OxyR Binding Research Articles

Reduced OxyR positively regulates the prodigiosin biosynthesis in Serratia marcescens FS14

OxyR, a LysR family transcriptional regulator, plays vital roles in bacterial oxidative stress response. In this study, we found that the deletion of oxyR not only inhibited the antioxidant capacity of S. marcescens FS14, but also decreased the production of prodigiosin. Further study revealed that OxyR activated the prodigiosin biosynthesis at the transcriptional level. Complementary results showed that not only the wild-type OxyR but also the reduced form OxyRC199S could activate the prodigiosin biosynthesis. We further demonstrated that reduced form of wild type OxyR could bind to the promoter of pig gene cluster, and identified the binding sites which is different from oxidized OxyR binding sites in E. coli. Our results demonstrated that OxyR in FS14 uses oxidized form to regulate the expression of the antioxidant related genes and utilizes reduced form to activate prodigiosin production. Further in silico analysis suggested that the activation of prodigiosin biosynthesis by reduced OxyR should be general in S. marcesencs. To our knowledge, this is the first report to show that OxyR uses the reduced form to activate the gene's expression, therefore, our results provide a novel regulation mechanism of OxyR.

Effects of OxyR regulator on oxidative stress, Apx toxin secretion and virulence of Actinobacillus pleuropneumoniae.

Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia, poses a significant threat to global swine populations due to its high prevalence, mortality rates, and substantial economic ramifications. Understanding the pathogen's defense mechanisms against host-produced reactive oxygen species is crucial for its survival, with OxyR, a conserved bacterial transcription factor, being pivotal in oxidative stress response. This study investigated the presence and role of OxyR in A. pleuropneumoniae serovar 1-12 reference strains. Transcriptomic analysis was conducted on an oxyR disruption mutant to delineate the biological activities influenced by OxyR. Additionally, specific assays were employed to assess urease activity, catalase expression, ApxI toxin secretion, as well as adhesion and invasion abilities of the oxyR disruption mutant on porcine 3D4/21 and PT cells. A mice challenge experiment was also conducted to evaluate the impact of oxyR inactivation on A. pleuropneumoniae virulence. OxyR was identified as a conserved regulator present in A. pleuropneumoniae serovar 1-12 reference strains. Transcriptomic analysis revealed the involvement of OxyR in multiple biological activities. The oxyR disruption resulted in decreased urease activity, elevated catalase expression, enhanced ApxI toxin secretion-attributed to OxyR binding to the apxIBD promoter-and reduced adhesion and invasion abilities on porcine cells. Furthermore, inactivation of oxyR reduced the virulence of A. pleuropneumoniae in a mice challenge experiment. The findings highlight the pivotal role of OxyR in influencing the virulence mechanisms of A. pleuropneumoniae. The observed effects on various biological activities underscore OxyR as an essential factor contributing to the pathogenicity of this bacterium.

Molecular Mechanisms of AhpC in Resistance to Oxidative Stress in Burkholderia thailandensis

Burkholderia thailandensis is a model organism for human pathogens Burkholderia mallei and Burkholderia pseudomallei. The study of B. thailandensis peroxiredoxin is helpful for understanding the survival, pathogenic infection, and antibiotic resistance of its homologous species. Alkyl hydroperoxide reductase subunit C (AhpC) is an important peroxiredoxin involved in oxidative damage defense. Here, we report that BthAhpC exhibits broad specificity for peroxide substrates, including inorganic and organic peroxides and peroxynitrite. AhpC catalyzes the reduction of oxidants using the N-terminal conserved Cys57 as a peroxidatic Cys and the C-terminal conserved Cys171 and Cys173 as resolving Cys. These three conserved Cys residues play critical roles in the catalytic mechanism. AhpD directly interacts with AhpC as an electron donor, and the conserved Cys residues in active site of AhpD are important for AhpC reduction. AhpC is directly repressed by OxyR as shown by identifying the OxyR binding site in the ahpC promoter with a DNA binding assay. This work sheds light on the function of AhpC in the peroxides and peroxynitrite damage response in B. thailandensis and homologous species.

