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J Bacteriol. Jan 2001; 183(2): 773–778.

Genetic and Physiological Characterization of ohr, Encoding a Protein Involved in Organic Hydroperoxide Resistance in Pseudomonas aeruginosa


The ohr (organic hydroperoxide resistance) gene product of Pseudomonas aeruginosa was essential for optimal resistance to organic hydroperoxides (OHPs) but not to hydrogen peroxide or paraquat. A Δohr mutant was hypersusceptible to OHPs in disk inhibition assays and showed enhanced killing by OHPs in liquid culture. The ohr gene product was demonstrated to contribute to the decomposition of OHPs. Transcription of ohr was induced up to 15-fold upon exposure to OHPs, and this induction was independent of OxyR. Somewhat enhanced ohr-lacZ activity was detected in mutant strains affected in ohr, ahpC, and oxyR, and this phenotype correlated with hypersusceptibility to OHPs, suggesting overlapping or compensatory functions of the ohr and ahpC gene products. A single transcriptional start site for ohr was determined, and ohr transcripts were abundant in cells treated with a sublethal dose of OHPs but not in cells treated with paraquat. An 84-bp portion upstream of the ohr mRNA start site was sufficient for ohr induction by OHPs. Thus, the ohr gene appears to encode an antioxidant enzyme that is not part of the OxyR regulon yet is specifically induced by OHPs.

Aerobic respiration in Pseudomonas aeruginosa leads to the production of toxic metabolic byproducts, including the superoxide anion (O2[center dot]), hydrogen peroxide (H2O2), the hydroxyl radical (HO[center dot]), and organic hydroperoxides (OHPs). Sublethal levels of O2[center dot] and H2O2 are detoxified by two superoxide dismutases (Fe-SOD and Mn-SOD) and two catalases (KatA and KatB), respectively (1, 37, 10). Although much is known of systems that combat O2[center dot] and H2O2 stress in P. aeruginosa and of regulators that facilitate such control (7, 10), little is known of those involved in resistance to OHPs. Bacterial OHP resistance is mediated by alkyl hydroperoxide reductases (Ahps) that detoxify the peroxide by reducing it to an alcohol (9, 21). In Salmonella enterica serovar Typhimurium, activity is mediated by a dimer composed of AhpC and AhpF subunits (14). Recently, a novel gene from Xanthomonas campestris restored wild-type resistance to t-butyl hydroperoxide (t-BHP) in an ahpCF mutant of Escherichia coli (11). This gene was designated ohr for OHP resistance and, strikingly, showed no significant homology to any known bacterial genes. In this study, we describe the ohr locus in P. aeruginosa, specifically focusing on its genetic regulation and role in OHP resistance.

Identification and cloning of a P. aeruginosa ohr homolog.

The amino acid sequence of the X. campestris Ohr protein was used to search the complete P. aeruginosa genome (http://www.pseudomonas.com). An ohr-like gene (Pa-ohr), composed of 426 bp encoding a predicted 14.5-kDa hydrophilic protein, was identified at bases 3203997 to 3204425. Pa-Ohr was 68% identical to the Ohr protein of X. campestris (GenBank accession no. AF036166) and 62% identical to an unknown protein from Acinetobacter calcoaceticus (GenBank accession no. Y09102) (data not shown). Using the available sequence information of the unfinished microbial genome projects, additional putative homologs of Ohr with at least 50% identity to Pa-Ohr were detected in several eubacteria, including Pseudomonas putida, Legionella pneumophila, Bacillus subtilis, Deinococcus radiodurans, Caulobacter crescentus, Shewanella putrefaciens, Vibrio cholerae, Enterococcus faecalis, and Staphylococcus aureus. Each of these proteins possessed two conserved cysteine residues that are postulated to participate in the proper function of Ohr (11).

Enhanced sensitivity of an ohr mutant to OHPs.

