Institute of Biotechnology, Forschungszentrum Jülich, D-52425 Jülich, Germany1
Institut de Génétique et Microbiologie, Bat. 360, Université Paris-Sud, Centre dOrsay, F-91405 Orsay Cedex, France2
Author for correspondence: Armel Guyonvarch. Tel: +33 1 69 15 63 41. Fax: +33 1 69 15 63 34. e-mail: armel{at}igmors.u-psud.fr
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ABSTRACT |
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Keywords: Corynebacterium glutamicum, lpd gene, initiation of transcription, initiation of translation
Abbreviations: DCIP, 2,6-dichlorophenolindophenol; INT, 2-(p-iodophenyl)-3-p-nitrophenyl-5-phenyltetrazolium chloride; LPD, lipoamide dehydrogenase
The GenBank accession number for the nucleotide sequence determined in this work is Y16642.
a Present address: Instituto de Ciencia e Tecnologia dos Alimentos, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil.
b Present address: University of Ulm, Department of Microbiology and Biotechnology, D-89069 Ulm, Germany.
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INTRODUCTION |
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The genes for LPD from several organisms have been cloned and sequenced. Only one copy of the gene has been found and analysed in most of the organisms so far studied; exceptions are Pseudomonas putida and Ralstonia eutropha (formerly Alcaligenes eutrophus), which contain three and two lpd genes, respectively (Hein & Steinbüchel, 1994 , 1996
; Palmer et al., 1991
). In E. coli, Bacillus subtilis and Zymomonas mobilis, the structural genes for LPD are linked to the genes encoding pyruvate dehydrogenase complex subunits E1 and/or E2 (Neveling et al., 1998
; Stephens et al., 1983
). In contrast, the lpd gene for the LPD enzymes of A. vinelandii (Westphal & de Kok, 1988
), Pseudomonas fluorescens (lpd) (Benen et al., 1989
) and one of the lpd genes of P. putida (lpdG) (Palmer et al., 1991
) are part of the 2-oxoglutarate dehydrogenase complex gene cluster. In R. eutropha, one lpd gene (pdhL) is located downstream of the pdhA and pdhB genes encoding pyruvate dehydrogenase E1 and E2 proteins (Hein & Steinbüchel, 1994
), and the other (odhL) downstream of the odhA and odhB genes encoding 2-oxoglutarate dehydrogenase E1o and E2 proteins (Hein & Steinbüchel, 1996
). In C. glutamicum, however, an lpd (or odhL) gene was not detected within the neighbourhood of the odhA gene (Usuda et al., 1996
) and thus the genetic organization of the C. glutamicum genes encoding LPD and the other subunits of the 2-oxoglutarate dehydrogenase complex is different from that of A. vinelandii, P. putida, P. fluorescens and R. eutropha. In the present study, we describe the cloning of the C. glutamicum lpd gene, its nucleotide sequence and the deduced amino acid sequence, and the sequence of the two nearest adjacent ORFs. We show homologous expression of the cloned gene and its transcriptional organization. We present the purification and biochemical analysis of the LPD enzyme. In this report, we also address the question of the in vivo participation of LPD in quinone redox cycling in C. glutamicum.
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METHODS |
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DNA hybridization experiments were performed as previously described (Eikmanns et al., 1994 ). Oligonucleotides for Southern hybridization were labelled with digoxigenin-dUTP or digoxigenin-dATP with the DIG Oligonucleotide Tailing Kit from Boehringer Mannheim. Labelling, hybridization, washing and detection were performed with the non-radioactive DIG DNA Labeling and Detection Kit from Boehringer Mannheim.
For DNA sequencing, appropriate subclones of pUC18lpd5·4 and pUC18lpd3·4 were created with the Erase-a-base kit from Promega, and sequenced by the dideoxy chain-termination method using the AutoRead sequencing kit from Pharmacia, with subsequent electrophoretic analysis using an ALF DNA sequencer from Pharmacia. Sequence data were compiled and analysed using programs at the EMBL (http://www.emblheidelberg.de) and NCBI (http://www.ncbi.nlm.nih.gov) websites.