OxyR and the hydrogen peroxide stress response in Caulobacter crescentus

Oxidative stress generated by hydrogen peroxide is faced by bacteria when encountering hostile environments. In order to define the physiological and regulatory networks controlling the oxidative stress response in the free-living bacterium Caulobacter crescentus, a whole transcriptome analysis of wild type and ΔoxyR strains in the presence of hydrogen peroxide for two different exposure times was carried out. The C. crescentus response to H2O2 includes a decrease of the assimilative sulfate reduction and a shift in the amino acid synthesis pathways into favoring the synthesis of histidine. Moreover, the expression of genes encoding enzymes for the depolymerization of polyhydroxybutyrate was increased, and the RpoH-dependent genes were severely repressed. Based on the expression pattern and sequence analysis, we postulate that OxyR is probably directly required for the induction of three genes (katG, ahpCF). The putative binding of OxyR to the ahpC regulatory region could be responsible for the use of one of two alternative promoters in response to oxidative stress. Nevertheless, OxyR is required for the expression of 103 genes in response to H2O2. Fur and part of its regulon were differentially expressed in response to hydrogen peroxide independently of OxyR. The non-coding RNA OsrA was upregulated in both strains, and an in silico analysis indicated that it may have a regulatory role. This work characterizes the physiological response to H2O2 in C. crescentus, the regulatory networks and differentially regulated genes in oxidative stress and the participation of OxyR in this process. It is proposed that besides OxyR, a second layer of regulation may be achieved by a small regulatory RNA and other transcriptional regulators.

Resistance to oxidative stress by inner membrane protein ElaB is regulated by OxyR and RpoS.

SummaryC‐tail anchored inner membrane proteins are a family of proteins that contain a C‐terminal transmembrane domain but lack an N‐terminal signal sequence for membrane targeting. They are widespread in eukaryotes and prokaryotes and play critical roles in membrane traffic, apoptosis and protein translocation in eukaryotes. Recently, we identified and characterized in Escherichia coli a new C‐tail anchored inner membrane, ElaB, which is regulated by the stationary phase sigma factor RpoS. ElaB is important for resistance to oxidative stress but the exact mechanism is unclear. Here, we show that ElaB functions as part of the adaptive oxidative stress response by maintaining membrane integrity. Production of ElaB is induced by oxidative stress at the transcriptional level. Moreover, elaB expression is also regulated by the key regulator OxyR via an OxyR binding site in the promoter of elaB. OxyR induces the expression of elaB in the exponential growth phase, while excess OxyR reduces elaB expression in an RpoS‐dependent way in the stationary phase. In addition, deletion of elaB reduced fitness compared to wild‐type cells after prolonged incubation. Therefore, we determined how ElaB is regulated under oxidative stress: RpoS and OxyR coordinately control the expression of inner membrane protein ElaB.

Characterization of 17β-hydroxysteroid dehydrogenase and regulators involved in estrogen degradation in Pseudomonas putida SJTE-1.

In bacteria, the enzyme catalyzing the transformation of 17β-estradiol is considered the key enzyme for its metabolism, whose enzymatic activity and regulatory network influence the biodegradation efficiency of this typical estrogen. In this work, a novel 17β-hydroxysteroid dehydrogenase (17β-HSD) was characterized from the estrogen-degrading strain Pseudomonas putida SJTE-1, and two regulators were identified. This 17β-HSD, a member of the short-chain dehydrogenase/reductase (SDR) superfamily, could be induced by 17β-estradiol and catalyzed the oxidization reaction at the C17 site of 17β-estradiol efficiently. Its Km value was 0.068mM, and its Vmax value was 56.26μmol/min/mg; over 98% of 17β-estradiol was oxidized into estrone in 5min, indicating higher efficiency than other reported bacterial 17β-HSDs. Furthermore, two genes (crgA and oxyR) adjacent to 17β-hsd were studied which encoded the potential CrgA and OxyR regulators. Overexpression of crgA could enhance the transcription of 17β-hsd, while that of oxyR resulted in the opposite effect. They could bind to the specific and different sites in the promoter region of 17β-hsd gene directly, and binding of OxyR could be released by 17β-estradiol. OxyR repressed the expression of 17β-hsd by its specific binding to the conserved motif of GATA-N9-TATC, while CrgA activated the expression of this gene through its binding to the motif of T-N11-A. Therefore, this 17β-HSD transformed 17β-estradiol efficiently and the two regulators regulated its expression directly. This work could promote the study of the enzymatic mechanism and regulatory network of the estrogen biodegradation pathway in bacteria.

