Thioredoxin-dependent hydroperoxide peroxidase activity of bacterioferritin comigratory protein (BCP) as a new member of the thiol-specific antioxidant protein (TSA)/Alkyl hydroperoxide peroxidase C (AhpC) family

Escherichia coli bacterioferritin comigratory protein (BCP), a putative bacterial member of the TSA/AhpC family, was characterized as a thiol peroxidase. BCP showed a thioredoxin-dependent thiol peroxidase activity. BCP preferentially reduced linoleic acid hydroperoxide rather than H(2)O(2) and t-butyl hydroperoxide with the use of thioredoxin as an in vivo immediate electron donor. The value of V(max)/K(m) of BCP for linoleic acid hydroperoxide was calculated to be 5-fold higher than that for H(2)O(2), implying that BCP has a selective capability to reduce linoleic acid hydroperoxide. Replacement of Cys-45 with serine resulted in the complete loss of thiol peroxidase activity, suggesting that BCP is a new bacterial member of TSA/AhpC family having a conserved cysteine as the primary site of catalysis. BCP exists as a monomer, and its functional Cys-45 appeared to exist as cysteine sulfenic acid. The expression level of BCP gradually elevated during exponential growth until mid-log phase growth, beyond which the expression level was decreased. BCP was induced 3-fold by the oxidative stress given by changing the growth conditions from the anaerobic to aerobic culture. Bcp null mutant grew more slowly than its wild type in aerobic culture and showed the hypersensitivity toward various oxidants such as H(2)O(2), t-butyl hydroperoxide, and linoleic acid hydroperoxide. The peroxide hypersensitivity of the null mutant could be complemented by the expression of bcp gene. Taken together, these data suggest that BCP is a new member of thioredoxin-dependent TSA/AhpC family, acting as a general hydroperoxide peroxidase.     

Control of singlet oxygen-induced oxidative damage in Escherichia coli

Singlet oxygen ((1)O(2)) is a highly reactive form of molecular oxygen that may harm living systems by oxidizing critical cellular macromolecules. The oxyR gene product regulates the expression of the enzymes and proteins that are needed for cellular protection against oxidative stress. In this study, the role of oxyR in cellular defense against a singlet oxygen was investigated using Escherichia coli oxyR mutant strains. Upon exposure to methylene blue and visible light, which generates singlet oxygen, the oxyR overexpression mutant was much more resistant to singlet oxygen-mediated cellular damage when compared to the oxyR deletion mutant in regard to growth kinetics, viability and protein oxidation. Induction and inactivation of major antioxidant enzymes, such as superoxide dismutase and catalase, were observed after their exposure to a singlet oxygen generating system in both oxyR strains. However, the oxyR overexpression mutant maintained significantly higher activities of antioxidant enzymes than did the oxyR deletion mutant. These results suggest that the oxyR regulon plays an important protective role in singlet oxygen-mediated cellular damage, presumably through the protection of antioxidant enzymes.     

In vitro and in vivo characterization of alkyl hydroperoxide reductase mutant strains of Helicobacter hepaticus

Mutant strains in the tsaA gene encoding alkyl hydroperoxide reductase were more sensitive to O(2) and to oxidizing agents (paraquat, cumene hydroperoxide and t-butylhydroperoxide) than the wild type, but were markedly more resistant to hydrogen peroxide. The mutant strains resistance phenotype could be attributed to a 4-fold and 3-fold increase in the catalase protein amount and activity, respectively compared to the parent strain. The wild type did not show an increase in catalase expression in response to sequential increases in O(2) exposure or to oxidative stress reagents, so an adaptive compensatory mutation has probably occurred in the mutants. In support of this, chromosomal complementation of tsaA mutants restored alkyl hydroperoxide reductase, but catalase was still up-expressed in all complemented strains. The katA promoter sequence was the same in all mutant strains and the wild type. Like its Helicobacter pylori counterpart strain, a H. hepaticus tsaA mutant contained more lipid hydroperoxides than the wild type strain. Hepatic tissue from mice inoculated with a tsaA mutant had lesions similar to those inoculated with the wild type, and included coagulative necrosis of hepatocytes. The liver and cecum colonizing abilities of the wild type and tsaA mutant were comparable. Up-expression of catalase in the tsaA mutants likely permits the bacterium to compensate (in colonization and virulence attributes) for the loss of an otherwise important oxidative stress-combating enzyme, alkyl hydroperoxide reductase. The use of erythromycin resistance insertion as a facile way to screen for gene-targeted mutants, and the chromosomal complementation of those mutants are new genetic procedures for studying H. hepaticus.     

Evidence against a direct antimicrobial role of H2O2 in the infection of plants by Erwinia chrysanthemi

We have investigated the role of bacterial resistance to oxidative stress in pathogenesis. The oxyR gene from the pathogenic bacterium Erwinia chrysanthemi has been characterized. It is closely related to that found in Escherichia coli (88% overall amino acid identity). An E. chrysanthemi oxyR mutant strain was constructed by marker exchange. After induction with a sublethal dose of H2O2, this mutant was more sensitive to H2O2 and showed reduced levels of catalase and glutathione reductase activities, compared with the wild type. The oxyR mutant was unable to form individual colonies on agar plates unless catalase was added exogenously. However, it retained full virulence in potato tubers and tobacco leaves. These results suggest that the host-produced H2O2 has no direct antimicrobial effect on the interaction of E. chrysanthemi with the two plant species.     

