1 Centre for Carbohydrate Bioengineering, TNO-RUG, University of Groningen, PO Box 14, 9750 AA Haren, The Netherlands
2 Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, PO Box 14, 9750 AA Haren, The Netherlands
3 Innovative Ingredients and Products Department, TNO Nutrition and Food Research, Rouaanstraat 27, 9723 CC Groningen, The Netherlands
Correspondence
L. Dijkhuizen
L.Dijkhuizen{at}biol.rug.nl
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ABSTRACT |
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The GenBank accession number for the L. reuteri 121 gene encoding levansucrase (lev) and its flanking regions is AF465251.
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INTRODUCTION |
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Lactobacillus polysaccharides and oligosaccharides are of special interest because they may contribute to human health due to their antitumour (De Roos & Katan, 2000), antiulcer (Oda et al., 1983
), immunomodulating (Schiffrin et al., 1995
), or cholesterol-lowering activity (Roberfroid, 1993
). Moreover, some strains (e.g. Lactobacillus reuteri) have been designated as probiotics, i.e. they may have beneficial effects on the host by improving the properties of the indigenous population of gastrointestinal micro-organisms (Havenaar & Huis in 't Veld, 1992
; Gibson et al., 1994
).
Previously, it was reported that L. reuteri 121 cultivated on media containing sucrose produced large amounts of both a glucan and a fructan polymer (van Geel-Schutten et al., 1999). The fructan polymer was a levan containing
(2
6)-linked fructosyl residues, with two major fractions in the estimated size distribution of 150 000 Da and larger than 2 000 000 Da (van Hijum et al., 2001
; van Geel-Schutten et al., 1999
).
Enzymes responsible for the synthesis of fructan polymers of the levan type are generally referred to as fructosyltransferases (FTF) or levansucrases (sucrose : 2,6--D-fructan 6-
-D-fructosyltransferase, EC 2.4.1.10). They catalyse the transfer of the fructosyl unit of sucrose to a number of acceptors including sucrose, water (resulting in hydrolysis) and fructan polymer. Levansucrases of Zymomonas mobilis and Bacillus species (Gunasekaran et al., 1995
; Perez-Oseguera et al., 1996
) have been studied in most detail. Levans are either linear or branched to various degrees at the C-1 position. The sizes of the bacterial levans vary from 20 kDa to several MDa. For lactic acid bacteria, fructan production by streptococci and several lactobacilli has been reported (Tieking et al., 2003
). Streptococcus salivarius strains produce branched levan polymers [containing up to 30 %
(2
1) branches] (Ebisu et al., 1975
; Hancock et al., 1976
; Simms et al., 1990
) whereas Streptococcus mutans JC-2 produces a fructan of the inulin type consisting mainly of
(2
1)-linked fructosyl units with 5 %
(2
6) branches (Rosell & Birkhed, 1974
; Ebisu et al., 1975
). Recently, inulosucrase genes from lactic acid bacteria were reported in L. reuteri 121 (van Hijum et al., 2002
) and Leuconostoc citreum (Olivares-Illana et al., 2002
, 2003
).
Recently we described the purification of the levansucrase protein responsible for levan formation in L. reuteri 121, and determination of amino acid sequences of peptide fragments (van Hijum et al., 2001). Here we report the isolation and characterization of the levansucrase gene from the same strain. The gene was expressed in E. coli and its enzyme product was characterized. Structural characterization of the levan produced by the purified recombinant enzyme showed that this levansucrase is responsible for levan synthesis by L. reuteri 121 cells grown on raffinose.
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METHODS |
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General molecular techniques.
L. reuteri total DNA was isolated according to Verhasselt et al. (1989) as modified by Nagy et al. (1995)
. General procedures for cloning, transformation, DNA manipulations and agarose gel electrophoresis were as described by van Hijum et al. (2002)
. DNA was amplified by PCR on a DNA Thermal Cycler 480 (Applied Biosystems). Pwo DNA polymerase (Roche Biochemicals) was used for standard PCRs and for construction of the expression plasmids. High-Fidelity DNA polymerase (Roche Biochemicals) was used for inverse PCR reactions. PCR oligonucleotides were purchased from Amersham Pharmacia Biotech. Southern hybridizations were performed as described by van Hijum et al. (2002)
. All methods were according to the manufacturer's instructions, unless otherwise stated.
