Centre for Molecular and Cellular Biology, The University of Queensland, Brisbane, QLD 4072, Australia1
Author for correspondence: John S. Mattick. Tel: +61 7 3365 4446. Fax: +61 7 3365 4388. e-mail: j.mattick{at}cmcb.uq.edu.au
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ABSTRACT |
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Keywords: type IV pili, fimbriae, twitching motility, surface translocation, Pseudomonas aeruginosa
The GenBank accession number for the sequence determined in this work is U93274.
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INTRODUCTION |
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Type IV fimbriae are flexible, filamentous surface appendages produced at the poles of the bacterial cell which mediate attachment to the host epithelial tissue and a form of surface translocation termed twitching motility. They also appear to act as receptors for certain bacteriophages. The mechanism of twitching motility has been proposed to be fimbrial retraction and extension (Bradley, 1980 ). Bacteria that exhibit twitching motility can be seen as rough, spreading colonies on agar plates under humid conditions, and as very fine zones of rapid colony expansion on smooth surfaces (Semmler et al., 1999
). This phenotype has been used to distinguish between bacteria which have functional fimbriae and those which possess a mutation in genes affecting fimbrial biogenesis or function (Hobbs et al., 1993
; Alm & Mattick, 1997
). Twitching motility also appears to be an important virulence factor as mutants which lack functional type IV fimbriae have reduced infectivity (Hazlett et al., 1991
; Comolli et al., 1999
). Twitching motility has also been shown to be involved in biofilm formation (OToole & Kolter, 1998
), which may be important during infection (Potera, 1999
; Costerton et al., 1999
).
Twitching motility and type IV fimbriae have been described in a wide range of bacteria, including P. aeruginosa, Neisseria gonorrhoeae, Neisseria meningitidis and other Neisseriaceae, various Moraxella species, Dichelobacter nodosus, Branhamella catarrhalis, Suttonella indologenes, Alteromonas putrefaciens, Pasteurella multocida, Xanthomonas maltophila, Kingella denitrificans and many others (Mattick et al., 1993 ). Related genes encoding the type IV fimbrial subunit and other components have also been found in a number of bacteria not previously recognized to possess type IV fimbriae, including Aeromonas spp. (Pepe et al., 1996
; Barnett et al., 1997
), Legionella pneumophila (Liles et al., 1998
; Stone & Abu Kwaik, 1998
), Pseudomonas syringae (Roine et al., 1998
) and Azoarcus spp. (Dorr et al., 1998
), the last two indicating that type IV fimbriae are important in bacterial colonization not only of animals but also of plants, fungi and protozoa. Type IV fimbriae have also been found in Myxococcus xanthus, where they have been shown to be required for social gliding motility, a process which appears to be functionally equivalent to twitching motility (Wu & Kaiser, 1995
; Semmler et al., 1999
).
Type IV fimbriae are filaments of about 6 nm in diameter which range up to several µm in length. They are primarily composed of a small (145160 aa) structural subunit (pilin or PilA in P. aeruginosa) with a characteristic highly conserved and highly hydrophobic amino-terminal region. This forms the core of the helical structure, whose outer face is comprised of the more hydrophilic and more variable domains of the subunit (Folkhard et al., 1981 ; Paranchych & Frost, 1988
; Dalrymple & Mattick, 1987
; Parge et al., 1990
; Forest & Tainer, 1997
).
The biogenesis and function of type IV fimbriae in P. aeruginosa is dependent on at least 35 genes which are located in several clusters on the chromosome. These include genes encoding the fimbrial subunit (PilA), a leader peptidase (PilD), ancillary proteins with pre-pilin-like leader sequences (PilE, PilV, PilW, PilX, FimT, FimU), inner and outer-membrane proteins (PilC, PilQ), nucleotide-binding proteins (PilB, PilT, PilU), other proteins whose functions are not clear (PilM-P, PilF, PilY1, PilY2, PilZ), the RpoN sigma factor, 2 two-component sensorregulator pairs (PilS/PilR and FimS/AlgR) and a complex chemosensory signal transduction system (PilG-L, ChpA-C) (for a recent review see Alm & Mattick, 1997 ). Here, we report the identification and characterization of a novel gene, fimV, which is also required for twitching motility.