The peptidoglycan-associated lipoprotein OprL helps protect a Pseudomonas aeruginosa mutant devoid of the transactivator OxyR from hydrogen peroxide-mediated killing during planktonic and biofilm culture

OxyR controls H(2)O(2)-dependent gene expression in Pseudomonas aeruginosa. Without OxyR, diluted (<10(7)/ml) organisms are easily killed by micromolar H(2)O(2). The goal of this study was to define proteins that contribute to oxyR mutant survival in the presence of H(2)O(2). We identified proteins in an oxyR mutant that were oxidized by using 2,4-dinitrophenylhydrazine for protein carbonyl detection, followed by identification using a two-dimensional gel/matrix-assisted laser desorption ionization-time of flight approach. Among these was the peptidoglycan-associated lipoprotein, OprL. A double oxyR oprL mutant was constructed and was found to be more sensitive to H(2)O(2) than the oxyR mutant. Provision of the OxyR-regulated alkyl hydroperoxide reductase, AhpCF, but not AhpB or the catalase, KatB, helped protect this strain against H(2)O(2). Given the sensitivity of oxyR oprL bacteria to planktonic H(2)O(2), we next tested the hypothesis that the biofilm mode of growth might protect such organisms from H(2)O(2)-mediated killing. Surprisingly, biofilm-grown oxyR oprL mutants, which (in contrast to planktonic cells) possessed no differences in catalase activity compared to the oxyR mutant, were sensitive to killing by as little as 0.5 mM H(2)O(2). Transmission electron microscopy studies revealed that the integrity of both cytoplasmic and outer membranes of oxyR and oxyR oprL mutants were compromised. These studies suggest that sensitivity to the important physiological oxidant H(2)O(2) in the exquisitely sensitive oxyR mutant bacteria is based not only upon the presence and location of OxyR-controlled antioxidant enzymes such as AhpCF but also on structural reinforcement by the peptidoglycan-associated lipoprotein OprL, especially during growth in biofilms.     

Hypochlorous acid stress in Escherichia coli: resistance, DNA damage, and comparison with hydrogen peroxide stress.

We have investigated the mechanisms of killing of Escherichia coli by HOCl by identifying protective functions. HOCl challenges were performed on cultures arrested in stationary phase and in exponential phase. Resistance to HOCl in both cases was largely mediated by genes involved in resistance to hydrogen peroxide (H2O2). In stationary phase, a mutation in rpoS, which controls the expression of starvation genes including those which protect against oxidative stress, renders the cells hypersensitive to killing by HOCl. RpoS-regulated genes responsible for this sensitivity were dps, which encodes a DNA-binding protein, and, to a lesser extent, katE and katG, encoding catalases; all three are involved in resistance to H2O2. In exponential phase, induction of the oxyR regulon, an adaptive response to H2O2, protected against HOCl exposure, and the oxyR2 constitutive mutant is more resistant than the wild-type strain. The genes involved in this oxyR-dependent resistance have not yet been identified, but they differ from those primarily involved in resistance to H2O2, including katG, ahp, and dps. Pretreatment with HOCl conferred resistance to H2O2 in an OxyR-independent manner, suggesting a specific adaptive response to HOCl. fur mutants, which have an intracellular iron overload, were more sensitive to HOCl, supporting the generation of hydroxyl radicals upon HOCl exposure via a Fenton-type reaction. Mutations in recombinational repair genes (recA or recB) increased sensitivity to HOCl, indicative of DNA strand breaks. Sensitivity was visible in the wild type only at concentrations above 0.6 mg/liter, but it was observed at much lower concentrations in dps recA mutants.     

Mutations in oxyR resulting in peroxide resistance in Xanthomonas campestris

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.     

A suppressor of the menadione-hypersensitive phenotype of a Xanthomonas campestris pv. phaseoli oxyR mutant reveals a novel mechanism of toxicity and the protective role of alkyl hydroperoxide reductase

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.     

Induction of the SOS response by hydrogen peroxide in various Escherichia coli mutants with altered protection against oxidative DNA damage.

The induction of the SOS response by H2O2 was measured in Escherichia coli by means of a sfiA::lacZ operon fusion. The effects of mutations in genes involved in DNA repair or DNA metabolism on the SOS response were investigated. We found that in an uvrA mutant, H2O2 induced the SOS response at lower concentrations than in the uvr+ parent strain, indicating that some lesions induced by H2O2 may be repaired by the uvrABC-dependent excision repair system. A nth mutation, yielding deficiency in thymine glycol DNA glycosylase, had no detectable effect on SOS induction, indicating that thymine glycol, a DNA lesion expected to be induced by H2O2, does not participate detectably in the induction of the SOS response by this chemical under our conditions. H2O2 still induced the SOS response in a dnaC(Ts) uvrA double mutant under conditions in which no DNA replication proceeds, suggesting that this chemical induces DNA strand breaks. Induction of the SOS response by H2O2 was also assayed in various mutants affected in genes suspected to be important for protection against oxidative stress. Mutations in the catalase genes, katE and katG, had only minor effects. However, in an oxyR deletion mutant, in which the adaptative response to H2O2 does not occur, SOS induction occurred at much lower H2O2 concentrations than in the oxyR+ parent strain. These results indicate that some enzymes regulated by the oxyR gene are, under our conditions, more important than catalase for protection against the H2O2-induced DNA damages which trigger the SOS response.