Novel organic hydroperoxide-sensing and responding mechanisms for OhrR, a major bacterial sensor and regulator of organic hydroperoxide stress.
ABSTRACT: 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.
Project description:Organic hydroperoxide reductase regulator (OhrR) in bacteria is a sensor for organic hydroperoxide stress and a transcriptional regulator for the enzyme organic hydroperoxide reductase (Ohr). In this study we investigated, using a GFP reporter system, whether Mycobacterium smegmatis OhrR has the ability to sense and respond to intracellular organic hydroperoxide stress. It was observed that M. smegmatis strains bearing the pohr-gfpuv fusion construct were able to express GFP only in the absence of an intact ohrR gene, but not in its presence. However, GFP expression in the strain bearing pohr-gfpuv with an intact ohrR gene could be induced by organic hydroperoxides in vitro and in the intracellular environment upon ingestion of the bacteria by macrophages; indicating that OhrR responds not only to in vitro but also to intracellular organic hydroperoxide stress. Further, the intracellular expression of pohr driven GFP in this strain could be abolished by replacing the intact ohrR gene with a mutant ohrR gene modified for N-terminal Cysteine (Cys) residue, suggesting that OhrR senses intracellular organic hydroperoxides through Cys residue. This is the first report demonstrating the ability of OhrR to sense intracellular organic hydroperoxides.
Project description:Organic hydroperoxides are oxidants generated during bacterial-host interactions. Here, we demonstrate that the peroxidase OhrA and its negative regulator OhrR comprise a major pathway for sensing and detoxifying organic hydroperoxides in the opportunistic pathogen Chromobacterium violaceum. Initially, we found that an ohrA mutant was hypersensitive to organic hydroperoxides and that it displayed a low efficiency for decomposing these molecules. Expression of ohrA and ohrR was specifically induced by organic hydroperoxides. These genes were expressed as monocistronic transcripts and also as a bicistronic ohrR-ohrA mRNA, generating the abundantly detected ohrA mRNA and the barely detected ohrR transcript. The bicistronic transcript appears to be processed. OhrR repressed both the ohrA and ohrR genes by binding directly to inverted repeat sequences within their promoters in a redox-dependent manner. Site-directed mutagenesis of each of the four OhrR cysteine residues indicated that the conserved Cys21 is critical to organic hydroperoxide sensing, whereas Cys126 is required for disulfide bond formation. Taken together, these phenotypic, genetic and biochemical data indicate that the response of C. violaceum to organic hydroperoxides is mediated by OhrA and OhrR. Finally, we demonstrated that oxidized OhrR, inactivated by intermolecular disulfide bond formation, is specifically regenerated via thiol-disulfide exchange by thioredoxin (but not other thiol reducing agents such as glutaredoxin, glutathione and lipoamide), providing a physiological reducing system for this thiol-based redox switch.
Project description:The organic hydroperoxide resistance protein Ohr has been identified in numerous bacteria where it functions in the detoxification of organic hydroperoxides, and expression of ohr is often regulated by a MarR-type regulator called OhrR. The genes annotated as BAB2_0350 and BAB2_0351 in the Brucella abortus 2308 genome sequence are predicted to encode OhrR and Ohr orthologs, respectively. Using isogenic ohr and ohrR mutants and lacZ promoter fusions, it was determined that Ohr contributes to resistance to organic hydroperoxide, but not hydrogen peroxide, in B. abortus 2308 and that OhrR represses the transcription of both ohr and ohrR in this strain. Moreover, electrophoretic mobility shift assays and DNase I footprinting revealed that OhrR binds directly to a specific region in the intergenic region between ohr and ohrR that shares extensive nucleotide sequence similarity with so-called "OhrR boxes" described in other bacteria. While Ohr plays a prominent role in protecting B. abortus 2308 from organic hydroperoxide stress in in vitro assays, this protein is not required for the wild-type virulence of this strain in cultured murine macrophages or experimentally infected mice.
