Paiboon Vattanaviboon

Novel Organic Hydroperoxide-Sensing and Responding Mechanisms for OhrR, a Major Bacterial Sensor and Regulator of Organic Hydroperoxide Stress

Xanthomonas campestris pv. phaseoli OhrR belongs to a major family of multiple-cysteine-containing bacterial organic hydroperoxide sensors and transcription repressors. Site-directed mutagenesis and subsequent in vivo functional analyses revealed that changing any cysteine residue to serine did not alter the ability of OhrR to bind to the P1 ohrR-ohr promoter but drastically affected the organic hydroperoxide-sensing and response mechanisms of the protein. Xanthomonas OhrR requires two cysteine residues, C22 and C127, to sense and respond to organic hydroperoxides. Analysis of the free thiol groups in wild-type and mutant OhrRs under reducing and oxidizing conditions indicates that C22 is the organic hydroperoxide-sensing residue. Exposure to organic hydroperoxides led to the formation of an unstable OhrR-C22 sulfenic acid intermediate that could be trapped by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole and detected by UV-visible spectral analysis in an oxidized C127S-C131S mutant OhrR. In wild-type OhrR, the cysteine sulfenic acid intermediate rapidly reacts with the thiol group of C127, forming a disulfide bond. The high-performance liquid chromatography-mass spectrometry analysis of tryptic fragments of alkylated, oxidized OhrR and nonreducing polyacrylamide gel electrophoresis analyses confirmed the formation of reversible intersubunit disulfide bonds between C22 and C127. Oxidation of OhrR led to cross-linking of two OhrR monomers, resulting in the inactivation of its repressor function. Evidence presented here provides insight into a new organic hydroperoxide-sensing and response mechanism for OhrRs of the multiple-cysteine family, the primary bacterial transcription regulator of the organic hydroperoxide stress response.

Bacterial Ohr and OsmC paralogues define two protein families with distinct functions and patterns of expression

Xanthomonas campestris Ohr (a protein involved in organic peroxide protection) and Escherichia coli OsmC (an osmotically inducible protein of unknown function) are related proteins. Database searches and phylogenetic analyses reveal that Ohr and OsmC homologues cluster into two related subfamilies of proteins widely distributed in both Gram-negative and Gram-positive bacteria. To determine if these two subfamilies are functionally distinct, ohr and osmC in Pseudomonas aeruginosa (a bacterium with one representative from each subfamily) were analysed. Only ohr mutants are hypersensitive to organic peroxide, and this phenotype can be restored by complementation with ohr but not osmC. In addition, expression of ohr was highly induced only by organic peroxides, and not by other oxidants or stresses. In contrast, osmC was induced by ethanol and osmotic stress. A similar pattern of regulation was observed for Ohr and OsmC homologues in the Gram-positive bacterium Deinococcus radiodurans, though uninduced expression was much higher and induction lower in this species. These data clearly support the conclusion that Ohr and OsmC define two functionally distinct subfamilies with distinct patterns of regulation.

Exposure to cadmium elevates expression of genes in the OxyR and OhrR regulons and induces cross-resistance to peroxide killing treatment in Xanthomonas campestris.

ABSTRACT Cadmium is an important heavy metal pollutant. For this study, we investigated the effects of cadmium exposure on the oxidative stress responses of Xanthomonas campestris, a soil and plant pathogenic bacterium. The exposure of X. campestris to low concentrations of cadmium induces cross-protection against subsequent killing treatments with either H2O2 or the organic hydroperoxide tert-butyl hydroperoxide (tBOOH), but not against the superoxide generator menadione. The cadmium-induced resistance to peroxides is due to the metals ability to induce increased levels of peroxide stress protective enzymes such as alkyl hydroperoxide reductase (AhpC), monofunctional catalase (KatA), and organic hydroperoxide resistance protein (Ohr). Cadmium-induced resistance to H2O2 is dependent on functional OxyR, a peroxide-sensing transcription regulator. Cadmium-induced resistance to tBOOH shows a more complex regulatory pattern. The inactivation of the two major sensor-regulators of organic hydroperoxide, OxyR and OhrR, only partially inhibited cadmium-induced protection against tBOOH, suggesting that these genes do have some role in the process. However, other, as yet unknown mechanisms are involved in inducible organic hydroperoxide protection. Furthermore, we show that the cadmium-induced peroxide stress response is mediated by the metals ability to predominately cause an increase in intracellular concentrations of organic hydroperoxide and, in part, H2O2. Analyses of various mutants of peroxide-metabolizing enzymes suggested that this increase in organic hydroperoxide levels is, at least in part, responsible for cadmium toxicity in Xanthomonas.

