Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, The University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, UK1
Author for correspondence: Robert K. Poole. Tel: +44 114 222 4447. Fax: +44 114 272 8697. e-mail: r.poole{at}sheffield.ac.uk
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ABSTRACT |
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Keywords: ubiquinone, Escherichia coli, superoxide, peroxide, oxidative stress
Abbreviations: HRP, horseradish peroxidase; SOD, superoxide dismutase; UQ, ubiquinone; UQH2, ubiquinol (fully reduced form)
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INTRODUCTION |
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In the facultatively anaerobic bacterium Escherichia coli, UQ with an 8-unit isoprenoid side-chain is an essential component of the aerobic respiratory chain, whereas an alternative quinone, menaquinone or MQ, is more functional in anaerobic respiration (Gennis & Stewart, 1996 ). The most commonly shown scheme for the sequence of carriers in the aerobic respiratory chains consists of a single UQ pool located immediately upstream of the oxidases and downstream of the respiratory dehydrogenases (e.g. Gennis & Stewart, 1996
). However, this scheme may be oversimplified, since we recently demonstrated that UQ also functions as electron carrier between cytochromes b and the terminal oxidases by using dual-wavelength spectrophotometry to monitor cytochrome reduction levels in a UQ-deficient strain (ubiCA) (Søballe & Poole, 1998
). The ubiCA operon encodes the enzymes chorismate lyase and 4-hydroxybenzoate transferase for the first two committed steps of UQ biosynthesis (Søballe & Poole, 1999
).
There remains controversy as to whether respiratory-chain quinones and quinone-like compounds such as menadione or anthracycline antibiotics might actually stimulate superoxide production (see Afanasev et al., 1990 ). In E. coli, it was recently demonstrated that NADH dehydrogenase II is a major source of superoxide and hydrogen peroxide production by autoxidation of its reduced FAD cofactor (Messner & Imlay, 1999
). Membranes from a UQ- and MQ-deficient mutant produced more superoxide and peroxide; this was attributed to electrons backing up at NADH dehydrogenase II. Sulfite reductase was found to be a second flavin-containing autoxidizable electron-transport enzyme of E. coli (Messner & Imlay, 1999
). However, in bacteria, it is still not clear whether, in addition to its respiratory roles, UQH2 acts as an antioxidant or pro-oxidant. E. coli does contain a quinone oxidoreductase (Qor) (Thorn et al., 1995
), which may be important in maintaining UQ in its fully reduced state by 2-electron reductions, but the role of this enzyme is unknown. In Saccharomyces cerevisiae, a UQ-deficient strain showed enhanced sensitivity to products of autoxidized polyunsaturated fatty acids, indicating a protective role of UQH2 (Do et al., 1996
).
Some studies directed at determining the roles of quinones in bacterial physiology (e.g. Imlay, 1995 ) have used UQ-deficient mutants isolated after chemical mutagenesis, which are recognized to be unstable or leaky. Therefore, we have used a stable knockout mutant having a deletion and insertion at the junction of the ubiC and ubiA genes (Søballe & Poole, 1998
) and tested the hypothesis that UQ in E. coli acts as an antioxidant in the cells defence against oxygen-derived radicals and oxidative stress in the cytoplasmic membrane.
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METHODS |
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Viable counts were performed after treating mid-exponential cultures of wild-type and ubi cells grown in MOPS/glucose (40 mM) medium with 0·03% (w/v) H2O2 or phleomycin (10 µg ml-1). Heat resistance was analysed by aerobic shock treatment at 52 °C. A portion (0·5 ml) of a mid-exponential culture was transferred to 4·5 ml MOPS/glucose medium, preincubated at 52 °C, and shaken at this temperature. When exposed to CuSO4 (5 mM) or linolenic acid (1 mM) the strains were grown in LB medium with xylose (40 mM). Samples of all the treated cultures were taken at appropriate time intervals and a dilution series (10-1 to 10-6) was performed in 0·9% NaCl. A portion (20 µl) of each dilution was spotted on LB agar plates and incubated at 37 °C overnight. DTT sensitivity studies were performed as described by Goldman et al. (1996a) . Wild-type and ubi mutant cells were streaked on nutrient agar plates containing various concentrations of DTT (0·824 mM) and grown at 30 °C for 2 d. To obtain aerobic growth curves, three flasks were inoculated with 1% of an overnight culture of wild-type or ubiCA cells in LB with glucose (40 mM). To one of the flasks was added H2O2 to a final concentration of 2·5 mM, whereas to another was added H2O2 (2·5 mM) plus cysteine to a final concentration of 0·83 mM.
