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Glutaredoxins (Grxs), ubiquitous redox enzymes belonging to the thioredoxin family, catalyze the reduction of thiol-disulfide exchange reactions in a glutathione-dependent manner. A Pseudomonas aeruginosa Δ grxD mutant exhibited hypersensitivity to oxidative stress-generating agents, such as paraquat (PQ) and cumene hydroperoxide (CHP). In vitro studies showed that P. aeruginosa GrxD acts as an electron donor for organic hydroperoxide resistance enzyme (Ohr) during CHP degradation. The ectopic expression of iron-sulfur cluster ([Fe-S]) carrier proteins, including ErpA, IscA, and NfuA, complements the function of GrxD in the Δ grxD mutant under PQ toxicity. Constitutively high expression of iscR , nfuA , tpx , and fprB was observed in the Δ grxD mutant. These results suggest that GrxD functions as a [Fe-S] cluster carrier protein involved in [Fe-S] cluster maturation. Moreover, the Δ grxD mutant demonstrates attenuated virulence in a Drosophila melanogaster host model. Altogether, the data shed light on the physiological role of GrxD in oxidative stress protection and virulence of the human pathogen, P. aeruginosa. IMPORTANCE Glutaredoxins (Grxs) are ubiquitous disulfide reductase enzymes. Monothiol Grxs, containing a CXXS motif, play an essential role in iron homeostasis and maturation of [Fe-S] cluster proteins in various organisms. We now establish that the human pathogen Pseudomonas aeruginosa GrxD is crucial for bacterial virulence, maturation of [Fe-S] clusters and facilitation of Ohr enzyme activity. GrxD contains a conserved signature monothiol motif (C 29 GFS), in which C29 is essential for its function in an oxidative stress protection. Our findings reveal the physiological roles of GrxD in oxidative stress protection and virulence of P. aeruginosa.

Cytolethal distending toxin (CDT) is a bacterial genotoxin that causes host cell cycle arrest and death. We previously employed a Saccharomyces cerevisiae model with inducible expression of the CDT catalytic subunit from Aggregatibacter actinomycetemcomitans, Aa CdtB, and showed that a wide variety of host factors play a role in facilitating the activity of CdtB. Our observation that a yeast H2B mutant defective in chromatin condensation was partially resistant to CdtB implies that chromatin structure may affect CDT function. In this study, we identified host chromatin regulatory genes required for CdtB cytotoxicity. We found that the deletion of HTZ1 or certain subunits of SWR, INO80, and SIR complexes increased cellular resistance to CdtB. We hypothesized that CdtB may interact with Htz1 or the chromatin, but immunoprecipitation experiments failed to detect physical interaction between CdtB and Htz1 or the chromatin. However, we observed reduced nuclear localization of CdtB in several mutants, suggesting that impaired nuclear translocation may, at least partly, explain the mechanisms of CdtB resistance. In addition, mutations in chromatin regulatory genes induce changes in the global gene expression profile, and these may indirectly affect CdtB toxicity. Our results suggest that decreased expression of endoplasmic reticulum (ER)-Golgi transport-related genes that may be involved in CdtB transport and/or increased expression of DNA repair genes may contribute to CdtB resistance. These results suggest that the functions of chromatin regulators may contribute to the activity of CDT in host cells.