OhrR, a transcription repressor that senses and responds to changes in organic peroxide levels in Xanthomonas campestris pv. phaseoli

We report the physiological role of OhrR as an organic peroxide sensor and transcription repressor in Xanthomonas campestris pv. phaseoli. In vivo exposure of X. campestris pv. phaseoli to either tert‐butyl or cumene hydroperoxides efficiently neutralized OhrR repression of expression from the OhrR‐regulated P1 promoter. H2O2 was a weak and non‐physiological inducer of the system while other oxidants and metabolites of organic peroxide metabolism did not induce the expression from the P1. Northern blotting results indicated a correlation between concentrations of tert‐butyl hydroperoxide used in the treatment and the induction of ohr (an OhrR‐regulated gene) expression. In addition, the levels of ohr mRNA in cultures induced by various concentrations of tert‐butyl hydroperoxide were reduced in cells with high levels of an organic peroxide metabolising enzyme (AhpC‐AhpF) but not in cells with high catalase levels suggesting that organic peroxide interacts with OhrR. DNA band shift experiments using purified OhrR and the P1 promoter fragment showed that organic peroxide treatment prevented binding of the protein to the P1 promoter by oxidation of OhrR, as the inhibition of binding to the P1 promoter was reversed by addition of a reducing agent, DTT. The highly conserved cysteine residue C22 of OhrR is required for organic peroxide inducible gene expression. A mutant protein, OhrRC22S can repress the P1 promoter activity but is insensitive to organic peroxide treatment. Thus, OhrR is the first transcription repressor characterized that appeared to evolve to physiologically sense organic peroxides.

A Xanthomonas Alkyl Hydroperoxide Reductase Subunit C (ahpC) Mutant Showed an Altered Peroxide Stress Response and Complex Regulation of the Compensatory Response of Peroxide Detoxification Enzymes

Alkyl hydroperoxide reductase subunit C (AhpC) is the catalytic subunit responsible for alkyl peroxide metabolism. A Xanthomonas ahpC mutant was constructed. The mutant had increased sensitivity to organic peroxide killing, but was unexpectedly hyperresistant to H(2)O(2) killing. Analysis of peroxide detoxification enzymes in this mutant revealed differential alteration in catalase activities in that its bifunctional catalase-peroxidase enzyme and major monofunctional catalase (Kat1) increased severalfold, while levels of its third growth-phase-regulated catalase (KatE) did not change. The increase in catalase activities was a compensatory response to lack of AhpC, and the phenotype was complemented by expression of a functional ahpC gene. Regulation of the catalase compensatory response was complex. The Kat1 compensatory response increase in activity was mediated by OxyR, since it was abolished in an oxyR mutant. In contrast, the compensatory response increase in activity for the bifunctional catalase-peroxidase enzyme was mediated by an unknown regulator, independent of OxyR. Moreover, the mutation in ahpC appeared to convert OxyR from a reduced form to an oxidized form that activated genes in the OxyR regulon in uninduced cells. This complex regulation of the peroxide stress response in Xanthomonas differed from that in other bacteria.

Regulation of the oxidative stress protective enzymes, catalase and superoxide dismutase in Xanthomonas — a review☆

Xanthomonas showed atypical regulation of catalase (Kat) and superoxide dismutase with respect to growth phase and response to various inducers. The highest levels of both enzymes were detected during early log phase of growth and declined as growth continued. This was in contrast to resistance levels to superoxides, H2O2 and organic peroxides, which reached maximum levels during stationary phase. Xanthomonas catalase was induced over six fold by superoxide generators and methyl methane sulfonate but weakly by H2O2. The regulation pattern of these enzymes could be important during plant/microbe interactions. To facilitate elucidation of Xanthomonas kat gene regulation, highly conserved regions of monofuctional Kat amino acid sequences were used to synthesize oligodeoxyribonucleotide primers for use in PCR reactions with Xanthomonas genomic DNA as templates. The Xanthomonas-specific PCR kat probe was used to isolate a functional kat from Xanthomonas campestris pv. phaseoli.

