The OxyR-regulated phnW gene encoding 2-aminoethylphosphonate:pyruvate aminotransferase helps protect Pseudomonas aeruginosa from tert-butyl hydroperoxide.
ABSTRACT: The LysR member of bacterial transactivators, OxyR, governs transcription of genes involved in the response to H2O2 and organic (alkyl) hydroperoxides (AHP) in the Gram-negative pathogen, Pseudomonas aeruginosa. We have previously shown that organisms lacking OxyR are rapidly killed by <2 or 500 mM H2O2 in planktonic and biofilm bacteria, respectively. In this study, we first employed a bioinformatic approach to elucidate the potential regulatory breadth of OxyR by scanning the entire P. aeruginosa PAO1 genome for canonical OxyR promoter recognition sequences (ATAG-N7-CTAT-N7-ATAG-N7-CTAT). Of >100 potential OxyR-controlled genes, 40 were strategically selected that were not predicted to be involved in the direct response to oxidative stress (e.g., catalase, peroxidase, etc.) and screened such genes by RT-PCR analysis for potentially positive or negative control by OxyR. Differences were found in 7 of 40 genes when comparing an oxyR mutant vs. PAO1 expression that was confirmed by ß-galactosidase reporter assays. Among these, phnW, encoding 2-aminoethylphosphonate:pyruvate aminotransferase, exhibited reduced expression in the oxyR mutant compared to wild-type bacteria. Electrophoretic mobility shift assays indicated binding of OxyR to the phnW promoter and DNase I footprinting analysis also revealed the sequences to which OxyR bound. Interestingly, a phnW mutant was more susceptible to t-butyl-hydroperoxide (t-BOOH) treatment than wild-type bacteria. Although we were unable to define the direct mechanism underlying this phenomenon, we believe that this may be due to a reduced efficiency for this strain to degrade t-BOOH relative to wild-type organisms because of modulation of AHP gene transcription in the phnW mutant.
Project description:BACKGROUND:Aerobically-grown bacteria can be challenged by hydrogen peroxide stress from endogenous aerobic metabolism and exogenously generated reactive oxygen species. Catalase (Kat), alkyl hydroperoxidase (Ahp), and glutathione peroxidase (Gpx) systems are major adaptive responses to H2O2 stress in bacteria. Stenotrophomonas maltophilia is a ubiquitous Gram-negative bacterium equipped with four Kats (KatA1, KatA2, KatMn, and KatE), one Ahp (AhpCF), and three Gpxs (Gpx1, Gpx2, and Gpx3). Here, we systematically investigated how the eight H2O2 scavenging genes differentially contribute to the low-micromolar levels of H2O2 generated from aerobic metabolism and high-millimolar levels of H2O2 from exogenous sources. METHODS:Gene expression was assessed and quantified by reverse transcription-PCR (RT-PCR) and real time quantitative PCR (qRT-PCR), respectively. The contribution of these enzymes to H2O2 stress was assessed using mutant construction and functional investigation. RESULTS:Of the eight genes, katA2, ahpCF, and gpx3 were intrinsically expressed in response to low-micromolar levels of H2O2 from aerobic metabolism, and the expression of katA2 and ahpCF was regulated by OxyR. AhpCF and KatA2 were responsible for alleviating aerobic growth-mediated low concentration H2O2 stress and AhpCF played a critical role for stationary-phase cells. KatA2 was upregulated to compensate for AhpCF in the case of ahpCF inactivation. After exposure to millimolar levels of H2O2, katA2 and ahpCF were upregulated in an OxyR-dependent manner. KatA2 was the critical enzyme for dealing with high concentration H2O2. Loss-of-function of KatA2 increased bacterial susceptibility to high concentration H2O2. CONCLUSIONS:AhpCF and KatA2 are key enzymes protecting S. maltophilia from hydrogen peroxide stress.