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METHODS |
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Construction of isogenic mutants.
Allelic exchange mutants were constructed of the genes usg-1 and hisT, which lie adjacent to fimV, using the sucrose selection system described previously (Schweizer, 1992 ; Alm & Mattick, 1996
). Briefly, these genes were subcloned into the vector pUK21 (forming pAS36 and pAS14, respectively). The tetracycline gene cartridge from pSM-TET was cloned into the PstI site within usg-1 and into a blunted NcoI/NdeI site in hisT to disrupt the genes. The resulting clones were then digested with SpeI, whose sites span the multiple cloning site of pUK21, and the disrupted genes were inserted into the suicide vector pRIC380. This vector carries the genes sacBR, which promote sensitivity to sucrose, and oriT, enabling conjugal transfer. The constructs were then transformed into the E. coli donor strain S17-1 in preparation for mating into P. aeruginosa. Following conjugation, the transconjugates were selected on 5% sucrose media containing tetracycline. This forces the excision of the plasmid whilst leaving the homologously recombined mutated gene in the chromosome. Mutants were confirmed using Southern analysis and examined using the subsurface twitching assay (see below).
Recombinant DNA techniques.
The preparation of plasmid DNA, restriction endonuclease digestion (New England Biolabs), ligation reactions, Southern blotting and radiolabelling of probe were carried out using standard protocols (Sambrook et al., 1989 ).
Sequence analysis.
Sequence templates were generated by a combination of subcloning and shotgun cloning strategies. The dsDNA was prepared for sequencing using a modified alkaline lysis method involving PEG precipitation (Applied Biosystems). Sequencing was performed using the Applied Biosystems PRISM system on a 373A automated sequencer. Nucleotide and predicted protein sequences were analysed using gapped BLAST (Altschul et al., 1997 ), SMART (Schultz et al., 1998
; Ponting et al., 1999
) and PSORT (Nakai & Horton, 1999
) programs.
Protein expression and analysis.
The FimV protein was subcloned into a P. aeruginosa expression plasmid, pUCPKS (Watson et al., 1996a ) and transformed into P. aeruginosa ADD1976, which contains a chromosomal T7 RNA polymerase gene under the control of an inducible lac promoter (Brunschwig & Darzins, 1992
). Protein expression was induced in the presence of [35S]methionine and analysed on 7·5% SDS-polyacrylamide gels as described previously (Alm & Mattick, 1995
).
Western blotting.
Bacterial cells from plates were resuspended to an OD600 of 1·0 in 50 mM sodium carbonate buffer pH 9·6. Samples (1 ml) were centrifuged and the cell pellet was resuspended in 100 µl sample buffer (60 mM Tris/HCl pH 6·8, 2% SDS, 10% glycerol, 5% ß-mercaptoethanol, 0·001% bromophenol blue). To remove DNA, the samples were centrifuged at 45000 r.p.m. for 90 min and the supernatant was heated to 100 °C for 5 min. Proteins in the samples were then separated by SDS-PAGE using a 15% polyacrylamide gel and a 5% stacking gel as described by Laemmli (1970) and transferred electrophoretically to Hybond-C nitrocellulose (Amersham) in the Tris/glycine system described by Towbin et al. (1979)
. Proteins were detected with anti-PilA antiserum (1:5000) followed by goat anti-rabbit immunoglobulin G conjugated to alkaline phosphatase (1:5000; Boehringer Mannheim).
ELISA.
