Department of Molecular Biology and Biotechnology, University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, UK1
Author for correspondence: David J. Kelly. Tel: +44 114 224414. Fax: +44 114 2728697. e-mail: d.kelly{at}sheffield.ac.uk
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ABSTRACT |
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Keywords: pyruvate carboxylase, pyruvate kinase, malic enzyme
Abbreviations: CAC, citric acid cycle; F1,6-BP, fructose 1,6-bisphosphate; G6-P, glucose 6-phosphate; MEZ, malic enzyme; OAA, oxaloacetate; PCK, phosphoenolpyruvate carboxykinase; PEP, phosphoenolpyruvate; PYC, pyruvate carboxylase; PYK, pyruvate kinase
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INTRODUCTION |
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The complete genome sequence of C. jejuni NCTC 11168 (Parkhill et al., 2000 ) has provided numerous insights into the metabolism and physiology of this organism. Although it is widely accepted that C. jejuni neither oxidizes nor ferments glucose (Smibert, 1984
), genes encoding enzymes of the EmbdenMeyerhof and pentose phosphate pathways are present in the genome (Parkhill et al., 2000
). Most of the reactions of the EmbdenMeyerhof pathway are reversible in vivo, with the notable exceptions of the 6-phosphofructokinase and pyruvate kinase (PYK) reactions (Fig. 1
), both of which constitute key control points. C. jejuni lacks a gene that could encode a 6-phosphofructokinase, which may account for the inability of the bacterium to catabolize glucose. However, a gene encoding fructose-1,6-bisphosphatase is present (Fig. 1
), which strongly suggests that the EmbdenMeyerhof pathway functions in gluconeogenesis. Nevertheless, a homologue of PYK (Cj0392c) has also been identified (Parkhill et al., 2000
). This is surprising, since PYK catalyses the physiologically irreversible conversion of phosphoenolpyruvate (PEP) and ADP to pyruvate and ATP at the final stage of the glycolytic pathway (Fig. 1
).
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Given the likely gluconeogenic role of the EmbdenMeyerhof pathway in C. jejuni, the presence and role of PYK is particularly enigmatic, as a futile cycle could result from the PYC, PCK and PYK reactions (Fig. 1). This study was undertaken to investigate the physiological function of the anaplerotic enzymes in C. jejuni and to determine the mode of regulation of the PYK. The phenotypic consequences of the mutational inactivation of PYK and the anaplerotic enzymes in C. jejuni are reported, permitting the assignment of definitive functions to several genes identified in the complete genome sequence (Parkhill et al., 2000
).
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METHODS |
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For growth assays, C. jejuni overnight starter cultures were prepared in BHI broth before inoculation into fresh BHI or MEM medium to which selected carbon sources at specified concentrations were added. Growth was monitored at 600 nm in an Amersham Pharmacia Biotech Ultrospec 2000 spectrophotometer.
DNA isolation and manipulation.
Plasmid DNA was isolated using the Qiagen Miniprep kit. C. jejuni chromosomal DNA was extracted using a modified SDS lysis procedure (Marmur, 1961 ). Standard techniques were employed for the cloning, transformation, preparation and restriction analysis of plasmid DNA from E. coli (Sambrook et al., 1989
).
Preparation of cell extracts and enzyme assays.
Cell-free extracts were prepared from 300 ml cultures grown to stationary phase. Cells were harvested in a Beckman Avanti J-25 I centrifuge (20 min, 4 °C, 8000 g), washed with 0·1 M Tris/HCl (pH 8·0) and then resuspended in 1 ml of the same buffer before sonication (MSE Soniprep 150, 6x10 s bursts, 12 µm amplitude). The crude extract was clarified by centrifugation (30 min, 14000 g) and the supernatant was retained for use on the same day.