Project description:When Escherichia coli grows on conventional substrates, it continuously generates 10 to 15 ?M/s intracellular H2O2 through the accidental autoxidation of redox enzymes. Dosimetric analyses indicate that scavenging enzymes barely keep this H2O2 below toxic levels. Therefore, it seemed potentially problematic that E. coli can synthesize a catabolic phenylethylamine oxidase that stoichiometrically generates H2O2. This study was undertaken to understand how E. coli tolerates the oxidative stress that must ensue. Measurements indicated that phenylethylamine-fed cells generate H2O2 at 30 times the rate of glucose-fed cells. Two tolerance mechanisms were identified. First, in enclosed laboratory cultures, growth on phenylethylamine triggered induction of the OxyR H2O2 stress response. Null mutants (?oxyR) that could not induce that response were unable to grow. This is the first demonstration that OxyR plays a role in protecting cells against endogenous H2O2. The critical element of the OxyR response was the induction of H2O2 scavenging enzymes, since mutants that lacked NADH peroxidase (Ahp) grew poorly, and those that additionally lacked catalase did not grow at all. Other OxyR-controlled genes were expendable. Second, phenylethylamine oxidase is an unusual catabolic enzyme in that it is localized in the periplasm. Calculations showed that when cells grow in an open environment, virtually all of the oxidase-generated H2O2 will diffuse across the outer membrane and be lost to the external world, rather than enter the cytoplasm where H2O2-sensitive enzymes are located. In this respect, the periplasmic compartmentalization of phenylethylamine oxidase serves the same purpose as the peroxisomal compartmentalization of oxidases in eukaryotic cells.
Project description:Genes encoding a homolog of Escherichia coli OxyR (oxyR) and an alkyl hydroperoxide reductase system (ahpC and ahpD) have been isolated from Streptomyces coelicolor A3(2). The ahpC and ahpD genes constitute an operon transcribed divergently from the oxyR gene. Expression of both ahpCD and oxyR genes was maximal at early exponential phase and decreased rapidly as cells entered mid-exponential phase. Overproduction of OxyR in Streptomyces lividans conferred resistance against cumene hydroperoxide and H2O2. The oxyR mutant produced fewer ahpCD and oxyR transcripts than the wild type, suggesting that OxyR acts as a positive regulator for their expression. Both oxyR and ahpCD transcripts increased more than fivefold within 10 min of H2O2 treatment and decreased to the normal level in 50 min, with kinetics similar to those of the CatR-mediated induction of the catalase A gene (catA) by H2O2. The oxyR mutant failed to induce oxyR and ahpCD genes in response to H2O2, indicating that OxyR is the modulator for the H2O2-dependent induction of these genes. Purified OxyR protein bound specifically to the intergenic region between ahpC and oxyR, suggesting its direct role in regulating these genes. These results demonstrate that in S. coelicolor OxyR mediates H2O2 induction of its own gene and genes for alkyl hydroperoxide reductase system, but not the catalase gene (catA), unlike in Escherichia coli and Salmonella enterica serovar Typhimurium.
Project description:Porphyromonas gingivalis is an anaerobic microorganism that inhabits the oral cavity, where oxidative stress represents a constant challenge. A putative transcriptional regulator associated with oxidative stress, an oxyR homologue, is known from the P. gingivalis W83 genome sequence. We used microarrays to characterize the response of P. gingivalis to H2O2 and examine the role of oxyR in the regulation of this response. Most organisms in which oxyR has been investigated are facultative anaerobes or aerobes. In contrast to the OxyR-regulated response of these microorganisms to H2O2, the main feature of the response in P. gingivalis was a concerted up-regulation of insertion sequence elements related to IS1 transposases. Common OxyR-regulated genes such as dps and ahpFC were not positively regulated in P. gingivalis in response to H2O2. However, their expression was dependent on the presence of a functional OxyR, as revealed by microarray comparison of an oxyR mutant to the wild type. Phenotypic characterization of the oxyR mutant showed that OxyR plays a role in both the resistance to H2O2 and the aerotolerance of P. gingivalis. Escherichia coli and other bacteria with more complex respiratory requirements use OxyR for regulating resistance to H2O2 and use a separate regulator for aerotolerance. In P. gingivalis, the presence of a single protein combining the two functions might be related to the comparatively smaller genome size of this anaerobic microorganism. In conclusion, these results suggest that OxyR does not act as a sensor of H2O2 in P. gingivalis but constitutively activates transcription of oxidative-stress-related genes under anaerobic growth.