The OxyR-regulated phnW gene encoding 2-aminoethylphosphonate:pyruvate aminotransferase helps protect Pseudomonas aeruginosa from tert-butyl hydroperoxide

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.

Expression of alkyl hydroperoxide reductase is regulated negatively by OxyR1 and positively by RpoE2 sigma factor in Azospirillum brasilense Sp7

OxyR proteins are LysR-type transcriptional regulators, which play an important role in responding to oxidative stress in bacteria. Azospirillum brasilense Sp7 harbours two copies of OxyR. The inactivation of the oxyR1, the gene organized divergently to ahpC in A. brasilense Sp7, led to an increased tolerance to alkyl hydroperoxides, which was corroborated by an increase in alkyl hydroperoxide reductase (AhpC) activity, enhanced expression of ahpC :lacZ fusion and increased synthesis of AhpC protein in the oxyR1::km mutant. The upstream region of ahpC promoter harboured a putative OxyR binding site, T-N11-A. Mutation of T, A or both in the T-N11-Amotif caused derepression of ahpC in A. brasilense suggesting that T-N11-A might be the binding site for a negative regulator. Retardation of the electrophoretic mobility of the T-N11-A motif harbouring oxyR1-ahpC intergenic DNA by recombinant OxyR1, under reducing as well as oxidizing conditions, indicated that OxyR1 acts as a negative regulator of ahpC in A. brasilense. Sequence of the promoter of ahpC, predicted on the basis of transcriptional start site, and an enhanced expression of ahpC:lacZ fusion in chrR2::km mutant background suggested that ahpC promoter was RpoE2 dependent. Thus, this study shows that in A. brasilense Sp7, ahpC expression is regulated negatively by OxyR1 but is regulated positively by RpoE2, an oxidative-stress-responsive sigma factor. It also shows that OxyR1 regulates the expression RpoE1, which is known to play an important role during photooxidative stress in A. brasilense.

OxyR-dependent formation of DNA methylation patterns in OpvABOFF and OpvABON cell lineages of Salmonella enterica.

Phase variation of the Salmonella enterica opvAB operon generates a bacterial lineage with standard lipopolysaccharide structure (OpvABOFF) and a lineage with shorter O-antigen chains (OpvABON). Regulation of OpvAB lineage formation is transcriptional, and is controlled by the LysR-type factor OxyR and by DNA adenine methylation. The opvAB regulatory region contains four sites for OxyR binding (OBSA-D), and four methylatable GATC motifs (GATC1–4). OpvABOFF and OpvABON cell lineages display opposite DNA methylation patterns in the opvAB regulatory region: (i) in the OpvABOFF state, GATC1 and GATC3 are non-methylated, whereas GATC2 and GATC4 are methylated; (ii) in the OpvABON state, GATC2 and GATC4 are non-methylated, whereas GATC1 and GATC3 are methylated. We provide evidence that such DNA methylation patterns are generated by OxyR binding. The higher stability of the OpvABOFF lineage may be caused by binding of OxyR to sites that are identical to the consensus (OBSA and OBSc), while the sites bound by OxyR in OpvABON cells (OBSB and OBSD) are not. In support of this view, amelioration of either OBSB or OBSD locks the system in the ON state. We also show that the GATC-binding protein SeqA and the nucleoid protein HU are ancillary factors in opvAB control.

