Download Three multidomain esterases from the cellulolytic

Microbiology (2000), 146, 1391–1397
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Three multidomain esterases from the
cellulolytic rumen anaerobe Ruminococcus
flavefaciens 17 that carry divergent dockerin
sequences
Vincenzo Aurilia,† Jennifer C. Martin, Sheila I. McCrae, Karen P. Scott,
Marco T. Rincon and Harry J. Flint
Author for correspondence : Harry J. Flint. Tel : j44 1224 716651. Fax : j44 1224 716687.
e-mail : h.flint!rri.sari.ac.uk
Rowett Research Institute,
Greenburn Road,
Bucksburn, Aberdeen
AB21 9SB, UK
Three enzymes carrying esterase domains have been identified in the rumen
cellulolytic anaerobe Ruminococcus flavefaciens 17. The newly characterized
CesA gene product (768 amino acids) includes an N-terminal acetylesterase
domain and an unidentified C-terminal domain, while the previously
characterized XynB enzyme (781 amino acids) includes an internal
acetylesterase domain in addition to its N-terminal xylanase catalytic domain.
A third gene, xynE, is predicted to encode a multidomain enzyme of 792 amino
acids including a family 11 xylanase domain and a C-terminal esterase domain.
The esterase domains from CesA and XynB share significant sequence identity
(44 %) and belong to carbohydrate esterase family 3 ; both domains are shown
here to be capable of deacetylating acetylated xylans, but no evidence was
found for ferulic acid esterase activity. The esterase domain of XynE, however,
shares 42 % amino acid identity with a family 1 phenolic acid esterase domain
identified from Clostridum thermocellum XynZ. XynB, XynE and CesA all
contain dockerin-like regions in addition to their catalytic domains, suggesting
that these enzymes form part of a cellulosome-like multienzyme complex. The
dockerin sequences of CesA and XynE differ significantly from those
previously described in R. flavefaciens polysaccharidases, including XynB,
suggesting that they might represent distinct dockerin specificities.
Keywords : esterase, cellulosome, Ruminococcus, rumen, dockerin
INTRODUCTION
Efficient breakdown of plant cell wall material by microorganisms requires not only glycosidase activities that
cleave polysaccharide chains, but also activities that
remove groups that are connected by ester linkages to
polysaccharides (Christov & Prior, 1993 ; Williamson et
al., 1998). Xylans are often heavily substituted with
acetyl groups and can also be connected to phenolic
acids, and hence to lignin, via ester linkages involving
arabinose substituents (Iiyama et al., 1994), while
pectins carry esterified methyl and acetyl groups
.................................................................................................................................................
† Present address : CNR-IABBAM, Via Argine 1085-80147 PonticelliNapoli, Italy.
The GenBank accession numbers for the sequences reported in this paper
are AJ238716 (cesA) and AJ272430 (xynE).
(Rombouts & Thibault, 1986 ; Searle-van Leeuwen et
al., 1992). There is evidence that acetyl substituents can
limit the attack of endoxylanases upon xylans (Wood &
McCrae, 1986 ; Biely et al., 1986 ; Puls et al., 1991) and
ferulic acid ester linkages may represent a significant
impediment to enzymic removal of polysaccharides from
lignified plant cell walls (Iiyama et al., 1994).
The rumen is a particularly active site for anaerobic
breakdown of a variety of plant cell wall material.
Among ruminal micro-organisms, phenolic acid esterase
activities have been reported from anaerobic fungi
(Borneman et al., 1991, 1992), and a gene encoding a
phenolic acid esterase has been isolated from Butyrivibrio fibrisolvens (Dalrymple et al., 1996). Acetylxylan esterases have been reported from rumen fungi
(Dalrymple et al., 1997) and from the rumen bacteria
Butyrivibrio fibrisolvens and Fibrobacter succinogenes
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V. A U R I L I A a n d O T H E R S
(McDermid et al., 1990 ; Hespell & O’Bryan-Shah,
1988). There is little evidence so far for the involvement
of esterase activities in cell wall degradation by Ruminococcus spp. (Akin et al., 1993), which represent one of
the most numerous groups of cellulolytic bacteria in the
rumen. Although it has been shown that Ruminococcus
albus can release phenolic monomers from plant material (Giraud et al., 1997), there have been no biochemical or molecular studies on esterases from ruminococci. We report here the specificities and relationships
of two catalytic domains from Ruminococcus flavefaciens enzymes that function as acetylesterases, and
also identify a third gene that encodes a multidomain
esterase. Recent evidence indicates that most plant cell
wall polysaccharidases in R. flavefaciens 17 carry
dockerin sequences, indicating that they are likely to
belong to cellulosome complexes (Kirby et al., 1997).