PYC and PCK activities were monitored in assays linked to the NADH-dependent reduction of OAA by malate dehydrogenase. Each 1 ml PYC assay mixture contained 0·1 M Tris/HCl (pH 8·0), 5 mM sodium pyruvate, 5 mM ATP, 50 mM NaHCO3, 5 mM MgCl2, 0·25 mM NADH, 2 U malate dehydrogenase (Sigma) and cell extract. The reaction was initiated by the addition of pyruvate, and the disappearance of NADH was monitored continuously at 340 nm. PCK activity was measured in 1 ml assays containing 100 mM MOPS (pH 7·0), 50 mM NaHCO3, 4 mM ADP, 2·5 mM MgCl2, 2 mM MnCl2, 0·25 mM NADH, 2 mM PEP, 2 U malate dehydrogenase and cell extract. MEZ activity was assayed in a 1 ml mixture containing 0·1 M Tris/HCl (pH 7·0), 2 mM MnCl2, 5 mM KCl, 2·5 mM NADP, 7·5 mM L-malate and cell extract. PYK activity was assayed in a mixture containing 0·1 M Tris/HCl (pH 7·0), 30 mM MgSO4, 10 mM ADP, 0·25 mM NADH, 5 mM PEP, 5 U lactate dehydrogenase (Sigma) and cell extract. The Km, Vmax and Hill coefficients of the enzymes were estimated by using the curve-fitting package Enzfitter from Biosoft.
Detection of biotinylated proteins.
Cell extracts from wild-type and pyc mutant cells, grown in BHI broth, were prepared as described above. Denatured extracted protein (0·3 mg) was loaded and separated by SDS-PAGE (12%, w/v, acrylamide), then electroblotted onto a PVDF membrane (Millipore) in a Bio-Rad Transblot cell at 250 mA for 16 h. Biotinylated polypeptides were detected using an avidinhorseradish peroxidase conjugate (Bio-Rad), according to the instructions of the manufacturer.
Insertional inactivation of the pycA, pycB, pyk, pckA and mez genes.
Primers PYCA-F and PYCA-R (Table 1) were designed to amplify a 1·4 kb fragment of the pycA homologue (Cj1037c) by PCR. This fragment was subsequently cloned into pGEM-T Easy (Promega). The aphA-3-containing SmaI fragment of pJMK30 (van Vliet et al., 1998
), which confers kanamycin resistance, was then cloned into the BglII site within the Cj1037c gene to generate pJV1. The C. jejuni pycB homologue (Cj0933c) was amplified with the primers PYCB-F and PYCB-R (Table 1
) and cloned into pGEM-T Easy. A chloramphenicol-acetyltransferase (CAT) cassette, originating from Campylobacter coli (Wang & Taylor, 1990
), was then cloned into a unique BstXI site located within the cloned pycB gene to generate pJV2. For the inactivation of the pckA homologue (Cj0932c), primers PCKA-F and PCKA-R (Table 1
) were used to amplify a 1·4 kb fragment of Cj0932c from chromosomal DNA and the resulting amplicon was cloned into pGEM-T Easy. The CAT cassette was then cloned into a unique BstEII site within the cloned Cj0932c fragment, yielding pJV3. A fragment of the pyk homologue (Cj0392c) was amplified with PYK-F and PYK-R (Table 1
). The resulting amplicon was cloned into pGEM-T Easy and the CAT cassette was inserted into the unique KpnI site within the cloned pyk gene; this construct was named pJV4.
