Unit of Mycology, Bacteriology and Nematology1 and Unit of Genomics2, Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK
Author for correspondence: Paul R. J. Birch. Tel: +44 1382 562731. Fax: +44 1382 562426. e-mail: pbirch{at}scri.sari.ac.uk
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ABSTRACT |
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Keywords: draft sequencing, plant pathogen, bacterial genomics, enterobacterium
Abbreviations: BAC, bacterial artificial chromomsome; Eca, Erwinia carotovora subsp. atroseptica; Ecc, Erwinia carotovora subsp. carotovora
The GenBank accession numbers for the 424 sequences determined in this work are BH614193 to BH614616.
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INTRODUCTION |
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The genus Erwinia includes several important phytopathogens such as E. amylovora, cause of fire-blight on apples, and the soft-rot erwinias E. chrysanthemi and E. carotovora (Alfano & Collmer, 1996 ). Additional interest in erwinias derives from their place in the Enterobacteriaciae and their close relationship with the model bacterium Escherichia coli. This affinity is confirmed by molecular taxonomy: Erwinia carotovora and Escherichia coli share greater than 95% 16S rDNA sequence identity (Hauben et al., 1998
). With the entire E. coli genome sequence available (Blattner et al., 1997
), and ongoing sequencing of several more enterobacterial species, there is now an excellent opportunity for comparisons of genome structure and content.
E. carotovora subspecies atroseptica (Eca) and E. carotovora. subsp. carotovora (Ecc) are economically important pathogens of crops, particularly potato, worldwide. Whereas Eca has a host-range restricted to potato, on which it causes both soft-rot of tubers and stem rot (blackleg), Ecc does not cause blackleg but elicits soft-rot on a wide range of hosts (Pérombelon & Salmond, 1995 ). E. carotovora degrades plant cell walls using a variety of extracellular enzymes (particularly pectic enzymes), secreted via the type I and type II pathways (Py et al., 1998
). This ability, along with phenotypic and molecular phylogenetic heterogeneity in the genus, has led to proposals to reclassify the soft-rot Erwinia spp. as Pectobacterium (Hauben et al., 1998
). Soft-rot can be considered a rather crude and opportunistic form of pathogenesis but the discovery of type III protein secretion systems and of diverse regulatory mechanisms controlling extracellular enzyme synthesis in both E. chrysanthemi and E. carotovora shows that soft-rot pathogenesis is more complex than previously thought (Bauer et al., 1994
; Barras et al., 1994
; Mukherjee et al., 1997
). The type III secretion system, encoded by hrp genes, is thought to allow the delivery of proteins directly into host cells via a pilus-like structure (Galán & Collmer, 1999
). The function of hrp genes in E. carotovora remains unclear.
Almost all genes known to influence virulence in E. carotovora have been identified by either direct gene cloning or transposon mutagenesis, and demonstration of their role in virulence has largely depended on simple plant assays, usually involving stem or tuber inoculation tests (Andersson et al., 1999 ; Hinton et al., 1989
; Pérombelon & Salmond, 1995
). Although this approach has been very successful over the past 15 years, there are many aspects of the plantErwinia interaction that are poorly understood. This is partly due to the limitations of the conventional routes of analysis. Relatively little is understood about the in planta behaviour of E. carotovora in terms of the genes involved in establishing infection, or the control of gene expression and temporal order of induction or repression of specific virulence genes and their regulators later in infection.