The three R. flavefaciens multidomain esterases discussed here, two of which carry xylanase domains
within the same polypeptide, also carry dockerin-like
sequences, suggesting that they may also be cellulosomeassociated.
METHODS
Gene isolation. About 2000 clones from a R. flavefaciens
λEMBL3 phage library were screened by detection of acetylesterase activity. The phages were transferred to Whatman
no.1 filter paper and incubated with β-naphthyl acetate (0n5 mg
ml−") and tetrazotized o-dianisidine (Blue Diazo) in 50 mM
sodium phosphate buffer pH 7n4. Two positive clones were
isolated and shown to be identical in their restriction enzyme
profiles. The clone pCesA.est is a plasmid subclone from one
phage that was subsequently shown to carry the coding region
for the N-terminal 390 amino acids of the cesA gene product.
Probes derived from pCesA.est were used to obtain overlapping plasmid clones by screening a pUCI3 plasmid library
of R. flavefaciens 17 in order to obtain the complete cesA gene
sequence. Correspondence between the cloned sequences and
the chromosomal cesA gene was confirmed by direct PCR
amplification using a primer set internal to cesA.
xynE was identified initially by PCR from chromosomal DNA
using the oligonucleotide JIIf [CCI(C\T)TI(A\G)TIGA(A\
G)TA(T\C)TA(T\C)ATIGTIG] (Kirby, 1996) as a family 11
domain-specific primer [corresponding to the amino acid
sequence P(L\F)(V\I\M)EYY(M\I)V] together with the reverse primer pESTr (CCICCCATIGARAAICC), designed to
recognize coding regions (corresponding to the amino acids
GFSMGG) of related family 1 esterases.
Construction of pXynB.est, expressing the esterase domain
of XynB. A 965 bp fragment was recovered from the plasmid
clone X1022, which carried the C-terminal coding sequences
of the xynB gene (Zhang et al., 1994 ; Zhang, 1992). Insertion
of this fragment into the SmaI site of pUC18, in the appropriate
orientation, resulted in an in-frame translational fusion with
the start of the lacZ gene. The predicted product carried the
first 10 amino acid residues of the lacZ product followed by
residues 435–755 of the XynB polypeptide. The XynB residues
correspond to the last 28 amino acids of a conserved
‘ stabilizing ’-type domain, the putative family 3 esterase
domain and the first 54 (out of 82) amino acids from the Cterminal XynB dockerin region (Kirby et al., 1997). For clarity,
this fusion construct is refered to here as pXynB.est, but was
previously designated XBest (Kirby et al., 1998). DNA
manipulations and hybridization procedures followed standard protocols (Sambrook et al., 1989).
DNA sequence analyses. DNA sequences were determined on
both strands using an ABI377 automated sequencer and
appropriate oligonucleotide primers. Computer analysis was
done with the UWGCG software available through the
Daresbury Seqnet and HGMP facilities (UK). Multiple alignments were done using   or , and phylogenetic
analysis through the  package.
Acetylxylan esterase assay. Escherichia coli DH5α or XL-1
Blue cells carrying the plasmid clones pCesA.est and
pXynB.est were pelleted by centrifuging for 10 min at 2000 g,
4 mC (Sorvall SS34). The pellet was washed with 10 ml buffer
A (50 mM sodium phosphate buffer pH 6n5, 2 mM DTT). The
pellet was finally resuspended in 2 ml of this buffer. The cells
were broken by sonication in a Soniprep 150 (SANYO
Gallenkamp PLC), using a 12 µm Exponential Microprobe
with three strokes of 1 min each, cooling on ice. The sonicate
was used for the enzyme determinations. Assays were performed in sodium phosphate buffer (50 mM) at pH 6n8 in the
presence of 1 % acetylated birchwood xylan : either native,
steam-extracted xylan (supplied by J. Puls, BFH, Hamburg,
Germany) or xylan chemically acetylated by the method of
Johnson et al. (1988). The acetate released was analysed
by HPLC using an Aminex HPX-87H Bio-Rad column
(300i7n8 mm) and a refractive index detector. Samples were
eluted with 4 mM H SO at 0n6 ml min−" and 35 mC. -Fucose
# standard.
%
was used as an internal
Reducing sugar release was
determined by the method of Lever (1977).
