The Role Of Azorhizobium Caulinodans ORS571 Ohr/ohrR System In ROS Resistance And Its Effect On Nodulation

When a symbiosis relationship formed between rhizobia and legumes,bacteria had to suffer from reactive oxygen species(ROS)produced by the host plant.Endogenous organic hydroperoxides,highly toxic,can be produced by the autoxidation of plant polyunsaturated fatty acids.Subsequently reacted with membrane proteins,speeded up membrane fluidity,eventually leading to DNA damage and even death.The generation rates of OHPs vary,as these rates depend on the degree of oxygen concentration.Although bacterial synthesized only saturated or monounsaturated fatty acids,neither of which are vulnerable to peroxidation chains.When interacted with plants or other eukaryotic organisms,the bacteria membrane easily exposed to OHPs produced by eukaryotes.In order to survive,multiple mechanisms been used to scavenge OHPs.The resistance to these oxygen radicals has been studied in various bacteria but for Azorhizobium caulinodans ORS571 still poorly understood.Among plant nodulating bacteria,the resistance to organic hydroperoxides(OHPs)only had been reported in Sinorhizobium meliloti 1021.Azorhizobium caulinodans ORS571 is a microsymbiont of the legume Sesbania rostrata,forming nodules not only on the roots but also on the stems.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 was important to study the OHPs resistance mechanisms of Azorhizobium caulinodans ORS571.We first reported AZC₂977 and AZC₃555 encodes respectively organic hydroperoxide resistance protein(Ohr)and its regulator OhrR,evaluated the role of ohr and ohrR genes on sensing and responding to OHPs stresses in A.caulinodans ORS571.First we constructed in frame deletion of each gene though double-crossover.In vitro,the inactivation of ohr significantly affects resistance to cumene and ter-butyl hydroperoxides but not to hydrogen peroxide.Indicates that Ohr specificly remove OHPs.In addition,we constructed ohr-lacZ transcriptional fusion expression carrier.The expression of ohr in ohrR deletion strains increased about 3 times,and it was specificity induced 2-fold by CuOOH in wild-type strains.The expression of ohr in reverse mutants behavior the same with wild-type strain,was also induced by CuOOH.The transcription of ohr was repressed by the MarR family regulator OhrR.However,inactivation of ohrR show exactly the opposite traits:the mutant strain sense to hydrogen peroxide,which was very different from other bacteria.We supposed that OhrR may regulate some gene related to hydrogen peroxide metabolism.We screened out a suspected strain by transposon tagging methods,but its relationship to OhrR remains to be further studied.The inactivation of ohr or ohrR did not affect competition with soil natural bacteria and root hair attachment,suggesting that both of the two genes did not affect the early interaction between A.caulinodans ORS571 and Sesbania rostrata.Nodulation assays found that the inactivation of ohr resulted in the decreasing of both root and stem noodle number.Indicated that ohr infected the nodulation ability of A.caulinodans ORS571.Organic hydroperoxides were found widely in animal and plant hosts.It was reported,some pathogens(such as Pseudomonas aeruginosa)reduced virulence when OhrR was inacted.This study demonstrated the significant role ohr and ohrR play in resistance against organic hydroperoxides in A.caulinodans ORS571.