Isolation of the levansucrase gene.
Based on the amino acid sequences (QVESNNYNGVAEVNTERQANGQI and VYSPLVSTLMASDEVE) of two peptide fragments of the L. reuteri 121 levansucrase (van Hijum et al., 2001), degenerate primers Deg1 and Deg2i were designed (Table 1
). PCR with Pwo DNA polymerase, these primers, and total DNA of L. reuteri 121 yielded an amplification product of 1385 bp (Fig. 1
, A), which was used to design primers for two inverse PCR steps: (i) N1i and N2, and (ii) C1i and C2 (Table 1
). L. reuteri 121 chromosomal DNA was digested with HincII and ligated, yielding circular DNA molecules. PCR with the ligation product as template and diverging primers (i) N1i and N2 yielded an amplicon of 1544 bp (Fig. 1
, B) and (ii) C1i and C2 yielded an amplicon of 1542 bp (Fig. 1
, C). The 1542 bp fragment was used to design inverse PCR primers IPBrevi and IPAfor (Table 1
). L. reuteri 121 chromosomal DNA was digested with HindIII and ligated. PCR with primers IPBrevi and IPAfor with the circular ligation product as template yielded an amplicon of 1700 bp (Fig. 1
, D). In total, a fragment of 4570 bp of L. reuteri 121 genomic DNA was cloned and sequenced (Fig. 1
).
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(ii) Nickel affinity purification.
Ni-NTA resin (500 µl; Qiagen) was used to bind protein from 26 ml cell extract (3·6 mg protein ml-1). The resin was washed with 5 ml demineralized water and 2·5 ml binding buffer (50 mM Na2HPO4/NaH2PO4, pH 8·0) prior to applying the cell extract. The suspension was gently shaken at 4 °C for 1 h. Unbound material was washed away with 2·5 ml binding buffer, and bound protein was eluted from the affinity resin with 2 ml binding buffer containing 200 mM imidazole and 1 mM -mercaptoethanol. The eluate was dialysed against phosphate buffer (5 mM, pH 8·0) and adjusted to a volume of 5 ml in Tris buffer (20 mM, pH 8·0).
(iii) Resource-Q column chromatography.
An anion-exchange column (Resource-Q; Amersham Pharmacia Biotech; 1 ml column volume; flow rate 1 ml min-1) was equilibrated with Tris buffer (20 mM, pH 8·0; A) and the sample (5 ml) was loaded on the column. The column was eluted with Tris buffer (20 mM, pH 8·0, 0·5 M NaCl; B) and eluted fractions, collected from 20 % B to 80 % B, were screened for levansucrase activity (glucose release from sucrose; see below). Positive fractions were run on SDS-PAGE and peak fractions containing one protein band were pooled (4 ml) and stored at 4 °C for further analysis.
Biochemical characterization of the recombinant levansucrase
(i) N-terminal amino acid sequencing.
This was performed as described previously (van Hijum et al., 2002).
(ii) Mass analysis.
Matrix-assisted laser desorption-ionization mass spectrometry was used to determine the protein molecular masses. The adjusted Ni-NTA eluate (5 µl; 100 µg ml-1) was mixed with matrix (5 µl; 20 mg sinapinic acid ml-1 in acetonitrile/0·1 % trifluoroacetic acid; 40/60, v/v), and 2 µl of the mixture was dried on a target. Spectra were recorded on a TofSpec MALDI E and SE spectrometer (Micromass).
(iii) Levansucrase activity assays.