SDS-PAGE and acetylesterase zymogram. SDS-PAGE was
based on the method described by Laemmli (1970). Protein
samples were denatured by boiling for 5 min in SDS loading
buffer (final concentration : 50 mM Tris\HCl pH 6n8, 100 mM
DTT, 2 % SDS, 10 % glycerol, 0n1 % bromophenol blue)
before applying to the gel. Molecular masses were estimated
by comparison with molecular mass standards (Sigma). After
electrophoresis the gel was washed in 50 mM Tris\HCl pH 7n5
and stained for acetylesterase activity in 50 mM Tris\HCl pH
7n5 containing 0n05 mg β-naphthyl acetate ml−" and 0n05 mg
Diazo Blue ml−". Coomassie blue was used to stain protein
bands. Growth of R. flavefaciens was as described previously
(Flint et al., 1993).
RESULTS
Zymogram analysis of acetylesterases from
R. flavefaciens 17
The production of acetylesterases by cultures of R.
flavefaciens 17 was confirmed by zymogram analysis.
Supernatant proteins from cultures grown on oat-spelt
xylan were separated by SDS-PAGE and tested for
activity against β-naphthyl acetate after renaturation as
shown in Fig. 1. The predominant band of acetylesterase
activity was around 80 kDa, with minor bands at 90 and
35 kDa.
Isolation of the R. flavefaciens cesA gene encoding a
new multidomain acetylesterase
An acetylesterase domain was identified previously in
the 90 kDa multidomain xylanase XynB (Kirby et al.,
1997). In order to isolate further genes encoding
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Multidomain esterases of R. flavefaciens
kDa
Evidence for an additional multidomain xylanase,
XynE, carrying a family 1 esterase catalytic domain
90
An additional new gene, designated xynE, was identified
in R. flavefaciens 17, initially by amplification of a 1n5 kb
chromosomal DNA sequence using a domain-specific
primer designed for family 11 xylanases (Kirby, 1996) in
conjunction with a primer designed to recognize family
1 esterases. The complete XynE product (792 amino
acids, accession number AJ272430) shows an N-terminal
signal peptide sequence followed by a region between
residues 26 and 421 of very high homology (99n5 %
amino acid identity) with XynB, corresponding to the
family 11 xylanase and adjacent ‘ stabilizing ’ domains
(Fig. 2). Thereafter XynE carries a dockerin-like sequence and a threonine-rich linker, followed by a Cterminal domain of 277 amino acid residues that shares
42 % amino acid identity with a family 1 esterase domain
from Clostridium thermocellum XynZ (Gre! pinet et al.,
1988) and 79 % amino acid identity with an unknown
portion of xylanase 1 from a Ruminococcus sp. (accession number Z49970 : C. Arakaki and co-workers).
80
35
.................................................................................................................................................
Fig. 1. Acetylesterases from culture supernatant of R.
flavefaciens 17. Polypeptides from the supernatant of a culture
grown with oat-spelt xylan as substrate were separated by SDSPAGE, followed by renaturation and detection of esterase
activity against β-naphthyl acetate (see Methods).
acetylesterases, a lambda phage library of R. flavefaciens
17 chromosomal DNA was screened for plaques showing activity against β-naphthyl acetate. Active plasmid
clones were obtained after subcloning from one positive
phage clone, and the sequence of a new gene responsible
for acetylesterase activity, designated cesA, was completed after isolation of further overlapping clones
obtained by hybridization screening of a pUC13 plasmid
library (see Methods). The predicted product of the cesA
gene is a 768 amino acid polypeptide starting with a
putative signal peptide sequence (40 amino acids) and a
predicted mature molecular mass of approximately
84 kDa (cesA accession number AJ238716 ; Fig. 2). The
domain that follows the signal peptide shares 44 %
amino acid identity with the acetylesterase domain of R.
flavefaciens 17 XynB, and 34 % amino acid identity with
the acetylesterase domain of BnaC from the rumen
anaerobic fungus Neocallimastix patriciarum (Zhang et
al., 1994 ; Dalrymple et al., 1997 ; Kirby et al., 1997) (Fig.