Project description:OxyR, a bacterial peroxide sensor, is a LysR-type transcriptional regulator (LTTR) that regulates the transcription of defense genes in response to a low level of cellular H2O2. Consisting of an N-terminal DNA-binding domain (DBD) and a C-terminal regulatory domain (RD), OxyR senses H2O2 with conserved cysteine residues in the RD. However, the precise mechanism of OxyR is not yet known due to the absence of the full-length (FL) protein structure. Here we determined the crystal structures of the FL protein and RD of Pseudomonas aeruginosa OxyR and its C199D mutant proteins. The FL crystal structures revealed that OxyR has a tetrameric arrangement assembled via two distinct dimerization interfaces. The C199D mutant structures suggested that new interactions that are mediated by cysteine hydroxylation induce a large conformational change, facilitating intramolecular disulfide-bond formation. More importantly, a bound H2O2 molecule was found near the Cys199 site, suggesting the H2O2-driven oxidation mechanism of OxyR. Combined with the crystal structures, a modeling study suggested that a large movement of the DBD is triggered by structural changes in the regulatory domains upon oxidation. Taken together, these findings provide novel concepts for answering key questions regarding OxyR in the H2O2-sensing and oxidation-dependent regulation of antioxidant genes.
Project description:Reactive oxygen species are common causes of cellular damages in all aerobic organisms. In Escherichia coli, the oxyR gene product is a positive regulator of the oxyR regulon that is induced in response to H2O2 stress. To identify genes involved in counteracting oxidative stress in plants, we transformed a delta oxyR mutant of E. coli with an Arabidopsis thaliana cDNA library and selected for clones that restored the ability of the delta oxyR mutant to grow in the presence of H2O2. Using this approach, we isolated a cDNA that has strong homology with the annexin super-gene family. The complemented mutant showed higher catalase activity. mRNA expression of the annexin gene in A. thaliana was higher in roots as compared with other organs and was also increased when the plants were exposed to H2O2 stress or salicylic acid. Based on the results presented in this study, we propose a novel physiological role for annexin in counteracting H2O2 stress.
Project description:Hydrogen peroxide (H2O2) is a strong oxidant capable of oxidizing cysteinyl thiolates, yet only a few cysteine-containing proteins have exceptional reactivity toward H2O2 One such example is the prokaryotic transcription factor OxyR, which controls the antioxidant response in bacteria, and which specifically and rapidly reduces H2O2 In this study, we present crystallographic evidence for the H2O2-sensing mechanism and H2O2-dependent structural transition of Corynebacterium glutamicum OxyR by capturing the reduced and H2O2-bound structures of a serine mutant of the peroxidatic cysteine, and the full-length crystal structure of disulfide-bonded oxidized OxyR. In the H2O2-bound structure, we pinpoint the key residues for the peroxidatic reduction of H2O2, and relate this to mutational assays showing that the conserved active-site residues T107 and R278 are critical for effective H2O2 reduction. Furthermore, we propose an allosteric mode of structural change, whereby a localized conformational change arising from H2O2-induced intramolecular disulfide formation drives a structural shift at the dimerization interface of OxyR, leading to overall changes in quaternary structure and an altered DNA-binding topology and affinity at the catalase promoter region. This study provides molecular insights into the overall OxyR transcription mechanism regulated by H2O2.
