A Xanthomonas alkyl hydroperoxide reductase subunit C (ahpC) mutant showed an altered peroxide stress response and complex regulation of the compensatory response of peroxide detoxification enzymes

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.     

Compensatory functions of two alkyl hydroperoxide reductases in the oxidative defense system of Legionella pneumophila

Legionella pneumophila expresses two catalase-peroxidase enzymes that exhibit strong peroxidatic but weak catalatic activities, suggesting that other enzymes participate in decomposition of hydrogen peroxide (H2O2). Comparative genomics revealed that L. pneumophila and its close relative Coxiella burnetii each contain two peroxide-scavenging alkyl hydroperoxide reductase (AhpC) systems: AhpC1, which is similar to the Helicobacter pylori AhpC system, and AhpC2 AhpD (AhpC2D), which is similar to the AhpC AhpD system of Mycobacterium tuberculosis. To establish a catalatic function for these two systems, we expressed L. pneumophila ahpC1 or ahpC2 in a catalase/peroxidase mutant of Escherichia coli and demonstrated restoration of H2O2 resistance by a disk diffusion assay. ahpC1::Km and ahpC2D::Km chromosomal deletion mutants were two- to eightfold more sensitive to H2O2, tert-butyl hydroperoxide, cumene hydroperoxide, and paraquat than the wild-type L. pneumophila, a phenotype that could be restored by trans-complementation. Reciprocal strategies to construct double mutants were unsuccessful. Mutant strains were not enfeebled for growth in vitro or in a U937 cell infection model. Green fluorescence protein reporter assays revealed expression to be dependent on the stage of growth, with ahpC1 appearing after the exponential phase and ahpC2 appearing during early exponential phase. Quantitative real-time PCR showed that ahpC1 mRNA levels were approximately 7- to 10-fold higher than ahpC2D mRNA levels. However, expression of ahpC2D was significantly increased in the ahpC1 mutant, whereas ahpC1 expression was unchanged in the ahpC2D mutant. These results indicate that AhpC1 or AhpC2D (or both) provide an essential hydrogen peroxide-scavenging function to L. pneumophila and that the compensatory activity of the ahpC2D system is most likely induced in response to oxidative stress.     

MntR modulates expression of the PerR regulon and superoxide resistance in Staphylococcus aureus through control of manganese uptake

The Staphylococcus aureus DtxR-like protein, MntR, controls expression of the mntABC and mntH genes, which encode putative manganese transporters. Mutation of mntABC produced a growth defect in metal-depleted medium and increased sensitivity to intracellularly generated superoxide radicals. These phenotypes resulted from diminished uptake of manganese and were rescued by the addition of excess Mn(II). Resistance to superoxide was incompletely rescued by Mn(II) for STE035 (mntA mntH), and the strain had reduced virulence in a murine abscess model of infection. Expression of mntABC was repressed by Mn(II) in an MntR-dependent manner, which contrasts with the expression of mntH that was not repressed in elevated Mn(II) and was decreased in an mntR mutant. This demonstrates that MntR acts as a negative and positive regulator of these loci respectively. PerR, the peroxide resistance regulon repressor, acts with MntR to control the expression of mntABC and manganese uptake. The expression of the PerR-regulated genes, katA (catalase), ftn (ferritin) and fur (ferric uptake regulator), was diminished in STE031 (mntR) when grown in excess Mn(II). Therefore, the control of Mn(II)-regulated members of the PerR regulon and the Fur protein is modulated by MntR through its control of Mn(II) uptake. The co-ordinated regulation of metal ion homeostasis and oxidative stress resistance via the regulators MntR, PerR and Fur of S. aureus is discussed.     

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.     

Role of a bacterial organic hydroperoxide detoxification system in preventing catalase inactivation

In the gastric pathogen Helicobacter pylori, catalase (KatA) and alkyl hydroperoxide reductase (AhpC) are two highly abundant enzymes that are crucial for oxidative stress resistance and survival of the bacterium in the host. Here we report a connection unidentified previously between the two stress resistance enzymes. We observed that the catalase in ahpC mutant cells in comparison with the parent strain is inactivated partially (approximately 50%). The decrease of catalase activity is well correlated with the perturbation of the heme environment in catalase, as detected by electron paramagnetic resonance spectroscopy. To understand the reason for this catalase inactivation, we examined the inhibitory effects of hydroperoxides on H. pylori catalase (either present in cell extracts or added to the purified enzyme) by monitoring the enzyme activity and the EPR signal of catalase. H. pylori catalase is highly resistant to its own substrate, without the loss of enzyme activity by treatment with a molar ratio of 1:3000 H2O2. However, it inactivated is by lower concentrations of organic hydroperoxides (the substrate of AhpC). Treatment with a molar ratio of 1:400 t-butyl hydroperoxide resulted in an inactivation of catalase by approximately 50%. UV-visible absorption spectra indicated that the catalase inactivation by organic hydroperoxides is caused by the formation of a catalytically incompetent compound II species. To further support the idea that organic hydroperoxides, which accumulate in the ahpC mutant cells, are responsible for the inactivation of catalase, we compared the level of lipid peroxidation found in ahpC mutant cells with that found in wild type cells. The results showed that the total amount of extractable lipid hydroperoxides in the ahpC mutant cells is approximately three times that in the wild type cells. Our findings reveal a novel role of the organic hydroperoxide detoxification system in preventing catalase inactivation.     