To determine the physiological role of Ohr in P. aeruginosa, a Δohr::Tc mutant was constructed from strain PAO1. Replacement of the ohr gene by a tetracycline resistance cassette was achieved through a biparental mating using E. coli SM10 harboring pEX100T-Δohr::Tc (Table (Table1),1), which allowed sucrose counterselection (16, 18). The deletion of the ohr locus ranged from 7 bp upstream of the ohr start codon to 80 bp downstream of the stop codon and was verified by Southern blotting (data not shown). Wild-type bacteria containing the control plasmid pUCP22 (23) and the Δohr::Tc mutant harboring either pUCP22 or the complementing plasmid pOHR593 (Table (Table1)1) were analyzed for their susceptibilities to oxidative stress-generating agents in disk inhibition assays as described before (13). The Δohr::Tc mutant was hypersusceptible to the organic peroxides t-BHP and cumene hydroperoxide (CHP) but not to H2O2 or paraquat, and this phenotype was fully complemented by plasmid pOHR593 containing the entire ohr gene (Fig. (Fig.1A).1A). The susceptibilities to CHP of the Δohr::Tc mutant and wild-type bacteria were also compared in liquid medium containing 3 mM CHP. The corresponding kill curves indicated that Δohr::Tc mutant cells were hypersusceptible to CHP (Fig. (Fig.1B).1B). Furthermore, wild-type or Δohr::Tc mutant cells containing plasmid pOHR593 were more resistant to killing by CHP than cells containing the pUCP22 vector control (Fig. (Fig.1B).1B). The calculated rates during the exponential killing by CHP, expressed in logarithmic units per hour, were 4.8 (PAO1/pUCP22), 7.4 (PAO1 Δohr::Tc/pUCP22), and 2.5 (PAO1/pOHR593 and PAO1 Δohr::Tc/pOHR593). These data strongly suggest a protective role for the ohr gene product in the defense against oxidative stress specifically imposed by OHPs. This hypothesis is supported by the finding that an ohr overproducer strain (PAO1/pOHR593) partially protected a cocultured Δohr mutant from killing by 1 mM CHP (Fig. (Fig.1C).1C). Furthermore, the contribution of the ohr gene product to the overall decomposition of OHP was assessed in ΔahpCF mutant background. The concentration of t-BHP diminished by >95% within 30 min when added to a culture of a ΔahpCF mutant but decreased only by 50% in a culture of Δohr ΔahpCF double mutant, indicating that the ohr gene product is involved in detoxifying OHPs (Fig. (Fig.1D).1D). The Δohr mutation did not affect the susceptibility to H2O2 or paraquat. In fact, the total enzymatic activities of catalase and of SOD in Δohr::Tc mutant cells were identical to the levels measured in wild-type cells. Total catalase activities were determined as described elsewhere (3) and were 71 ± 12 U mg−1 (PAO1) and 72 ± 9 U mg−1ohr::Tc). Total SOD activities were measured as previously described (6) and were 98 ± 3.5 U mg−1 (PAO1) and 95 ± 2.4 U mg−1ohr::Tc).

Strains and plasmids used in this study
FIG. 1
Susceptibility of a P. aeruginosa ohr mutant to oxidative stress agents. (A) Inhibition assay of PAO1/pUCP22, PAO1 Δohr::Tc/pUCP22, and PAO1 Δohr::Tc/pOHR593 growth around disks containing 10 μl of 1% (114 mM) t-BHP, 20% ...

Inducible expression of ohr.

