1 Institute of Biological Sciences, University of Tsukuba, Ibaraki, 305-8577, Japan
2 Institute of Community Medicine, University of Tsukuba, Ibaraki, 305-8577, Japan
3 Institute of Basic Medical Sciences, University of Tsukuba, Ibaraki, 305-8577, Japan
Correspondence
Toshiko Ohta
tohta{at}sakura.cc.tsukuba.ac.jp
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ABSTRACT |
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Supplementary data showing that staphylococcal QorA does not predominantly catalyse the two-electron reduction of PQ is available at http://mic.sgmjournals.org
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INTRODUCTION |
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The same kinds of ROS are also produced under aerobic conditions and can damage essential biomolecules such as DNA, protein and lipids. To confront these ROS, S. aureus produces antioxidant enzymes including superoxide dismutase (SOD) (Clements et al., 1999; Valderas & Hart, 2001
) and catalase (Sanz et al., 2000
). SOD is a major important enzyme in scavenging superoxide anions, which are reduced to hydrogen peroxide. Subsequently, hydrogen peroxide is broken down to water by catalase. The existence of the other oxidative stress responsive proteins, e.g., alkyl hydroperoxide reductase and metallo-regulated gene A (Horsburgh et al., 2001a
) was reported, and a putative quinone oxidoreductase gene was recently discovered in the whole genome sequence of S. aureus (Kuroda et al., 2001
).
Although membrane-bound quinone oxidoreductases (Qor) are well recognized in bacteria, few examples of soluble Qors are yet characterized. The membrane-bound Qors such as NADH-ubiquinone oxidoreductase are involved in a respiratory chain (Friedrich, 1998). This enzyme is NADH-dependent, requires either FMN or FAD and may also possess ironsulfur clusters as cofactors. The soluble Qors are widespread in both prokaryotes and eukaryotes (Persson et al., 1994
). The eukaryotic soluble Qors are further divided into two functionally distinct groups, DT-diaphorase and
-Crystallin. DT-diaphorase is a flavo-enzyme that catalyses an NAD(P)H-dependent two-electron reduction of quinone. This reduction process, which produces hydroquinone, is suggested to involve the detoxification of quinone (Ernster, 1987
). On the other hand,
-Crystallin is a non-flavo-enzyme and catalyses the NADPH-dependent one-electron reduction of quinone to produce the semiquinone radical. This radical is readily oxidized back to quinone in the presence of oxygen, resulting in the generation of superoxide anions. Rao et al. (1992)
suggested the involvement of
-Crystallin in oxidative stress response by mediating hexose monophosphate shunt activity through NADPH homeostasis in the guinea pig. In the plant Arabidopsis thaliana, three
-Crystallin homologues were identified as genes that complemented the sensitive phenotype to thiol-oxidizing drug diamide in a yeast yap1 mutant that was hypersensitive to oxidative stress (Babiychuk et al., 1995
). Mano et al. (2000)
reported that one of the three
-Crystallin homologous genes, Arabidopsis P1, encoded an NADPH: diamide oxidoreductase and demonstrated that the activity was similar to
-Crystallin. Although these reports suggest that DT-diaphorese and
-Crystallin play important roles in the oxidative stress response, the catalytic activity and gene expression profile of the bacterial soluble Qors are still unknown.
Because S. aureus is equipped with two SODs (SodA and SodM) (Clements et al., 1999; Valderas & Hart, 2001
) and shows a relatively high catalase activity (Yumoto et al., 1999
), this organism has a high ability to conquer oxidative stresses. These features of S. aureus are utilized to infect its host. Therefore, it is important to understand oxidative stress response factors of S. aureus and if possible to find a clue to prevent infection by this pathogen. Therefore we paid attention to SA1989, qor homologue, which was identified by N315 genome sequencing analysis (Kuroda et al., 2001
). We examined if the product of the qor homologue has Qor activity, and if staphylococcal Qor catalyses a one-electron or a two-electron reduction of PQ (9,10-phenanthrenequinone). Here, we have reported the manner of transcription of the putative qor and its catalytic activity as a Qor. The expression of qor was enhanced under the oxidative stress condition. Moreover, the recombinant protein had an activity as an NADPH-dependent reductase, and superoxide anion generation during PQ reduction. Therefore, it indicates that staphylococcal Qor has a similar activity to
-Crystallin.