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RESULTS |
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Enzymic properties of the pycA, pycB, pyk and mez mutants
The wild-type strain and the mutants were assayed for their PYC, PYK, PCK and MEZ activities, to investigate whether mutations in the pyk, pycA, pycB and mez genes affected enzyme activity at the metabolic junction around pyruvate. The results are displayed in Table 2. PYC activity was completely abolished in the pycA and pycB mutants. PYK activity was reduced somewhat in the pyc mutants, with this effect being more pronounced with the pycB mutant (approximately twofold lower than that of the wild-type). Whereas PCK activity was not altered in the pycA mutant, the pycB mutant displayed an approximately ninefold reduction in PCK activity. This indicates that the pycB mutation is polar, affecting the expression of pckA (which lies 13 bp downstream of the pycB gene). MEZ activity was reduced fourfold in the pycA mutant, although MEZ activity in the pycB mutant was similar to that of the wild-type. The reason for this discrepancy is unclear. As shown in Table 2
, the pyk mutant was devoid of PYK activity. Although MEZ and PYC activities in the pyk mutant were comparable to those in the wild-type, PCK activity was reduced by approximately threefold. It should be noted that although PEP and ADP are substrates for the PYK reaction they are also substrates for PCK in PEP carboxylation (Fig. 1
), but the latter reaction also requires bicarbonate. Hence, the PYK assay is specific and this is confirmed by the lack of any residual activity in the pyk mutant. The possibility that PYK activity interferes with the PCK assay is precluded by the fact that the pyk mutant displays decreased (not increased) PCK activity. MEZ activity was abolished in the mez mutant (Table 2
), thus confirming the identity of Cj1287c as the gene that encodes MEZ. PYC, PYK and PCK activities remained largely unaltered in this strain. Extracts of the wild-type strain were devoid of pyruvate orthophosphate dikinase activity, which catalyses the reversible, ATP-dependent conversion of pyruvate to PEP. Furthermore, no PEP synthase activity was detected in wild-type cell extracts. PEP synthase catalyses the conversion of pyruvate and ATP to PEP, AMP and phosphate, and is thought to function in gluconeogenesis. Thus, PCK appears to be the only means of synthesizing PEP, which is consistent with the failure to isolate a pckA mutant.
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PCK activity displayed an absolute dependence on the presence of divalent metal cations. Both Mg2+ and Mn2+ were required for maximal activity. When ATP and AMP were substituted for ADP, PCK activity was not detected. ATP and AMP strongly inhibited PCK activity in the presence of 4 mM ADP (Fig. 6c). Moreover, 2-oxoglutarate and glutamate also inhibited PCK activity (Fig. 6c
). The bicarbonate concentration dependency of PCK activity deviated from MichaelisMenten kinetics, being linear at concentrations of up to 140 mM. PCK observed hyperbolic kinetics when the initial velocity was plotted against PEP concentration (Table 3
). Effector studies and kinetics for PCK were also performed on the pyk mutant and gave similar data (not shown) to those obtained for the wild-type strain, ruling out the possibility of interference of the PCK activity by PYK.
The MEZ activity of C. jejuni NCTC 11168 was strictly dependent on NADP; no activity was detected with NAD as a cofactor. Furthermore, no activity was measurable with D-malate. MEZ displayed sigmoidal kinetics when L-malate was the variable substrate. The affinity of the enzyme for malate was very low, with an apparent Km of 8·97 mM (Table 3). The Hill coefficient (Table 3
) was indicative of positive co-operative interactions between the substrate and the enzyme. MEZ activity was demonstrated to be dependent on the presence of divalent metal cations. Co2+, Ni2+ or Mg2+, but not Ca2+, could substitute for Mn2+. MEZ activity from several organisms has been shown to be stimulated by
and K+ (Gourdon et al., 2000
; Driscoll & Finan, 1996
). Similarly, the MEZ activity of C. jejuni was increased three- and twofold in the presence of K+ and
, respectively. In addition, MEZ activity was not affected by glutamate or AMP, whereas ATP, PEP and F1,6-BP markedly inhibited activity. G6-P, on the other hand, enhanced MEZ activity (Fig. 6d
).