Campylobacter jejuni Contains Two Fur Homologs: Characterization of Iron-Responsive Regulation of Peroxide Stress Defense Genes by the PerR Repressor

Expression of the peroxide stress genes alkyl hydroperoxide reductase (ahpC) and catalase (katA) of the microaerophile Campylobacter jejuni is repressed by iron. Whereas iron repression in gram-negative bacteria is usually carried out by the Fur protein, previous work showed that this is not the case in C. jejuni, as these genes are still iron repressed in a C. jejuni fur mutant. An open reading frame encoding a Fur homolog (designated PerR for "peroxide stress regulator") was identified in the genome sequence of C. jejuni. The perR gene was disrupted by a kanamycin resistance cassette in C. jejuni wild-type and fur mutant strains. Subsequent characterization of the C. jejuni perR mutants showed derepressed expression of both AhpC and KatA at a much higher level than that obtained by iron limitation, suggesting that expression of these genes is controlled by other regulatory factors in addition to the iron level. Other iron-regulated proteins were not affected by the perR mutation. The fur perR double mutant showed derepressed expression of known iron-repressed genes. Further phenotypic analysis of the perR mutant, fur mutant, and the fur perR double mutant showed that the perR mutation made C. jejuni hyperresistant to peroxide stress caused by hydrogen peroxide and cumene hydroperoxide, a finding consistent with the high levels of KatA and AhpC expression, and showed that these enzymes were functional. Quantitative analysis of KatA expression showed that both the perR mutation and the fur mutation had profound effects on catalase activity, suggesting additional non-iron-dependent regulation of KatA and, by inference, AhpC. The PerR protein is a functional but nonhomologous substitution for the OxyR protein, which regulates peroxide stress genes in other gram-negative bacteria. Regulation of peroxide stress genes by a Fur homolog has recently been described for the gram-positive bacterium Bacillus subtilis. C. jejuni is the first gram-negative bacterium where non-OxyR regulation of peroxide stress genes has been described and characterized.     

A peroxide-induced zinc uptake system plays an important role in protection against oxidative stress in Bacillus subtilis

In Bacillus subtilis, hydrogen peroxide (H2O2) induces expression of the PerR regulon including catalase (KatA), alkyl hydroperoxide reductase and the DNA-binding protein MrgA. We have identified the P-type metal-transporting ATPase ZosA (formerly YkvW) as an additional member of the perR regulon. Expression of zosA is induced by H2O2 and repressed by the PerR metalloregulatory protein, which binds to two Per boxes in the promoter region. Physiological studies implicate ZosA in Zn(II) uptake. ZosA functions together with two Zur-regulated uptake systems and one known efflux system to maintain Zn(II) homeostasis. ZosA is the major pathway for zinc uptake in cells growing with micromolar levels of Zn(II) that are known to repress the two Zur-regulated transporters. A perR mutant is sensitive to high levels of zinc, and this sensitivity is partially suppressed by a zosA mutation. ZosA is important for resistance to both H2O2 and the thiol-oxidizing agent diamide. This suggests that increased intracellular Zn(II) may protect thiols from oxidation. In contrast, catalase is critical for H2O2 resistance but does not contribute significantly to diamide resistance. Growth of cells with elevated zinc significantly increases resistance to high concentrations of H2O2, and this effect requires ZosA. Our results indicate that peroxide stress leads to the upregulation of a dedicated Zn(II) uptake system that plays an important role in H2O2 and disulphide stress resistance.     

Aerotolerance and peroxide resistance in peroxidase and PerR mutants of Streptococcus pyogenes

Survival in aerobic conditions is critical to the pathogenicity of many bacteria. To investigate the means of aerotolerance and resistance to oxidative stress in the catalase-negative organism Streptococcus pyogenes, we used a genomics-based approach to identify and inactivate homologues of two peroxidase genes, encoding alkyl hydroperoxidase (ahpC) and glutathione peroxidase (gpoA). Single and double mutants survived as well as the wild type under aerobic conditions. However, they were more susceptible than the wild type to growth suppression by paraquat and cumene hydroperoxide. In addition, we show that S. pyogenes demonstrates an inducible peroxide resistance response when treated with sublethal doses of peroxide. This resistance response was intact in ahpC and gpoA mutants but not in mutants lacking PerR, a repressor of several genes including ahpC and catalase (katA) in Bacillus subtilis. Because our data indicate that these peroxidase genes are not essential for aerotolerance or induced resistance to peroxide stress in S. pyogenes, genes for a novel mechanism of managing peroxide stress may be regulated by PerR in streptococci.     

