhpC/TSA family protein（Bcp）,evaluated the role of ahpC and bcp genes on sensing and responding to ROS stresses in A.caulinodans ORS571.First we constructed in-frame deletion of each gene through double-crossover.In vitro,the inactivation of ahpC,bcp significantly affects resistance to H2O2,but not to CuOOH and t-BOOH.Among the three peroxide,the genes have more catalase activity,but less organic hydrogen peroxidase activity.We constructed translational lacZ fusions in ahpC.This fusion was used to monitor hydrogen peroxide levels as sensed by A.caulinodans during the course of an infection in wild type and oxyR.We showed that ther e was no relationship between oxyR and ahpC expression in H2O2-independent fashion.From our research,ahpC was sensitive to organic hydrogen peroxidase,while ohr specificly removes OHPs.In our study,we showed that the expression of ohr in ahpC deletion strain increased about 1.5 times,and it was specificity induced 2-fold by CuOOH in A ahpC strain.The results indicated that the two genes have complementary functions when expsosed to CuOOH.Further more,we studied the root-hair attachment,the nodulation and nitrogenase activity in symbiosis,our results revealed that the nodulation and nitrogenase activity of ahpC,bcp mutants were lower than that of wild type,the inactivation of ahpC,bcp did not affect root hair attachment.Based on these data,ahpC and bcp are not only important for antioxidant defense,but also critical for nodule formation and nitrogen fixation during interaction with plant hosts.