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METHODS |
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Materials.
2-Methyl-1,4-naphthoquinone (menadione) was obtained from Wako and 9,10-phenanthrenequinone (PQ) was purchased from Sigma. 9,10-Diacetoxyphenanthrene (DAP) was synthesized as follows: PQ (0·1 g) was dissolved in 15 ml tetrahydrofuran and then mixed with Zn dust (50 mg), followed by 0·5 ml acetic anhydride, 1 drop water and 3 drops triethylamine. The mixture was stirred at room temperature for 20 min, then an additional 50 mg Zn dust, 1 drop water and 3 drops triethylamine were added and the mixture was heated under reflux for 1 h. After cooling, the mixture was diluted with chloroform (40 ml), washed with water, saturated with NaHCO3 and then dried over MgSO4. The solvent was removed in vacuo to leave a pale yellow solid (0·13 g). The product was recrystallized in benzene and a white solid (45 mg) obtained was identified as DAP by 1H- and 13C-NMR, and HPLC/electrospray ionization-mass spectrometry. Acetylated cytochrome C was synthesized as reported previously (Kumagai et al., 2000).
Preparation of total RNA and Northern blotting.
A 1 ml aliquot of overnight culture of S. aureus was inoculated into 100 ml M9 medium. The exponential-phase culture (OD600 0·50·6) was subjected to appropriate shocks. The shocked culture was harvested by centrifugation at 10 000 g and 4 °C for 1 min. Cells were resuspended in 700 µl 10 mM Tris/HCl (pH 8·0), 1 mM EDTA (pH 8·0) containing 10 µg lysostaphin, and were incubated at 37 °C for 2 min. Then 1 % SDS, 0·3 M sodium acetate (pH 4·8) and an equal volume of phenol (pH 4·8) were added to the cell suspension, followed by five cycles of freezing and thawing (at -80 °C and 65 °C). After centrifugation at 10 000 g for 10 min at 4 °C, the supernatant was ethanol precipitated at -80 °C for 30 min. After washing with 70 % ethanol, the pellet was resuspended in 10 mM Tris/HCl (pH 8·0) and 1 mM EDTA (pH 8·0). The obtained total RNA was used for Northern blot analyses and primer extensions. The Northern blots were performed as follows. Total RNA (10 µg) was loaded onto 1 % formaldehyde agarose gels and electrophoresed in 1x MOPS buffer. The separated RNAs were transferred to Hybond-N+ (Amersham Biosciences). The blot was pre-hybridized with rapid-hybri buffer (Amersham Biosciences) at 65 °C for 15 min and then a 32P-labelled DNA probe was added for hybridization at 65 °C for 2 h. The hybridized blot was washed with 2x SSC/0·1 % SDS at room temperature for 15 min, and with 1x SSC/0·1 % SDS and 0·1x SSC/0·1 % SDS at 65 °C for 15 min each. The blot was autoradiographed with BAS-5000 MAC imaging analyser (Fuji Film). The DNA fragments for probes were amplified by PCR using the chromosomal DNA of N315 as templates.
Primer extension analysis.
Primer extension reactions were performed by incubating 10 or 20 µg RNA and 5 pmol 5'-FITC-labelled primer (5'-ATCCTCTTGAGGCATGCCTTCT-3' for qorA; 5'-ATCCATGTAAGAACAATGTTGGAATT-3' for SA1990 and 5'-ATCATCGTTTTCTGGCGTTGGCT-3' for SA1988, respectively) in a reverse transcription mixture (RT mixture) containing 50 mM Tris/HCl (pH 8·3), 75 mM KCl, 3 mM MgCl2, 1 mM DTT and 0·5 mM dNTPs. In the case of qorA, the RT mixtures were incubated at 70 °C for 10 min, 55 °C for 20 min and 25 °C for 10 min. In the case of SA1990 and SA1988, the RT mixtures were incubated at 70 °C for 10 min, 58 °C for 20 min and 25 °C for 10 min. Subsequently, 200 U Superscript II reverse transcriptase (Invitrogen) was added to the RT mixtures, which were then incubated at 42 °C for 30 min. The extended products were ethanol precipitated and analysed on 6 % urea denaturing polyacrylamide sequencing gels. The upstream regions of the three genes were cloned into pUC119 (Yanisch-Perron et al., 1985) and sequenced with a Thermo Sequenase fluorescence-labelled primer cycle sequencing kit (Amersham Biosciences) using the same oligonucleotides that had been used for the primer extension reactions. Signals were detected with a FluoroImager image analyser (Amersham Biosciences).