Genome sequencing offers an alternative approach to better understanding of Erwinia pathogenicity. However, the expense and resources required for whole-genome sequencing projects are often prohibitive. By the nature of the strategies required (Frangeul et al., 1999 ), a large proportion of the cost and effort will inevitably be directed to generation of redundant sequence information (from multiple coverage random sequencing) and gap closure. In this study we used a sample-sequencing strategy for gene discovery that is focused on a particular region of the Eca genome thought to contain putative pathogenicity genes, and which is contained on two large, overlapping DNA fragments cloned into a bacterial artificial chromosome (BAC) vector. Although sample sequencing from whole bacterial genomes has been described previously (e.g. McLelland & Wilson, 1998
; Viprey et al., 2000
), sample sequencing of a targeted region of a bacterial genome has not. The contiguous BAC clones were chosen as they hybridize to the hrpN gene from Ecc, and should thus span a hrp gene cluster. We address the following questions. 1. Based on the structure of hrp gene clusters sequenced in other species (Galán & Collmer, 1999
), does one-fold coverage sample sequencing of the BAC clones provide data on all predicted ORFs from the Eca hrp gene cluster? 2. What genes flank the Eca hrp gene cluster and how are they ordered? 3. Is one-fold sample sequencing of contiguous BACs a cost-effective route to provide sequence information from a high percentage of ORFs that may feed directly into downstream gene functional analyses? 4. What comparisons can be made between the genomes of Eca and the model bacterium and animal pathogen E. coli?
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METHODS |
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High-molecular-mass Eca DNA preparation.
A 30 ml culture of Eca was grown to an OD600 of 0·9 in LB at 27 °C with shaking (300 r.p.m.). The culture was cooled on ice for 15 min and cells were harvested by centrifugation at 2500 r.p.m. for 10 min at 4 °C. Cells were resuspended in 4 ml buffer 1 (200 mM NaCl, 10 mM Tris/HCl pH 7·2, 100 mM EDTA), harvested and resuspended in 1 ml buffer 2 (200 mM NaCl, 10 mM Tris/HCl pH 7·2, 10 mM EDTA). Cells were incubated at 37 °C for 5 min then 42 °C for 5 min before 1·5 ml of molten 1·5% LMP agarose in distilled H2O equilibrated to 42 °C was added and mixed. This was aliquoted into 100 µl plug moulds and cooled to room temperature. Plugs were removed and incubated in 20 ml GET (50 mM glucose, 10 mM EDTA, 25 mM Tris/HCl pH 8·0, 2·5 mg lysozyme ml-1) at 4 °C for 24 h. GET was replaced with 50 ml EPS (0·5 M EDTA pH 9·2, 1% w/v Sarcosyl, 1 mg proteinase K ml-1) at 50 °C and incubated for 24 h at 50 °C with gentle shaking, washed in EPS and incubated for a further 24 h at 50 °C. Plugs were washed twice in 10 mM Tris/HCl pH 8·0, 10 mM EDTA, 1 mM PMSF for 1 h at room temperature, four times in 30 ml 10 mM Tris/HCl pH 8·0, 10 mM EDTA at 50 °C, once in 30 ml 0·5 M EDTA pH 9·2 at 50 °C for 1 h and once in 0·05 M EDTA pH 8·0 for 1 h at 4 °C. Plugs were stored in 0·05 M EDTA pH 8·0 at 4 °C until use.
BAC library construction.
Five 100 µl plugs were chopped to a slurry with a clean razor blade. The slurry was washed with 1 ml 0·1% Triton X-100, centrifuged briefly and the supernatant removed. To 50 µl slurry were added 7 µl 40 mM spermidine, 7 µl 10x endonuclease buffer and 0·7 µl 10 mg BSA ml-1 followed by incubation for 30 min on ice. HindIII enzyme (0·05 U per tube in 5 µl) was added and the samples were incubated for 30 min on ice then 30 min at 37 °C. The reaction was stopped with 7 µl 0·5 M EDTA pH 8·0. The entire sample was loaded on a 1% w/v low-melting-point (LMP) agarose gel and electrophoresed on a Bio-Rad CHEF Mapper for 18 h, 6 V cm-1, 20 s switch time and 120° included angle at 11 °C, alongside suitable high-molecular-mass markers. Gel slices containing DNA fragments >100 kb were excised from the adjacent lanes (without exposure to ethidium bromide or UV) and washed three times in TE buffer for 30 min at 4 °C. Gel slices (100 mg) were incubated in 1 ml TES buffer (TE containing 50 mM NaCl) for 1 h. TES was removed and the gel slices melted at 65 °C for 10 min, then 40 °C for 10 min. One microlitre of ß-agarase (New England Biolabs) was added and the mix was incubated at 40 °C for a further 1 h. DNA concentration was estimated by comparison with lambda concentration standards. Finally, Erwinia DNA was ligated into HindIII-digested, dephosphorylated pBeloBAC11 (Kim et al., 1996 ) (100 ng DNA, 10 ng vector, 1x ligation buffer [including ATP], 1·33 U DNA ligase, volume 100 µl) overnight at 12 °C. Ligations were desalted by drop dialysis and 1 µl aliquots were electroporated into electrocompetent E. coli DH10B cells (prepared by standard methods) using a Bio-Rad E. coli Pulser. Transformants were diluted 1:20 with SOC (Sambrook et al., 1989
) and shaken gently at 37 °C for 1 h prior to spread-plating on LBIX. After overnight incubation at 37 °C, recombinant (white) colonies were picked and insert sizes estimated by CHEF gel analysis after digestion with NotI. Clones were transferred to two 384-well microtitre plates with 70 µl freezing medium (LB with 36 mM K2HPO4, 13·2 mM KH2PO4, 1·7 mM sodium citrate, 0·4 mM MgSO4, 6·8 mM (NH4)2SO4, 4·4%, v/v, glycerol), grown overnight at 37 °C and stored at -80 °C.