This was based on a method described by Engvall & Perlmann (1972) . The cells were resuspended in 50 mM sodium carbonate buffer pH 9·6 at an OD600 of 1·0 and 200 µl of suspension was loaded into wells of a 96-well ELISA plate. After overnight incubation at 4 °C, the wells were washed with PBS (137 mM NaCl, 2 mM KCl, 10 mM NaHPO4, pH 7·4) containing 0·1% Tween 20, blocked with 3% BSA for 1 h and then exposed to an anti-PilA antibody at a starting dilution of 1:500 for 2 h at 37 °C. After removal of antisera, the wells were again washed with PBS containing 0·1% Tween 20. Goat anti-rabbit immunoglobulin G conjugated with alkaline phosphatase was then added (1:5000) and the mixture incubated for 2 h at 37 °C. Detection was carried out using 20 mg p-nitrophenyl phosphate (Sigma) ml-1 in 1 M Tris buffer pH 8·0 and the plate was read at 405 nm using an ELISA reader (Bio-Rad).
Elastase assay.
Aliquots (2 µl) of overnight broth cultures were inoculated onto the surface of LB agar plates containing 0·1% elastin (Sigma). After incubation at 37 °C for 23 d, plates were examined for zones of proteolytic clearing surrounding the colonies.
Twitching motility assay.
Twitching motility was assayed as described previously (Alm & Mattick, 1995 ). Briefly, the P. aeruginosa strain to be tested was stab-inoculated through a 1% agar plate, and after overnight growth at 37 °C the zone of twitching motility between the agar and Petri dish interface was visualized by staining with Coomassie brilliant blue R250.
Light still and video microscopy.
Light microscopy was performed as described previously (Semmler et al., 1999 ). Sterile microscope slides were submerged in molten GelGro media to obtain a thin layer of media coating the slide. The slides were allowed to set in a horizontal position and air-dried briefly prior to use. The slides were then inoculated with a small loopful of bacteria taken from an overnight plate culture. A sterile glass coverslip was placed over the point of inoculation and the slide transferred to a large Petri dish containing a moist tissue and sealed with Nescofilm (Bando Chemical Industries) to maintain humid conditions. Incubation times ranged from 26 h at 37 °C.
Slide cultures were examined using a Zeiss Axioskop 50 microscope with Nomarski facilities at x200 to x400 magnification. Video microscopy was performed in a room heated to 30 °C. Video images were recorded over a period of 24 h at speeds of either 1 field per 3·22 s, 1 field per 0·66 s or real time (1 field per 1/50 s) using a JVC TK-CI38IEG video camera connected to a Sanyo TLS-S2500P time-lapse video recorder. Video images were edited and converted to Quick-time movies using Avid Videoshop version 3.0 and can be viewed at http://www.cmcb.uq.edu.au/cmcb/PUBS/twitch.html.
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RESULTS |
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DNA flanking the site of insertion of the transposon in each of these mutants was cloned using a marker rescue approach that involved digestion of mutant chromosomal DNA with restriction enzymes (either EcoRI or HindIII) which cut once within the transposon beyond the tetracycline resistance marker, ligation into pBluescript II KS(+) and recovery of tetracycline-resistant E. coli colonies. The DNA adjacent to the transposon insertion in each of the mutants was then sequenced using a primer which is specific for the inverted repeats of Tn5-B21 (Hobbs et al., 1993 ). Database searches with these sequences revealed that the transposon mutants were unique and not located in previously characterized genes.
Cloned chromosomal DNA flanking the site of transposon insertion in S76 was used for Southern analysis of mutant genomic DNA. This analysis demonstrated that the transposon insertions in mutants S4, S76, S359 and S361 were located in the same 10 kb HindIII and 6 kb EcoRI restriction fragments, whereas the S125 insertion site was not located within these fragments. The cloned DNA from S76 was also used to screen a reference PAO1 cosmid library (Ratnaningsih et al., 1990 ), from which we identified three cosmids (pMO010323, pMO011618 and pMO012140) covering the region. Restriction mapping of these cosmids and further Southern analysis showed that the transposon insertion site of the S125 mutant was located in 3·5 kb HindIII and 1 kb EcoRI fragments contained within these cosmids, adjacent to the restriction fragments containing the other four insertions (Fig. 1
).