Preparation of membranes.
This was performed as described by Søballe & Poole (1998) . Protein concentrations were determined by the method of Markwell et al. (1978)
.
Superoxide anion detection.
The rate of production during reactions in vitro was measured as the superoxide dismutase (SOD)-sensitive rate of cytochrome c reduction (Imlay & Fridovich, 1991
) in a Beckman DU 650 spectrophotometer. The 1 ml reaction cuvette contained 50 mM potassium phosphate buffer, 20 µM cytochrome c, and wild-type or ubi membranes (200 µg protein ml-1, final concentration). Reduction of cytochrome c was initiated by the addition of 100 µM NADH (final concentration) and monitored spectrophotometrically at 550 nm. Duplicate reactions were performed with the addition of 250 units SOD (Sigma). The extent of cytochrome reduced was calculated using an absorption coefficient,
, of 21·0 mM-1 cm-1.
H2O2 production.
The amount of H2O2 produced by respiring membranes was measured using the scopoletin assay in a Hitachi F-2500 fluorescence spectrophotometer in time-scanning mode with excitation at 350 nm and emission at 460 nm (Loshen et al., 1971 ). The reaction mix (2·5 ml) in phosphate-buffered saline (PBS) buffer contained membranes (200 µg protein ml-1, wild-type or ubiCA) and 0·76 µM horseradish peroxidase (HRP). The fluorescent substrate scopoletin was added to a final concentration of 0·2 µM and the reaction was initiated by addition of 25 mM glucose. The amount of H2O2 generated was determined from a standard curve, in which the H2O2 concentration (00·15 M) was directly proportional to the quenching of scopoletin fluorescence. When indicated, water-soluble ubiquinone (UQ-1 or UQ-2, purchased from Sigma) was added to a final concentration of 0·22·0 µM (from a 10 mM stock in 1:1 ethanol/water) before the reaction was initiated by the addition of 5 mM glycerol.
Determination of intracellular H2O2 production was based on the assumption that free diffusion of H2O2 through the cell membrane allows an equilibrium to occur after about 15 min (Gonzalez-Flecha & Demple, 1994 ). Cells from 2·5 ml of a culture of the wild-type or ubiCA strain, grown in LB to the exponential phase (50 Klett units), were harvested and resuspended in 25 ml phosphate-buffered saline. Samples were taken at 5 min intervals and spun briefly in a microfuge before assaying the H2O2 content of the supernatant using the above fluorometric assay.
Catalase assay.
Cell pellets were washed in 100 mM potassium phosphate buffer (pH 7·0) and disrupted by sonication (five periods of 1 min each). Cell debris and unbroken cells were removed by centrifugation in a microfuge for 30 min at 13000 r.p.m. The protein concentration of the supernatant extract was determined by the method of Markwell et al. (1978) . The catalase activity was measured by monitoring
A at 240 nm in time-scanning mode in a Beckman DU 650 spectrophotometer (Gonzalez-Flecha & Demple, 1994
). The 1 ml UV-cuvette contained 510 µg supernatant protein in potassium phosphate buffer and the reaction was initiated by adding H2O2 to a final concentration of 5 mM. The initial rate of decomposition of H2O2 was determined using an
240 of 43·6x10-3 mM-1 cm-1. One unit of catalase activity is defined as the change in H2O2 concentration (mM) per min.
ß-Galactosidase assays.
Assays were carried out at room temperature as described before (Søballe & Poole, 1997 ). Each culture was assayed in triplicate; results were confirmed in at least two independent experiments.
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RESULTS AND DISCUSSION |
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UQ limits accumulation of H2O2 in vitro
H2O2 production in membranes from wild-type and ubiCA cells was measured using a HRP-dependent assay, in which the quenching of the fluorescent substrate scopoletin is directly proportional to the production of H2O2 (not shown). The initial rate of H2O2 production was 37% lower in the ubiCA mutant compared to the wild-type (Table 2). However, in the ubiCA membranes, all the available scopoletin was quenched within 6 min (Fig. 2b
), whereas the substrate was only 50% quenched in the wild-type strain in the same time period (Fig. 2a
); overall, there was a twofold increase in the accumulation of H2O2 in the ubiCA mutant under these assay conditions (Fig. 2
, Table 2
).