Molecular mechanism involved in the response to hydrogen peroxide stress in Acinetobacter oleivorans DR1.

Two-dimensional gel electrophoresis was conducted to investigate the effect of H2O2 on whole protein expression in Acinetobacter oleivorans DR1. Functional classification of 13 upregulated proteins using MALDI-TOF mass spectrometry showed relationships with oxidative stress, energy production and conversion, nucleotide and amino acid metabolism, membrane-related, ion transport, and chaperone-related functions. Alignment of OxyR-binding regions from Pseudomonas aeruginosa and Escherichia coli with promoters of identified proteins revealed that only ahpC, ahpF, and trxB (thioredoxin-disulfide reductase) genes, along with a newly found oprC (putative outer membrane receptor protein) gene, have OxyR-binding sites. The oxyR and ahpC mutants were more sensitive to H2O2 and showed growth defects in both nutritional and n-hexadecane-amended media. Four catalases present in the genome of A. oleivorans DR1 were not detected, which led us to confirm the expression and activity of those catalases in the presence of H2O2. The expression patterns of the four catalase genes differed at different concentrations of H2O2. Interestingly, the promoters of both known OxyR-controlled katG gene (AOLE_17390) and putative small catalase gene (AOLE_09800) have OxyR-binding sites. Gel-shift assay confirmed OxyR binding to the promoter regions of newly identified OxyR-controlled genes encoding OprC and a putative catalase. Hierarchical expression and OxyR-binding of several OxyR-controlled genes suggested that concentration is an important factor in inducing the set of genes under H2O2 stress.

Regulation of Catalase-Peroxidase KatG Is OxyR Dependent and Fur Independent in Caulobacter crescentus

Most organisms that grow in the presence of oxygen possess catalases and/or peroxidases, which are necessary for scavenging the H(2)O(2) produced by aerobic metabolism. In this work we investigate the pathways that regulate the Caulobacter crescentus katG gene, encoding the only enzyme with catalase-peroxidase function in this bacterium. The transcriptional start site of the katG gene was determined, showing a short 5' untranslated region. The katG regulatory region was mapped by serial deletions, and the results indicate that there is a single promoter, which is responsible for induction at stationary phase. An oxyR mutant strain was constructed; it showed decreased katG expression, and no KatG protein or catalase-peroxidase activity was detected in stationary-phase cell extracts, implying that OxyR is the main positive regulator of the C. crescentus katG gene. Purified OxyR protein bound to the katG regulatory region between nucleotides -42 and -91 from the transcription start site, as determined by a DNase I footprinting assay, and a canonical OxyR binding site was found in this region. Moreover, OxyR binding was shown to be redox dependent, given that only oxidized proteins bound adjacent to the -35 sequence of the promoter and the katG P1 promoter was activated by OxyR in an H(2)O(2)-dependent manner. On the other hand, this work showed that the iron-responsive regulator Fur does not regulate C. crescentus katG, since a fur mutant strain presented wild-type levels of katG transcription and catalase-peroxidase production and activity, and the purified Fur protein was not able to bind to the katG regulatory region.

Global responses of Aliivibrio salmonicida to hydrogen peroxide as revealed by microarray analysis

Aliivibrio salmonicida causes “cold-water vibriosis” (or “Hitra disease”) in fish, including marine-reared Atlantic salmon. During development of the disease the bacterium will encounter macrophages with antibacterial activities such as production of damaging reactive oxygen species (ROS). To defend itself the bacterium will presumably start producing detoxifying enzymes, reducing agents, and proteins involved in DNA and protein repair systems. Even though responses to oxidative stress are well studied for a few model bacteria, little work has been done in general to explain how important groups of pathogens, like members of the Vibrionaceae family, can survive at high levels of ROS. We have used bioinformatic tools and microarray to study how A. salmonicida responds to hydrogen peroxide (H 2O 2). First, we used the recently published genome sequence to predict potential binding sites for OxyR (H 2O 2 response regulator). The computer-based search identified OxyR sites associated with 20 single genes and 8 operons, and these predictions were compared to experimental data from Northern blot analysis and microarray analysis. In general, OxyR binding site predictions and experimental results are in agreement. Up- and down regulated genes are distributed among all functional gene categories, but a striking number of ≥ 2 fold up regulated genes encode proteins involved in detoxification and DNA repair, are part of reduction systems, or are involved in carbon metabolism and regeneration of NADPH. Our predictions and –omics data corroborates well with findings from other model bacteria, but also suggest species-specific gene regulation.