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Cloning and sequence analysis of the fimV region
The 6 kb EcoRI (pASP6) and 3·5 kb HindIII (pASB351) fragments (Fig. 1) were subcloned from pMO011618 and sequenced. The complete sequence of 8 kb of this region (GenBank accession no. U93274) identified six ORFs (Fig. 1
), three of which were previously characterized genes from P. aeruginosa: leuB (3-isopropylmalate dehydrogenase); asd (aspartate-ß-semialdehyde dehydrogenase); and orfA (Hoang et al., 1997
; Hoang & Schweizer, 1997
). Our sequence analysis of this region revealed a frameshift error in the previously reported sequence of orfA. The revised sequence shows that this putative gene encodes a protein which shows significant homology to the product of the unknown genes termed usg-1 from Azotobacter vinelandii (50% identity and 66% similarity over 333 aa) and E. coli (36% identity and 53% similarity over 339 aa). In light of this we have renamed orfA as usg-1. Interestingly, the Usg-1 proteins are also predicted to belong to the family of aspartate-ß-semialdehyde dehydrogenases and in fact are homologous to the asd gene products from Vibrio cholerae and Vibrio mimicus, as well as Shewanella sp. and L. pneumophila. These enzymes catalyse the second step in the common biosynthetic pathway leading from aspartate to the cell wall precursor meso-diaminopimelate, lysine, methionine, isoleucine and threonine. Two of the remaining ORFs, which we have termed orfB and hisT, showed significant homology to genes characterized in other bacteria. The product of orfB (187 aa) shows close strong similarity to the hypothetical proteins YafE from E. coli (58% identity and 68% similarity over 182 aa) and YcgJ from Bacillus subtilis (37% identity and 54% similarity over 166 aa), and to putative methyltransferases from a number of bacterial species including Lactococcus lactis, Bacillus stearothermophilus, Streptomyces hygroscopicus, Microcococcus luteus and E. coli. HisT shows strong homology to pseudouridylate synthetases (involved in tRNA modification) from a broad spectrum of bacterial species (Arps & Winkler, 1987
). Sequence analysis of the region downstream of hisT indicated that the previously characterized gene trpF (phosphoribosyl anthranilate isomerase) (Murata, 1996
) is located immediately downstream of hisT.
The remaining ORF in this region was found to contain all five transposon insertions (Fig. 1). This ORF, designated fimV, is 2·8 kb in size and has an overall G+C content of 67·5 mol%, in agreement with the estimated 67 mol% for the P. aeruginosa genome as a whole (West & Iglewski, 1988
). Further analysis of the fimV sequence showed a decrease in rare codon usage within the ORF and a high G+C bias (81·7%) in the third codon position, suggestive of a likely coding region (West & Iglewski, 1988
).
BLAST search analyses at NCBI revealed that FimV shows regions of homology with the recently described protein TspA of N. meningitidis (Kizil et al., 1999 ). Analysis of the Unfinished Microbial Genomes databases at NCBI also identified homologies between FimV and predicted proteins from N. gonorrhoeae (TspA equivalent), L. pneumophila, V. cholerae and Shewanella putrefaciens (Fig. 2
). FimV also shows significant homology with the predicted product of a second P. aeruginosa gene identified in the unfinished genome sequences (Fig. 2
). Interestingly, except for Shewanella putrefaciens, these bacterial species are all known to possess type IV fimbriae. Further analysis of the Shewanella putrefaciens genome sequences indicated that this organism should also be capable of producing type IV fimbriae as it possesses many close homologues of P. aeruginosa proteins required for type IV fimbrial biogenesis (including homologues of PilA-F, PilM-Q, PilT and PilU). It appears therefore that fimV is specific to type-IV-fimbriate bacteria.