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DISCUSSION |
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Bacterial and eukaryotic PYCs described to date are homotetramers (4) of 110130 kDa, with the exception of the Pseudomonas citronellolis, Azotobacter vinelandii and Methanobacterium thermoautotrophicum PYCs, which have an
4ß4 structure. The
subunit (65 kDa), encoded by pycB, carries the biotin moiety and contains all of the catalytically active sites necessary for the PYC reaction. The ß subunit (45 kDa), encoded by pycA, offers conformational stability within the enzymes core (Goss et al., 1981
; Mukhopadhyay et al., 1998
). In contrast to the
4 PYCs, all of which require or are stimulated by acetyl-CoA, the
4ß4 enzymes are fully active in the absence of this effector (Cohen et al., 1979
; Mukhopadhyay et al., 1998
). The presence of two genes that encode the 65 kDa and 54 kDa subunits of PYC, the lack of a 65 kDa biotinylated band in the pycB mutant and the inability of acetyl-CoA to activate the PYC of C. jejuni suggest that this enzyme is most likely to be of the
4ß4 type. The
4ß4 PYCs also appear to be insensitive to the other characteristic metabolic inhibitors described thus far for this enzyme, including L-aspartate and glutamate which are potent inhibitors of some eukaryotic, bacterial and fungal PYCs (Mukhopadhyay & Purwantini, 2000
; Modak & Kelly, 1995
; Fier & Suzuki, 1969
). The presence of a PEP carboxylase, which is regulated by acetyl-CoA and L-aspartate (Liao & Atkinson, 1971
; OBrien et al., 1977
), in organisms possessing
4ß4 PYCs is noteworthy. Moreover, PYC content in these organisms is a function of the carbon source used for growth (OBrien et al., 1977
; Scrutton & Taylor, 1974
). It seems reasonable to conclude, therefore, that fine control is not involved in the regulation of pyruvate carboxylation in these species. C. jejuni, on the other hand, has only a single enzyme (PYC) with the capacity to catalyse net synthesis of OAA from a three-carbon precursor, and therefore might be expected to display markedly different regulatory properties. In accord with this supposition, the C. jejuni enzyme was found to be potently inhibited by L-aspartate and by glutamate, both of which could potentially be catabolized by aspartate aminotransferase to produce OAA, the product of the PYC reaction.
Almost all of the bacterial PYKs are activated by phosphorylated sugars and adenosine mono- or diphosphates, the latter of which are indicative of a low cellular energy charge. F1,6-BP, the product of the reaction catalysed by 6-phosphofructokinase, allosterically regulates PYK via feed-forward activation, whereas ATP, the product of the PYK reaction itself, regulates via feed-back inhibition. The PYK of C. jejuni NCTC 11168 is typical of a glycolytic enzyme with respect to its activation by F1,6-BP, G6-P and AMP, and its inhibition by ATP. Moreover, the higher specific activity of PYK compared to that of PYC, PCK and MEZ is notable. These findings are surprising in view of the fact that C. jejuni does not catabolize glucose, as reported by Smibert (1984) . Determination of glucose utilization using nuclear magnetic resonance assays of culture supernatants has confirmed the inability of C. jejuni to metabolize glucose (J. Velayudhan, S. Hall and D. J. Kelly, unpublished data). This may be attributed to the absence of 6-phosphofructokinase, which is a key regulatory enzyme for sugar degradation via the EmbdenMeyerhof pathway (Fig. 1
). The role of PYK in C. jejuni could be to metabolize an as yet unidentified substrate in a pathway that bypasses 6-phosphofructokinase, allowing for ATP synthesis by substrate-level phosphorylation. Substrates of the pentose phosphate pathway are one possibility. The presence of homologues for the non-oxidative branch of the pentose phosphate pathway in the C. jejuni genome could allow growth on pentoses. However, neither ribose, xylose nor arabinose was capable of supporting the growth of C. jejuni in MEM
(J. Velayudhan and D. J. Kelly, unpublished data). In congruence with the activation of the PYK of C. jejuni by F1,6-BP, primary sequence comparisons reveal that residue 403 of the PYK is a serine. In non-regulated PYKs this residue is strictly conserved as a glutamic acid, which may lead to ineffective binding of F1,6-BP (Jurica et al., 1998
). Furthermore, residues Thr378-Ser-Ser-Gly-Lys-Ser383 in the PYK of C. jejuni correspond to a well-defined phosphate-binding pocket in the yeast PYK, at which the 6'-phosphate of F1,6-BP makes a series of hydrogen bonds to the side chains of these residues.