Reactive chlorine species (RCS), particularly hypochlorous acid (HOCl), are powerful antimicrobial oxidants generated by biological pathways and chemical syntheses. Pseudomonas aeruginosa is an important opportunistic pathogen that has adapted mechanisms for protection and survival in harsh environments, including RCS exposure. Based on previous transcriptomic studies of HOCl exposure in P. aeruginosa , we found that the expression of PA0565 , or rcsA , which encodes an alkyl hydroperoxidase D-like protein, exhibited the highest induction among the RCS-induced genes. In this study, rcsA expression was dominant under HOCl stress and greatly increased under HOCl-related stress conditions. Functional analysis of RcsA showed that the distinguishing core amino acid residues Cys60, Cys63, and His67 were required for the degradation of sodium hypochlorite (NaOCl), suggesting an extended motif in the AhpD family. After allelic exchange mutagenesis in the P. aeruginosa rcsA , the P. aeruginosa rcsA deletion mutant showed significantly decreased HOCl resistance. Ectopic expression of P. aeruginosa rcsA led to significantly increased NaOCl resistance in Escherichia coli Moreover, the pathogenicity of the rcsA mutant decreased dramatically in both Caenorhabditis elegans and Drosophila melanogaster host model systems compared to the wild type (WT). Finally, the Cys60, Cys63, and His67 variants of RcsA were unsuccessful at complementing phenotypes of the rcsA mutant. Overall, our data indicate the importance of P. aeruginosa RcsA in defense against HOCl stress under disinfections and during infections of hosts, which involves the catalytic Cys60, Cys63, and His67 residues. IMPORTANCE Pseudomonas aeruginosa is a common pathogen that is a major cause of serious infections in many hosts. Hypochlorous acid (HOCl) is a potent antimicrobial agent found in household bleach and is a widely used disinfectant. P. aeruginosa has evolved adaptive mechanisms for protection and survival during HOCl exposure. We identified P. aeruginosa rcsA as a HOCl-responsive gene encoding an antioxidant protein that may be involved in HOCl degradation. RcsA has a distinguishing core motif containing functional Cys60, Cys63, and His67 residues. P. aeruginosa rcsA plays an important role in bleach tolerance, with expression of P. aeruginosa rcsA in Escherichia coli also conferring HOCl resistance. Interestingly, RcsA is required for full virulence in worm and fruit fly infection models, indicating a correlation between mechanisms of bleach toxicity and host immunity during infection. This provides new insights into the mechanisms used by P. aeruginosa to persist in harsh environments such as hospitals., (Copyright © 2020 American Society for Microbiology.)

Unlabelled: The Agrobacterium tumefaciens C58 genome harbors an operon containing the dmeR (Atu0890) and dmeF (Atu0891) genes, which encode a transcriptional regulatory protein belonging to the RcnR/CsoR family and a metal efflux protein belonging to the cation diffusion facilitator (CDF) family, respectively. The dmeRF operon is specifically induced by cobalt and nickel, with cobalt being the more potent inducer. Promoter-lacZ transcriptional fusion, an electrophoretic mobility shift assay, and DNase I footprinting assays revealed that DmeR represses dmeRF transcription through direct binding to the promoter region upstream of dmeR A strain lacking dmeF showed increased accumulation of intracellular cobalt and nickel and exhibited hypersensitivity to these metals; however, this strain displayed full virulence, comparable to that of the wild-type strain, when infecting a Nicotiana benthamiana plant model under the tested conditions. Cobalt, but not nickel, increased the expression of many iron-responsive genes and reduced the induction of the SoxR-regulated gene sodBII Furthermore, control of iron homeostasis via RirA is important for the ability of A. tumefaciens to cope with cobalt and nickel toxicity., Importance: The molecular mechanism of the regulation of dmeRF transcription by DmeR was demonstrated. This work provides evidence of a direct interaction of apo-DmeR with the corresponding DNA operator site in vitro The recognition site for apo-DmeR consists of 10-bp AT-rich inverted repeats separated by six C bases (5'-ATATAGTATACCCCCCTATAGTATAT-3'). Cobalt and nickel cause DmeR to dissociate from the dmeRF promoter, which leads to expression of the metal efflux gene dmeF This work also revealed a connection between iron homeostasis and cobalt/nickel resistance in A. tumefaciens., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Unlabelled: Agrobacterium tumefaciens has a cluster of genes (Atu3178, Atu3179, and Atu3180) encoding an ABC-type transporter, here named troA, troB, and troC, respectively, which is shown here to be a zinc-specific uptake system. Reverse transcription (RT)-PCR analysis confirmed that troA, troB, and troC are cotranscribed, with troC as the first gene of the operon. The yciC (Atu3181) gene is transcribed in the opposite orientation to that of the troCBA operon and belongs to a metal-binding GTPase family. Expression of troCBA and yciC was inducible under zinc-limiting conditions and was controlled by the zinc uptake regulator, Zur. Compared to the wild type, the mutant strain lacking troC was hypersensitive to a metal chelator, EDTA, and the phenotype could be rescued by the addition of zinc, while the strain with a single yciC mutation showed no phenotype. However, yciC was important for survival under zinc limitation when either troC or zinT was inactivated. The periplasmic zinc-binding protein, ZinT, could not function when TroC was inactivated, suggesting that ZinT may interact with TroCBA in zinc uptake. Unlike many other bacteria, the ABC-type transporter ZnuABC was not the major zinc uptake system in A. tumefaciens However, the important role of A. tumefaciens ZnuABC was revealed when TroCBA was impaired. The strain containing double mutations in the znuA and troC genes exhibited a growth defect in minimal medium. A. tumefaciens requires cooperation of zinc uptake systems and zinc chaperones, including TroCBA, ZnuABC, ZinT, and YciC, for survival under a wide range of zinc-limiting conditions., Importance: Both host and pathogen battle over access to essential metals, including zinc. In low-zinc environments, physiological responses that make it possible to acquire enough zinc are important for bacterial survival and could determine the outcome of host-pathogen interactions. A. tumefaciens was found to operate a novel pathway for zinc uptake in which ZinT functions in concert with the high-affinity zinc importer TroCBA., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Pseudomonas aeruginosa PAO1 has two differentially expressed methionine sulfoxide reductase genes: msrA (PA5018) and msrB (PA2827). The msrA gene is expressed constitutively at a high level throughout all growth phases, whereas msrB expression is highly induced by oxidative stress, such as sodium hypochlorite (NaOCl) treatment. Inactivation of either msrA or msrB or both genes (msrA msrB mutant) rendered the mutants less resistant than the parental PAO1 strain to oxidants such as NaOCl and H2O2. Unexpectedly, msr mutants have disparate resistance patterns when exposed to paraquat, a superoxide generator. The msrA mutant had a higher paraquat resistance level than the msrB mutant, which had a lower paraquat resistance level than the PAO1 strain. The expression levels of msrA showed an inverse correlation with the paraquat resistance level, and this atypical paraquat resistance pattern was not observed with msrB. Virulence testing using a Drosophila melanogaster model revealed that the msrA, msrB, and, to a greater extent, msrA msrB double mutants had an attenuated virulence phenotype. The data indicate that msrA and msrB are essential genes for oxidative stress protection and bacterial virulence. The pattern of expression and mutant phenotypes of P. aeruginosa msrA and msrB differ from previously characterized msr genes from other bacteria. Thus, as highly conserved genes, the msrA and msrB have diverse expression patterns and physiological roles that depend on the environmental niche where the bacteria thrive.