Construction of expression vector for GSTQorA and purification of its product.
The gene encoding qorA was amplified from S. aureus N315 (Hiramatsu et al., 1991) by PCR using the following synthetic primers, which were designed to contain the intact ShineDalgarno (SD) sequence of qorA: 5'-ATAAGTAAAAGCTTCAAATAAA-3' and 5'-TGAACAAATTTGCTGCAGATAAGA-3'. The PCR product was digested with HindIII and PstI, blunt-ended and cloned into pGEX-2T (Amersham Biosciences) to obtain pGQA, which contains a GSTQorA fusion and an intact QorA expression vector. Strain EQA01 was constructed by transformation of pGQA into E. coli BL21. Strain EQA01 was grown at 37 °C overnight in 3 ml LB broth containing 50 µg ampicillin ml-1. Cells were then cultured in 100 ml of the same medium at 37 °C and 150 r.p.m. At the exponential phase (OD600 0·50·6) of growth, GSTQorA fusion protein was induced by addition of 1 mM IPTG (final concentration). After 2 h, cells were harvested by centrifuging them at 2000 g at 4 °C for 10 min. All of the following purification steps were performed at 4 °C. Cell pellets were washed with 50 mM Tris/HCl (pH 7·5) containing 5 mM EDTA. After washing, the pellets were resuspended in 50 mM Tris/HCl (pH 7·5) containing 5 mM EDTA and 1 mM PMSF, and were then sonicated twice using a BioRaptor (COSMO BIO) for 3 min each. The cell lysate was then centrifuged at 10 000 g for 20 min. The resulting supernatants were mixed gently with 100 µl Glutathione Sepharose 4B beads (Amersham Biosciences) for 1 h. The beads were collected by centrifugation at 500 g for 5 min. The beads were washed with 50 mM Tris/HCl (pH 7·5) containing 5 mM EDTA and GSTQorA protein was eluted with 10 mM reduced glutathione containing 50 mM Tris/HCl (pH 7·5) and 5 mM EDTA. Thrombin-treated GSTQorA protein was prepared by incubating the bead-bound GSTQorA with 0·5 mg thrombin ml-1 containing 50 mM Tris/HCl (pH 8·0), 150 mM NaCl and 2·5 mM CaCl2 at room temperature for 1 h, and then the supernatant fractions were collected. To stop the thrombin digestion, 1 mM EGTA was added. The concentration of protein was measured using the Bradford method. Obtained proteins were analysed by 12·5 % SDS-PAGE.
Amino acid sequencing.
The N-terminal sequencing was done with the protein sequencer ABI 470A (Applied Biosystems). Following SDS-PAGE, the proteins were transferred onto a PVDF membrane. The Coomassie blue stained bands were cut out and applied to the sequencer without Polybrene treatment, as described previously (Ohta et al., 1991).
Measurement of NADPH consumption.
The reaction mixture (1 ml) contained the desired concentration of quinone, 0·1 mM NADPH, 50 mM Tris/HCl (pH 7·5), 0·2 % Tween-20 and purified enzyme. The reaction was initiated by the addition of quinone. The rate of NADPH oxidation at 25 °C over 5 min was monitored by the decrease in absorbance at 340 nm and was calculated by using an extinction coefficient of 6·22 mM-1 cm-1, employing a U-3200 spectrophotometer (Hitachi).
Determination of one-electron reduction of PQ.
It is well known that quinones undergo one-electron reduction by enzymes to yield semiquinone radicals which react readily with molecular oxygen to yield superoxide anions (Ernster, 1987). For this reason, the semiquinone radical of PQ was determined by measuring the generation of superoxide anions during reduction of PQ by QorA as described previously (Kumagai et al., 2000
). The reaction mixture (1·5 ml) contained 20 µM PQ, 0·1 mM NADPH, 0·1 M potassium phosphate buffer (pH 7·4), 25 µM acetylated cytochrome C and purified enzyme with or without bovine erythrocyte SOD (1000 U). The reaction was initiated by addition of PQ. The rate of reduction of acetylated cytochrome C at 25 °C for 5 min was monitored by the increase in absorbance at 550 nm. Superoxide anion generation was calculated as nmol ml-1 of the superoxide dismutase-inhibitable reduction of acetylated cytochrome C by using an extinction coefficient of 21·1 mM-1 cm-1.