The inability to construct a pckA mutant and the detrimental effect of reduced PCK activity on the growth of the polar pycB mutant indicates that PCK activity is indispensable for the growth of C. jejuni. The paucity of alternative enzymes that can function in the capacity of PEP synthesis, as indicated by enzyme assays, suggests that OAA decarboxylation by PCK is crucial for precursor synthesis for anabolic pathways that leave central metabolism upstream of PEP. The direction in which PCK operates is probably determined by the ratio of adenine nucleotide pools and the steady-state concentrations of OAA and PEP, hence the sensitivity of the C. jejuni PCK to adenosine phosphates. In addition, higher intracellular PEP pools may account for the decreased PCK activity observed in the pyk mutant. The inability of the pycB mutant, which possesses low levels of PCK, to grow on malate as a sole carbon source is consistent with this enzyme functioning predominantly as a gluconeogenic OAA decarboxylase. Bacillus subtilis and E. coli pck mutants are, similarly, unable to grow on CAC intermediates, although they grow normally on glucose (Diesterhaft & Freese, 1973 ; Goldie & Sanwal, 1980
). The carboxylation reaction was not saturable at bicarbonate concentrations up to 140 mM, precluding a role for PCK in PEP carboxylation, as the organism is unlikely to encounter such high bicarbonate concentrations under physiological conditions.
A role in NADPH generation for biosynthesis is a possible function for MEZ, similar to the MEZs of Corynebacterium glutamicum and Aspergillus nidulans (Gourdon et al., 2000 ; Wynn & Ratledge, 1997
). However, the very low affinity of the MEZ of Campylobacter jejuni for malate and the comparable phenotype of the mez mutant to that of the wild-type is suggestive of a minor anaplerotic role for this enzyme in C. jejuni.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Cohen, N. D., Duc, J. A., Beegen, H. & Utter, M. F. (1979). Quaternary structure of pyruvate carboxylase from Pseudomonas citronellolis. J Biol Chem 254, 9262-9269.[Abstract]
Diesterhaft, M. D. & Freese, E. (1973). Role of pyruvate carboyxlase, phosphoenolpyruvate carboxykinase, and malic enzyme during growth and sporulation of Bacillus subtilis. J Biol Chem 248, 6062-6070.
Driscoll, B. T. & Finan, T. M. (1996). NADP+-dependent malic enzyme of Rhizobium meliloti. J Bacteriol 178, 2224-2231.[Abstract]
Ferrero, R. L., Cussac, V., Courcoux, P. & Labigne-Roussel, A. (1992). Construction of isogenic urease-negative mutants of Helicobacter pylori by allelic exchange. J Bacteriol 174, 4212-4217.[Abstract]
Fier, H. A. & Suzuki, I. (1969). Pyruvate carboxylase of Aspergillus niger: kinetic study of a biotin containing carboxylase. Can J Biochem 47, 697-710.[Medline]
Goldie, A. H. & Sanwal, B. D. (1980). Genetic and physiological characterisation of Escherichia coli mutants deficient in phosphoenolpyruvate carboxykinase activity. J Bacteriol 141, 1115-1121.[Medline]
Gornicki, P., Scappino, L. A. & Haselkorn, R. (1993). Genes for two subunits of acetyl coenzyme A carboxylase of Anabaena sp. strain PCC 7120: biotin carboxylase and biotin carboxyl carrier protein. J Bacteriol 175, 5268-5272.[Abstract]
Goss, J. A., Cohen, N. D. & Utter, M. F. (1981). Characterisation of the subunit structure of pyruvate carboxylase from Pseudomonas citronellolis. J Biol Chem 256, 11819-11825.
Gourdon, P., Baucher, M.-F., Lindley, N. D. & Guyonvarch, A. (2000). Cloning of the malic enzyme gene from Corynebacterium glutamicum and role of the enzyme in lactate metabolism. Appl Environ Microbiol 66, 2981-2987.