Cloning of the lpd gene.
To clone lpd, two degenerate oligonucleotides (TGYATYCCHTCBAARGCNCTDCTG and CAYGCNCAYCCHACBCTDTCBGARGCA) were designed on the basis of highly conserved regions in lipoamide dehydrogenases (Benen et al., 1989 ; Westphal & de Kok, 1988
; Stephens et al., 1983
), and according to the codon preference in Corynebacterium-related species (Malumbres et al., 1993
). C. glutamicum chromosomal DNA was digested with BamHI and fragments in the size range 4·86·0 kb were purified and ligated into the BamHI site of pUC18. From this partial genomic DNA bank, one plasmid, namely pUC18lpd5·4, containing a 5·4 kb BamHI fragment that hybridized with the two probes, was selected. The C. glutamicum lpd gene was further localized within a 3 kb BamHISphI fragment. The restriction map of the 3 kb BamHISphI fragment is shown in Fig. 1
. From a KpnI-generated partial genomic DNA bank, plasmid pUC18lpd3·4 was isolated as described previously. The isolated 3·4 kb KpnI fragment covers the complete lpd gene and in addition about 1·7 kb upstream of it (Fig. 1
). Using plasmid pUC18lpd3·4, the nucleotide sequence of the 1710 bp upstream of the lpd gene was determined.
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For Northern hybridization, 10 µg RNA was separated on an agarose gel containing 17% formaldehyde and transferred onto a nylon membrane. An lpd antisense RNA probe was prepared by ligating the 1·1 kb ScaISacI (Fig. 1) fragment isolated from plasmid pUC18lpd5·4 into plasmid pGEM-4Z, linearization of the resulting plasmid with BamHI, and synthesizing digoxigenin-dUTP-labelled RNA using T7 RNA polymerase and the Labelling Kit from Boehringer Mannheim. Hybridization (at 46 °C, in the presence of 50% formamide), washing and detection were performed with the Nucleic Acid Detection Kit from Boehringer Mannheim. The size marker was a 0·369·5 kb RNA ladder from Promega.
Primer extension experiments were performed as previously described (Eikmanns et al., 1994 ). The oligonucleotides used as primers (lpd1: 5'-GGCCGGCTCCGAGTACT-3'; and lpd2: 5'-TGCACGGATGGCGGAGACAT-3') are complementary to the sequence from position 232 to 251 in Fig. 2
, and from position 260 to 280 (not on Fig. 2
), respectively. RNA was isolated either from C. glutamicum wild-type strain or from C. glutamicum transformed with pEKOlpd5·4. For exact localization of the transcriptional start site, sequencing reactions using plasmid pEKOlpd5·4 and the same oligonucleotide used for primer extensions were co-electrophoresed.
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Enzyme assays.
To determine enzyme activities, crude extracts were prepared as described by Merkamm & Guyonvarch (2001) . The protein concentration was determined by the Biuret method (Gornall et al., 1949
), using bovine serum albumin as the standard.
The LPD activity in cell extracts was assayed photometrically as described by Guest & Creaghan (1974) . One unit of activity was defined as 1 µmol NADH formed min-1. Reverse LPD activity was also measured photometrically at 30 °C. One unit of activity was defined as 1 µmol NADH oxidized min-1.
2-Oxoglutarate dehydrogenase and pyruvate dehydrogenase complex activities were determined photometrically according to the method described by Guest & Creaghan (1974) . One unit of activity was defined as 1 µmol NADH formed min-1.
NADH:NAD+ transhydrogenation was assessed by the reduction of thio-NAD+ using the method of Fioravanti (1981) . NADH-duroquinone reductase activity of the purified LPD was measured as described by Owen et al. (1980)
. In both cases, one unit of activity was defined as 1 µmol NADH oxidized min-1. LPD-mediated NADH diaphorase activity, as well as NADH-linked DCIP (2,6-dichlorophenolindophenol)- and INT [2-(p-iodophenyl)-3-p-nitrophenyl-5-phenyltetrazolium chloride]-reducing activities of LPD were determined by activity staining after non-denaturing gel electrophoresis of the purified LPD protein essentially after Walker et al. (1997)
and Walker & Fioravanti (1995)
.