Project description:Pseudomonas aeruginosa ohrR and ospR are gene homologs encoding oxidant sensing transcription regulators. OspR is known to regulate gpx, encoding a glutathione peroxidase, while OhrR regulates the expression of ohr that encodes an organic peroxide specific peroxiredoxin. Here, we show that ospR mediated gpx expression, like ohrR and ohr, specifically responds to organic hydroperoxides as compared to hydrogen peroxide and superoxide anion. Furthermore, the regulation of these two systems is interconnected. OspR is able to functionally complement an ohrR mutant, i.e. it regulates ohr in an oxidant dependent manner. In an ohrR mutant, in which ohr is derepressed, the induction of gpx expression by organic hydroperoxide is reduced. Likewise, in an ospR mutant, where gpx expression is constitutively high, oxidant dependent induction of ohr expression is reduced. Moreover, in vitro binding assays show that OspR binds the ohr promoter, while OhrR binds the gpx promoter, albeit with lower affinity. The binding of OhrR to the gpx promoter may not be physiologically relevant; however, OspR is shown to mediate oxidant-inducible expression at both promoters. Interestingly, the mechanism of OspR-mediated, oxidant-dependent induction at the two promoters appears to be distinct. OspR required two conserved cysteines (C24 and C134) for oxidant-dependent induction of the gpx promoter, while only C24 is essential at the ohr promoter. Overall, this study illustrates possible connection between two regulatory switches in response to oxidative stress.
Project description:Fatty acid hydroperoxides are involved in host-pathogen interactions. In both plants and mammals, polyunsaturated fatty acids are liberated during infection and enzymatically oxidized to the corresponding toxic hydroperoxides during the defensive oxidative burst that is designed to thwart the infection. The bacterial transcription factor OhrR (organic hydroperoxide reductase regulator) is oxidized by organic hydroperoxides, as a result of which the ohr gene encoding organic hydroperoxide reductase is induced. This enzyme converts the hydroperoxides to less toxic alcohols. We show here that OhrR from Burkholderia thailandensis represses expression of ohr Gene expression is induced by cumene hydroperoxide and to a lesser extent by inorganic oxidants; however, Ohr contributes to degradation only of the organic hydroperoxide. B. thailandensis OhrR, which binds specific sites in both ohr and ohrR promoters, as evidenced by DNase I footprinting, belongs to the 2-Cys subfamily of OhrR proteins, and its oxidation leads to reversible disulfide bond formation between conserved N- and C-terminal cysteines in separate monomers. Oxidation of the N-terminal Cys is sufficient for attenuation of DNA binding in vitro, with complete restoration of DNA binding occurring on addition of a reducing agent. Surprisingly, both overexpression of ohr and deletion of ohr results in enhanced survival on exposure to organic hydroperoxide in vitro While ?ohr cells are more virulent in a Caenorhabditis elegans model of infection, ?ohrR cells are less so. Taken together, our data suggest that B. thailandensis OhrR has several unconventional features and that both OhrR and organic hydroperoxides may contribute to virulence.
Project description:The organic hydroperoxide stress resistance regulator (OhrR) is a MarR type of transcriptional regulator that primarily regulates the expression of organic hydroperoxide reductase (Ohr) in bacteria. In mycobacteria, the genes encoding these proteins exist in only a few species, which include the fast-growing organism Mycobacterium smegmatis. To delineate the roles of Ohr and OhrR in defense against oxidative stress in M. smegmatis, strains lacking the expression of these proteins were constructed by deleting the ohrR and ohr genes, independently and together, through homologous recombination. The OhrR mutant strain (MS?ohrR) showed severalfold upregulation of Ohr expression, which could be observed at both the transcript and protein levels. Similar upregulation of Ohr expression was also noticed in an M. smegmatis wild-type strain (MSWt) induced with cumene hydroperoxide (CHP) and t-butyl hydroperoxide (t-BHP). The elevated Ohr expression in MS?ohrR correlated with heightened resistance to oxidative stress due to CHP and t-BHP and to inhibitory effects due to the antituberculosis drug isoniazid (INH). Further, this mutant strain exhibited significantly enhanced survival in the intracellular compartments of macrophages. In contrast, the strains lacking either Ohr alone (MS?ohr) or both Ohr and OhrR (MS?ohr-ohrR) displayed limited or no resistance to hydroperoxides and INH. Additionally, these strains showed no significant differences in intracellular survival from the wild type. Electrophoretic mobility shift assays (EMSAs) revealed that the overexpressed and purified OhrR interacts with the ohr-ohrR intergenic region with a greater affinity and this interaction is contingent upon the redox state of the OhrR. These findings suggest that Ohr-OhrR is an important peroxide stress response system in M. smegmatis.