Determination of two-electron reduction of PQ.
Because 9,10-dihydroxyphenanthrene, a two-electron reduction product of PQ, is a labile catechol metabolite, we determined 9,10-dihydroxyphenanthrene as its diacetoxy derivative. Reduction of PQ to 9,10-dihydroxyphenanthrene was determined as its acetoxy derivative, 9,10-diacetoxyphenanthrene (DAP), by HPLC (K. Taguchi, A. K. Cho, R. Koizumi, N. Shimojo & Y. Kumagai, unpublished). The reaction mixture (1·5 ml) contained 20 µM PQ, 0·1 mM NADPH, purified enzyme and 0·1 M potassium phosphate buffer (pH 7·4), unless otherwise noted. The reaction was initiated by addition of PQ. Incubation was carried out at 25 °C for 2 min. A portion of the reaction mixture (0·5 ml) was transferred to a centrifuge tube containing acetic anhydride (20 µl) and heated at 80 °C for 5 min: under these conditions, 9,10-dihydroxyphenanthrene formed from PQ was successively converted into DAP. Then the mixture was mixed with 170 µl 10 % trichloroacetate (final 2·5 %) and centrifuged at 14 000 g for 5 min. The supernatant (40 µl) was subjected to HPLC analysis. Separation of 9,10-diacetoxyphenanthrene from PQ was performed on YMC-Pack ODS-AM-303 a YMC-Pack ODS-AM (250x4·6 mm internal diameter, 5 µm particle size; YMC) with a Shimadzu LC-10AT pump and SPD-10A UV-VIS detector (Kyoto). Elution was accomplished with acetonitrile/1 % CH3COOH (3 : 2, v/v) at a flow rate of 1 ml min-1. Detection was performed at 255 nm. Peak height was determined by a Chromatorecorder 11 (System Instruments). Under these conditions retention times of PQ and DAP were 6·8 and 10·6 min, respectively.
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RESULTS |
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Oxidative stress enhances the expression of genes in the qorA operon
When S. aureus establishes an infection in its host, attacks by ROS produced from the host defence systems are lightened by oxidative stress responsive proteins such as SODs and catalase. Available reports of soluble Qors indicate that they are important factors in the response to oxidative stress. Accordingly, to analyse the expression of qorA, a Northern blot analysis was conducted in the presence of the redox-cycling agent, PQ. Signals of 2·6 kb and 1·4 kb were detected, using a qorA gene specific probe, and each signal was clearly enhanced by exposing to PQ in a time-dependent manner. The 2·6 kb signal of qorA was regarded as the transcript co-transcribed with nearby genes such as SA1990 or SA1988 (Fig. 2b). Signals of 2·6 kb were also observed in hybridization with both SA1990 and SA1988 specific probes. In SA1990, two signals (2·6 kb and 1·2 kb) were detected and accumulated by PQ exposure. In SA1988, the 2·6 kb and 1·2 kb signals were also induced by addition of PQ. Besides, a termination structure was identified downstream of SA1988 (Fig. 2b
), and loose stemloops were found downstream of SA1990 and qorA. These results suggested that the qorA gene is expressed as a polycistronic transcript with both SA1990 and SA1988. It should be noted that the expression of lacG, which is located upstream of SA1990, was not enhanced in the presence of PQ (data not shown).