Expression of ohr was studied by using plasmid pPZ-Pohr-99 containing a transcriptional fusion of the ohr promoter to the promoterless lacZ reporter gene. β-Galactosidase activity was detectable at low levels (<0.5 U mg−1) in cell extracts from untreated PAO1/pPZ-Pohr-99 cultures. This basal ohr-lacZ expression was only marginally affected by the carbon source added to M9-based medium. The presence of 1% citrate resulted in a twofold increase, while 1% corn oil resulted in a twofold decrease compared to reporter activity measured in M9-1% glucose cultures (data not shown). Reporter activity increased up to 15-fold upon treatment of bacteria with 0.03 to 0.30 mM CHP (Fig. (Fig.2A).2A). Up to fivefold greater β-galactosidase activity was detected in cells exposed to 0.10 or 0.30 mM t-BHP (Fig. (Fig.2B).2B). Due to cell death, CHP or t-BHP concentrations equal to or higher than 1 mM did not result in such a response (data not shown). In contrast, the addition of H2O2 (1 mM) and the O2[center dot] generating compound paraquat (0.35 mM), both of which lead to induction of other antioxidant genes in P. aeruginosa, such as katB, ahpB, and ahpCF, at these sublethal concentrations (13), or the presence of a high iron concentration [100 μM Fe(III)], did not result in higher ohr-lacZ expression (data not shown). Thus, ohr expression appeared to respond specifically to OHPs. The reason for the observed difference in maximal ohr induction upon exposure to either CHP or t-BHP is unclear. It may be a consequence of different uptake efficiencies or reactivities of these two compounds. In fact, CHP is poorly water soluble and therefore may readily bind to the cell surface, while the more water-soluble t-BHP remains dissolved in the medium. The expression of ohr in response to OHP exposure was further studied by analyzing extracts of PAO1/pPZ-Pohr-99 at various time points after the addition of 100 μM CHP to mid-exponential-phase cultures (A600 = 0.5). A roughly twofold increase in β-galactosidase activity was observed after 5 min. Then the response was linear until 30 min postexposure (Fig. (Fig.2C),2C), with a calculated β-galactosidase production rate of 0.20 U mg−1 min−1. After 30 min, β-galactosidase activity reached a plateau, presumably due to a new steady-state level of ohr expression or due to complete detoxification of CHP, as shown above.

FIG. 2
Response of ohr-lacZ expression to OHPs. (A) Induction by CHP. P. aeruginosa PAO1 harboring pPZ-Pohr-99 was grown aerobically in 50 ml of M9–0.4% glucose medium at 37°C and split into 2.5-ml subcultures at mid-exponential phase ...

Role of Ohr in compensating for an absence of Ahps.

Since ohr expression was inducible by OHPs, Ohr may function similarly to Ahps. Therefore, expression of ohr was monitored using pPZ-Pohr-99 in various mutant strains affected in single or multiple ahp genes that were described earlier (13). Basal expression of ohr was threefold higher in the Δohr::Tc mutant than in wild-type cells, and induction by low concentrations of CHP (10 and 30 μM) also resulted in higher activities (Fig. (Fig.3A).3A). In the ΔahpCF::Gm mutant, ohr expression was somewhat higher than in wild-type cells, and in the Δohr::Tc ΔahpCF::Gm double mutant, ohr was expressed at higher levels than in the single mutants, suggesting an additive effect (Fig. (Fig.3A).3A). Similarly, ΔoxyR::Gm mutant cells, which are affected in the production of at least AhpB, AhpC-AhpF, and KatB-AnkB (13), expressed ohr at higher levels than wild-type cells. In contrast, a ΔahpA::Tc ΔahpB::Gm double mutant had a somewhat higher basal expression of ohr but did not respond to CHP to the extent observed in the other mutants. The susceptibilities of these mutant strains to CHP were determined using a disk inhibition assay as described above (Fig. (Fig.3B).3B). Strikingly, the susceptibilities correlated well with the observed ohr-lacZ activities, with the Δohr::Tc ΔahpCF::Gm double mutant being the most susceptible. Taken together, the data suggest overlapping functions of Ohr and AhpC-AhpF. However, the AhpA, AhpB, AhpC-AhpF, and Ohr proteins of P. aeruginosa have not yet been characterized biochemically, especially regarding their potential for use of multiple substrates. Some of the observed ohr expression levels and the CHP susceptibilities of the mutant strains may be caused by compensatory induction of alternate antioxidant genes, as has been described for other bacteria (2, 15) and for P. aeruginosa (13). Specifically, mutants affected in either ohr, ahpC, or both exhibited higher ohr expression and induction levels, presumably due to higher oxidative stress in these backgrounds, while the ahpA ahpB double mutant showed lower ohr induction, since it may respond to this defect with a compensatory upregulation of ahpC-ahpF.

