Organic Hydroperoxide Induces Prodigiosin Biosynthesis in Serratia sp. ATCC 39006 in an OhrR-Dependent Manner.

The biosynthesis of prodigiosin in the model prodigiosin-producing strain, Serratia sp. ATCC 39006, is significantly influenced by environmental and cellular signals. However, a comprehensive regulatory mechanism for this process has not been well established. In the present study, we demonstrate that organic hydroperoxide activates prodigiosin biosynthesis in an OhrR-dependent manner. Specifically, the MarR-family transcriptional repressor OhrR (Ser39006_RS05455) binds to its operator located far upstream of the promoter region of the prodigiosin biosynthesis operon (319 to 286 nucleotides [nt] upstream of the transcription start site) and negatively regulates the expression of prodigiosin biosynthesis genes. Organic hydroperoxide disassociates the binding between OhrR and its operator, thereby promoting the prodigiosin production. Moreover, OhrR modulates the resistance of Serratia sp. ATCC 39006 to organic hydroperoxide by regulating the transcription of its own gene and the downstream cotranscribed ohr gene. These results demonstrate that OhrR is a pleiotropic repressor that modulates the prodigiosin production and the resistance of Serratia sp. ATCC 39006 to organic hydroperoxide stress. IMPORTANCE Bacteria naturally encounter various environmental and cellular stresses. Organic hydroperoxides generated from the oxidation of polyunsaturated fatty acids are widely distributed and usually cause lethal oxidative stress by damaging cellular components. OhrR is known as a regulator that modulates the resistance of bacteria to organic hydroperoxide stress. In the current study, organic hydroperoxide disassociates OhrR from the promoter of prodigiosin biosynthesis gene cluster, thus promoting transcription of pigA to -O genes. In this model, organic hydroperoxide acts as an inducer of prodigiosin synthesis in Serratia sp. ATCC 39006. These results improve our understanding of the regulatory network of prodigiosin synthesis and serve as an example for identifying the cross talk between the stress responses and the regulation of secondary metabolism.

Organic Hydroperoxide Resistance Gene ohr (VPA1681) Confers Protection against Organic Peroxides in the Presence of Alkyl Hydroperoxide Reductase Genes in Vibrio parahaemolyticus.

The marine foodborne enteropathogen Vibrio parahaemolyticus contains the chief organic peroxide reductases AphC1-AhpC2 and a putative organic hydroperoxide resistance enzyme (Ohr; VPA1681) against different peroxides. This study investigated the function of the Ohr under the presence of AhpC1-AhpC2 in this pathogen by gene mutation. Experimental results demonstrated that the ohr gene product was a weak scavenger of H2O2 only in the mutant strains that lacked the peroxide sensor/regulator oxyR and ahpC1-ahpC2 genes. The Ohr of V. parahaemolyticus was highly effective at scavenging organic peroxide, as demonstrated by assaying the defective changes in the Δohr mutant strain and determining the detoxifying activity of the purified recombinant V. parahaemolyticus Ohrvp protein in the reduced form. The Ohr and AhpC1-AhpC2 exhibited similar functions against organic peroxides; however, only the ΔahpC1ΔahpC2 mutant strain showed a significant increase in susceptibility to several disinfectants, organic acids, and antibiotics compared with the wild-type strain. The transcription of the ohr gene depended on exogenous cumene hydroperoxide (cumene) stress and was markedly enhanced in the ΔohrR (VPA1682) mutant strains. This study revealed the organic hydroperoxide reductase activity of the Ohr in V. parahaemolyticus, and its role probably depends on sophisticated regulation by OhrR. IMPORTANCE Vibrio parahaemolyticus is the most prevalent foodborne pathogen in Taiwan and some other coastal Asian countries, and its antioxidative activity contributes to the tolerance of this bacterium to different environmental stresses. This study reports on the function of the organic hydroperoxide resistance gene (ohr; VPA1681) and its gene regulator, ohrR (VPA1682), in this pathogen. The strain with the ohr gene had effective protection against organic peroxide, and the recombinant Ohrvp was active in its reduced form. The function of Ohr was significant mostly in strains in which the function of AhpC1-AhpC2 was limited. The ohrR repressor of the ohr gene was effective at low concentrations of organic peroxide. Other common Vibrio species that contain homologous ohr, ohrR, ahpC1, and ahpC2 genes, which are phylogenetically close to those of V. parahaemolyticus, may share similar functions to those revealed in this study.

Ethanol in Combination with Oxidative Stress Significantly Impacts Mycobacterial Physiology.