Hybridization screening of the BAC library.
BAC library clones were transferred to two nylon membranes (saturated with LB) using a sterile 384-pin plastic replicator. The membranes were then placed on LB agar, incubated at 37 °C overnight to allow colony growth and DNA was blotted by standard methods (Sambrook et al., 1989 ).
The primers hrpN-1 and hrpN-2 were selected from the Ecc hrpN gene (GenBank accession no. L78834, 679 bp to 1022 bp). Other primer pairs were selected from BAC end or subclone sequences generated in this study. PCRs were performed using either Ecc (SCRI 193) or BAC clones 2B8 or 1C22 DNA as template, Boerhinger Taq polymerase and reaction mix with a thermal profile of 94 °C for 2 min followed by 35 cycles of 94 °C for 30 s, 61·4 °C for 30 s and 72 °C for 1 min. PCR products were purified (Promega Wizard PCR Prep), 32P-labelled using High-Prime kit (Boehringer), purified through a Nick Column (Pharmacia) and hybridized under high stringency (Sambrook et al., 1989 ) to the BAC library.
End-sequencing BAC clones.
BAC DNA for end-sequencing was obtained using the method of Kelley et al. (1999) . Sequencing reactions were performed using a Big Dye Sequencing Kit (Perkin Elmer) with approx. 0·5 µg BAC DNA, 2 pmol primer (T7 or SP6) and 4 mM added MgCl2 per reaction. The thermal profile was 98 °C for 2 min followed by 100 cycles of 96 °C for 30 s, 50 °C for 20 s and 60 °C for 4 min.
BAC subcloning.
BAC DNA was prepared from 4 l of culture using large-scale plasmid preps (Qiagen) according to the manufacturers instructions. The DNA was incubated overnight with PlasmidSafe (Cambiolab), using at least 20 µl enzyme per 100 µg DNA, to remove E. coli chromosomal DNA. Nebulization was performed using 3050 µg DNA in 2 ml aliquots in a pre-chilled Sidestream Nebuliser (Medic-Aid) on ice. Two samples were nebulized, one at 1 bar for 90 s in 10% glycerol, 10 mM Tris/HCl pH 8, 5 mM NaCl and the other at 1 bar for 20 s in 10 mM Tris/HCl pH 8, 5 mM NaCl using H2 or N2 gas. Nebulized DNA was transferred to 2 ml microtubes, precipitated with 2-propanol, redissolved in 250 µl 1x T4 DNA polymerase buffer containing 50 U T4 DNA polymerase and 500 nM dNTPs, and incubated for 30 min at room temperature for end repair. Following electrophoresis, gel slices containing DNA of the desired size fractions were excised and digested with Gelase (Cambiolab) overnight, according to the manufacturers instructions. Following sample extraction with phenol/chloroform and 2-propanol precipitation, samples were phosphorylated using T4 kinase according to the manufacturers instructions (Gibco). DNA samples were phenol/chloroform extracted twice, ethanol precipitated, dissolved in 3050 µl TE and quantified by comparison with standards. Alternatively, DNA was fragmented by partial digestion using Sau3A1 (approx. 40 ng BAC DNA with 1 U enzyme for 30 min). DNA fragments of 600800 bp were size-fractionated for cloning.