Establishing and Maintaining Sequestration of Dam Target Sites for Phase Variation ofagn43inEscherichia coli

Phase variation of the outer membrane protein Ag43 encoded by agn43 in Escherichia coli is controlled by an epigenetic mechanism. Sequestration of the regulatory region from Dam-dependent methylation has to be established and maintained throughout a generation to obtain and maintain the OFF phase. This work shows that hemimethylated DNA, which is formed by the passage of the DNA replication fork in an ON-phase cell, can be sequestered from methylation by OxyR binding, which is thus a key event for the switch from ON to OFF. No evidence was found that the protein SeqA, which also binds to the region, is involved in sequestration. To facilitate the dissection of this process further, a novel approach was introduced that does not alter the sequence of the regulatory region or the cellular concentration of Dam or OxyR, which consists of inserting auxiliary OxyR binding sites upstream of the regulatory region. Using this strategy, it was shown that the ON-to-OFF switch frequency can be modulated without changing the OFF-to-ON frequency. The data support a model in which in an ON-phase cell, the subcellular OxyR availability at the replication fork as it passes through the agn43 regulatory region is key for initiating an ON-to-OFF switch. In contrast, this availability is not a determining factor for the switch from OFF to ON. This finding shows that different variables affect these two stochastic events. This provides new insight into the events determining the stochastic nature of epigenetic phase variation.

OxyR is involved in the expression of thioredoxin reductase TrxB inPseudomonas putida

OxyR regulates the expression of a peroxiredoxin (AhpC) and two catalases (KatA and KatB), which play roles in peroxide protection, at the transcription level in Pseudomonas putida KT2442. Proteome analysis indicated significantly increased amounts of the enzymes AhpC, KatA, KatB, and a peroxiredoxin reductase (AhpF) in the oxyR1 mutant cells; these increases reflected the upregulation of the expression of the genes encoding these enzymes. Additionally, although the effect of oxyR1 mutation on the trxB transcript level was not clearly evident, it increased the amount of thioredoxin reductase (TrxB) by fivefold. Primer extension analysis revealed that trxB was constitutively transcribed from the P1 site; however, hydrogen peroxide treatment lowered the transcription of trxB from P1 but induced its transcription from P2. Adjacent to the -35 base of the P2 initiation site, sequences similar to those involved in the proposed OxyR binding in Escherichia coli were found in a region to which OxyR was shown to bind. These observations suggest that in P. putida, OxyR regulates TrxB expression by promoting trxB transcription from the P2 site when oxidative stresses lowered the transcription from the constitutive P1 site.

Submicromolar hydrogen peroxide disrupts the ability of Fur protein to control free‐iron levels in Escherichia coli