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DISCUSSION |
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All of the three genes, qorA, SA1990 and SA1988, responded to the redox-cycling agent PQ (Fig. 2). Especially strong responses in qorA and SA1990 were observed (Figs 2 and 3
). Moreover, judging from profiles of expression and sizes, the 2·6 kb signal detected by the qorA probe probably corresponded to two mRNAs, i.e., the co-transcripts of SA1990qorA and qorASA1988. Because PQ is one of the substrates for QorA, the enhancement of gene expression of qorA is understood. As for SA1988, since it shares homology with qorA, it may also encode one of the quinone oxidoreductases. As regard to SA1990, the ORF was 867 bp long and encoded putative polypeptides, similar to a conserved hypothetical protein. The predicted molecular mass and pI of the SA1990 protein were 32·7 kDa and 10·36, respectively, and it contained 32 lysine residues. The high pI implies that SA1990 is a DNA-binding protein, and it may play a role in the oxidative stress response. This is the first demonstration of oxidative stress induction of the qor gene in bacteria. In S. aureus, either the PerR box or Fur box is responsible for transcriptional activation under oxidative stress (Horsburgh et al., 2001a
, b
). However, those regulatory sequences were not found at the determined promoter regions of qorA, SA1990 or SA1988 (Fig. 3
). This finding indicates the existence of an unknown regulatory system, which remains to be clarified by future studies. To demonstrate the role of qorA under oxidative conditions, we have constructed a qorA disruptant, and measured growth rate and NADPH consumption in the mutant. Compared with the parent strain, however, no significant changes were detected in this mutant (data not shown).
Our construct expressed GSTQorA and intact staphylococcal QorA, which were bound to each other throughout the purification steps. It suggested that staphylococcal QorA forms a multimer under physiological conditions. Other kinds of Qor proteins also form a dimer in E. coli (Thron et al., 1995) and a tetramer in the guinea pig (
-Crystallin) (Huang et al., 1987
). From a crystallization analysis of E. coli Qor, it is revealed that each subunit has an NAD(P)H-binding site, and a quinone binding site, which is thought to be located in a cleft between the co-enzyme and catalytic domains. Edwards et al. (1996)
reported that in spite of the low sequence identity between E. coli Qor, Thermoplasma acidophilum glucose dehydrogenase and horse liver alcohol dehydrogenase, these three enzymes shared a three-dimensional structural fold. They also reported that subunit interactions between those enzymes led to a significantly larger change of deviations in catalytic domain and NAD(P)H-binding domain. Because the deviations would ease substrate access, the catalytic activity of the multimer should be higher than that of the monomer. Based on this suggestion, although the three-dimensional structure of QorA is unknown, it can be said that multimer formation may increase the efficiency of activity. A non-redundant structural database search suggested that QorA did not possess FMN or FAD binding motifs. In fact, the oxidation of NADPH in the presence of PQ was not accelerated by the addition of FAD (data not shown).
Quinones are enzymically reduced via two different pathways and the overall pathway is illustrated in Fig. 8. (1) Semiquinone radicals are formed via a one-electron reduction pathway (e.g., NADPH-cytochrome P450 reductase, NADH-cytochrome b5 reductase,
-Crystallin, etc.) (Fig. 8
, arrow 1), and (2) hydroquinones are formed by a two-electron reduction pathway (e.g., NAD(P)H-quinone oxidoreductase) (Fig. 8
, arrow 4) (Ernster, 1987
). Semiquinone radicals react with molecular oxygen, resulting in the generation of superoxide anions (Fig. 8
, arrow 2) and lead to redox cycling. As shown in Table 2
, hydroquinone formation accounted for less than 10 % of superoxide generation during reduction of PQ by GSTQorA. Therefore, staphylococcal QorA predominantly catalyses the one-electron reduction of quinone. In this case, the stoichiometric ratio of NADPH consumption and superoxide anion generation should be 1:2 (Rao et al., 1992
). However, the estimated ratio in the present study with QorA was approximately 1: 0·5 (Table. 2)
. Theoretically, if the one-electron reduction potential (
) of a quinone is below -155 mV, which is the
for
, then the semiquinone radical will undergo redox cycling and generate superoxide anions. However, the
of PQ/PQ·- is -124 mV, which is less negative than the
of
. In the latter, superoxide anions reduce quinone to the semiquinone radical at fairly high rates, resulting in a low steady-state level accumulation of superoxide anions (Cadenas, 1995
). Thus, generated superoxide would be expected to readily react with PQ and the substantial superoxide anion levels would decrease in the reaction mixture. As a consequence, the PQ radical would be accumulated (Iyanagi, 1987
). Alternatively, the accumulated PQ radical may turn to hydroquinone of PQ (Fig. 8
, arrow 3).
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ACKNOWLEDGEMENTS |
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Received 6 June 2002;
revised 25 September 2002;
accepted 28 October 2002.