3). This indicates that the CesA and XynB domains both
belong to carbohydrate esterase family 3, whose members show features in common with lipases (Dalrymple
et al., 1997 ; Upton & Buckley, 1995 ; see also http :\\
afmb.cnrs-mrs.fr\" pedro\CAZY\db.html). The esterase domain in CesA is followed by a region of approximately 80 amino acids that resembles dockerins found
previously in three R. flavefaciens polysaccharidases
(Kirby et al., 1997). Finally CesA carries a C-terminal
domain (418 amino acids) of unknown function that
showed no significant matches with other proteins in
database searches.
Relationships of dockerin-like regions from
R. flavefaciens esterases
Five dockerin sequences have now been identified in
enzymes of R. flavefaciens 17, including those from the
CesA, XynE and XynB multidomain esterases (Fig. 4).
All five include at least one potential calcium-binding
motif, and show limited homology with dockerins from
cellulolytic Clostridium spp. (Bayer et al., 1998a). A
phylogenetic comparison including typical type I (LicB)
and type II (CipA) dockerins from C. thermocellum is
shown in Fig. 5. It is clear that the dockerins from the
two newly reported esterases CesA and XynE diverge
significantly from those of XynB, XynD and EndA.
More specifically, they differ at residues 10 and 11
within their putative calcium-binding motifs (Fig. 4) ;
these two residues have been proposed by Pages et al.
(1997) to be particularly important in cohesin-binding
selectivity of dockerins from Clostridium spp. The R.
flavefaciens dockerins do not cluster closely with C.
thermocellum dockerins of type I or type II, and appear
to represent a distinct group, or groups (Fig. 5).
Specificity of acetylesterase domains from enzymes
of R. flavefaciens 17
The specificities of the acetylesterase domains from
XynB and CesA, expressed in E. coli, were studied in
truncated subclones that lack the N-terminal xylanase
domain, in the case of XynB, or the unknown Cterminal domain in the case of CesA (Fig. 2, Table 1).
Both domains showed greater activity against βnaphthyl acetate than against β-naphthyl propionate,
although the relative activities against β- and α-naphthyl
acetate differed. Neither enzyme showed activity against
methyl ferulate or against MUTMAC [4-methylumbelliferyl-7-(p-trimethylammonium cinnamate) chloride],
indicating that they lack ferulic acid esterase activity.
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(a)
(b)
.....................................................................................................
Fig. 2. (a) Domain structures of CesA, XynB
and XynE from Ruminococcus flavefaciens
17. (b) Truncated products encoded by
plasmids pCesA.est (C-terminal truncation of
CesA) and pXynB.est (N-terminal truncation
of XynB) (see Methods).
.....................................................................................................
Fig. 3. Multiple alignment (CLUSTAL V) of
acetylesterase domains of CesA, XynB and
Neocallimastix patriciarum BnaC. The three
amino acids thought to be involved in
catalysis in enzymes of this family (Cygler et
al., 1993) (see text) are shown in bold and
marked $ above the sequence.
.....................................................................................................
Fig. 4. Dockerin-like regions from R.
flavefaciens CesA and XynE compared with
those from R. flavefaciens EndA, XynB and
XynD. Identical residues conserved in the R.
flavefaciens enzymes and in dockerins from
Clostridium thermocellum LicB (Schimming
et al., 1992) and CipA (Gerngross & Demain,
1993) are indicated in bold. Asterisks
indicate residues corresponding to the
conserved positions 1, 3, 5, 9 and 12
identified in the calcium-binding motifs
(shown by the solid line and flanking spaces)
of C. thermocellum and C. cellulolyticum
dockerins by Pages et al. (1997).
Both enzymes were able to release acetate from
chemically acetylated xylan (55n3 and 27n8 µmol acetate
released per mg protein for XynB and CesA, respectively,
in 24 h incubations) or from native, steam-extracted
xylan (92n8 and 54n7 µmol acetate released per mg
protein for XynB and CesA, respectively, in 24 h).
Neither enzyme was able to release detectable amounts
of acetate from sugar beet pulp (results not shown).