Jurica, M. S., Mesecar, A., Heath, P. J., Shi, W., Nowak, T. & Stoddard, B. L. (1998). The allosteric regulation of pyruvate kinase by fructose-1,6-bisphosphate. Structure 6, 195-210.[Medline]
Kelly, D. J. (2001). The physiology and metabolism of Campylobacter jejuni and Helicobacter pylori. J Appl Microbiol 90, 16S-24S.
Ketley, J. M. (1997). Pathogenesis of enteric infection by Campylobacter. Microbiology 143, 5-21.
Li, S.-J. & Cronan, J. E.Jr (1992). The gene encoding the biotin carboxylase subunit of Escherichia coli acetyl-CoA carboxylase. J Biol Chem 267, 855-863.
Liao, C.-L. & Atkinson, D. E. (1971). Regulation of the phosphoenolpyruvate branchpoint in Azotobacter vinelandii: phosphoenolpyruvate carboxylase. J Bacteriol 106, 31-36.[Medline]
Marmur, J. (1961). A procedure for the isolation of deoxyribonucleic acid from microorganisms. J Mol Biol 3, 208-218.
Mattevi, A., Bolognesi, M. & Valentini, G. (1996). The allosteric regulation of pyruvate kinase. FEBS Lett 389, 15-19.[Medline]
Modak, H. V. & Kelly, D. J. (1995). Acetyl-CoA-dependent pyruvate carboxylase from the photosynthetic bacterium Rhodobacter capsulatus: rapid and efficient purification using dye-ligand affinity chromatography. Microbiology 141, 2619-2628.[Abstract]
Mukhopadhyay, B. & Purwantini, E. (2000). Pyruvate carboxylase from Mycobacterium smegmatis: stabilisation, rapid purification, molecular and biochemical characterisation and regulation of the cellular level. Biochim Biophys Acta 1475, 191-206.[Medline]
Mukhopadhyay, B., Stoddard, S. F. & Wolfe, R. S. (1998). Purification, regulation, and molecular and biochemical characterisation of pyruvate carboxylase from Methanobacterium thermoautotrophicum strain H*. J Biol Chem 273, 5155-5166.
OBrien, R. W., Chuang, D. T., Taylor, B. L. & Utter, M. F. (1977). Novel enzymic machinery for the metabolism of oxaloacetate, phosphoenolpyruvate, and pyruvate in Pseudomonas citronellolis. J Biol Chem 252, 1257-1263.[Abstract]
Parkhill, J., Wren, B. W., Mungall, K. & 18 other authors (2000). The genome sequence of the foodborne pathogen Campylobacter jejuni reveals hypervariable sequences. Nature 403, 665668.[Medline]
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Scrutton, M. C. & Taylor, B. L. (1974). Isolation and characterisation of pyruvate carboxylase from Azotobacter vinelandii OP. Arch Biochem Biophys 164, 641-654.[Medline]
Smibert, R. M. (1984). Genus Campylobacter Sebald and Véron 1963, 907AL. In Bergeys Manual of Systematic Bacteriology , pp. 111-117. Edited by N. R. Krieg & J. G. Holt. Baltimore, MD:Williams & Wilkins.
Tauxe, R. V. (1992). Epidemiology of Campylobacter jejuni infections in the United States and other industrialized nations. In Campylobacter jejuni: Current Status and Future Trends, , pp. 9-19. Edited by I. Nachamkin, M. J. Blaser & L. S. Tompkins. Washington, DC:American Society for Microbiology.
van Vliet, A. H. M., Wooldridge, K. G. & Ketley, J. M. (1998). Iron-responsive gene regulation in a Campylobacter jejuni fur mutant. J Bacteriol 180, 5291-5298.
Wang, Y. & Taylor, D. E. (1990). Chloramphenicol resistance in Campylobacter coli: nucleotide sequence, expression, and cloning vector construction. Gene 94, 23-28.[Medline]
Wynn, J. P. & Ratledge, C. (1997). Malic enzyme is a major source of NADPH for lipid accumulation by Aspergillus nidulans. Microbiology 143, 253-257.
Received 14 August 2001;
revised 19 October 2001;
accepted 5 November 2001.