Superoxide dismutase and catalase activities were determined as described by Merkamm & Guyonvarch (2001) .
Purification of LPD.
Cell extracts for the purification of LPD were prepared as described above except that the cells were resuspended in 1 ml 50 mM Tris/HCl buffer (pH 7·0) for every 100 ml of culture. All purification steps were carried out at 4 °C. After the addition of 5 units DNase I ml-1, 15 mg RNaseA ml-1 and 100 mM PMSF, the cell extract was subjected to ultracentrifugation at 183000 g for 2 h. The supernatant was then loaded onto a Toyopearl HW65 column and the enzyme was eluted with 50 mM sodium phosphate buffer (pH 7·5) with a flow rate of 0·5 ml min-1. From the start of the gradient, fractions were collected and tested for LPD activity. The active fractions were pooled and run on an Octyl Sepharose 4 Fast Flow column equilibrated with 5 mM sodium phosphate buffer containing 1 M ammonium sulfate (pH 7·5). Proteins were eluted with a linear gradient of 10 M ammonium sulfate in 5 mM sodium phosphate buffer (pH 7·5). Fractions were collected and tested for LPD activity and for enzyme purity.
SDS-PAGE analysis.
SDS-PAGE was performed according to the method of Laemmli (1970) followed by Coomassie brilliant blue R-250-staining. Protein standards were phosphorylase b (Mr 94000), albumin (Mr 67000), ovalbumin (Mr 43000), carbonic anhydrase (Mr 30000), trypsin inhibitor (Mr 20100) and
-lactalbumin (Mr 14400).
N-terminal sequence analysis.
Purified LPD was separated by SDS-PAGE, transferred to a PVDF membrane and sequenced with an Applied Biosystems 477A sequencer equipped with a Blott cartridge and a model 120 on-line high-pressure liquid chromatograph.
Nucleotide sequence accession number.
The GenBank accession number of the nucleotide sequence of the 3025 bp BamHISphI fragment encompassing the entire lpd gene is Y16642.
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RESULTS |
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Another approach was therefore tested, using DNADNA hybridization as described in Methods. Southern hybridization was performed to confirm that the cloned 5·4 kb BamHI and 3·4 kb KpnI fragments originated from C. glutamicum (data not shown) and that they corresponded to fragments within the genome with no detectable structural alterations.
Expression of the cloned gene
For expression of the cloned gene in E. coli and in C. glutamicum, the 5·4 kb BamHI fragment was ligated into the C. glutamicumE. coli shuttle vector pEKO, resulting in plasmid pEKOlpd5·4. This vector was introduced into E. coli DH5 and C. glutamicum, and the LPD activities of the transformants and of the parental strains were determined. Whereas the recombinant E. coli cells showed no difference in their LPD activity when compared to the host strain cells, C. glutamicum(pEKOlpd5·4) showed an approximately 12-fold higher specific activity compared with the parental wild-type strain [1·85 and 0·16 units (mg protein)-1, respectively in crude cell extracts]. This result proves that the isolated fragment contains a functional lpd gene and indicates the presence of a promoter in front of this gene. The fact that the 5·4 kb BamHI fragment did not confer LPD activity to E. coli shows that the C. glutamicum lpd gene is not expressed in E. coli and explains the failure to obtain this gene by heterologous complementation of the E. coli mutant JRG301.
The effect of lpd overexpression in C. glutamicum on the specific activities of the 2-oxoglutarate dehydrogenase and pyruvate dehydrogenase complexes was studied. We determined these activities in cell extracts of C. glutamicum(pEKOlpd5·4) and C. glutamicum wild-type. Both specific activities were equivalent in the recombinant strain and in the parental strain [2-oxoglutarate dehydrogenase complex: 0·03 and 0·05 units (mg protein)-1; and pyruvate dehydrogenase complex: 0·03 and 0·04 units (mg protein)-1, respectively].
