OxyR,an important oxidative stress regulator to phenazines production and hydrogen peroxide resistance in Pseudomonas chlororaphis GP72

Pseudomonas chlororaphis GP72 is an important plant growth-promoting rhizobacteria (PGPR) with a wide-spectrum antibiotic activity toward several soil-borne pathogens. The adaption of this strain to different environmental oxidative stress and redox phenazine pigment by the predicted regulator OxyR were investigated. The deletion of oxyR led to a significant reduction of the viability, production of three phenazine derivatives and resistance to hydrogen peroxide and paraquat on the KB agar plates. However, the mutant ΔoxyR grew better with shorter delay. In addition, the mutant ΔoxyR showed an increased resistance to hydrogen peroxide, which occurred at the concentration varying from 1.0 mM to 5.0 mM in the KB broth, as compared with the wild type. In addition, the biofilm formation ability was obviously enhanced and influenced by the different oxidants in the mutant. Quantitative RT-PCR experiments indicated that the expression of katG, ahpC, ahpD and phzE were increased in the oxyR mutant background in response to hydrogen peroxide. katG was mainly responsible for the enhanced resistance to hydrogen peroxide. The loss of oxyR is suggested to benefit the hydrogen peroxide inducible gene expression. Thus, OxyR is an important global regulator that regulates multiple pathways to enhance the survival of P. chlororaphis GP72 exposed to different oxidative stresses.     

Polyamines as modulators of gene expression under oxidative stress in Escherichia coli

Activity of enzymes of polyamine synthesis and contents of their products increased in E. coli cells in response to oxidative stress caused by addition of hydrogen peroxide to an exponentially growing culture. Putrescine and spermidine added to the culture medium in physiological concentrations significantly increased expression of genes oxyR and katG responsible for defense against oxidative stress, whereas cadaverine had no effect. The role of polyamines as modulators of the gene expression was confirmed by experiments with an inhibitor of polyamine synthesis, 1,3-diaminopropane, which decreased the level of cell polyamines and thus abolished the ability of the cell to induce oxyR expression under oxidative stress. A genetic method gave similar results: under oxidative stress mutants with disorders in polyamine synthesis displayed a significantly decreased level of induction of the oxyR and katG genes, and this level was recovered on addition of putrescine. In the presence of inhibitors of DNA-gyrase, nalidixic acid and novobiocin, the oxyR expression depended on the extent of DNA supercoiling. Putrescine decreased the inhibitory effects of nalidixic acid and novobiocin, and this confirmed its properties of a stimulator of DNA supercoiling. Resistance to rifampicin was studied to exemplify the mutation rate under oxidative stress. Putrescine decreased twofold the level of mutations and increased the number of viable cells in the culture exposed to oxidative stress.     

Role of Putrescine in the Regulation of the Expression of the Oxidative Stress Defense Genes of Escherichia coli

The role of putrescine in the adaptive response of Escherichia coligrown aerobically in synthetic M9 medium with glucose to the H₂O₂-induced oxidative stress was studied. Under oxidative stress, the expression of the single-copy reporter gene fusions oxyR"::lacZand katG"::lacZwas found to undergo biphasic changes, which were most pronounced in glucose-starved E. colicells. The concentration-dependent activating effect of putrescine on the expression of the OxyR regulon genes was maximum when theoxyRgene was inhibited by high concentrations of hydrogen peroxide.     

Proline Metabolism Increases katG Expression and Oxidative Stress Resistance in Escherichia coli

The oxidation of l-proline to glutamate in Gram-negative bacteria is catalyzed by the proline utilization A (PutA) flavoenzyme, which contains proline dehydrogenase (PRODH) and Δ¹-pyrroline-5-carboxylate (P5C) dehydrogenase domains in a single polypeptide. Previous studies have suggested that aside from providing energy, proline metabolism influences oxidative stress resistance in different organisms. To explore this potential role and the mechanism, we characterized the oxidative stress resistance of wild-type and putA mutant strains of Escherichia coli. Initial stress assays revealed that the putA mutant strain was significantly more sensitive to oxidative stress than the parental wild-type strain. Expression of PutA in the putA mutant strain restored oxidative stress resistance, confirming that depletion of PutA was responsible for the oxidative stress phenotype. Treatment of wild-type cells with proline significantly increased hydroperoxidase I (encoded by katG) expression and activity. Furthermore, the ΔkatG strain failed to respond to proline, indicating a critical role for hydroperoxidase I in the mechanism of proline protection. The global regulator OxyR activates the expression of katG along with several other genes involved in oxidative stress defense. In addition to katG, proline increased the expression of grxA (glutaredoxin 1) and trxC (thioredoxin 2) of the OxyR regulon, implicating OxyR in proline protection. Proline oxidative metabolism was shown to generate hydrogen peroxide, indicating that proline increases oxidative stress tolerance in E. coli via a preadaptive effect involving endogenous hydrogen peroxide production and enhanced catalase-peroxidase activity.     