Sucrose conversion by levansucrase yields (a) fructose, which is (partly) built into the growing polymer, and (b) glucose, in a 1 : 1 ratio to the amount of sucrose converted. In control experiments the glucose formed reflected the total amount of sucrose utilized, since the residual sucrose (measured by hydrolysing sucrose with invertase and enzymically measuring the free glucose and fructose), fructan (measured by a mild 0·5 M trifluoroacetic acid hydrolysis followed by the enzymic detection of fructose), free glucose and free fructose formed added up to the amount of sucrose added to the reaction mixture (results not shown). Based on the above-mentioned experiments, the amount of glucose formed reflects the total amount of sucrose utilized by the enzyme (total activity). The amount of fructose formed is a measure of the hydrolytic activity of the enzyme (transfer of fructosyl units to water). The amount of glucose minus the amount of free fructose reflects the transferase activity (the transfer of fructosyl units to an acceptor other than water). Glucose and fructose were measured enzymically as described by van Hijum et al. (2001). Levansucrase activity was measured in a sodium acetate buffer (25 mM; pH 5·4) with 100 mM sucrose and 1 mM calcium chloride at 50 °C, unless stated otherwise. The optimal temperature and pH for L. reuteri 121 Lev (at 3 µg ml-1) total enzyme activity (glucose release from sucrose) were determined from 20 to 55 °C and pH 3·0 to 6·5 (from pH 5·5 to 6·5 a 25 mM MES buffer was used), respectively. One unit of enzyme activity is defined as the release of 1 µmol glucose or fructose min-1. All experiments were performed in triplicate and, where appropriate, the results are presented as the means±SEM. The Sigma Plot program (version 4.0) was used for curve fitting of the data, either with the standard MichaelisMenten formula: [y=(axx)/(c+x)], the three-parameter Hill formula: [y=(axx)b/(cb+xb)], or a MichaelisMenten formula with a substrate inhibition constant: [y=(axx)/(c+x+(x2/d))]. In these formulae, y is the specific activity (U mg-1), x is the substrate concentration (mM sucrose), a is the Vmax (U mg-1), b is the Hill factor, c is the Km (mM sucrose; K50 in the case of Hill-type kinetics), and d is the substrate inhibition constant (mM sucrose).
(iv) Levansucrase activity assays in SDS-PAGE gels.
Protein (approx. 5 µg) was run in duplicate on SDS-PAGE. A duplicate part of the gel was stained with Coomassie brilliant blue to identify the position of the proteins in the gel. Protein was cut from the corresponding unstained duplicate part of the gel. To determine enzyme activity in gel slices, protein was renatured by adding a sodium acetate buffer containing sucrose and 0·5 % (v/v) Triton X-100, and incubated at 50 °C. Glucose and fructose formation in the samples were determined as described by van Hijum et al. (2001).
Fructan analysis
(i) Fructan production and purification.
Reaction products of FTF were produced at the optimal growth temperature of strain 121 (37 °C), by incubating the purified levansucrase in a sodium acetate buffer (25 mM, pH 5·4; 1 mM CaCl2) with 100 g sucrose l-1, at 37 °C for 16 h. For comparison, fructan produced by L. reuteri cells grown overnight on MRSr was used. Polymer was precipitated and cleaned as described by van Hijum et al. (2001).
(ii) Molecular mass and methylation analysis.
Polymer characteristics (i) molecular mass by HPSEC/MALLS and (ii) the fructose linkage type by methylation were determined as described by van Hijum et al. (2001).
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RESULTS |
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ORF1 contained a putative start codon (TTG, encoding a formylmethionine at position 1193), with a perfect ShineDalgarno ribosome-binding site (AGGAGG) 8 bp upstream. Furthermore, two putative promoter sequences could be identified, according to the consensus promoter sequences described for Lactobacillus genes (Pouwels & Leer, 1993): (i) 238 bp upstream of the formylmethionine the sequences TTGTAA (-35) and TATAAA (-10) with a spacer region of 11 nucleotides, (ii) 199 bp upstream of the formylmethionine the sequences TTGATA (-35) and TAATAAA (-10) with a spacer region of 12 nucleotides. A strong terminator hairpin structure (
G -22·6 kcal mol-1) was found between ORF1 (68 nucleotides downstream) and ORF3 (172 bp downstream). The hairpin comprised a stem of 18 bp and a loop of 11 unpaired bases.