Youn & Kang (2000) demonstrated that LPD-overexpressing strains of Streptomyces seoulensis have an enhanced sensitivity to menadione, due to the in vivo participation of LPD in quinone redox cycling. We thus tested whether an increased level of LPD in C. glutamicum contributes to such redox cycling. Sensitivity of LPD-overexpressing and control cells to menadione and superoxide-generating compounds was tested by growth inhibition tests on plates with inhibitor-soaked disks as described in Methods. The LPD-overexpressing strain exhibited an enhanced sensitivity towards menadione. There was no marked difference between strains with regard to sensitivity to paraquat or hydrogen peroxide. This indicated that the increased sensitivity of the LPD-overexpressing strain did not result from an increased susceptibility to general oxidative stress. To refine the results, growth of C. glutamicum(pEKO) and C. glutamicum(pEKOlpd5·4) was challenged on BHI plates containing various concentrations of menadione. When cells were harvested in mid-exponential phase (OD570 4), growth of C. glutamicum(pEKO) was completely inhibited by 800 µM menadione, whereas C. glutamicum(pEKOlpd5·4) was inhibited by 550 µM menadione. Cells harvested in stationary phase (OD570 16) were more resistant, full inhibition being obtained with 850 and 750 µM menadione, respectively. The effect of LPD overexpression on the expression levels of the antioxidant enzymes superoxide dismutase and catalase was investigated in cells during the exponential phase of growth and during stationary phase. In all conditions, catalase activity was constant (250280 units mg-1). There was no difference between strains with regard to superoxide dismutase, but activity increased from 57 units mg-1 during the exponential phase to 2629 units mg-1 during the stationary phase. These results are consistent with the production of a menadione semiquinone radical anion by LPD in C. glutamicum, detoxified by superoxide dismutase, as described in S. seoulensis.
Nucleotide sequence of the lpd locus
The nucleotide sequence of the 3025 bp BamHISphI fragment was determined from both strands by the dideoxy chain-termination method (GenBank accession number Y16642). Computer analysis revealed two ORFs extending from bp 211 to 1618 (ORF1) and from bp 1830 to 2688 (ORF2). ORF1 and ORF2 exhibited a codon usage matching that of moderately expressed C. glutamicum genes (Malumbres et al., 1993 ). Database searches with the deduced polypeptides of these two ORFs revealed that the amino acid sequence encoded by ORF1 shows significant identity to known LPD polypeptides (see below), whereas the amino acid sequence encoded by ORF2 shows no significant similarity to any known gene. These results indicated that ORF1 represents the lpd gene from C. glutamicum. The predicted lpd gene product consists of 469 amino acids with a Mr of 50619, which is in good agreement with the size of already known LPD proteins from other organisms (Neveling et al., 1998
; Walker et al., 1997
; Westphal & de Kok, 1988
). From comparison data, the translational initiation site was predicted to be the GTG codon starting at nucleotide 211. To confirm this, the N-terminal amino acid sequence of purified LPD from C. glutamicum was determined. The amino acid sequence obtained was T-E-H-Y-D-V-V-V-L-G-A-G-P-G; thus the N-terminal sequence corresponds to the predicted translational start of lpd at nucleotide 211. The initial formyl-methionine residue predicted to be present from the DNA sequence was not found in the purified enzyme, indicating that it is removed by processing (Ben-Bassat & Bauer, 1987
). GenBank and SWISS-PROT database searches with the deduced amino acid sequence revealed that the amino acid sequence, the predicted secondary structure and the regions of functional significance of the C. glutamicum LPD are highly similar to those of LPD proteins from a number of Gram-positive and Gram-negative bacteria.
The lpd gene is not preceded by a typical ribosome-binding site, but is followed by a structure resembling a rho-independent transcription terminator (Rosenberg & Court, 1979 ). According to the rules of Tinoco et al. (1973)
, this palindromic structure should be capable of forming a stemloop with a
G value of -34·2 kcal mol-1 (-144 kJ mol-1) at 25 °C.