In aerobic environments, mutants of Escherichia coli that lack peroxidase and catalase activities (Hpx(-)) accumulate submicromolar concentrations of intracellular H(2)O(2). We observed that in defined medium these strains constitutively expressed members of the Fur regulon. Iron-import proteins, which Fur normally represses, were fully induced. H(2)O(2) may antagonize Fur function by oxidizing the Fur:Fe(2+) complex and inactivating its repressor function. This is a potential problem, as in iron-rich environments excessive iron uptake would endanger H(2)O(2)-stressed cells by accelerating hydroxyl-radical production through the Fenton reaction. However, the OxyR H(2)O(2)-response system restored Fur repression in iron-replete Luria-Bertani medium by upregulating the synthesis of Fur protein. Indeed, when the OxyR binding site upstream of fur was disrupted, Hpx(-) mutants failed to repress transporter synthesis, and they exhibited high levels of intracellular free iron. Mutagenesis and bacteriostasis resulted. These defects were eliminated by mutations or chelators that slowed iron import, confirming that dysregulation of iron uptake was the root problem. Thus, aerobic organisms must grapple with a conundrum: how to monitor iron levels in oxidizing environments that might perturb the valence of the analyte. The induction of Fur synthesis by the OxyR response comprises one evolutionary solution to that problem.

Mutational Analysis To Define an Activating Region on the Redox-Sensitive Transcriptional Regulator OxyR

The OxyR transcription factor is a key regulator of the Escherichia coli response to oxidative stress. Previous studies showed that OxyR binding to a target promoter enhances RNA polymerase binding and vice versa, suggesting a direct interaction between OxyR and RNA polymerase. To identify the region of OxyR that might contact RNA polymerase, we carried out alanine scanning and random mutagenesis of oxyR. The combination of these approaches led to the identification of several mutants defective in the activation of an OxyR target gene. A subset of the mutations map to the DNA-binding domain, other mutations appear to affect dimerization of the regulatory domain, while another group is suggested to affect disulfide bond formation. The two mutations, D142A and R273H, giving the most dramatic phenotype are located in a patch on the surface of the oxidized OxyR protein and possibly define an activating region on OxyR.

OxyR regulated the expression of two major catalases, KatA and KatB, along with peroxiredoxin, AhpC in Pseudomonas putida

OxyR is known to activate/repress the expression of the oxyR regulon, which consists of several genes, which play important antioxidant role in Escherichia coli. To elucidate the role of OxyR in Pseudomonas putida KT2442, the oxyR1 mutation that caused the upregulation of ahpC in a toluene-resistant variant strain was introduced, because no null mutants in oxyR were isolated. This mutation was shown to cause the accumulation of a catalase (KatA) along with AhpC throughout the growth, and of a RpoS-dependent catalase/peroxidase (KatB) in the stationary phase. Following the identification of the transcription start site of two catalase genes, sequences similar to those involved in the proposed OxyR binding for E. coli were found upstream from each of the promoter regions of katA and katB, as well as ahpC. Purified OxyR was shown to bind to these sequences, under both reduced and oxidized states. Moreover, the oxyR1 mutation increased the transcription levels of these genes. These results are consistent with the conclusion, distinct from those observed in an opportunistic pathogen Pseudomonas aeruginosa, that OxyR controlled expression of all the principal peroxide-degrading enzymes in P. putida. The mutation did not cause any notable changes in the transcriptional levels of several antioxidant genes, including those of glutathione reductase, glutaredoxins and thioredoxins, which would involve maintenance of the cellular thiol-disulfide balance, suggesting that the transcriptional regulation of these antioxidant genes should be different from that of katA, katB and ahpC in P. putida.

Superoxide dismutase-encoding gene of the obligate anaerobe Porphyromonas gingivalis is regulated by the redox-sensing transcription activator OxyR