FIG. 3
Role of ohr in mutant strains affected in Ahp reductase genes. (A) Expression of ohr-lacZ in various mutant strains. Cultures of PAO1, single mutants (Δohr::Tc, ΔahpCF::Gm, and ΔoxyR::Gm), and double mutants (Δohr::Tc Δ ...

A genetic analysis of the ohr promoter.

The transcriptional start site of ohr was mapped to a single T at bp −56 relative to the ATG translational start codon. Specific ohr transcripts were detected using an ohr riboprobe covering the 5′ end of the ohr transcript in RNase protection assays as described elsewhere (13). Total RNA of untreated wild-type cells contained barely detectable levels of ohr mRNA, while a dramatic increase in ohr mRNA was detected in cells that had been exposed to 100 μM CHP for 30 min (Fig. (Fig.4).4). The presence of 100 μM paraquat did not affect the level of ohr mRNA. An identical pattern of ohr expression was observed in ΔoxyR mutant cells. These findings indicate that ohr induction occurred at the transcriptional level and was independent of OxyR, which is in agreement with the results obtained using ohr-lacZ reporter fusions. To determine whether other known regulators of the P. aeruginosa oxidative stress machinery were involved in ohr regulation, ohr-lacZ expression was also measured in mutant strains affected in soxR, rpoS, and rhlR lasR. In these cases, induction by 100 or 300 μM CHP was not affected (data not shown). Therefore, ohr gene expression appeared to be upregulated by a novel mechanism involving a yet-to-be-identified positive or negative factor responding specifically to OHPs. As a first step toward the identification of such a regulatory factor, the ohr promoter was analyzed using a series of pPZ-Pohr-lacZ fusions containing systematically shorter upstream sequences (Fig. (Fig.5).5). A sequence of at least 84 bp upstream of the mapped transcriptional start site was required for full induction of the ohr promoter (pPZ-Pohr-89). A 6-bp-shorter promoter fragment (pPZ-Pohr-83) resulted in normal basal expression but responded only marginally to the presence of CHP. Constructs containing only 59 bp or less of upstream sequence did not result in any detectable ohr-lacZ expression, although they contained the −35 and −10 promoter elements (Fig. (Fig.5).5). These results demonstrate that the 84-bp sequence located upstream of the mRNA start site was essential for the stress response. This sequence contained inverted repeats (CAAATC-N7-GATTTG) with the potential to form a stem-loop structure (ΔG0 = −3.9 kcal mol−1) and contained two short direct repeats (TTAT) spaced 21 bp apart. Deletion of the upstream TTAT element resulted in the loss of inducibility (Fig. (Fig.5).5). The spacing of the two TTAT motifs suggested a location on the same face of the DNA, spaced two turns apart, and may represent the target site of a positive activator of ohr. Similar cis-acting regulatory elements have been demonstrated to play a crucial role in antioxidant gene activation by the well-characterized OxyR protein (13, 20, 22). We therefore postulate the existence of a trans-acting regulatory protein involved in ohr regulation, and future approaches to identify such a regulator could include the screening of a mutant library for the lack of ohr-lacZ induction or affinity purification of a putative regulator on its immobilized target sequence. Such studies are currently under way.

FIG. 4
RNase protection assay of ohr mRNA. A riboprobe specific for the 5′ portion of ohr (ohr rp) was generated by runoff in vitro transcription from the T7 promoter on pCRII-ohr-220 and used to detect ohr transcripts. RNA was isolated from wild-type ...
FIG. 5
Analysis of the ohr promoter. A series of pPZ-Pohr-lacZ fusions containing the indicated portions of the ohr promoter and upstream sequence was constructed. Their reporter activities were measured in triplicate cultures of wild-type cells in the absence ...


This work was supported by a grant from the National Institute of Allergy and Infectious Diseases (AI-15490) to M.L.V. and was supported in part by a Public Health Service grant (AI-40541) to D.J.H. and a Cystic Fibrosis Grant (HASSET97PO) to D.J.H.


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