From plasmid pUClpd3·4, the nucleotide sequence of the 1710 bp upstream of the lpd gene was determined. Computer analysis revealed an ORF (ORF3) transcribed in the same direction as the lpd gene, starting 1137 bp and ending 492 bp upstream of the translational start site of the lpd gene. ORF3 encompasses 645 bp and is predicted to code for a polypeptide of 215 amino acids with a Mr of 22325. The deduced amino acid sequence showed very low (22·3%) identity to the E. coli ß-galactoside acetyltransferase (Hediger et al., 1985 ).
Transcriptional analysis of the lpd gene
Northern (RNA) hybridization experiments were performed in order to analyse the size of the lpd transcript. For this purpose, total RNA from C. glutamicum and C. glutamicum(pEKOlpd5·4) was isolated, size-fractionated, transferred onto a nylon membrane and hybridized to a lpd-specific digoxigenin-UTP-labelled antisense RNA probe. Hybridization to the lpd probe resulted in signals at 1·45 kb (data not shown). This size corresponds approximately to that of the structural lpd gene and indicates that the C. glutamicum lpd gene is monocistronic.
To identify the transcriptional start site upstream of lpd, a ribonuclease protection assay was performed with 50 µg total RNA isolated from C. glutamicum and [35S]dCTPS-labelled antisense RNA derived from the 0·77 kb BamHIEcoRI fragment at the 5' end of lpd (Fig. 1
). The signal obtained was in the size range of 550 bp and corresponded approximately to the region 210230 nt in Fig. 2
. Since the signal did not allow sufficiently precise assignment to a specific nucleotide, primer extension experiments with AMV reverse transcriptase and [35S]dATP
S were performed. Using an oligonucleotide primer (lpd1) covering codons 714 (nt 232251 in Fig. 2
) and 50 µg total RNA from C. glutamicum or from C. glutamicum(pEKOlpd5·4), signals were obtained (Fig. 3
, lanes 1 and 3, respectively), which correspond to the G211 residue in Fig. 2
. This result was confirmed in an independent experiment with another primer (lpd2, data not shown). This shows that transcription of the C. glutamicum lpd gene starts at the lpd translational start. Analysis of DNA sequence upstream of the transcriptional initiation site of lpd revealed the presence of motifs (Fig. 2
) with similarity to consensus -10 and -35 regions of C. glutamicum promoters (Patek et al., 1996
).
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The absorption spectra of reduced C. glutamicum LPD revealed the occurrence of flavin. As in the case of other LPD proteins (Walker et al., 1997 ), maxima were apparent at approximately 350 and 450 nm, while the minima occurred at 400 nm.
As described by Walker et al. (1997) for the mitochondrial LPD from Hymenolepsis diminuta, the purified enzyme from C. glutamicum readily catalysed a reversible NADH:NAD+ transhydrogenation, with an equimolar transfer of protons [1·102 µmol NADH oxidized per µmol thio-NAD+ reduced, and a specific activity of 0·765 units (mg protein)-1]. By activity staining after non-denaturing gel electrophoresis, the enzyme was shown to have an NADH diaphorase activity and to promote electron transfer from NADH to redox-active compounds such as INT and DCIP. In addition, the purified enzyme showed an NADH-quinone reductase activity [1·661 units (mg protein)-1] as described for LPD from E. coli by Owen et al. (1980)
.
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DISCUSSION |
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DNADNA hybridization data indicated the presence of one unique lpd gene in C. glutamicum. This is consistent with data for most other prokaryotic organisms, with the exception of P. putida and R. eutropha, for which three and two lpd genes, respectively, at different loci on the chromosome, have been found (Palmer et al., 1991 ).