Here, we investigate the mycobacterial response to the combined stress of an organic oxidant (cumene hydroperoxide [CHP]) and a solvent (ethanol). To understand the interaction between the two stressors, we treated Mycobacterium smegmatis cells to a range of ethanol concentrations (2.5% to 10% [vol/vol]) in combination with a subinhibitory concentration of 1 mM CHP. It was observed that the presence of CHP increases the efficacy of ethanol in inducing rapid cell death. The data further suggest that ethanol reacts with the alkoxy radicals to produce ethanol-derived peroxides. These radicals induce significant membrane damage and lead to cell lysis. The ethanol-derived radicals were primarily recognized by the cells as organic radicals, as was evident by the differential upregulation of the ohr-ohrR genes that function in cells treated with the combination of ethanol and CHP. The role of organic peroxide reductase, Ohr, was further confirmed by the significantly higher sensitivity of the deletion mutant to CHP and the combined stress treatment of CHP and ethanol. Moreover, we also observed the sigma factor σB to be important for the cells treated with ethanol alone as well as the aforementioned combination. A ΔsigB mutant strain had significantly higher susceptibility to the stress conditions. This finding was correlated with the σB-dependent transcriptional regulation of ohr and ohrR In summary, our data indicate that the combination of low levels of ethanol and organic peroxides induce ethanol-derived organic radicals that lead to significant oxidative stress on the cells in a concentration-dependent manner.IMPORTANCE Bacterial response to a combination of stresses can be unexpected and very different compared with that of an individual stress treatment. This study explores the physiological and transcriptional response of mycobacteria in response to the combinatorial treatment of an oxidant with the commonly used solvent ethanol. The presence of a subinhibitory concentration of organic peroxide increases the effectiveness of ethanol by inducing reactive peroxides that destroy the membrane integrity of cells in a significantly short time span. Our work elucidates a mechanism of targeting the complex mycobacterial membrane, which is its primary source of intrinsic resistance. Furthermore, it also demonstrates the importance of exploring the effect of various stress conditions on inducing bacterial clearance.

Distinct Roles of Shewanella oneidensis Thioredoxin in Regulation of Cellular Responses to Hydrogen and Organic Peroxides.

The thioredoxin (Trx) and glutaredoxin (Grx) antioxidant systems are deeply involved in bacterial response to oxidative stress, but to date, we know surprisingly little about the roles of these systems in response to reactive oxygen species (ROS) other than hydrogen peroxide (H2O2). In this study, we used Shewanella oneidensis, an environmental bacterium, as a research model to investigate the roles of Trx and Grx in oxidative stress response because it has functionally intertwined ROS responsive regulators OxyR and OhrR. We found that Trx1 is the major thiol/disulfide redox system and that in its absence a Grx system becomes essential under normal conditions. Although overshadowed by Trx1 in the wild type, Trx2 can fully replace Trx1 in physiology when overproduced. Trx1 is required for OxyR to function as a repressor but, more importantly, plays a critical role in the cellular response to organic peroxide (OP) by mediating the redox status of OhrR but not OP scavenger OhrA. While none of the trx and grx genes are OxyR dependent, trxA and trxC are affected by OhrR indirectly. Additional data suggest that depletion of glutathione is likely the cue to trigger induced expression of trxA and trxC These findings underscore the particular importance of Trx in the bacterial OP stress response.IMPORTANCE The Trx and Grx systems are deeply involved in bacterial responses to H2O2-induced oxidative stress. However, little is known about their roles in response to other ROS, such as organic peroxides (OPs). In this study, we used S. oneidensis as a research model to investigate the interplay between Trx/Grx and OxyR/OhrR. We show that Trxs mediate the redox status of transcriptional OP-responding regulator OhrR. Although none of the trx or grx genes are directly controlled by OxyR or OhrR, expression of trxA and trxC is induced by tert-butyl hydroperoxide (t-BHP). We further show that the trxA and trxC genes respond to effects of glutathione (GSH) depletion rather than oxidation. These findings underscore the particular importance of Trx in the bacterial OP stress response.

Global Transcriptional Response to Organic Hydroperoxide and the Role of OhrR in the Control of Virulence Traits in Chromobacterium violaceum.

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.

The Arsenal of Leptospira Species against Oxidants.

Reactive oxygen species (ROS) are byproducts of oxygen metabolism produced by virtually all organisms living in an oxic environment. ROS are also produced by phagocytic cells in response to microorganism invasion. These highly reactive molecules can damage cellular constituents (proteins, DNA, and lipids) and exhibit antimicrobial activities when present in sufficient amount. Consequently, microorganisms have evolved defense mechanisms to counteract ROS-induced oxidative damage. Leptospira are diderm bacteria form the Spirochaetes phylum. This genus is diverse, encompassing both free-living non-pathogenic bacteria as well as pathogenic species responsible for leptospirosis, a widespread zoonotic disease. All leptospires are exposed to ROS in the environment, but only pathogenic species are well-equipped to sustain the oxidative stress encountered inside their hosts during infection. Importantly, this ability plays a pivotal role in Leptospira virulence. In this review, we describe the ROS encountered by Leptospira in their different ecological niches and outline the repertoire of defense mechanisms identified so far in these bacteria to scavenge deadly ROS. We also review the mechanisms controlling the expression of these antioxidants systems and recent advances in understanding the contribution of Peroxide Stress Regulators in Leptospira adaptation to oxidative stress.

Ohr - OhrR, a neglected and highly efficient antioxidant system: Structure, catalysis, phylogeny, regulation, and physiological roles.

