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.     

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.     

The katA catalase gene is regulated by OxyR in both free-living and symbiotic Sinorhizobium meliloti

The characterization of an oxyR insertion mutant provides evidences that katA, which encodes the unique H2O2-inducible HPII catalase, is regulated by OxyR not only in free-living Sinorhizobium meliloti but also in symbiotic S. meliloti. Moreover, oxyR is expressed independently of exogenous H2O2 and downregulates its own expression in S. meliloti.     

Characterization of the OxyR regulon of Neisseria gonorrhoeae

OxyR regulates the expression of the majority of H(2)O(2) responses in Gram-negative organisms. In a previous study we reported the OxyR-dependent derepression of catalase expression in the human pathogen Neisseria gonorrhoeae. In the present study we used microarray expression profiling of N. gonorrhoeae wild-type strain 1291 and an oxyR mutant strain to define the OxyR regulon. In addition to katA (encoding catalase), only one other locus displayed a greater than two-fold difference in expression in the wild type : oxyR comparison. This locus encodes an operon of two genes, a putative peroxiredoxin/glutaredoxin (Prx) and a putative glutathione oxidoreductase (Gor). Mutant strains were constructed in which each of these genes was inactivated. A previous biochemical study in Neisseria meningitidis had confirmed function of the glutaredoxin/peroxiredoxin. Assay of the wild-type 1291 cell free extract confirmed Gor activity, which was lost in the gor mutant strain. Phenotypic analysis of the prx mutant strain in H(2)O(2) killing assays revealed increased resistance, presumably due to upregulation of alternative defence mechanisms. The oxyR, prx and gor mutant strains were deficient in biofilm formation, and the oxyR and prx strains had decreased survival in cervical epithelial cells, indicating a key role for the OxyR regulon in these processes.     

Alkyl Hydroperoxide Reductase Is the Primary Scavenger of Endogenous Hydrogen Peroxide in Escherichia coli

Hydrogen peroxide is generated during aerobic metabolism and is capable of damaging critical biomolecules. However, mutants of Escherichia coli that are devoid of catalase typically exhibit no adverse phenotypes during growth in aerobic media. We discovered that catalase mutants retain the ability to rapidly scavenge H(2)O(2) whether it is formed internally or provided exogenously. Analysis of candidate genes revealed that the residual activity is due to alkyl hydroperoxide reductase (Ahp). Mutants that lack both Ahp and catalase could not scavenge H(2)O(2). These mutants excreted substantial amounts of H(2)O(2), and they grew poorly in air. Ahp is kinetically a more efficient scavenger of trace H(2)O(2) than is catalase and therefore is likely to be the primary scavenger of endogenous H(2)O(2). Accordingly, mutants that lack Ahp accumulated sufficient hydrogen peroxide to induce the OxyR regulon, whereas the OxyR regulon remained off in catalase mutants. Catalase still has an important role in wild-type cells, because the activity of Ahp is saturated at a low (10(-5) M) concentration of H(2)O(2). In contrast, catalase has a high K(m), and it therefore becomes the predominant scavenger when H(2)O(2) concentrations are high. This arrangement is reasonable because the cell cannot provide enough NADH for Ahp to rapidly degrade large amounts of H(2)O(2). In sum, E. coli does indeed generate substantial H(2)O(2), but damage is averted by the scavenging activity of Ahp.     

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.     

Increasing the oxidative stress response allows Escherichia coli to overcome inhibitory effects of condensed tannins

Tannins are plant-derived polyphenols with antimicrobial effects. The mechanism of tannin toxicity towards Escherichia coli was determined by using an extract from Acacia mearnsii (Black wattle) as a source of condensed tannins (proanthocyanidins). E. coli growth was inhibited by tannins only when tannins were exposed to oxygen. Tannins auto-oxidize, and substantial hydrogen peroxide was generated when they were added to aerobic media. The addition of exogenous catalase permitted growth in tannin medium. E. coli mutants that lacked HPI, the major catalase, were especially sensitive to tannins, while oxyR mutants that constitutively overexpress antioxidant enzymes were resistant. A tannin-resistant mutant was isolated in which a promoter-region point mutation increased the level of HPI by 10-fold. Our results indicate that wattle condensed tannins are toxic to E. coli in aerobic medium primarily because they generate H(2)O(2). The oxidative stress response helps E. coli strains to overcome their inhibitory effect.     

A protease-resistant catalase, KatA, released upon cell lysis during stationary phase is essential for aerobic survival of a Pseudomonas aeruginosa oxyR mutant at low cell densities

A Pseudomonas aeruginosa oxyR mutant was dramatically sensitive to H(2)O(2), despite possessing wild-type catalase activity. Oxygen-dependent oxyR phenotypes also included an inability to survive aerobic serial dilution in Luria broth and to resist aminoglycosides. Plating the oxyR mutant after serial dilution in its own spent culture supernatant, which contained the major catalase KatA, or under anaerobic conditions allowed for survival. KatA was resistant to sodium dodecyl sulfate, proteinase K, pepsin, trypsin, chymotrypsin and the neutrophil protease cathepsin G. When provided in trans and expressed constitutively, the OxyR-regulated genes katB, ahpB, and ahpCF could not restore both the serial dilution defect and H(2)O(2) resistance; only oxyR itself could do so. The aerobic dilution defect could be complemented, in part, by only ahpB and ahpCF, suggesting that the latter gene products could possess a catalase-like activity. Aerobic Luria broth was found to generate approximately 1.2 microM H(2)O(2) min(-1) via autoxidation, a level sufficient to kill serially diluted oxyR and oxyR katA bacteria and explain the molecular mechanism behind the aerobic serial dilution defect. Taken together, our results indicate that inactivation of OxyR renders P. aeruginosa exquisitely sensitive to both H(2)O(2) and aminoglycosides, which are clinically and environmentally important antimicrobials.