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Effects of UQ deficiency on oxidative stress in vivo: peroxide levels, catalase activity and expression of katG and sodA
The above results demonstrate that isolated membranes from a UQ-deficient strain accumulate substantially more superoxide and peroxide than do membranes containing a normal complement of UQ. To determine if this has physiological consequences for growth, or whether the additional flux of reactive oxygen species can be accommodated by cytoplasmic SOD and catalase activities, we determined the effects of the ubiCA knockout in intact cells.
The steady-state intracellular H2O2 concentration, which was determined after allowing diffusion of H2O2 into PBS buffer, was increased 1·8-fold in the ubiCA mutant (Table 2). These results mimic the
and H2O2 measurements in membranes and support a protective role of UQ in oxygen radical scavenging.
We also assayed levels of total catalase activity in unfractionated cell extracts. Rates of H2O2 conversion were twofold higher in the ubiCA cells compared to wild-type levels (Table 2).
E. coli possesses a bifunctional catalase-peroxidase (HPI, KatG) and a second monofunctional catalase, HPII. HPI, together with an alkyl hydroperoxide reductase, many other proteins with roles in resisting peroxide stress, and a small untranslated RNA are regulated by the OxyR protein in response to peroxide (Demple, 1991 ; Rosner & Storz, 1997
). The intracellular concentration of H2O2 in E. coli is normally maintained around 0·10·2 µM during aerobic growth and catalase activity is regulated to compensate for changes in H2O2 production rates (Gonzalez-Flecha & Demple, 1995
). Thus, katG transcription is a useful measure of intracellular peroxide levels (Gonzalez-Flecha & Demple, 1995
). Table 3
shows that expression of
(katGlacZ) increased 2·2-fold in a wild-type strain when induced with H2O2 under our experimental conditions. To seek confirmation that the presence of UQ affects H2O2 levels in vivo, the effect of introducing the ubiCA allele into the
(katGlacZ) strain was studied. The basal level of katG expression was 2·1-fold higher in the ubi background, but induction with H2O2 still increased the expression a further 1·3-fold (Table 3
).
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A ubiCA mutant is hypersensitive to mediators of oxidative stress
In view of the elevated levels of superoxide and peroxide measured in ubiCA cells, the ability of cells to tolerate additional exogenous oxidative stress mediated by H2O2 or CuSO4 was examined (Fig. 4). The ubiCA mutant appeared to be hypersensitive to treatment with 0·03% H2O2 and its viability was reduced about 16-fold relative to the wild-type strain after 1 h (Fig. 4a
).
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Copper ions (Cu2+/Cu1+) participate in a redox-cycle, resulting in the generation of superoxide anion. They have also been reported to catalyse the conversion of H2O2 to ·OH in vitro (Kimura & Nishioka, 1997 ). When treated with CuSO4, the viability of the ubiCA mutant was reduced 10-fold relative to the wild-type strain after 90 min (Fig. 4b
). Likewise, the viability of the ubiG mutant, HW271, was reduced 4·4-fold after 30 min compared to its isogenic wild-type (not shown). These results indicate the importance of the presence of UQ for protection against oxidative stresses generated by H2O2 or CuSO4. Sensitivity to oxidative stress in a UQ-deficient mutant of the fission yeast Schizosaccharomyces pombe has also been reported recently (Suzuki et al., 1997
).
Cysteine is an amino acid with antioxidant properties due to the presence of the thiol group (Suzuki et al., 1997 ). Sensitivity of the ubiCA mutant to H2O2 (2·5 mM) could be abolished by the addition of this compound to the growing cells (Fig. 5b
). In contrast, the presence of H2O2 or cysteine did not affect growth of the wild-type cells (Fig. 5a
). Likewise, cysteine or glutathione restores growth of a UQ-deficient (dps) mutant of fission yeast in minimal medium (Suzuki et al., 1997
).
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The effects of heat shock at 52 °C on the viability of wild-type and ubiCA strains are shown in Fig. 6(c). The ubiCA mutant proved to be extremely resistant to the lethal effects of heat shock. After 48 min at 52 °C the surviving fraction of the ubiCA mutant was reduced 4·5-fold, whereas that of the wild-type was reduced about 170-fold (Fig. 6c
).