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Multidomain esterases of R. flavefaciens
Table 2. Action of isolated acetylesterase and xylanase
domains upon steam-extracted birchwood xylan
.................................................................................................................................................
CesA.est and XynB.est refer to the cloned acetylesterase
domains of CesA and XynB, respectively, expressed in E. coli
(see Fig. 2). pXynD (referred to as L9XRHH by Flint et al.,
1993) expresses the XynD xylanase from R. flavefaciens 17.
Values for acetate release and reducing sugar release are means
of three and six determinations, respectively ; standard
deviations are given in parentheses.
Source of enzyme
Acetate released
in 24 h (µmol
per assay)*
XynD
CesA.est
XynB.est
XynD, CesA.est
XynD, XynB.est
0n34 (0n06)
3n23 (0n16)
3n34 (0n18)
5n04 (0n20)
5n28 (0n25)
.................................................................................................................................................
Fig. 5. Phylogenetic tree showing the relationships of five
dockerins from R. flavefaciens 17 enzymes (CesA, XynE, XynB,
XynD and EndA) and typical type I (LicB) and type II (CipA)
dockerins from Clostridium thermocellum. A variable 8 amino
acid region between the two conserved dockerin motifs in
XynB and XynD (Fig. 4) was omitted from the analysis. The tree
shown is an unrooted neighbour-joining tree.
Table 1. Activity of extracts from E. coli cells carrying
pXynB.est or pCesA.est upon soluble esterase substrates
.................................................................................................................................................
See Fig. 2 for the truncated products encoded by pXynB.est and
pCesA.est. Activity was not detected in incubations performed
with similar concentrations of crude protein from the
untransformed E. coli host strain DH5α. Values represent
means of duplicate assays ; individual values differed from the
mean by 10 % or less.
Substrate
Activity [µmol substrate
cleaved min−1
(mg crude protein)−1]
XynB.est
α-Naphthyl acetate
β-Naphthyl acetate
β-Naphthyl propionate
Methyl ferulate
MUTMAC*
4n85
2n25
1n08
0n05
0n05
CesA.est
0n31
0n78
0n14
0n05
0n05
* MUTMAC,
4-methylumbelliferyl-7-(p-trimethylammonium
cinnamate) chloride.
The possibility of synergy between the debranching
activity of the acetylesterase and cleavage of the main
xylan chain by xylanases was studied with native steamextracted acetylated xylan as substrate (Table 2).
Xylanase activity was provided by the cloned R.
flavefaciens xylanase XynD, whose N-terminal xylanase
domain shares 95 % sequence identity with the corresponding domain from XynB (Flint et al., 1993). A
small degree of synergy (approx. 50 %) was observed
with respect to acetate release for each of the esterase
domains acting with the xylanase, indicating that the
esterases may be slightly more active in deacetylating
xylooligosaccharides than the polysaccharide. A small
Reducing sugar
release (µmol
xylose equivalents
per assay)
0n50 (0n06)
0n05
0n05
0n68 (0n07)
0n73 (0n04)
* Assays were performed in a final volume of 303 µl, containing
the following amounts of total E. coli protein : CesA.est,
0n059 mg ; XynB.est, 0n036 mg ; XynD, 0n42 mg. Incubation was
for 24 h at 37 mC in the presence of sodium azide (see Methods).
degree of synergy was also observed with respect to
reducing sugar release, consistent with some interference
of xylanase action by acetyl substituents (Biely et al.,
1986 ; Wood & McCrae, 1986). The degree of acetylation of the steam-extracted xylan used here was
approximately 15 %, and it is possible that greater
degrees of synergy would be observed with more heavily
substituted substrates.
The specificity of the XynE esterase domain has not yet
been determined.
DISCUSSION
This work establishes for the first time that Ruminococcus spp. possess esterases that are likely to play an
important role in plant cell wall degradation in the
rumen. More specifically, we have identified three genes
in R. flavefaciens 17 whose products contain esterase
domains : XynB (predicted mature size 87 kDa), CesA
(84 kDa) and XynE (88 kDa).
The exact roles of the XynB, XynE and CesA esterases in
plant cell wall breakdown have yet to be fully established. We have shown that the XynB and CesA
activities are capable of removing acetyl groups from
acetylated xylans. Since neither domain requires the
simultaneous presence of an endoxylanase, both
enzymes appear capable of acting on the polysaccharide,
although the slight enhancement of acetate release in the
presence of xylanase suggests that they may be more
active on oligosaccharides than on the polysaccharide.