A high-copy-number cloning vector, pGEM3zf (Promega), was digested with HincII or BamHI (for insertion of sheared or Sau3AI-digested fragments respectively) and then dephosphorylated with shrimp alkaline phosphatase (New England Biolabs). Ligations were performed using 2050 ng vector with 20100 ng of insert DNA and 1 µl aliquots were used to transform electro-competent E. coli DH10B cells as described previously. Recombinant colonies were individually transferred to 1·3 ml TB in a 96-deep-well block. Blocks were covered with a gas-permeable seal and shaken for approximately 22 h prior to harvesting cells (3000 g for 7 min). The supernatant was removed and plasmid DNA was extracted by alkaline lysis using a Biomek 2000 robot, according to the University of Oklahoma Advanced Center for Genome Technology (http://www.genome.ou.edu/Biomek2000_dsisol_v1.html). DNA was dissolved in 50 µl H2O and 1 or 2 µl aliquots were used in sequencing reactions (Big Dye sequencing kit, Perkin Elmer) with SP6 or T7 primers (protocol at http://www.genome.ou.edu/big_dyes_plasmid.html). Reaction products were analysed using an ABI Prism 377 DNA Sequencer with 96-lane upgrade, according to the manufacturers instructions. Sequences were edited to remove vector sequence and regions of poor quality and searched using BLASTX or BLASTN (Altschul et al., 1997 ) against the GenBank nr database (http://www.ncbi.nlm.nih.gov/).
Investigating the presence of sequences 95100% similar to E. coli by PCR.
Primers were chosen from 20 subclone sequences that were 95100% similar at the nucleotide level to E. coli (Table 1). Each primer pair was tested by PCR (as previously but with an annealing temperature of 60 °C or 64 °C) using either Eca or E. coli cells to provide DNA template.
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RESULTS |
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The remaining 364 sequences from 2B8, yielding a total of 118150 bp, and 256 sequences from 1C22, yielding a total of 99537 bp, represented a slight excess of the target one-fold coverage in each case. The sequences were assembled into contigs using CAP3 software (http://genome.cs.mtu.edu/cap/cap3.html). Only 84 sequences did not fall into contigs (singletons). In contrast, 536 sequences fell into 143 contigs, which ranged in size from 52 bp to 2346 bp, with a mean size of 651 bp. Taken together, the contigs and singletons comprise 119325 bp of independent sequence, approximately 60% of the predicted region covered by the BAC clones.
BLASTX/N searches were conducted on all contigs and singletons, and these sequences were compiled into categories according to the organism providing the strongest database match (Fig. 1). Sequences were categorized as no match if the BLAST search found no similar sequences. The no match category would be expected to include non-coding sequences in addition to novel ORFs. Perhaps unsurprisingly, given that the locus contains a putative hrp gene cluster, similarities to Erwinia genes constituted a significant percentage of the sequences from each BAC. In addition, a high percentage of sequences from each BAC showed no similarities to database sequences. The majority of strong matches to sequences from enterobacteria, including E. coli, were found on 2B8. Conversely, the majority of sequences with strongest similarities to genes in X. fastidiosa, the only plant pathogen to be completely sequenced, were found on 1C22 (Fig. 1
).
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In addition to hrpdsp genes, sequences similar to Erwinia hec, pec, pel and peh genes were found (Table 3). In E. chrysanthemi, the hecAB genes are located within the hrp cluster (Kim et al., 1998
). PCR with primers designed to anneal to each of the hec-like Eca sequences generated amplification products from 1C22 but not from 2B8. As the entire hrp cluster resides on 2B8, it thus appears that the hecAB genes of Eca are located outside the hrp cluster and are therefore arranged differently in Eca and E. chrysanthemi.