The ubiCA mutation causes sensitivity to DTT
Like ubi mutants, mutants (cydAB or cydDC) unable to synthesize the quinol oxidase, cytochrome bd, have a pleiotropic phenotype, including sensitivity to H2O2 (Wall et al., 1992 ; Goldman et al., 1996b
). In addition, certain reducing compounds can suppress the temperature-sensitive phenotype of both cydAB and cydDC mutants. DTT was found to inhibit Cyd- mutants at 3 and 10 mM, concentrations at which the isogenic wild-type strains were resistant (Goldman et al., 1996a
, b
). A firm explanation for these effects is not available, but it has been suggested (Goldman et al., 1996a
) that the thiol periplasmic environment of a cydC mutant is oxidized in comparison to the wild-type strain. Using the same assay as Goldman et al. (1996a)
, we found that the ubiCA mutant was sensitive to 8 mM DTT, whereas the wild-type was resistant to DTT at this concentration (not shown). Thiol hypersensitivity has also been reported for ubi mutants isolated after chemical mutagenesis (Zeng et al., 1998
). Thus the DTT sensitivity is not due to the ability to assemble cytochrome bd per se, but to respiratory chain function. We hypothesize that ubi mutants are sensitive to DTT because of the requirement for the respiratory chain to oxidize the essential redox-active CXXC motif of DsbB. This membrane protein in turn oxidizes the active-site cysteines in DsbA, the disulfide bond formation factor in the periplasm (Kobayashi & Ito, 1999
). Indeed, ubiA menA mutants and hemA mutants do accumulate a reduced form of DsbA (Kobayashi et al., 1997
) and, intriguingly, dsbA/dsbB mutants are sensitive to DTT (Missiakas & Raina, 1997
). These findings and the work of Bader et al. (1999)
clearly link the function of the respiratory chain with maintenance of an appropriate redox environment in the periplasm.
Conclusions
The importance of UQ as a component of E. coli respiratory chains terminated by oxygen and nitrate is well established. The present studies using a defined knockout allele of ubiCA clearly demonstrate additional roles for UQ in limiting the accumulation of superoxide and peroxide. This contradicts the view that quinones might constitute an important source of superoxide by virtue of the spontaneous autoxidation of the radical form (for references, see Afanasev et al., 1990 ). Skulachev (1997)
has suggested that respiration that does not involve the Q-cycle a mechanism that can produce long-lived semiquinone (QH·) might serve as a defence against reactive oxygen species. Further protection from superoxide production in functioning respiratory chains might be afforded by high rates of electron transfer to the terminal oxidase, thereby avoiding excessive electronegativity of respiratory carriers (Papa et al., 1997
) and the potential for undesirable single-electron donation to oxygen. Both these conditions appear to be met in E. coli, in which a Q cycle need not be invoked (Poole & Ingledew, 1987
) and in which operation of cytochrome bd, which is not a proton pump, allows very rapid rates of respiration with a phenomenally high apparent affinity for oxygen (Km about 5 nM; Dmello et al., 1996
). Both cyanide (Imlay, 1995
) and quinone deficiency (Messner & Imlay, 1999
) increase superoxide production in membrane vesicles, possibly by electron leakage from an upstream component such as NADH dehydrogenase II.
The complex phenotype of ubi mutants is not surprising and arises from at least two important aspects of UQ function. First, UQ is able to limit accumulation of superoxide and peroxide due to its ability to rapidly abstract electrons from upstream dehydrogenases and transfer them to the oxidases. Second, reduced UQ is able to react with superoxide in vitro (Nakayama et al., 1997 ) and functions as an antioxidant in scavenging oxygen radicals such as perferryls or lipid peroxyl radicals (Ernster & Dallner, 1995
). These studies do not really distinguish between these two mechanisms. Consequent damage in ubi mutants by lipid peroxidation may therefore explain the oxidative-stress-sensitive phenotype and poor growth, as well as the changes in membrane permeability and heat tolerance. Finally, other phenotypes such as sensitivity to DTT are more likely a consequence of impaired respiratory chain function.
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ACKNOWLEDGEMENTS |
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Received 11 August 1999;
revised 13 December 1999;
accepted 15 December 1999.