The ability to maintain intracellular concentrations of toxic reactive oxygen species (ROS) within safe limits is essential for all aerobic life forms. In bacteria, as well as other organisms, ROS are produced during the normal course of aerobic metabolism, necessitating the constitutive expression of ROS scavenging systems. However, bacteria can also experience transient high-level exposure to ROS derived either from external sources, such as the host defense response, or as a secondary effect of other seemingly unrelated environmental stresses. Consequently, transcriptional regulators have evolved to sense the levels of ROS and coordinate the appropriate oxidative stress response. Three well-studied examples of these are the peroxide responsive regulators OxyR, PerR, and OhrR. OxyR and PerR are sensors of primarily H(2)O(2), while OhrR senses organic peroxide (ROOH) and sodium hypochlorite (NaOCl). OxyR and OhrR sense oxidants by means of the reversible oxidation of specific cysteine residues. In contrast, PerR senses H(2)O(2) via the Fe-catalyzed oxidation of histidine residues. These transcription regulators also influence complex biological phenomena, such as biofilm formation, the evasion of host immune responses, and antibiotic resistance via the direct regulation of specific proteins.

The Pseudomonas aeruginosa PAO1 thiol peroxidase homolog (Tpx) belongs to a family of enzymes implicated in the removal of toxic peroxides. We have shown the expression of tpx to be highly inducible with redox cycling/superoxide generators and diamide and weakly inducible with organic hydroperoxides and hydrogen peroxide (H(2)O(2)). The PAO1 tpx pattern is unlike the patterns for other peroxide-scavenging genes in P. aeruginosa. Analysis of the tpx promoter reveals the presence of a putative IscR binding site located near the promoter. The tpx expression profiles in PAO1 and the iscR mutant, together with results from gel mobility shift assays showing that purified IscR specifically binds the tpx promoter, support the role of IscR as a transcriptional repressor of tpx that also regulates the oxidant-inducible expression of the gene. Recombinant Tpx has been purified and biochemically characterized. The enzyme catalyzes thioredoxin-dependent peroxidation and can utilize organic hydroperoxides and H(2)O(2) as substrates. The Δtpx mutant demonstrates differential sensitivity to H(2)O(2) only at moderate concentrations (0.5 mM) and not at high (20 mM) concentrations, suggesting a novel protective role of tpx against H(2)O(2) in P. aeruginosa. Altogether, P. aeruginosa tpx is a novel member of the IscR regulon and plays a primary role in protecting the bacteria from submillimolar concentrations of H(2)O(2).