BLAST searches (http://www.ncbi.nlm.nih.gov/blast/) with the deduced Lev amino acid sequence showed highest similarity with: L. reuteri 121 inulosucrase (Inu; AF459437; 56 % identity and 86 % similarity in 768 amino acids), S. mutans FTF (P11701; 48 % identity and 65 % similarity in 773 amino acids), and S. salivarius FTF (Q55242; 48 % identity and 66 % similarity in 735 amino acids). Lev contained the core regions of Glycoside Hydrolase family 68 of levansucrase and invertases (Fig. 2; 41 % identity and 55 % similarity in amino acid residues 187 to 640; Pfam entry at 02435; http://pfam.wustl.edu/) and family 32 of invertases, levanases and inulinases (Fig. 2
; 24 % identity and 36 % similarity in amino acid residues 274 to 437; Smart entry at 00640; http://smart.embl-heidelberg.de/).
A striking feature of the Lev protein is the presence of direct repeats in the N- and C-terminal regions (Fig. 2). BLAST searches with the amino acid sequences of these repeats yielded no significant similarity with any known protein sequence. These repeats were not observed in the amino acid sequences of Inu and other FTFs from Gram-positive bacteria (Fig. 2
) or FTFs from Gram-negative bacteria. The C-terminal amino acid sequence of the Lev protein contained a proline-rich putative spacer region (Fig. 2
; 72 amino acids with 13 proline residues). Furthermore, a Gram-positive LPXTG cell-wall anchor was identified (Fig. 2
; Pfam entry PF00746 at http://pfam.wustl.edu/).
The isolated DNA fragment also contained ORF2, encoding a putative protein of 272 amino acids (from ATG start codon at position 133; Fig. 1), and ORF3, encoding a putative protein of 134 amino acids (from ATG start codon at position 4299; Fig. 1
). BLAST searches with the translated amino acid sequence of ORF2 showed highest similarity to hypothetical protein NMA1791 from Neisseria meningitidis (AL162757; 41 % identity and 60 % similarity in 263 amino acids). BLAST searches with the deduced amino acid sequence of ORF3 revealed highest similarity with a transposase from Lactobacillus casei (CAA05973; 74 % identity and 89 % similarity in 59 amino acids) and to transcription termination factor Rho from Neisseria gonorrhoeae (Q06447). Conserved domain database searches (http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml) showed the C-terminal domain of the translated protein from ORF3 to have homology to Rve, an integrase core domain protein of HIV-1 (Pfam entry at 00665).
Recombinant enzyme expression and purification
Cell extracts of E. coli Top10 harbouring the four Lev derivatives (Lev, LevHis, Lev773 and Lev
773His) clearly possessed sucrase activity (glucose release from sucrose) when incubated in a buffer with sucrose as substrate. The highest sucrase activity with all four constructs was observed when E. coli cells were incubated overnight with 0·02 % arabinose (approx. 11 000 U l-1 in the cell extracts). No activity was detected without arabinose induction. The Lev and LevHis proteins showed smearing on SDS-PAGE gels (results not shown), whereas distinct bands were observed with the Lev
773 and Lev
773His proteins on SDS-PAGE gels. Lev
773His was selected for further purification using the polyhistidine tag.