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.     

Challenging Xanthomonas campestris with low levels of arsenic mediates cross-protection against oxidant killing

Xanthomonas encounters highly toxic reactive oxygen species (ROS) from many sources, such as those generated by plants against invading bacteria, other soil bacteria and from aerobic respiration. Thus, conditions that alter intracellular ROS levels such as exposure to toxic metalloids would have profound effects on bacterial physiology. Here, we report that exposure of Xanthomonas campestris pv. phaseoli (Xp) to low levels of arsenic induces physiological cross-protection against killing by H(2)O(2) and organic hydroperoxide but not a superoxide generator. Cross-protection against H(2)O(2) and organic hydroperoxide toxicity was due to increased expression of genes encoding major peroxide-metabolizing enzymes such as alkyl hydroperoxide reductase (AhpC), catalase (KatA) and organic hydroperoxide resistance protein (Ohr). Arsenic-induced protection against H(2)O(2) and organic hydroperoxide requires the peroxide stress response regulators, OxyR and OhrR, respectively. Moreover, analyses of double mutants of the major H(2)O(2) and organic hyproperoxide-scavenging enzymes, Xp ahpC katA and Xp ahpC ohr, respectively, suggested the existence of unidentified OxyR- and OhrR-regulated genes that are involved in arsenic-induced resistance to H(2)O(2) and organic hyproperoxide killing in Xp. These arsenic-induced physiological alterations could play an important role in bacterial survival both in the soil environment and during plant-pathogen interactions.     

Oxidative stress response and its role in sensitivity to isoniazid in mycobacteria: characterization and inducibility of ahpC by peroxides in Mycobacterium smegmatis and lack of expression in M. aurum and M. tuberculosis.

Mycobacterium tuberculosis is a natural mutant with inactivated oxidative stress regulatory gene oxyR. This characteristic has been linked to the exquisite sensitivity of M. tuberculosis to isonicotinic acid hydrazide (INH). In the majority of mycobacteria tested, including M. tuberculosis, oxyR is divergently transcribed from ahpC, a gene encoding a homolog of the subunit of alkyl hydroperoxide reductase that carries out substrate peroxide reduction. Here we compared ahpC expression in Mycobacterium smegmatis, a mycobacterium less sensitive to INH, with that in two highly INH sensitive species, M. tuberculosis and Mycobacterium aurum. The ahpC gene of M. smegmatis was cloned and characterized, and the 5' ends of ahpC mRNA were mapped by S1 nuclease protection analysis. M. smegmatis AhpC and eight other polypeptides were inducible by exposure to H2O2 or organic peroxides, as determined by metabolic labeling and Western blot (immunoblot) analysis. In contrast, M. aurum displayed differential induction of only one 18-kDa polypeptide when exposed to organic peroxides. AhpC could not be detected in this organism by immunological means. AhpC was also below detection levels in M. tuberculosis H37Rv. These observations are consistent with the interpretation that ahpC expression and INH sensitivity are inversely correlated in the mycobacterial species tested. In further support of this conclusion, the presence of plasmid-borne ahpC reduced M. smegmatis susceptibility to INH. Interestingly, mutations in the intergenic region between oxyR and ahpC were identified and increased ahpC expression observed in deltakatG M. tuberculosis and Mycobacterium bovis INH(r) strains. We propose that mutations activating ahpC expression may contribute to the emergence of INH(r) strains.     

Analysis of growth phase regulated KatA and CatE and their physiological roles in determining hydrogen peroxide resistance in Agrobacterium tumefaciens

Agrobacterium tumefaciens possesses two catalases, a bifunctional catalase-peroxidase, KatA and a homologue of a growth phase regulated monofunctional catalase, CatE. In stationary phase cultures and in cultures entering stationary phase, total catalase activity increased 2-fold while peroxidase activity declined. katA and catE were found to be independently regulated in a growth phase dependent manner. KatA levels were highest during exponential phase and declined as cells entered stationary phase, while CatE was detectable at early exponential phase and increased during stationary phase. Only small increases in H2O2 resistance levels were detected as cells entering stationary phase. The katA mutant was more sensitive to H2O2 than the parental strain during both exponential and stationary phase. Inactivation of catE alone did not significantly change the level of H2O2 resistance. However, the katA catE double mutant was more sensitive to H2O2 during both exponential and stationary phase than either of the single catalase mutants. The data indicated that KatA plays the primary role and CatE acts synergistically in protecting A. tumefaciens from H2O2 toxicity during all phases of growth. Catalase-peroxidase activity (KatA) was required for full H2O2 resistance. The expression patterns of the two catalases in A. tumefaciens reflect their physiological roles in the protection against H2O2 toxicity, which are different from other bacteria.