In Xanthomonas campestris pv. campestris, SoxR likely functions as a sensor of redox-cycling drugs and as a transcriptional regulator. Oxidized SoxR binds directly to its target site and activates the expression of xcc0300, a gene that has protective roles against the toxicity of redox-cycling compounds. In addition, SoxR acts as a noninducible repressor of its own expression. X. campestris pv. campestris requires SoxR both for protection against redox-cycling drugs and for full virulence on a host plant. The X. campestris model of the gene regulation and physiological roles of SoxR represents a novel variant of existing bacterial SoxR models.

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 repressor's 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 repressor's 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.

Cytolethal distending toxin (CDT) is a bacterial toxin that induces G(2)/M cell cycle arrest, cell distension, and/or apoptosis in mammalian cells. It is produced by several Gram-negative species and may contribute to their pathogenicity. The catalytic subunit CdtB has homology with DNase I and may act as a genotoxin. However, the mechanism by which CdtB leads to cell death is not yet clearly understood. Here, we used Saccharomyces cerevisiae as a model to study the molecular pathways involved in the function of CdtB from Aggregatibacter actinomycetemcomitans, a cause of aggressive periodontitis. We show that A. actinomycetemcomitans CdtB (AaCdtB) expression induces S/G(2) arrest and death in a DNase-catalytic residue and nuclear localization-dependent manner in haploid yeasts. Yeast strains defective in homologous recombination (HR) repair, but not other DNA repair pathways, are hypersensitive to AaCdtB, suggesting that HR is required for survival upon CdtB expression. In addition, yeast does not harbor the substrate for the other activity proposed for CdtB function, which is phosphatidylinositol-3,4,5-triphosphate phosphatase. Thus, these results suggest that direct DNA-damaging activity alone is sufficient for CdtB toxicity. To investigate how CdtB induces cell death, we examined the effect of CdtB in yeast strains with mutations in apoptotic regulators. Our results suggest that yeast death occurs independently of the yeast metacaspase gene YCA1 and the apoptosis-inducing factor AIF1 but is partially dependent on histone H2B serine 10 phosphorylation. Therefore, we report here the evidence that AaCdtB causes DNA damage that leads to nonapoptotic death in yeast and the first mutation that confers resistance to CdtB.

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.

The copper resistance determinant copARZ, which encodes a CPx-type copper ATPase efflux protein, a transcriptional regulator, and a putative intracellular copper chaperone, was functionally characterized for the phytopathogenic bacterium Agrobacterium tumefaciens. These genes are transcribed as an operon, and their expression is induced in response to increasing copper and silver ion concentrations in a copR-dependent fashion. Analysis of the copARZ promoter revealed a putative CopR binding box located within the spacer of the -35 and -10 promoter motifs. In vitro, purified CopR could specifically bind to the box. The inactivation of the copARZ operon or copZ reduces the level of resistance to copper but not to other metal ions. Also, the copARZ operon mutant shows increased sensitivity to the superoxide generators menadione and plumbagin. In addition, the loss of functional copZ does not affect the ability of copper ions to induce the copARZ promoter, indicating that CopZ is not involved in the copper-sensing mechanism of CopR. Altogether, the results demonstrate a crucial role for the copARZ operon as a component of the copper resistance machinery in A. tumefaciens.

The analysis of genetics and physiological functions of Agrobacterium tumefaciens RirA (rhizobial iron regulator) has shown that it is a transcription regulator and a repressor of iron uptake systems. The rirA mutant strain (NTLrirA) overproduced siderophores and exhibited a highly constitutive expression of genes involved in iron uptake (fhuA, irp6A, and fbpA) compared to that of the wild-type strain (NTL4). The deregulation in the iron control of iron uptake in NTLrirA led to iron overload in the cell, which was supported by the observation that the NTLrirA mutant was more sensitive than wild-type NTL4 to an iron-activated antibiotic, streptonigrin. The NTLrirA mutant was more sensitive than the parental strain to oxidants, including hydrogen peroxide, organic hydroperoxide, and a superoxide generator, menadione. However, the addition of an iron chelator, 2,2'-dipyridyl, reversed the mutant hypersensitivity to H(2)O(2) and organic hydroperoxide, indicating the role of iron in peroxide toxicity. Meanwhile, the reduced level of superoxide dismutase (SodBIII) was partly responsible for the menadione-sensitive phenotype of the NTLrirA mutant. The NTLrirA mutant showed a defect in tumorigenesis on tobacco leaves, which likely resulted from the increased sensitivity of NTLrirA to oxidants and the decreased ability of NTLrirA to induce virulence genes (virB and virE). These data demonstrated that RirA is important for A. tumefaciens during plant-pathogen interactions.