Novel roles of ohrR-ohr in Xanthomonas sensing, metabolism, and physiological adaptive response to lipid hydroperoxide.

Lipid hydroperoxides are highly toxic to biological systems. Here, the Xanthomonas campestris pv. phaseoli sensing and protective systems against linoleic hydroperoxide (LOOH) were investigated by examining the phenotypes, biochemical and regulatory characteristics of various Xanthomonas mutants in known peroxide resistance pathways. Analysis of LOOH resistance levels indicates that both alkyl hydroperoxide reductase (AhpC) and organic hydroperoxide resistance enzyme (Ohr) have important and nonredundant roles in the process. Nonetheless, inactivation of ohr leads to a marked reduction in LOOH resistance levels. The regulatory characteristics of an ohr mutant add further support to its primary role in LOOH protection. Northern analysis shows that LOOH had differential effects on induction of ahpC and ohr expression with the latter being more sensitive to the inducer. Analysis of the ahpC and ohr promoters confirmed that the LOOH-dependent induction of these promoters is mediated by the transcription regulators OxyR and OhrR, respectively. Using the in vivo promoter assays and the in vitro gel mobility shift assay, we show that LOOH directly oxidized OhrR at the sensing residue Cys-22 leading to its inactivation. In addition, physiological analysis shows that pretreatment of X. campestris pv. phaseoli with a sublethal dose of LOOH induced high levels of resistance to subsequent exposure to lethal concentrations of LOOH. This novel LOOH-induced adaptive response requires a functional ohrR-ohr operon. These data illustrate an important novel physiological role for the ohrR-ohr system in sensing and inactivating lipid hydroperoxides.

Analyses of the Regulatory Mechanism and Physiological Roles of Pseudomonas aeruginosa OhrR, a Transcription Regulator and a Sensor of Organic Hydroperoxides

ohrR encodes an organic hydroperoxide sensor and a transcriptional repressor that regulates organic hydroperoxide-inducible expression of a thiol peroxidase gene, ohr, and itself. OhrR binds directly to the operators and represses transcription of these genes. Exposure to an organic hydroperoxide leads to oxidation of OhrR and to subsequent structural changes that result in the loss of the repressors ability to bind to the operators that allow expression of the target genes. Differential induction of ohrR and ohr by tert-butyl hydroperoxide suggests that factors such as the repressors dissociation constants for different operators and the chemical nature of the inducer contribute to OhrR-dependent organic hydroperoxide-inducible gene expression. ohrR and ohr mutants show increased and decreased resistance to organic hydroproxides, respectively, compared to a parental strain. Moreover, the ohrR mutant had a reduced-virulence phenotype in the Pseudomonas aeruginosa-Caenorhabditis elegans pathogenicity model.

The Catalase-Peroxidase KatG Is Required for Virulence of Xanthomonas campestris pv. campestris in a Host Plant by Providing Protection against Low Levels of H2O2

Xanthomonas campestris pv. campestris katG encodes a catalase-peroxidase that has a role in protecting the bacterium against micromolar concentrations of H(2)O(2). A knockout mutation in katG that causes loss of catalase-peroxidase activity correlates with increased susceptibility to H(2)O(2) and a superoxide generator and is avirulent in a plant model system. katG expression is induced by oxidants in an OxyR-dependent manner.

Multiple Superoxide Dismutases in Agrobacterium tumefaciens: Functional Analysis, Gene Regulation, and Influence on Tumorigenesis

Agrobacterium tumefaciens possesses three iron-containing superoxide dismutases (FeSods) encoded by distinct genes with differential expression patterns. SodBI and SodBII are cytoplasmic isozymes, while SodBIII is a periplasmic isozyme. sodBI is expressed at a high levels throughout all growth phases. sodBII expression is highly induced upon exposure to superoxide anions in a SoxR-dependent manner. sodBIII is expressed only during stationary phase. Analysis of the physiological function of sods reveals that the inactivation of sodBI markedly reduced levels of resistance to a superoxide generator, menadione. A mutant lacking all three Sod enzymes is the most sensitive to menadione treatment, indicating that all sods contribute at various levels towards the overall menadione resistance level. Sods also have important roles in A. tumefaciens virulence toward a host plant. A sodBI but not a sodBII or sodBIII mutant showed marked reduction in its ability to induce tumors on tobacco leaf discs, while the triple sod null mutant is avirulent.