The Role Of Azorhizobium Caulinodans KatG And OxyR On ROS Resistance And Symbiotic Fixation

Reactive oxygen species（ROS）such as superoxide anions（O2-）,hydroxyl radicals（HO·）,and hydrogen peroxide（H22O2）are produced by the incomplete reduction of molecular oxygen in any living cells,which can damage lipids,proteins,and DNA in growing cells.For rhizobia that form symbiotic nodules on a host legume,ROS are encountered during the whole steps of legume-rhizobia symbiosis,when plants generate ROS in order to defend against invading pathogens.These ROS signals trigger production of Nod factors and subsequent development of nodules and N2 fixation activity.Thus,rhizobium must cope with and tolerate ROS before and after symbiosis.It has been reported that catalase is the most common enzyme,which is in charge of resisting inorganic peroxide.Compared to the root nodules formed by Sesbania rostrala and Azorhizobium caulinodans ORS571,the stem nodules have higher efficiency of nitrogen fixation with greater oxidative stress.Therefore,the study of A.caulinodans to cope with ROS is a very good model.In order to study how A.caulinodans modulates ROS resistance,we performed bioinformatics analysis on the A.caulinodans ORS571 genome and found katG（azc₃290）and oxyR（azc₄162）.At first we constructed katG and oxyR in-frame deletion mutants and monitored the growth behavior of wild type,ΔkatG andΔoxyR cells in TY liquid medium in vitro.And there was no significant difference in growth rate among the three strains,indicating that ΔkatG,and ΔoxyR do not affect viability of the cell under normal growth conditions.To define roles of KatG and OxyR in conferring H2O2 resistance,we tested wild type,ΔkatG and ΔoxyR strains using H2O2 disc diffusion plate and H2O2 killing assays.The results showed that ΔkatG is the most sensitive to H2O2 among the three strains and ΔoxyR showed slightly more sensitivity to H2O2 than wild type.Moreover,Expression of katG and oxyR in trans partially restored this defect.Together,these data indicate that OxyR and KatG are involved in A.caulinodans ROS resistance.In many bacteria,catalase expression is controlled by the transcriptional activator OxyR,such as Escherichia coli.To examine whether the catalase gene katG is regulated by OxyR,we constructed a katG-lacZ transcriptional fusion reporter integrated into the katG locus by homologous recombination to measure katG expression in wild type and ΔoxyR strains.As a result,katG gene is disrupted.We found that katG expression was abrogated in AoxyR compared to wild type,but interestingly H2O2 was not necessary for induction of the katG gene under the growth conditions tested.So expression of katG was upregulated in the presence of oxyR,but not induced by H2O2.The cells in the exponential and stationary were both experimented and get the same conclusion.And complementation of plasmid-borne oxyR partially restored katG expression in ΔoxyR.To analyze how OxyR-regulated katG contributes to symbiosis,we examined the number of nodules and measured the nitrogen fixation capacity.We found that A 50%decrease in nodule number was counted in both A katG and AoxyR compared to WT.Also,a strong decrease（60%）of the nitrogen fixation capacity was observed both in AkatG and AoxyR.Thus,katG and oxyR are involved in nodulation and nitrogen fixing activity in A.caulinodans.To further study which gene is influenced by katG or oxyR mutants in the process of symbiosis,we constructed PnodA/nifA-lcacZ translational plasmid to detect their expression,respectively.It has been known the expression of nodA can be induced by naringenin.We test its expression with or without naringenin,with or without H2O2 in WT,AkatG and AoxyR.It shows that deletions in katG or oxyR did not affect nodA expression.Furthermore,addition of H2O2 did not induce nodA.As for nifA,which can be induced by nitrogen deficiency medium.Still katG and oxyR deletion didn’t affact nifA expression in vitro and H2O2 has nothing to do with it.In a word,deletion of katG and oxyR did not affact nodAlnifA expression in this condition.To summarize,katG and oxyR are involved in nodulation and nitrogen fixing activity in A.caulinodans and their deletions could affect nodulation and nitrogen fixation in symbiosis.Also,katG is upregulated by OxyR and has essential role in ROS resistance in free-living condition.