As shown by N-terminal amino acid sequencing of the purified enzyme and by mapping of the transcriptional start site, the start of transcription and translation of the C. glutamicum lpd gene occurs at the same position (Fig. 2). Transcriptional and translational start at the same position was previously reported for actinomycete genes (Horinouchi et al., 1987
), for some bacteriophage genes that are expressed in E. coli (Ptashne et al., 1976
) and for the thrC gene of C. glutamicum (Han et al., 1990
). In E. coli, Sprengart et al. (1990)
have shown that initiation of translation could involve base pairing between the 16S rRNA and downstream sequences of mRNAs. This base pairing involves the anti-downstream box 3'-AGUACUUAGUGUUUC-5' at position 14831469 in the E. coli 16S rRNA and explains the expression of the lambda cI gene in E. coli (7 bases paired out of 15). In coryneform bacteria, the equivalent to the E. coli anti-downstream box should have the sequence 3'-AGCGGCUAGGGUGGA-5' at positions 14271441 of the 16S rRNA (Amador et al., 1999
). Base pairing could occur between this box and downstream sequences of the lpd (8 bases out of 15, Fig. 2
) and thrC (7 bases out of 15) mRNAs. The fact that there is no obvious E. coli anti-downstream box complementary sequence in the C. glutamicum lpd and thrC mRNAs may explain why the lpd and thrC (Han et al., 1990
) genes of C. glutamicum are not expressed in E. coli. However, base pairing between the C. glutamicum anti-downstream box and the lambda cI mRNA is possible since these sequences are at least partially complementary (5 bases out of 15) and accordingly, cI has been shown to be expressed in E. coli as well as in C. glutamicum (Labarre et al., 1993
). The abundance of the LPD protein in C. glutamicum cells suggests that in this organism the initiation of translation involving the putative anti-downstream box may be very efficient.
In E. coli, a complex co-regulation of the respective genes ensures the synthesis of all the subunits involved in the pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase multienzyme complexes in appropriate amounts (Quail et al., 1994 ). In E. coli, even if LPD (0·4% of total proteins) is synthesized in significant excess of its theoretical demand in wild-type cells (Smith & Neidhardt, 1983
), both activities are lowered when LPD is overproduced (Reed et al., 1975
). Reduced activities were interpreted as the result of an unbalanced assembly of E1o/E2o/E3 or E1p/E2p/E3 complexes, because of an enhanced competition between E1 (pyruvate decarboxylase or 2-oxoglutarate decarboxylase) dimers and overproduced E3 (LPD) dimers for space on the surface of E2 cores during the assembly process. This decrease does not appear in C. glutamicum. This is not that surprising in the case of 2-oxoglutarate dehydrogenase if, as suggested by Usuda et al. (1996)
, the ODHA protein from C. glutamicum is a bifunctional enzyme with E1o and E2o activities. In this case, excess LPD cannot of course compete with E1o dimers for assembly of complexes. It would then be interesting to analyse the corresponding protein involved in the pyruvate dehydrogenase complex. Nevertheless, in C. glutamicum, from the amount of LPD in wild-type cells (at least 0·3% of cytoplasmic proteins), an excess of its synthesis for the 2-oxoglutarate dehydrogenase and pyruvate dehydrogenase complexes is still arguable.
In E. coli (Owen et al., 1980 ) and in S. seoulensis (Youn & Kang, 2000
), LPD in excess was proposed to serve for the reduction of membrane-bound quinones. In H. diminuta, NADH:NAD+ transhydrogenation by LPD was also proposed to reflect a mechanism for the movement of reducing equivalents across the membrane (Walker et al., 1997
). Since purified LPD from C. glutamicum has an NADH-quinone reductase activity and a transhydrogenase activity, and since LPD-overexpressing C. glutamicum cells exhibit enhanced sensitivity to menadione as described for S. seoulensis (Youn & Kang, 2000
), an involvement of LPD in reducing equivalent transport in the membrane of C. glutamicum is probable. This implication regarding cellular energetics may explain in part the failure to obtain lpd-deficient mutants in C. glutamicum (data not shown). Confirmation of these findings will require additional studies with site-directed lpd mutants affected in synthesis level, assembly or activity characteristics.
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ACKNOWLEDGEMENTS |
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The authors are grateful to Dr M. A. Blight for critical reading of the manuscript and corrections.
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Received 5 February 2001;
revised 26 March 2001;
accepted 2 April 2001.