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Journal of Bacteriology, January 2001, p. 773-778, Vol. 183, No. 2
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.2.773-778.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Genetic and Physiological Characterization of
ohr, Encoding a Protein Involved in Organic Hydroperoxide
Resistance in Pseudomonas aeruginosa
Urs A.
Ochsner,1,*
Daniel J.
Hassett,2 and
Michael
L.
Vasil1
Department of Microbiology, University of
Colorado Health Sciences Center, Denver, Colorado
80262,1 and Department of Molecular
Genetics, Biochemistry and Microbiology, University of Cincinnati
College of Medicine, Cincinnati, Ohio
45267-05242
Received 7 August 2000/Accepted 30 October 2000
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ABSTRACT |
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 
ohr 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.
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TEXT |
Aerobic respiration in
Pseudomonas aeruginosa leads to the production of toxic
metabolic byproducts, including the superoxide anion
(O2·
), hydrogen peroxide
(H2O2), the hydroxyl radical (HO·), and
organic hydroperoxides (OHPs). Sublethal levels of
O2· and H2O2 are
detoxified by two superoxide dismutases (Fe-SOD and Mn-SOD) and two
catalases (KatA and KatB), respectively (1, 3-7, 10).
Although much is known of systems that combat
O2· 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
ohr::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-ohr::Tc (Table
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 ohr::Tc mutant harboring either
pUCP22 or the complementing plasmid pOHR593 (Table 1) were analyzed for
their susceptibilities to oxidative stress-generating agents in disk
inhibition assays as described before (13). The
ohr::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.
1A). The susceptibilities to CHP of the ohr::Tc mutant
and wild-type bacteria were also compared in liquid medium containing 3 mM CHP. The corresponding kill curves indicated that
ohr::Tc mutant cells were hypersusceptible to CHP
(Fig. 1B). Furthermore, wild-type or ohr::Tc mutant
cells containing plasmid pOHR593 were more resistant to killing
by CHP than cells containing the pUCP22 vector control (Fig. 1B). The calculated rates during the exponential killing by CHP, expressed in
logarithmic units per hour, were 4.8 (PAO1/pUCP22), 7.4 (PAO1 ohr::Tc/pUCP22), and 2.5 (PAO1/pOHR593 and
PAO1 ohr::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 ohr mutant from killing by 1 mM CHP (Fig. 1C).
Furthermore, the contribution of the ohr gene product to the
overall decomposition of OHP was assessed in ahpCF mutant
background. The concentration of t-BHP diminished by >95%
within 30 min when added to a culture of a ahpCF mutant
but decreased only by 50% in a culture of ohr ahpCF
double mutant, indicating that the ohr gene product is involved in detoxifying OHPs (Fig. 1D). The ohr mutation
did not affect the susceptibility to H2O2 or
paraquat. In fact, the total enzymatic activities of catalase and of
SOD in ohr::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 mg1 (PAO1) and 72 ± 9 U mg1
(ohr::Tc). Total SOD activities were measured as
previously described (6) and were 98 ± 3.5 U
mg1 (PAO1) and 95 ± 2.4 U mg1
(ohr::Tc).
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FIG. 1.
Susceptibility of a P. aeruginosa ohr mutant
to oxidative stress agents. (A) Inhibition assay of PAO1/pUCP22, PAO1
ohr::Tc/pUCP22, and PAO1
ohr::Tc/pOHR593 growth around disks containing 10 µl of 1% (114 mM) t-BHP, 20% (1.25 M) CHP, 0.5% (145 mM) H2O2, or 0.5% (20 mM) paraquat. Normalized
amounts of cells (0.2 A600 unit, roughly 6 × 108 cells) grown in M9-0.4% glucose medium were mixed
with 3 ml of M9 top agarose and poured onto M9 agar. The zones of
growth inhibition were measured after incubation for 16 h at
37°C. The error bars represent the standard deviations of
quadruplicate assays. (B) Killing of P. aeruginosa by CHP.
PAO1/pUCP22, PAO1/pOHR593, PAO1 ohr::Tc/pUCP22,
and PAO1 ohr::Tc/pOHR593 were grown at 37°C
to mid-exponential phase (A600 = 0.5) and
then exposed to a sublethal concentration (300 µM) of CHP for 15 min,
after which CHP was added to a final concentration of 3 mM. The number
of viable cells was determined in serially diluted samples taken every
15 min postexposure. The experiment was performed separately four
times, and one representative set of data is shown. (C) Killing
of the ohr mutant in coculture with an ohr
overproducer strain. Mid-log-phase PAO1/pOHR593 and
ohr::Tc cells were washed, and 0.2 A600 unit of each ml1 (roughly
6 × 108 cells ml1) were cocultured in
fresh antibiotic-free M9 medium containing 1 mM CHP. Viable cells of
the two strains in the mixed culture were determined by plating on agar containing carbenicillin
(PAO1/pOHR593) or tetracycline (PAO1 ohr::Tc).