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In P. aeruginosa, fimV is situated between the genes usg-1 and hisT (Fig. 1). Interestingly, in E. coli usg-1 is known to be located directly upstream of and within an operon with hisT (Arps & Winkler, 1987
). The difference therefore between the E. coli and P. aeruginosa genomic organization is the presence of fimV between usg-1 and hisT. A search of the E. coli genome shows that it does not contain a homologue of FimV. We constructed allelic exchange mutants of the P. aeruginosa genes usg-1 and hisT to determine whether (like fimV) either might be involved in type IV fimbrial biogenesis or function (Fig. 1
). Following confirmation by Southern blotting, the mutants PAKusg-1::TcR and PAKhisT::TcR were examined for their ability to exhibit twitching motility using the subsurface twitching assay and light microscopy. Both retained wild-type twitching motility (data not shown). Further analysis of the sequences surrounding the fimV homologue genes in the other type IV fimbriate bacteria revealed that Shewanella putrefaciens has a similar genetic arrangement to that of P. aeruginosa, with the fimV homologue situated between genes encoding homologues of Usg-1 and HisT. The V. cholerae genetic arrangement also has the fimV homologue situated upstream of hisT. The conserved genetic arrangement in these type-IV-fimbriate species perhaps further supports our hypothesis that these genes encode proteins with analogous functions to FimV.
T7 expression of FimV
The protein encoded by fimV was examined using a T7 expression system. fimV was cloned as a 3·161 kb Ppu10I/BspHI fragment covering the predicted coding region of fimV (from -86 bp to the stop codon) into the broad-host-range T7 expression vectors pUCPKS/SK (Fig. 1). The gene was cloned both in the forward and reverse direction (pASE280 and pASE281, respectively) relative to the T7 promoter. These constructs were transformed into P. aeruginosa ADD1976, which contains a chromosomal T7 RNA polymerase gene under lac promoter control. A unique band was observed in the pASE280 expressed lane that was not found in the other samples (Fig. 3
). The size of this band was estimated to be 145 kDa, which was 47 kDa greater than that predicted from the sequence for FimV (98 kDa). The fidelity of the insert including the stop codon was confirmed by restriction enzyme profiling and end sequence analysis. To try to locate the source of the anomaly we then generated truncations of the acidic C terminus of FimV, by removing 455 bp (pASE230) and 1005 bp (pASE18a) from the end of the fimV gene, with in-frame stop codons close downstream in the vector (Fig. 1
). However, both truncations still produced proteins with similar anomalies in electrophoretic migration (Fig. 3
). This suggests that the anomaly may not be caused by the highly acidic C terminus but rather resides in the N-terminal two-thirds of the protein, perhaps due to some other undetermined secondary structure or possibly covalent modification of the protein in this region.
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We assessed quantitatively the degree of sensitivity of each of the fimV mutants for infection by the type-IV-fimbriae-specific bacteriophage PO4. All mutants demonstrated wild-type titres for this phage (data not shown). These data suggest that the S4 group of fimV mutants may fall into the same class as mutants of pilG, pilI and pilJ, which also appear incapable of producing assembled pilus filaments but remain sensitive to fimbriae-specific bacteriophage. It has been proposed that these strains form a preliminary structure or pre-pilus complex consisting of an exposed pilus tip at the cell surface to which the phage can bind and subsequently infect (Darzins, 1993 , 1994
).
Complementation of fimV
The plasmid pASE281 containing fimV cloned downstream of the lac promoter (which is constitutively active in P. aeruginosa) was used for complementation studies of the fimV mutants. Subsurface twitching assays showed that the non-motile S4 mutant had twitching motility restored by pASE281 but not to wild-type levels. Instead, the zones remained small and irregular (Fig. 4e). S125 was also not complemented to wild-type twitching motility but appeared to exhibit exaggerated medusa-like structures erupting from the centre of the colony (Fig. 4f
). PAK containing this construct also resulted in a reduction and aberration of the wild-type twitching zone (Fig. 4d
). These results suggested that the overexpression of fimV may be interfering with normal twitching motility both in the mutants and in wild-type PAK.
To test this possibility, fimV was cloned into pMMB207, a vector that has an inducible tac promoter (carries the repressor lacI), to generate plasmid pASM281 (Fig. 1). By varying the concentrations of IPTG, the levels of expressed FimV could therefore be altered. Using a concentration range of 010 mM IPTG, the twitching zones from the S4 and S125 mutants and PAK were observed. The results showed that between 0·01 mM and 0·03 mM IPTG there was complete restoration of normal twitching motility in the mutants and the wild-type was unaffected (Fig. 5
). (We presume that the twitching motility observed in the mutants at 0 mM IPTG was due to leakage of the tac promoter.) Levels of IPTG above 0·03 mM resulted in reduced and aberrant twitching zones in both wild-type and the mutants (Fig. 5
). These data confirm that the loss of fimV is responsible for the loss of twitching motility in the mutants, that overexpression of fimV causes aberrant twitching motility and that a specific level of FimV expression is required for normal motility.