The Lev773His protein was purified to homogeneity from E. coli cell extracts by two column chromatography steps (Table 2
). The yield of protein after purification was relatively low due to loss of protein in the washing steps of the Ni-NTA column. In E. coli cell-free extracts and Ni-NTA fractions, a second, smaller and less abundant protein band was found next to the dominant protein band. The smaller band had an apparent size of 75 000 Da, smaller than the calculated molecular mass (84 676 Da) of Lev
773His. SDS-PAGE of cell extract, Ni-NTA and resource-Q fractions showed that the dominant protein band had an apparent size of 110 000 Da (results not shown), larger than the calculated molecular mass (84 676 Da) of Lev
773His. Similar sizes and ratios as the Lev
773His protein were observed for Lev
773. Mass spectrometry analysis of the adjusted Ni-NTA eluate showed that the protein running at 110 000 Da had a mass of 84 772 Da and that the protein running at 75 000 Da had a mass of 63 841 Da. The N-terminal amino acid sequence of the protein running at 110 000 (MDQVES) corresponded to the lev translated amino acid sequence starting at position 37 (Fig. 2
). The N-terminal amino acid sequence of the protein running at 75 000 Da (MPATYTVDA) corresponded to the translated amino acid sequence of lev starting from an alternative start codon (ATG) at position 1877 (amino acid residue 229 in Fig. 2
). The deduced molecular mass of the Lev protein variant translated from the alternative start codon was 63 891 Da, corresponding to the size of the smaller protein determined by mass spectrometry. An imperfect ribosome-binding site (AAGGAA; at position 1863) and no consensus promoter sequence could be identified. We conclude that the L. reuteri 121 lev gene contains a second start codon that is recognized by E. coli. Staining for sucrase activity in SDS-PAGE gels showed a clearly positive activity band for the Lev
773His protein, whereas the N-terminally truncated Lev protein showed no detectable activity.
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Recombinant enzyme characterization
The purified Lev773His enzyme showed highest activity (glucose release from sucrose; total activity) at 50 °C and around pH 4·55·5. The enzyme was almost inactive without addition of 1 mM Ca2+ (2·28±0·19 % residual activity); no cations other than calcium could restore enzyme activity. Lev enzyme activity was (almost) completely inhibited by Hg+, Fe3+ and Cu2+. Partial inhibition was observed with Fe2+ (Table 3
).
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DISCUSSION |
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The C-terminal domain of Lev consists of a proline-rich region, an LPXTG motif, a stretch of hydrophobic residues, and finally three positively charged amino acids (Fig. 2). We reported a similar LPXTG motif and C-terminal topology for the inulosucrase from L. reuteri (van Hijum et al., 2002
). No other FTFs have been reported to carry a C-terminal LPXTG motif. For the cell-wall-associated FTF of S. salivarius, however, the presence of a hydrophobic membrane-spanning region and a putative spacer region rich in serine and threonine residues was reported (Rathsam & Jacques, 1998
). The presence of a C-terminally located cell-wall-anchoring motif suggests that the levansucrase protein in L. reuteri 121 is cell-wall-associated. This is in accordance with previous observations, showing that the L. reuteri levansucrase activity occurs cell-associated as well as a free supernatant protein (van Geel-Schutten et al., 1999
; van Hijum et al., 2001
).
The biochemical properties and the products formed by the Lev773His enzyme and the levansucrase purified from L. reuteri (van Hijum et al., 2001
) are comparable. Obviously, the C-terminal truncation of Lev from amino acid 773 onwards (Fig. 2
) and the addition of a C-terminal Myc epitope and polyhistidine tag did not have significant effects on the products formed. The full-length recombinant Lev protein (LevHis), containing the membrane-spanning region, showed smearing on SDS-PAGE gels, suggesting that the C-terminal membrane-spanning region in E. coli interfered with protein expression or protein stability. Similar effects of the C-terminal domain were observed for the L. reuteri Inu (van Hijum et al., 2002
). With the HPSEC/MALLS method two major fractions with molecular masses of 20 000 Da (97 %, w/w) and (34)x106 Da (3 %, w/w) were found for fructan polymers produced by both L. reuteri and Lev
773His. Earlier we reported molecular masses of 150 000 and more than 2x106 Da when using gel-filtration chromatography (van Hijum et al., 2001
). The discrepancy between these results can be explained by an increased accuracy with the HPSEC/MALLS method (Blennow et al., 2001
; Turquois & Gloria, 2000
).
The estimated affinities of the Lev enzyme for sucrose in the various reactions are comparable to values found for other levansucrases. The Kcat values of Lev for the release of both glucose (=147±3 s-1) and fructose (
=117±7 s-1) from sucrose are clearly higher than those of the S. salivarius FTF (
=63·5±3·6 s-1 and
=28·9±1·2 s-1, respectively). This corresponded with the high hydrolytic activity that we also observed for the purified L. reuteri levansucrase (van Hijum et al., 2001
). The Lev enzyme apparently transfers the fructosyl unit of sucrose relatively efficiently to water. Nevertheless, the enzyme produced significant amounts of levan both in vivo (van Hijum et al., 2001
) and in vitro (this study).