Project description:Oxidative stresses triggered by reactive oxygen species (ROS) that damage various cellular components are unavoidable for virtually all living organisms. In defense, microorganisms have evolved sophisticated mechanisms to sense, respond to, and battle against ROS. Shewanella oneidensis, an important research model for applied and environmental microbes, employs OxyR to mediate the response to H2O2 by derepressing the production of the major H2O2 scavenger KatB as a major means toward these goals. Surprisingly, despite enhanced H2O2 degradation, the oxyR mutant carries a viability deficiency phenotype (plating defect), which can be suppressed by the addition of exogenous iron species. Experiments showed that the defect was not due to iron starvation. Rather, multiple lines of evidence suggested that H2O2 generated abiotically in lysogeny broth (LB) is responsible for the defect by quickly killing mutant cells. We then showed that the iron species suppressed the plating defect by two distinct mechanisms, either as an H2O2 scavenger without involving living cells or as an environmental cue to stimulate an OxyR-independent response to help cells cope with oxidative stress. Based on the suppression of the plating defect by overproduction of H2O2 scavengers in vivo, we propose that cellular components that are vulnerable to H2O2 and responsible for the defect may reside outside the cytoplasm.In bacteria, OxyR is the major regulator controlling the cellular response to H2O2. The loss of OxyR results in reduced viability in many species, but the underlying mechanism is unknown. We showed in S. oneidensis that this defect was due to H2O2 generated abiotically in LB. We then showed that this defect could be corrected by the addition of Fe(2+) or catalase to the LB or increased intracellular production of catalase. Further analyses revealed that Fe(2+) was able not only to decompose H2O2 directly but also to stimulate the activity of OxyR-independent H2O2-scavenging enzymes. Our data indicate that iron species play a previously underappreciated role in protecting cells from H2O2 in environments.
Project description:Most Gram-negative bacteria respond to excessive levels of H2O2 using the peroxide-sensing transcriptional regulator OxyR, which can induce the expression of antioxidant genes to restore normality. Vibrio vulnificus has two distinct OxyRs (OxyR1 and OxyR2), which are sensitive to different levels of H2O2 and induce expression of two different peroxidases, Prx1 and Prx2. Although OxyR1 has both high sequence similarity and H2O2 sensitivity comparable with that of other OxyR proteins, OxyR2 exhibits limited sequence similarity and is more sensitive to H2O2 To investigate the basis for this difference, we determined crystal structures and carried out biochemical analyses of OxyR2. The determined structure of OxyR2 revealed a flipped conformation of the peptide bond before Glu-204, a position occupied by glycine in other OxyR proteins. Activity assays showed that the sensitivity to H2O2 was reduced to the level of other OxyR proteins by the E204G mutation. We solved the structure of the OxyR2-E204G mutant with the same packing environment. The structure of the mutant revealed a dual conformation of the peptide bond before Gly-204, indicating the structural flexibility of the region. This structural duality extended to the backbone atoms of Gly-204 and the imidazole ring of His-205, which interact with H2O2 and invariant water molecules near the peroxidatic cysteine, respectively. Structural comparison suggests that Glu-204 in OxyR2 provides rigidity to the region that is important in H2O2 sensing, compared with the E204G structure or other OxyR proteins. Our findings provide a structural basis for the higher sensitivity of OxyR2 to H2O2 and also suggest a molecular mechanism for bacterial regulation of expression of antioxidant genes at divergent concentrations of cellular H2O2.
Project description:In Escherichia coli hosts, hydrogen peroxide is one of the factors that may cause induction of lambda prophage. Here, we demonstrate that H2O2-mediated lambda prophage induction is significantly enhanced in the oxyR mutant host. The mRNA levels for cI gene expression were increased in a lambda lysogen in the presence of H2O2. On the other hand, stimulation of the p(M) promoter by cI857 overproduced from a multicopy plasmid was decreased in the DeltaoxyR mutant in the presence of H2O2 but not under normal growth conditions. The purified OxyR protein did bind specifically to the p(M) promoter region. This binding impaired efficiency of interaction of the cI protein with the OR3 site, while stimulating such a binding to OR2 and OR1 sites, in the regulatory region of the p(M) promoter. We propose that changes in cI gene expression, perhaps in combination with moderately induced SOS response, may be responsible for enhanced lambda prophage induction by hydrogen peroxide in the oxyR mutant. Therefore, OxyR seems to be a factor stimulating lambda prophage maintenance under conditions of oxidative stress. This proposal is discussed in the light of efficiency of induction of lambdoid prophages bearing genes coding for Shiga toxins.