Investigation Of Oxidative Stress Regulator OxyR In Pseudomonas Chororaphis GP72

Pseudomonas chlororaphis GP72is an important plant growth-promotingrhizobacteria （PGPR） with wide-spectrum antibiotic activity towards severalsoil-borne pathogens, it produces three phenazine antibiotics. The mechanismof phenazines against phytopathogens is the accumulation of toxic reactiveoxygen species （ROS） including superoxide （O-2） and hydrogen peroxide（H₂O₂）. However, little is known about how P. chlororaphis GP72adapts tosuch oxidative stress environment produced by its own abundant phenazines.This mechanism will benefit P. chlororaphis GP72to increase phenazinesproduction and play the role in biocontrol. The bioinformatic analysis of P.chlororaphis GP72genome sequence predicts only one oxidative stressresponse （OSR） regulator, OxyR, which was reported to influence theoxidative stress and antibiotics production. In this work, we evaluated therole of the predicted oxidative stress regulator OxyR in P. chlororaphis GP72for the first time and found that the viability, motility, phenazines production,resistance to different oxidants, biofilm formation and related genes expression were influenced by oxyR gene mutation.First, oxyR gene sequences of high affinity strains were aligned and theprimers were designed according to the conserved regions for the cloning ofoxyR from P. chlororaphis GP72. There are two highly conserved cysteines,which are essential to hydrogen peroxide-resistance, suggesting that OxyR isassociated with oxidative stress in P. chlororaphis GP72.Next, oxyR gene mutation, complementation and overexpression strains wereconstructed. We investigated their growth, morphology, phenazinesproduction and found that deletion of oxyR gene leaded to the reduction ofthe viability on LB agar plates, motility, production of three phenazinederivatives in P. chlororaphis GP72. The reduction of the viability can berescued by the addition of exogenous catalase on LB agar plates. It indicatesthat OxyR is important to oxidative stress and phenazines biosynthesis.Then, considering the antibiotics mechanism of phenazines and the role ofoxidative stress regulation, the growth and biofilm formation of oxyR genemutant were investigated in presence of paraquat （the superoxide-generatingchemical） and hydrogen peroxide. We found that the resistance to hydrogenperoxide and paraquat decreased on the KB agar plates. However, in KBbroth, the oxyR gene mutant grew better with a shorter delay and showed anincreased resistance to hydrogen peroxide occurred at the concentration varying form1.0mM to5.0mM. In addition, the ability of biofilm formationwas obviously enhanced and influenced by the different oxidants in themutant. It further shows the key role of OxyR in oxidative stress in in P.chlororaphis GP72.At last, qRT-PCR experiments indicated that the expression of katG, ahpC,ahpD and phzE were increased in the oxyR mutant background in response tohydrogen peroxide. katG was mainly responsible for the enhanced resistanceto hydrogen peroxide. It suggests that loss of oxyR benefits the hydrogenperoxide inducible gene expression. Thus, OxyR is an important globalregulator that regulates multiple pathways to enhance the survival of P.chlororaphis GP72exposed to different oxidative stress.