Also shown is the kill curve for ohr::Tc cells in a
monoculture at a comparable cell density
(A600 = 0.4). (D) Decomposition of
t-BHP. An ohr mutant in an ahpCF
background and its parental strain were grown to stationary phase in M9
medium and then exposed to 300 µM t-BHP. Hydroperoxide
concentrations were determined at 10-min intervals by mixing 100 µl
of supernatant with 900 µl of a freshly prepared solution of 25 mM
sulfuric acid, 100 µM xylenol orange, and 100 µM ferrous sulfate in
methanol-water (9:1), allowing the color to develop for 15 min, and
measuring the A560. A standard curve was
previously established using known concentrations of t-BHP
(data not shown).
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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. 
-Galactosidase activity was
detectable at low levels (<0.5 U mg1) 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.
2A). Up to fivefold greater
-galactosidase activity was detected in cells exposed to 0.10 or 0.30 mM t-BHP (Fig. 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· 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
-galactosidase activity was observed after 5 min. Then the response
was linear until 30 min postexposure (Fig. 2C), with a calculated
-galactosidase production rate of 0.20 U mg1
min1. After 30 min, -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.
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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 (A600 = 0.5), to
which increasing concentrations of CHP ranging from 0 to 10 mM were
added. The cells were shaken for 1 h, and the -galactosidase
reporter activities were determined as previously described
(13). (B) Induction by t-BHP. Cells were grown
as for panel A and exposed to increasing concentrations of
t-BHP prior to analysis of -galactosidase reporter
activity. (C) Time response curve of ohr-lacZ expression.
Mid-exponential-phase cultures (A600 = 0.5)
of PAO1/pPZ-Pohr-99 in M9 medium at 37°C were
exposed to 100 µM CHP, and -galactosidase activities were
determined in samples taken at the indicated time points. The values
are the means of triplicate experiments.
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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 ohr::Tc mutant than in
wild-type cells, and induction by low concentrations of CHP (10 and 30 µM) also resulted in higher activities (Fig.
3A). In the ahpCF::Gm
mutant, ohr expression was somewhat higher than in wild-type
cells, and in the ohr::Tc ahpCF::Gm
double mutant, ohr was expressed at higher levels than in
the single mutants, suggesting an additive effect (Fig. 3A). Similarly,
oxyR::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 ahpA::Tc
ahpB::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. 3B). Strikingly, the susceptibilities correlated
well with the observed ohr-lacZ activities, with the
ohr::Tc ahpCF::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.
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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
(ohr::Tc, ahpCF::Gm, and
oxyR::Gm), and double mutants
(ohr::Tc ahpCF::Gm and
ahpA::Tc ahpB::Gm) harboring
pPZ-Pohr-99 were grown in 15 ml of M9 medium at
37°C and split into 2.5-ml subcultures at mid-exponential phase
(A600 = 0.5), to which various
concentrations (0, 10, and 30 µM) of CHP were added. The cells were
harvested 30 min postexposure and -galactosidase activities were
determined. The error bars show the standard deviations of triplicate
experiments. (B) Growth inhibition of mutant strains by CHP. The PAO1
wild type and the indicated single and double mutants were assayed for
their susceptibilities to 20% (1.25 M) CHP in a disk inhibition assay
as described for Fig. 1.
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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. 4). The presence of 100 µM
paraquat did not affect the level of ohr mRNA. An identical
pattern of ohr expression was observed in oxyR
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. 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. 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
(G0 = 3.9 kcal mol1) and
contained two short direct repeats (TTAT) spaced 21 bp apart. Deletion
of the upstream TTAT element resulted in the loss of inducibility (Fig.
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.
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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 cells and from
oxyR::Gm mutant cells grown aerobically to
mid-exponential phase in M9 medium at 37°C. Parallel cultures were
treated with 100 µM CHP or 100 µM paraquat (PQ) as indicated.
Normalized samples (20 µg) of total RNA were used. Aliquots of the
same RNA batches were subjected to control hybridizations to a
riboprobe specific for the constitutively expressed omlA
gene (12), as shown in the boxed part at the bottom.
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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 or presence of 100 µM CHP.
Beside the indicated 35 and 10 promoter elements and the mapped
mRNA start site, elements potentially involved in ohr
regulation are shown, including two TTAT motifs (boxed) and a stem-loop
structure (dashed arrows). Also shown are the translational start of
ohr and the end of the coding sequence of the unrelated
upstream gene.
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ACKNOWLEDGMENTS |
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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FOOTNOTES |
*
Corresponding author. Mailing address:
Department of Microbiology, University of Colorado Health Sciences
Center, Denver, CO 80262. Phone: (303) 315-5093. Fax: (303)
315-6785. E-mail: urs.ochsner{at}UCHSC.edu.
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Journal of Bacteriology, January 2001, p. 773-778, Vol. 183, No. 2
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.2.773-778.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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