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.

Exposure of Agrobacterium tumefaciens to menadione, cumene hydroperoxide, and diamide strongly induced trxA expression. The trxA mutant showed a reduction in the aerobic growth rate and plating efficiency and was cytochrome c oxidase negative. Atypically, the mutant has decreased resistance to menadione but an increased H2O2 resistance phenotype.

In Agrobacterium tumefaciens, the balance between acquiring enough iron and avoiding iron-induced toxicity is regulated in part by Fur (ferric uptake regulator). A fur mutant was constructed to address the physiological role of the regulator. Atypically, the mutant did not show alterations in the levels of siderophore biosynthesis and the expression of iron transport genes. However, the fur mutant was more sensitive than the wild type to an iron chelator, 2,2'-dipyridyl, and was also more resistant to an iron-activated antibiotic, streptonigrin, suggesting that Fur has a role in regulating iron concentrations. A. tumefaciens sitA, the periplasmic binding protein of a putative ABC-type iron and manganese transport system (sitABCD), was strongly repressed by Mn(2+) and, to a lesser extent, by Fe(2+), and this regulation was Fur dependent. Moreover, the fur mutant was more sensitive to manganese than the wild type. This was consistent with the fact that the fur mutant showed constitutive up-expression of the manganese uptake sit operon. Fur(At) showed a regulatory role under iron-limiting conditions. Furthermore, Fur has a role in determining oxidative resistance levels. The fur mutant was hypersensitive to hydrogen peroxide and had reduced catalase activity. The virulence assay showed that the fur mutant had a reduced ability to cause tumors on tobacco leaves compared to wild-type NTL4.

Sinorhizobium meliloti hpdA, which encodes the herbicide target 4-hydroxyphenylpyruvate dioxygenase, is positively regulated by HpdR. Gel mobility shift and DNase I footprinting analyses revealed that HpdR binds to a region that spans two conserved direct-repeat sequences within the hpdR-hpdA intergenic space. HpdR-dependent hpdA transcription occurs in the presence of 4-hydroxyphenylpyruvate, tyrosine, and phenylalanine, as well as during starvation.

Inactivation of Agrobacterium tumefaciens soxR increases sensitivity to superoxide generators. soxR expression is highly induced by superoxide stress and is autoregulated. SoxR also directly regulates the superoxide-inducible expression of atu5152. Taken together, the physiological role of soxR and the mechanism by which it regulates expression of target genes make the A. tumefaciens SoxR system different from other bacterial systems.

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.

The genes involved in organic hydroperoxide protection in Agrobacterium tumefaciens were functionally evaluated. Gene inactivation studies and functional analyses have identified ohr, encoding a thiol peroxidase, as the gene primarily responsible for organic hydroperoxide protection in A. tumefaciens. An ohr mutant was sensitive to organic hydroperoxide killing and had a reduced capacity to metabolize organic hydroperoxides. ohr is located next to, and is divergently transcribed from, ohrR, encoding a sensor and transcription regulator of organic hydroperoxide stress. Transcription of both ohr and ohrR was induced by exposure to organic hydroperoxides but not by exposure to other oxidants. This induction required functional ohrR. The results of gel mobility shift and DNase I footprinting assays with purified OhrR, combined with in vivo promoter deletion analyses, confirmed that OhrR regulated both ohrR and ohr by binding to a single OhrR binding box that overlapped the ohrR and ohr promoters. ohrR and ohr are both required for the establishment of a novel cumene hydroperoxide-induced adaptive response. Inactivation or overexpression of other Prx family genes (prx1, prx2, prx3, bcp1, and bcp2) did not affect either the resistance to, or the ability to degrade, organic hydroperoxide. Taken together, the results of biochemical, gene regulation and physiological studies support the role of ohrR and ohr as the primary system in sensing and protecting A. tumefaciens from organic hydroperoxide stress.