Oxidative Stress Response and Characterization of the oxyR-ahpC and furA-katG Loci in Mycobacterium marinum

Oxidative stress response in pathogenic mycobacteria is believed to be of significance for host-pathogen interactions at various stages of infection. It also plays a role in determining the intrinsic susceptibility to isoniazid in mycobacterial species. In this work, we characterized the oxyR-ahpC and furA-katG loci in the nontuberculous pathogen Mycobacterium marinum. In contrast to Mycobacterium smegmatis and like Mycobacterium tuberculosis and Mycobacterium leprae, M. marinum was shown to possess a closely linked and divergently oriented equivalents of the regulator of peroxide stress response oxyR and its subordinate gene ahpC, encoding a homolog of alkyl hydroperoxide reductase. Purified mycobacterial OxyR was found to bind to the oxyR-ahpC promoter region from M. marinum and additional mycobacterial species. Mobility shift DNA binding analyses using OxyR binding sites from several mycobacteria and a panel of in vitro-generated mutants validated the proposed consensus mycobacterial recognition sequence. M. marinum AhpC levels detected by immunoblotting, were increased upon treatment with H₂O₂, in keeping with the presence of a functional OxyR and its binding site within the promoter region of ahpC. In contrast, OxyR did not bind to the sequences upstream of the katG structural gene, and katG expression did not follow the pattern seen with ahpC. Instead, a new open reading frame encoding a homolog of the ferric uptake regulator Fur was identified immediately upstream of katG in M. marinum. The furA-katG linkage and arrangement are ubiquitous in mycobacteria, suggesting the presence of additional regulators of oxidative stress response and potentially explaining the observed differences in ahpC and katG expression. Collectively, these findings broaden our understanding of oxidative stress response in mycobacteria. They also suggest that M. marinum will be useful as a model system for studying the role of oxidative stress response in mycobacterial physiology, intracellular survival, and other host-pathogen interactions associated with mycobacterial diseases.     

Regulation of the katG-dpsA operon and the importance of KatG in survival of Burkholderia pseudomallei exposed to oxidative stress

Homologues of the catalase-peroxidase gene katG and the gene for the non-specific DNA binding protein dpsA were identified downstream of oxyR in Burkholderia pseudomallei. Northern experiments revealed that both katG and dpsA are co-transcribed during oxidative stress. Under conditions where the katG promoter is not highly induced, dpsA is transcribed from a second promoter located within the katG-dpsA intergenic region. A katG insertion mutant was found to be hypersensitive to various oxidants. Analysis of katG expression in the oxyR mutant indicates that OxyR is a dual function regulator that represses the expression of katG during normal growth and activates katG during exposure to oxidative stress. Both reduced and oxidized OxyR were shown to bind to the katG promoter.     

RpoS and oxidative stress conditions regulate succinyl‐CoA: 3‐ketoacid‐coenzyme A transferase (SCOT) expression in Burkholderia pseudomallei

Burkholderia pseudomallei, a pathogenic gram‐negative bacterium, causes the severe human disease melioidosis. This organism can survive in eukaryotic host cells by escaping reactive oxygen species via the regulation of stress responsive sigma factors, including RpoS. In B. pseudomallei, RpoS has been reported to play a role in the oxidative stress response through enhanced activity of OxyR and catalase. In this study, the RpoS dependent oxidative stress responsive system was further characterized using comparative proteomic analysis. The proteomic profiles of wild‐type B. pseudomallei following exposure to H₂O₂ and between wild‐type and the rpoS mutant strains were analyzed. Using stringent criteria, 13 oxidative responsive proteins, eight of which are regulated by RpoS, were identified with high confidence. It was observed that ScoA, a subunit of the SCOT enzyme not previously shown to be involved directly in the oxidative stress response, is significantly down‐regulated after hydrogen peroxide treatment. ScoA and ScoB have been predicted to be organized in a single operon using computational methods: in this study it was confirmed by RT‐PCR that these genes are indeed co‐transcribed as a single mRNA. The present study is the first to report a role for RpoS in the down‐regulation of SCOT expression in response to oxidative stress in B. pseudomallei.     

DRA0336, another OxyR homolog,involved in the antioxidation mechanisms in <Emphasis Type="Italic">Deinococcus radiodurans</Emphasis>

A novel OxyR (DR0615) with one conserved cysteine that senses hydrogen peroxide in Deinococcus radiodurans had been identified in our previous work. Comparative genomics revealed that D. radiodurans possesses another OxyR homolog, OxyR₂ (DRA0336). In this study, we constructed the deletion mutant of oxyR ₂ and the double mutant of both the OxyR homologs to investigate the role of OxyR in response to oxidative stress in D. Radiodurans. Deletion of oxyR ₂ resulted in an obviously increased sensitivity to hydrogen peroxide, and the double mutant for oxyR and oxyR ₂ was significantly more sensitive than any of the two single mutants. The total catalase activity of the double mutant was lower than that of any of the single mutants, and reactive oxygen species (ROS) accumulated to a greater extent. DNA microarray analysis further suggested that oxyR ₂ was involved in antioxidation mechanisms. Site-direct mutagenesis and complementation analysis revealed that C₂₂₈ in OxyR₂ was essential. This is the first report of the presence of two OxyR in one organism. These results suggest that D. radiodurans OxyR and OxyR₂ function together to protect the cell against oxidative stress.     

Influence of the Escherichia coli oxyR gene function on λ prophage maintenance

In Escherichia coli hosts, hydrogen peroxide is one of the factors that may cause induction of λ prophage. Here, we demonstrate that H₂O₂-mediated λ prophage induction is significantly enhanced in the oxyR mutant host. The mRNA levels for cI gene expression were increased in a λ lysogen in the presence of H₂O₂. On the other hand, stimulation of the p _M promoter by cI857 overproduced from a multicopy plasmid was decreased in the ΔoxyR mutant in the presence of H₂O₂ but not under normal growth conditions. The purified OxyR protein did bind specifically to the p _M promoter region. This binding impaired efficiency of interaction of the cI protein with the OR3 site, while stimulating such a binding to OR2 and OR1 sites, in the regulatory region of the p _M promoter. We propose that changes in cI gene expression, perhaps in combination with moderately induced SOS response, may be responsible for enhanced λ prophage induction by hydrogen peroxide in the oxyR mutant. Therefore, OxyR seems to be a factor stimulating λ prophage maintenance under conditions of oxidative stress. This proposal is discussed in the light of efficiency of induction of lambdoid prophages bearing genes coding for Shiga toxins.