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In addition to the hecAB orthologues, there are other sequences from 1C22 with similarity to adhesin, haemagglutanin and haemolysin genes from mammalian pathogens such as Neisseria meningitidis and Pseudomonas aeruginosa, and the plant pathogen X. fastidiosa (Simpson et al., 2000 ).
Sample sequencing allows a comparison of genome structure between Eca and E. coli
A number of Eca sequences show strongest similarities to E. coli ORFs. These sequences, and their position in the E. coli genome, are shown in Table 4. The E. coli genome (4·6 Mb) has been divided into 100 minutes. Each minute thus represents approximately 46 kb. Eca sequence similarities were observed to genes throughout the E. coli genome, from 23 to 99 min. Assuming a similar genome size, we would predict the region of the Eca genome covered by the two overlapping BAC clones to be no more than 5 min. These results suggest rearrangement between the genomes. Nevertheless, the majority of E. coli-like sequences in 2B8 reside between 27 and 36·5 min on the E. coli chromosome. In particular, the ORFs ychM, chaA, chaC and goaG are adjacent to nar genes (involved in nitrite/nitrate metabolism) in both Eca and E. coli, suggesting some conservation in gene order between these enterobacteria. In addition, ORFs b1598, ydgB, rstA and rstB are all clustered between 36 and 36·3 mins in the E. coli genome and appear to also be clustered in the Eca genome.
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DISCUSSION |
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An Eca BAC library was made, and is being used to generate a physical map of the Eca genome (results not shown). A region of two overlapping BACs, predicted to span an entire hrpdsp gene cluster, was identified by a combination of BAC end sequencing and hybridization probing. The hrpdsp gene cluster in E. amylovora has been sequenced and comprises 30 ORFs, covering approximately 30 kb of DNA. Despite the independent sequence coverage obtained after contiging (119325 bp) only representing 60% of the region spanned by the BACs, 27 (90%) of the predicted 30 Eca hrpdsp ORFs were identified. Thirteen of these ORFs (approx. 9 kb) are included in the overlap between the BACs, where two-fold sequence coverage would be expected. Nevertheless, one-fold sequencing of 2B8 alone still revealed 25 (86%) of the 29 hrpdsp genes on this BAC. The three ORFs that were not initially identified, hrpA, hrpB and hrpO, were relatively small (<446 bp) and thus less likely to be detected by one-fold sample sequencing. PCR amplification across the regions predicted to contain these ORFs revealed that they were indeed present in Eca. Extrapolation of this PCR strategy facilitated a detailed map of the Eca hrpdsp gene cluster and showed that, whereas the Eca cluster is similar in content and arrangement to that of E. amylovora, it is different to that in the more closely related soft-rot potato pathogen E. chrysanthemi. Moreover, it is different to the hrpdsp cluster of E. herbicola, which lacks the hrpW and hrpW-chaperone genes identified here (Mor et al., 2001 ). In addition, genes encoding two Hrp-dependent effector molecules identified in this work, dspE and hrpW, have not yet been observed in other soft-rot erwinias. The conservation of the hrpdsp gene clusters between E. amylovora and Eca has thus provided a useful test of the number of ORFs that may be detected and identified by low-level draft sequencing of overlapping BAC clones.