Ohrs (organic hydroperoxide resistance proteins) are antioxidant enzymes that play central roles in the response of microorganisms to organic peroxides. Here, we describe recent advances in the structure, catalysis, phylogeny, regulation, and physiological roles of Ohr proteins and of its transcriptional regulator, OhrR, highlighting their unique features. Ohr is extremely efficient in reducing fatty acid peroxides and peroxynitrite, two oxidants relevant in host-pathogen interactions. The highly reactive Cys residue of Ohr, named peroxidatic Cys (Cp), composes together with an arginine and a glutamate the catalytic triad. The catalytic cycle of Ohrs involves a condensation between a sulfenic acid (Cp-SOH) and the thiol of the second conserved Cys, leading to the formation of an intra-subunit disulfide bond, which is then reduced by dihydrolipoamide or lipoylated proteins. A structural switch takes place during catalysis, with the opening and closure of the active site by the so-called Arg-loop. Ohr is part of the Ohr/OsmC super-family that also comprises OsmC and Ohr-like proteins. Members of the Ohr, OsmC and Ohr-like subgroups present low sequence similarities among themselves, but share a high structural conservation, presenting two Cys residues in their active site. The pattern of gene expression is also distinct among members of the Ohr/OsmC subfamilies. The expression of ohr genes increases upon organic hydroperoxides treatment, whereas the signals for the upregulation of osmC are entry into the stationary phase and/or osmotic stress. For many ohr genes, the upregulation by organic hydroperoxides is mediated by OhrR, a Cys-based transcriptional regulator that only binds to its target DNAs in its reduced state. Since Ohrs and OhrRs are involved in virulence of some microorganisms and are absent in vertebrate and vascular plants, they may represent targets for novel therapeutic approaches based on the disruption of this key bacterial organic peroxide defense system.

Regulation of tert-Butyl Hydroperoxide Resistance by Chromosomal OhrR in A. baumannii ATCC 19606.

In this study, we show that Acinetobacter baumannii ATCC 19606 harbors two sets of ohrR-ohr genes, respectively encoded in chromosomal DNA and a pMAC plasmid. We found no significant difference in organic hydroperoxide (OHP) resistance between strains with or without pMAC. However, a disk diffusion assay conducted by exposing wild-type, ∆ohrR-C, C represented gene on chromosome, or ∆ohr-C single mutants, or ∆ohrR-C∆ohr-C double mutants to tert-butyl hydroperoxide (tBHP) found that the ohrR-p-ohr-p genes, p represented genes on pMAC plasmid, may be able to complement the function of their chromosomal counterparts. Interestingly, ∆ohr-C single mutants generated in A. baumannii ATCC 17978, which does not harbor pMAC, demonstrated delayed exponential growth and loss of viability following exposure to 135 µg of tBHP. In a survival assay conducted with Galleria mellonella larvae, these mutants demonstrated almost complete loss of virulence. Via an electrophoretic mobility shift assay (EMSA), we found that OhrR-C was able to bind to the promoter regions of both chromosomal and pMAC ohr-p genes, but with varying affinity. A gain-of-function assay conducted in Escherichia coli showed that OhrR-C was not only capable of suppressing transformed ohr-C genes but may also repress endogenous enzymes. Taken together, our findings suggest that chromosomal ohrR-C-ohr-C genes act as the major system in protecting A. baumannii ATCC 19606 from OHP stresses, but the ohrR-p-ohr-p genes on pMAC can provide a supplementary protective effect, and the interaction between these genes may affect other aspects of bacterial viability, such as growth and virulence.

Ohr and OhrR Are Critical for Organic Peroxide Resistance and Symbiosis in Azorhizobium caulinodans ORS571.

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.

MarR Family Transcription Factors from Burkholderia Species: Hidden Clues to Control of Virulence-Associated Genes.

Species within the genus Burkholderia exhibit remarkable phenotypic diversity. Genomic plasticity, including genome reduction and horizontal gene transfer, has been correlated with virulence traits in several species. However, the conservation of virulence genes in species otherwise considered to have limited potential for infection suggests that phenotypic diversity may not be explained solely on the basis of genetic diversity. Instead, differential organization and control of gene regulatory networks may underlie many phenotypic differences. In this review, we evaluate how regulation of gene expression by members of the multiple antibiotic resistance regulator (MarR) family of transcription factors may contribute to shaping the physiological diversity of Burkholderia species, with a focus on the clinically relevant human pathogens. All Burkholderia species encode a relatively large number of MarR proteins, a feature common to bacteria that must respond to environmental changes such as those associated with host invasion. However, evolution of gene regulatory networks has likely resulted in orthologous transcription factors controlling disparate sets of genes. Adaptation to, and survival in, diverse habitats, including a human or plant host, is key to the success of Burkholderia species as (opportunistic) pathogens, and recent reports suggest that control of virulence-associated genes by MarR proteins features prominently among the survival strategies employed by these species. We suggest that identification of MarR regulons will contribute significantly to clarification of virulence determinants and phenotypic diversity.

Organic Peroxide-Sensing Repressor OhrR Regulates Organic Hydroperoxide Stress Resistance and Avermectin Production in Streptomyces avermitilis.

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.