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In view of the complementation data, which suggested that fimV function is controlled by gene dosage, the effect of overexpressing fimV in the wild-type, in the non-motile fimV mutant S4 and in S125 was also examined. Transformation of pASE281 into the mutants partially restored the raft and lattice-type structures typical of twitching motility, but also resulted in dramatic cell elongation (Fig. 6d), which was also observed when fimV was overexpressed in the wild-type (not shown). The most spectacular phenotype was observed in S125 with pASE281 which exhibited grossly elongated cells, and a medusa-like phenotype at the colony level (see Fig. 4f
). These effects were not observed in controls with vector alone, indicating that the elongated phenotype was not an artefact of carbenicillin selection (data not shown).
Electron microscopy of cells taken from the colony edges confirmed that overexpression of fimV in PAK, S4 and S125 produces cells that are grossly elongated, often with lengths up to 50100 times normal (data not shown). Interestingly, only a small percentage of the population demonstrated this abnormality, and cells at the centre of the colony, away from the twitching edge, appeared largely normal. This suggests that only those cells which are actively in twitching mode (see Semmler et al., 1999 ) are affected by abnormally high levels of FimV.
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DISCUSSION |
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Although the precise role of FimV has not yet been determined, the examination of the mutant phenotypes has revealed that this protein is essential for twitching motility. Four of the five fimV mutants (S4, S76, S359, S361) are incapable of twitching motility. Western analysis and ELISA data showed that these mutants continue to produce the PilA subunit but do not produce surface-assembled fimbriae, suggesting a lesion in the process of fimbrial assembly. The fifth mutant, S125, produces a small, irregular twitching zone, demonstrates impairment of cellular motility during twitching and expresses a reduced number of fimbriae on the cell surface. The differences observed between S125 and the other mutants is likely to be due to the sites of transposon insertions, which in S125 is located ~230 bp from the stop codon of fimV. We predict that this mutant produces a truncated form of FimV, presumably with partial function, whilst the others do not produce a stable fimV product.
Our complementation data indicates that a precise level of FimV is required for normal twitching motility. Microscopic examinations show that overexpression of fimV results in the formation of dramatically elongated cells, probably accounting for the observed defects in twitching motility when FimV is produced at high levels. The presence of a putative peptidoglycan-binding domain in the N-terminus of FimV and its homologues may give some clue as to the function of these proteins. The elongated cell phenotype obtained when fimV is overexpressed may be due to interference by high levels of FimV during the remodelling of the peptidoglycan layer in the process of cell division and/or fimbrial biogenesis, perhaps indicating some direct association of FimV with peptidoglycan components. It is also of interest to note that fimV is located downstream of the genes asd and usg-1, the products of which are predicted to be involved in the production of the cell wall precursor meso-diaminopimelate. We propose that FimV may be involved in remodelling of the peptidoglycan layer to enable assembly of the type IV fimbrial structure and associated machinery. Interestingly, upstream of the pilMQ operon of P. aeruginosa is a gene, ponA, encoding a high-molecular-mass penicillin-binding protein (PBP-1A), one of a class of proteins involved in the formation and maintenance of the peptidoglycan layer (Martin et al., 1993 ). A related gene (fimD) is also observed in the same operon as the fimbrial subunit genes (fimA and fimZ) in class II strains of D. nodosus (Hobbs et al., 1991
). Taken together, these observations suggest that cell wall structure and local remodelling is important for type IV fimbrial biogenesis and/or function at the pole of the cells.
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ACKNOWLEDGEMENTS |
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Received 23 December 1999;
revised 29 February 2000;
accepted 7 March 2000.