In addition to the hrp and dsp genes, pel, peh, pec and hec genes were identified. E. chrysanthemi is the only Erwinia species previously reported to possess pecSM genes, which encode repressors of pel and cel gene expression (Praillet et al., 1997 ) and inducers of peh gene expression (Nasser et al., 1999
). The hecAB genes have also only previously been observed in E. chrysanthemi; they are of unknown function but show strong sequence similarity to haemolysin genes and are predicted to be co-regulated with operons involved in pectinolysis (Kim et al., 1998
). A haemolysin-like gene in Pseudomonas putida is involved in adhesion of the bacterium to plant seeds (Espinosa-Urgel et al., 2000
). In addition to the hec-like sequences, several other sequences were observed with strong similarities to adhesin, haemagglutinin and haemolysin genes in mammalian pathogens and the plant pathogen X. fastidiosa. This finding is perhaps surprising, as X. fastidiosa is not an enterobacterium, has no known pathogenicity factors in common with Erwinia (it lacks pectinolytic and hrp genes) and is considered a specialized plant pathogen rather than a saprophyte or opportunistic pathogen. All of the haemagglutinin- and haemolysin-like sequences were obtained from the 1C22 BAC clone, although they seem to lie at different loci along the length of this BAC. A number of sequences similar to rhizobacterium genes were observed, including attachment and opine catabolism genes from Agrobacterium tumefaciens, and opine catabolism genes from rhizobacteria. These sequences were all located on 2B8 and the sequences similar to agtA and mocR were shown by hybridization to reside on a single EcoRI fragment and a single SalI fragment, both of approximately 23 kb in size. It is thus possible that they are derived from a single operon. Agrobacterium and other rhizobacteria induce opine synthesis in planta to provide a specialized nutrient source (Kemp, 1982
; Murphy et al., 1993
). Although opine catabolism is known in other bacteria, including Pseudomonas spp. (Beauchamp et al., 1991
; Gardener & de Bruijn, 1998
), it has not been reported in Erwinia spp. or other enterobacteria.
The functions of all of the Eca sequences identified above are unknown. However, Rantakari et al. (2001) have recently shown that type III secretion plays a role in pathogenesis in Ecc. As Eca is amenable to genetic manipulation, the information gained from sample sequencing could feed directly into gene functional studies through, for example, PCR identification of specific gene knock-outs from a Tn5 mutation library or microarrays for genome-scale analyses of gene expression.
Comparisons of genome organization between Eca and E. coli
Erwinia spp. are the closest plant-pathogenic relatives to the model bacterium and animal pathogen E. coli. Comparisons between the genomes of Eca and E. coli will thus contribute to our understanding of evolution within the Enterobacteriaceae and may also indicate common or distinct mechanisms in animal and plant pathogenesis. In a preliminary assessment of structural organization between Eca and E. coli, we compared the locations of only those sequences showing the strongest similarity to E. coli ORFs. Approximately 10% of the Eca sequences showed strongest similarity to those of E. coli. The majority of these were obtained from clone 2B8 and comprise the nar gene equivalents of these two species. The close association of ychM, chaA, chaC and goaG gene orthologues to nar genes in both E. coli and Eca suggests a degree of conservation in genome organization. Moreover, the b1598, ydgB, rstA and rstB ORFs are apparently clustered in E. coli and Eca. The order of orthologous genes on the chromosomes of different enterobacteria is usually conserved (Brunder & Karch, 2000 ), or else large regions may be rearranged but with conserved gene order within each region (Liu & Sanderson, 1996
). However, many Eca sequences from the sampled BACs were strongly similar to sequences located throughout the E. coli genome, indicating considerably different gene order between these species. Such a lack of conservation in genome organization has been reported between the closely related Bacillus cereus and Bacillus subtilis (Økstad et al., 1999
).
In conclusion, sample sequencing of overlapping BAC clones from a region of interest has yielded numerous novel candidate pathogenicity sequences and allowed a preliminary comparison of structural organization between the genomes of Eca and E. coli. Moreover, a multiple genome coverage BAC library of Eca, in conjunction with Southern blotting, facilitated the generation of a physical map across the 200 kb genomic region spanned by 2B8 and 1C22. This will allow comparisons with other erwinias to assess conservation of gene order, content, and the relative location of the hrpdsp cluster.
Information from a high percentage of ORFs may be obtained by one-fold sample sequencing, as was demonstrated by the identification of 90% of expected hrpdsp genes. As a physical map of Eca develops, this approach could be extended to the entire genome. One-fold sequence coverage of a minimum tiling path of BACs from around the genome would rapidly generate information for high-throughput analyses of gene expression and function at relatively low cost: a strategy that could be applied to other bacteria where genomic information is lacking and where its acquisition is limited by funds and resources.
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ACKNOWLEDGEMENTS |
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Received 18 October 2001;
revised 8 January 2002;
accepted 14 January 2002.