A methionine sulfoxide reductase gene (msrA) from Xanthomonas campestris pv. phaseoli has unique expression patterns and physiological function. msrA expression is growth dependent and is highly induced by exposure to oxidants and N-ethylmaleimide in an OxyR- and OhrR-independent manner. An msrA mutant showed increased sensitivity to oxidants but only during stationary phase.

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.

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 metal's 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 metal's 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.

We isolated menadione-resistant mutants of Xanthomonas campestris pv. phaseoli oxyR (oxyR(Xp)). The oxyRR2(Xp) mutant was hyperresistant to the superoxide generators menadione and plumbagin and was moderately resistant to H(2)O(2) and tert-butyl hydroperoxide. Analysis of enzymes involved in oxidative-stress protection in the oxyRR2(Xp) mutant revealed a >10-fold increase in AhpC and AhpF levels, while the levels of superoxide dismutase (SOD), catalase, and the organic hydroperoxide resistance protein (Ohr) were not significantly altered. Inactivation of ahpC in the oxyRR2(Xp) mutant resulted in increased sensitivity to menadione killing. Moreover, high levels of expression of cloned ahpC and ahpF in the oxyR(Xp) mutant complemented the menadione hypersensitivity phenotype. High levels of other oxidant-scavenging enzymes such as catalase and SOD did not protect the cells from menadione toxicity. These data strongly suggest that the toxicity of superoxide generators could be mediated via organic peroxide production and that alkyl hydroperoxide reductase has an important novel function in the protection against the toxicity of these compounds in X. campestris.

Analysis of the sequence immediate upstream of ohr revealed an open reading frame, designated ohrR, with the potential to encode a 17-kDa peptide with moderate amino acid sequence homology to the MarR family of negative regulators of gene expression. ohrR was transcribed as bicistronic mRNA with ohr, while ohr mRNA was found to be 95% monocistronic and 5% bicistronic with ohrR. Expression of both genes was induced by tert-butyl hydroperoxide (tBOOH) treatment. High-level expression of ohrR negatively regulated ohr expression. This repression could be overcome by tBOOH treatment. In vivo promoter analysis showed that the ohrR promoter (P1) has organic peroxide-inducible, strong activity, while the ohr promoter (P2) has constitutive, weak activity. Only P1 is autoregulated by OhrR. ohr primer extension results revealed three major primer extension products corresponding to the 5' ends of ohr mRNA, and their levels were strongly induced by tBOOH treatment. Sequence analysis of regions upstream of these sites showed no typical Xanthomonas promoter. Instead, the regions can form a stem-loop secondary structure with the 5' ends of ohr mRNA located in the loop section. The secondary structure resembles the structure recognized and processed by RNase III enzyme. These findings suggest that the P1 promoter is responsible for tBOOH-induced expression of the ohrR-ohr operon. The bicistronic mRNA is then processed by RNase III-like enzymes to give high levels of ohr mRNA, while ohrR mRNA is rapidly degraded.

Bacillus subtilis displays a complex adaptive response to the presence of reactive oxygen species. To date, most proteins that protect against reactive oxygen species are members of the peroxide-inducible PerR and sigma(B) regulons. We investigated the function of two B. subtilis homologs of the Xanthomonas campestris organic hydroperoxide resistance (ohr) gene. Mutational analyses indicate that both ohrA and ohrB contribute to organic peroxide resistance in B. subtilis, with the OhrA protein playing the more important role in growing cells. Expression of ohrA, but not ohrB, is strongly and specifically induced by organic peroxides. Regulation of ohrA requires the convergently transcribed gene, ohrR, which encodes a member of the MarR family of transcriptional repressors. In an ohrR mutant, ohrA expression is constitutive, whereas expression of the neighboring ohrB gene is unaffected. Selection for mutant strains that are derepressed for ohrA transcription identifies a perfect inverted repeat sequence that is required for OhrR-mediated regulation and likely defines an OhrR binding site. Thus, B. subtilis contains at least three regulons (sigma(B), PerR, and OhrR) that contribute to peroxide stress responses.

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.