Conversely, the slight stimulation of reducing sugar
release by a cloned R. flavefaciens xylanase in the
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presence of the acetylesterase suggests that the removal
of acetyl groups makes the substrate more accessible to
xylanase hydrolysis. The association of the XynB
esterase with a family 11 xylanase in the same polypeptide makes it highly plausible that deacetylation of
xylans is the primary role of this domain. The role of the
CesA enzyme, however, may be quite different and the
function of its C-terminal domain has yet to be
established. The lack of detectable deacetylating activity
against sugar beet pulp does not rule out a role of the Nterminal domain of CesA in deacetylation of particular
regions of pectins or pectin breakdown products (Searlevan Leeuwen et al., 1992 ; Shevchik & HugouvieuxCotte-Pattat, 1997) and this possibility deserves further
study. The specificity of the esterase domain identified in
XynE has yet to be investigated.
In general, esterase domains present in carbohydrateactive enzymes are quite diverse with respect to their
primary amino acid sequences and are currently grouped
into at least seven different families (see http :\\
afmb.cnrs-mrs.fr\" pedro\CAZY\db.html). The acetylesterase domains from R. flavefaciens 17 XynB and
CesA appear to be the first active esterase domains
belonging to family 3 to be reported in bacterial
enzymes. The putative esterase domain identified here in
R. flavefaciens 17 XynE, on the other hand, belongs to
family 1. Family 1 domains have been reported previously in multidomain xylanases from several bacterial
species and include enzymes that show both acetylesterase and phenolic acid esterase activities (Bartolome!
et al., 1997). In addition, NodB-homologous domains
belonging to esterase family 4, which can show acetylesterase activity (Laurie et al., 1997), have been found in
enzymes from Cellulomonas fimi, Cellvibrio mixtus and
Streptomyces lividans (Shareck et al., 1995), and in the
XynA xylanase from Ruminococcus albus (database
accession number U43089 : T. Nagamine and coworkers). To date, therefore, representatives of three
different esterase families (1, 3 and 4) have been shown
to occur in xylanases from Ruminococcus spp.
In addition to their catalytic domains, the three R.
flavefaciens esterases considered here all carry dockerinlike regions. These dockerins are assumed to be involved
in binding to corresponding cohesin domains present in
a scaffolding protein or proteins in a cellulosome
multienzyme complex (Bayer et al., 1998b) since evidence has recently been obtained for a scaffolding
protein in R. flavefaciens 17 (Ding et al., 1999 ; S.-Y.
Ding and others, unpublished). The likely occurrence of
esterase activities in addition to hydrolytic activities in
cellulosomal complexes has been suggested elsewhere
for Clostridium thermocellum (Blum et al., 1998).
Interestingly, we show here that the CesA and XynE
dockerins diverge significantly in their sequences from
each other and from those reported previously in XynB,
EndA and XynD, including differences in the amino acid
positions implicated in the cohesin selectivity of
clostridial dockerins (Pages et al., 1997). This divergence
raises the possibility that more than one dockerin–
cohesin specificity might be involved in the assembly of
the putative R. flavefaciens cellulosome complexes.
CesA and XynE might, for example, bind to different
cohesins compared with XynB, XynD and EndA, with
consequences for the temporal and spatial positioning of
these polypeptides on the cell surface. In C. thermocellum only one type of dockerin–cohesin interaction is
involved in the binding of enzyme subunits to the
scaffolding protein, but a second is involved in attachment of the complex to cell surface anchoring
proteins (Salamitou et al., 1994 ; Bayer et al., 1998a).
Understanding of the functional significance of the
dockerin divergence seen in R. flavefaciens enzymes
must await the outcome of detailed studies on dockerin–
cohesin interactions in this species.
ACKNOWLEDGEMENTS
This work was supported by the Scottish Executive Rural
Affairs Department. We are grateful for support via the EU
TMR Fellowship scheme to V. A. We are also grateful to
Jurgen Puls for the kind gift of steam-extracted birch wood
xylan and to Brian Dalrymple, Gary Williamson and Ed Bayer
for valuable discussion.
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.................................................................................................................................................
Received 27 September 1999 ; revised 5 March 2000 ; accepted 13 March
2000.
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