A striking feature of the Lev773His protein is its high optimal temperature of 50 °C, and that at 50 °C a shift occurs from MichaelisMenten to kinetics best described by the Hill equation. Speculatively, with increasing temperatures, the enzyme can use sucrose more efficiently as acceptor than (oligo) fructan molecules. Only for the L. reuteri 121 Inu has a temperature optimum of 50 °C been reported (van Hijum et al., 2002
, 2003
); other FTFs show lower optimal temperatures. Regardless of the optimal temperature, no Hill-type kinetics has to our knowledge been observed for FTFs previously, except for Inu (van Hijum et al., 2003
). The Hill factors calculated from activities at 50 °C for the Lev
773His (total activity and transferase reactions) were lower than 1. This was also observed for the Inu enzyme (van Hijum et al., 2003
) and indicates a negative cooperativity for these reactions. With Hill-type kinetics it is assumed that there is more than one binding site present in the enzyme and/or multimeric forms of the enzyme. For Hill-type kinetics, a positive cooperativity indicates a positive interaction of binding sites present in the enzyme and/or multimers. Alternatively a negative cooperativity indicates a negative interaction of enzyme binding sites and/or multimers. In FTFs, it is not known how many binding sites are present for substrate and product binding due to the lack of detailed structural protein information. Multimeric forms of FTFs were reported only for the levansucrase from Actinomyces viscosus T14 (Pabst et al., 1979
). Thus, we cannot draw conclusions on the nature of the negative cooperativity suggested by the best-fit for the total and transferase activities at 50 °C observed in this L. reuteri Lev enzyme.
Two non-levan-producing mutants of L. reuteri 121 have been described (strains 35-5 and K24), isolated during continuous culture experiments (van Geel-Schutten et al., 1999). Under the growth conditions applied, levansucrase activity became lost in a few generations, suggesting that the lev gene in L. reuteri 121 is located on a transposable element, or on a plasmid. Interestingly, ORF3 (Fig. 1
) shows strong homology to a transposase from a Lactobacillus casei strain (CAA05973). Transposable elements have been described for a number of lactic acid bacteria (Davidson et al., 1996
). When comparing genomic maps of Lactococcus lactis MG1363 and Streptococcus thermophilus, a large number of inversions and translocations are present. These genomic rearrangements are partly attributed to the presence of mobile elements in the genomes of lactic acid bacteria such as transposons (Davidson et al., 1996
). In view of the presence of ORF3 downstream of ORF1, the possible location of lev on a transposable element, flanked by recognition sequences for a transposase encoded by ORF3, warrants further investigation. No transposon insertion sequences could be identified in the DNA sequence flanking ORF1. Therefore, the mechanism of inactivation of the levansucrase activity in L. reuteri 121 mutants remains unclear.
This is believed to be the first report of the identification of a Lactobacillus levansucrase gene (lev) and the characterization of the recombinant protein. The L. reuteri 121 levansucrase is most closely related to L. reuteri Inu and to levansucrases of streptococci, based on biochemical characteristics and sequence homologies. L. reuteri 121 levan contains significantly lower amounts of (2
1) branches than levans produced by Streptococcus spp. The L. reuteri 121 levansucrase is unusual in displaying a relatively high rate of sucrose hydrolysis. The lev gene was successfully expressed in E. coli, enabling production of relatively larger amounts of levansucrase and its levan polymer. Our current studies focus on a detailed biochemical and structural characterization of the L. reuteri Lev and Inu enzymes, to identify features that determine (i) the percentage of
(2
1
6) branches, (ii) product size, (iii) the
(2
1) versus
(2
6) product specificity, and (iv) hydrolysis versus transglycosylation specificity.
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ACKNOWLEDGEMENTS |
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Received 28 July 2003;
revised 31 October 2003;
accepted 1 December 2003.