Project description:Azorhizobium caulinodans is a symbiotic nitrogen-fixing bacterium that forms both root and stem nodules on Sesbania rostrata. During nodule formation, bacteria have to withstand organic peroxides that are produced by plant. Previous studies have elaborated on resistance to these oxygen radicals in several bacteria; however, to the best of our knowledge, none have investigated this process in A. caulinodans. In this study, we identified and characterised the organic hydroperoxide resistance gene ohr (AZC_2977) and its regulator ohrR (AZC_3555) in A. caulinodans ORS571. Hypersensitivity to organic hydroperoxide was observed in an ohr mutant. While using a lacZ-based reporter system, we revealed that OhrR repressed the expression of ohr. Moreover, electrophoretic mobility shift assays demonstrated that OhrR regulated ohr by direct binding to its promoter region. We showed that this binding was prevented by OhrR oxidation under aerobic conditions, which promoted OhrR dimerization and the activation of ohr. Furthermore, we showed that one of the two conserved cysteine residues in OhrR, Cys11, was critical for the sensitivity to organic hydroperoxides. Plant assays revealed that the inactivation of Ohr decreased the number of stem nodules and nitrogenase activity. Our data demonstrated that Ohr and OhrR are required for protecting A. caulinodans from organic hydroperoxide stress and play an important role in the interaction of the bacterium with plants. The results that were obtained in our study suggested that a thiol-based switch in A. caulinodans might sense host organic peroxide signals and enhance symbiosis.
Project description:The osmotically inducible protein OsmC, like its better-characterized homolog, the organic hydroperoxide protein Ohr, is involved in defense against oxidative stress caused by exposure to organic hydroperoxides. The crystal structure of Escherichia coli OsmC reported here reveals that the protein is a tightly folded domain-swapped dimer with two active sites located at the monomer interface on opposite sides of the molecule. We demonstrate that OsmC preferentially metabolizes organic hydroperoxides over inorganic hydrogen peroxide. On the basis of structural and enzymatic similarities, we propose that the OsmC catalytic mechanism is analogous to that of the Ohr proteins and of the structurally unrelated peroxiredoxins, directly using highly reactive cysteine thiol groups to elicit hydroperoxide reduction.
Project description:A major pathway for the detoxification of organic hydroperoxides, such as cumene hydroperoxide (CHP), involves the MarR family transcriptional regulator OhrR and the peroxidase OhrA. However, the effect of these peroxides on the global transcriptome and the contribution of the OhrA/OhrR system to bacterial virulence remain poorly explored. Here, we analyzed the transcriptome profiles of Chromobacterium violaceum exposed to CHP and after the deletion of ohrR, and we show that OhrR controls the virulence of this human opportunistic pathogen. DNA microarray and Northern blot analyses of CHP-treated cells revealed the upregulation of genes related to the detoxification of peroxides (antioxidant enzymes and thiol-reducing systems), the degradation of the aromatic moiety of CHP (oxygenases), and protection against other secondary stresses (DNA repair, heat shock, iron limitation, and nitrogen starvation responses). Furthermore, we identified two upregulated genes (ohrA and a putative diguanylate cyclase with a GGDEF domain for cyclic di-GMP [c-di-GMP] synthesis) and three downregulated genes (hemolysin, chitinase, and collagenase) in the ohrR mutant by transcriptome analysis. Importantly, we show that OhrR directly repressed the expression of the putative diguanylate cyclase. Using a mouse infection model, we demonstrate that the ohrR mutant was attenuated for virulence and showed a decreased bacterial burden in the liver. Moreover, an ohrR-diguanylate cyclase double mutant displayed the same virulence as the wild-type strain. In conclusion, we have defined the transcriptional response to CHP, identified potential virulence factors such as diguanylate cyclase as members of the OhrR regulon, and shown that C. violaceum uses the transcriptional regulator OhrR to modulate its virulence.
Project description:The bacterium Streptomyces avermitilis is an industrial-scale producer of avermectins, which are important anthelmintic agents widely used in agriculture, veterinary medicine, and human medicine. During the avermectin fermentation process, S. avermitilis is exposed to organic peroxides generated by aerobic respiration. We investigated the role of MarR-family transcriptional regulator OhrR in oxidative stress response and avermectin production in S. avermitilis. The S. avermitilis genome encodes two organic hydroperoxide resistance proteins: OhrB1 and OhrB2. OhrB2 is the major resistance protein in organic peroxide stress responses. In the absence of organic peroxide, the reduced form of OhrR represses the expression of ohrB2 gene by binding to the OhrR box in the promoter region. In the presence of organic peroxide, the oxidized form of OhrR dissociates from the OhrR box and the expression of ohrB2 is increased by derepression. OhrR also acts as a repressor to regulate its own expression. An ohrR-deletion mutant (termed DohrR) displayed enhanced avermectin production. Our findings demonstrate that OhrR in S. avermitilis represses avermectin production by regulating the expression of pathway-specific regulatory gene aveR. OhrR also plays a regulatory role in glycolysis and the pentose phosphate (PP) pathway by negatively controlling the expression of pykA2 and ctaB/tkt2-tal2-zwf2-opcA2-pgl.