During plant-microbe interactions and in the environment, Xanthomonas campestris pv. phaseoli is likely to be exposed to high concentrations of multiple oxidants. Here, we show that simultaneous exposures of the bacteria to multiple oxidants affects cell survival in a complex manner. A superoxide generator (menadione) enhanced the lethal effect of an organic peroxide (tert-butyl hydroperoxide) by 1, 000-fold; conversely, treatment of cells with menadione plus H(2)O(2) resulted in 100-fold protection compared to that for cells treated with the individual oxidants. Treatment of X. campestris with a combination of H(2)O(2) and tert-butyl hydroperoxide elicited no additive or protective effect. High levels of catalase alone are sufficient to protect cells against the lethal effect of menadione plus H(2)O(2) and tert-butyl hydroperoxide plus H(2)O(2). These data suggest that H(2)O(2) is the lethal agent responsible for killing the bacteria as a result of these treatments. However, increased expression of individual genes for peroxide (alkyl hydroperoxide reductase, catalase)- and superoxide (superoxide dismutase)-scavenging enzymes or concerted induction of oxidative stress-protective genes by menadione gave no protection against killing by a combination of menadione plus tert-butyl hydroperoxide. However, X. campestris cells in the stationary phase and a spontaneous H(2)O(2)-resistant mutant (X. campestris pv. phaseoli HR) were more resistant to killing by menadione plus tert-butyl hydroperoxide. These findings give new insight into oxidant killing of Xanthomonas spp. that could be generally applied to other bacteria.

A spontaneous Xanthomonas campestris pv. phaseoli H(2)O(2)-resistant mutant emerged upon selection with 1 mM H(2)O(2). In this report, we show that growth of this mutant under noninducing conditions gave high levels of catalase, alkyl hydroperoxide reductase (AhpC and AhpF), and OxyR. The H(2)O(2) resistance phenotype was abolished in oxyR-minus derivatives of the mutant, suggesting that elevated levels and mutations in oxyR were responsible for the phenotype. Nucleotide sequence analysis of the oxyR mutant showed three nucleotide changes. These changes resulted in one silent mutation and two amino acid changes, one at a highly conserved location (G197 to D197) and the other at a nonconserved location (L301 to R301) in OxyR. Furthermore, these mutations in oxyR affected expression of genes in the oxyR regulon. Expression of an oxyR-regulated gene, ahpC, was used to monitor the redox state of OxyR. In the parental strain, a high level of wild-type OxyR repressed ahpC expression. By contrast, expression of oxyR5 from the X. campestris pv. phaseoli H(2)O(2)-resistant mutant and its derivative oxyR5G197D with a single-amino-acid change on expression vectors activated ahpC expression in the absence of inducer. The other single-amino-acid mutant derivative of oxyR5L301R had effects on ahpC expression similar to those of the wild-type oxyR. However, when the two single mutations were combined, as in oxyR5, these mutations had an additive effect on activation of ahpC expression.

We constructed and characterized a Xanthomonas campestris pv. phaseoli oxyR mutant. The mutant was hypersensitive to H2O2 and menadione killing and had reduced aerobic plating efficiency. The oxidants' induction of the catalase and ahpC genes was also abolished in the mutant. Analysis of the adaptive responses showed that hydrogen peroxide-induced protection against hydrogen peroxide was lost, while menadione-induced protection against hydrogen peroxide was retained in the oxyR mutant. These results show that X. campestris pv. phaseoli oxyR is essential to peroxide adaptation and revealed the existence of a novel superoxide-inducible peroxide protection system that is independent of OxyR.

We have isolated a new organic hydroperoxide resistance (ohr) gene from Xanthomonas campestris pv. phaseoli. This was done by complementation of an Escherichia coli alkyl hydroperoxide reductase mutant with an organic hydroperoxide-hypersensitive phenotype. ohr encodes a 14.5-kDa protein. Its amino acid sequence shows high homology with several proteins of unknown function. An ohr mutant was subsequently constructed, and it showed increased sensitivity to both growth-inhibitory and killing concentrations of organic hydroperoxides but not to either H2O2 or superoxide generators. No alterations in sensitivity to other oxidants or stresses were observed in the mutant. ohr had interesting expression patterns in response to low concentrations of oxidants. It was highly induced by organic hydroperoxides, weakly induced by H2O2, and not induced at all by a superoxide generator. The novel regulation pattern of ohr suggests the existence of a second organic hydroperoxide-inducible system that differs from the global peroxide regulator system, OxyR. Expression of ohr in various bacteria tested conferred increased resistance to tert-butyl hydroperoxide killing, but this was not so for wild-type Xanthomonas strains. The organic hydroperoxide hypersensitivity of ohr mutants could be fully complemented by expression of ohr or a combination of ahpC and ahpF and could be partially complemented by expression ahpC alone. The data suggested that Ohr was a new type of organic hydroperoxide detoxification protein.