Inspection of the genomic DNA sequence of the oral anaerobe Porphyromonas gingivalis reveals that the micro-organism possesses the peroxide-sensing transcription activator OxyR, but not the superoxide-sensing transcription factor SoxR. Investigatation of oxidative-stress-responsive proteins in P. gingivalis by two-dimensional gel electrophoresis showed that two proteins were predominantly upregulated in oxidative conditions. In a P. gingivalis oxyR mutant these two proteins were not induced by treatment with hydrogen peroxide under aerobic conditions. By N-terminal amino acid sequencing, the two proteins were found to be superoxide dismutase and alkyl hydroperoxide reductase, encoded by sod and ahpC, respectively. Northern blot and lacZ fusion analyses revealed that P. gingivalis sod and ahpC were positively regulated by OxyR. Primer extension analysis located the promoter regions of sod and ahpC, and putative -35 boxes of these promoters were found immediately adjacent to their putative OxyR-binding sequences. Moreover, the promoter regions of sod and ahpC had the ability to bind P. gingivalis OxyR protein. These results demonstrate that P. gingivalis sod is one of the OxyR regulons, suggesting that OxyR functions as an intracellular redox sensor rather than a peroxide sensor in this organism. A sod gene of Bacteroides fragilis, which is taxonomically related to P. gingivalis, is inducible by redox stresses but not controlled by its OxyR. A DNA fragment including the B. fragilis sod promoter region could bind the P. gingivalis OxyR protein; however, a putative OxyR binding sequence within the DNA fragment was 14 bases distant from a putative -35 box of its promoter.

Genetic and physiological analysis of the major OxyR-regulated katA from Xanthomonas campestris pv. phaseoli

katA encodes the major catalase that accounts for 90 % of the total catalase activity present in Xanthomonas campestris pv. phaseoli. katA is located upstream of an ORF designated ankA encoding a cytoplasmic membrane protein homologous to eukaryotic ankyrin. Transcriptional analysis of katA and ankA identified two katA transcripts: a major monocistronic katA transcript and a minor bicistronic katA-ankA transcript. KatA expression was induced in the presence of various oxidants including H2O2, organic hydroperoxides and the superoxide-generating agent menadione, in an OxyR-dependent manner. Analysis of the katA promoter region showed a putative OxyR binding site located upstream of an Escherichia coli-like sigma70 -35 region that is likely to be responsible for transcription activation in response to oxidant treatment. Gel mobility shift experiments confirmed that purified OxyR specifically binds to the katA promoter. A katA mutant was highly sensitive to H2O2 during both the exponential and stationary phases of growth. This phenotype could be complemented by functional katA, confirming the essential role of the gene in protecting X. campestris from H2O2 toxicity. Unexpectedly, inactivation of ankA also significantly reduced resistance to H2O2 and the phenotype could be complemented by plasmid-borne expression of ankA. Physiological analyses showed that katA plays an important role in, but is not solely responsible for, both the adaptive and menadione-induced cross-protective responses to H2O2 killing in X. campestris.

Induction of the sufA operon encoding Fe-S assembly proteins by superoxide generators and hydrogen peroxide: involvement of OxyR, IHF and an unidentified oxidant-responsive factor.

A promoter (sufAp), inducible by various oxidants, directs transcription of the sufABCDSE operon encoding an alternative Fe-S cluster assembly system in Escherichia coli. Superoxide generators and H2O2 induced expression of sufA-lacZ even in DeltasoxRS and DeltaoxyR mutants, suggesting participation of an additional regulator(s) in oxidant induction of the sufA operon. Through deletion and linker scanning mutagenesis, we found three cis-acting oxidant-responsive elements (OREs). ORE-I lies between -236 and -197 nucleotides from the transcription start site, overlapping extensively with the OxyR binding site reported previously. ORE-II (-156 to -127) was found to be the site of IHF action. ORE-III (-56 to -35) had no predictable binding sites for known regulators. Gel mobility shift assays with a 50 bp DNA probe containing ORE-III revealed the presence of an ORE-III-specific factor that binds only when cells are treated with oxidants. S1 mapping analysis revealed that phenazine methosulphate (PMS) and H2O2 induced sufA expression by more than 40-fold. In a DeltaoxyR mutant, sufA was still induced more than 10-fold. Fur, a ferric uptake regulator that negatively regulates this operon in response to iron availability, did not mediate the oxidant induction. Deletion of the suf operon caused cells to be more sensitive to superoxide-generating agents without affecting sensitivity to H2O2. From these results, we propose that the oxidant induction of the sufA operon is mediated through OxyR, IHF, plus an unidentified oxidant-responsive factor, and that the suf gene products are needed to defend cells against oxidative stress caused by superoxide generators.