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Journal of Bacteriology, January 1999, p. 338-340, Vol. 181, No. 1
Microbial Ingredients Section, NIZO Food
Research, 6710 BA Ede, The Netherlands
Received 26 June 1998/Accepted 14 October 1998
We used homologous and heterologous expression of the
glycosyltransferase genes of the Lactococcus lactis NIZO
B40 eps gene cluster to determine the activity and
substrate specificities of the encoded enzymes and established the
order of assembly of the trisaccharide backbone of the
exopolysaccharide repeating unit. EpsD links glucose-1-phosphate from
UDP-glucose to a lipid carrier, EpsE and EpsF link glucose from
UDP-glucose to lipid-linked glucose, and EpsG links galactose from
UDP-galactose to lipid-linked cellobiose. Furthermore, EpsJ appeared to
be involved in EPS biosynthesis as a galactosyl phosphotransferase or
an enzyme which releases the backbone oligosaccharide from the lipid carrier.
Many bacteria are known to produce
polysaccharides, which can either be excreted into the environment as
exopolysaccharides (EPSs), form a capsule around the cell as capsular
polysaccharides, or be attached to the cell membrane as the O antigens
of lipopolysaccharides. The biosynthesis of polysaccharides that
consist of repeating units includes their assembly on a lipid carrier
by sequential transfer of monosaccharides from nucleotide sugars by
glycosyltransferases (GTFs) and the subsequent polymerization and
export of these repeating units (15, 17).
Although numerous bacterial gene clusters involved in cell surface
polysaccharide biosynthesis have been described, only a few of these
have been analyzed for the function of their GTF genes. Homologous
expression has been used to study the GTF genes involved in O-antigen
synthesis from different serogroups of Salmonella enterica
(8). Mutations in the different GTF genes involved in
Rhizobium meliloti EPS biosynthesis have been generated, and the lipid-linked intermediates which accumulated in permeabilized cells
of the mutant bacteria were analyzed by thin-layer chromatography (TLC)
to infer the biosynthetic step catalyzed by each enzyme (12). Furthermore, heterologous complementation has been
used for the functional analysis of GTF genes of
Sphingomonas spp. and Rhizobium leguminosarum
(10). The involvement of streptococcal GTF genes in capsule
biosynthesis has been studied for type III of Streptococcus
group B and Streptococcus pneumoniae serotype 14 (5, 6,
13). For serotype 14, these genes have been shown to be essential
for the synthesis of the repeating unit by their expression in
Escherichia coli (5, 6). Finally, the
eps gene cluster of Streptococcus thermophilus
coding for an unknown number of GTFs has been demonstrated to be
involved in EPS biosynthesis by heterologous expression in a
plasmid-free Lactococcus lactis strain (14).
L. lactis NIZO B40 produces an extracellular
phosphopolysaccharide with a repeating unit consisting of
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Exopolysaccharide Biosynthesis in Lactococcus
lactis NIZO B40: Functional Analysis of the Glycosyltransferase
Genes Involved in Synthesis of the Polysaccharide Backbone
ABSTRACT
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4)[
-L-Rhap-(12)][-D-Galp-1-PO4-3]-
-D-Galp-(14)--D-Glcp-(14)--D-Glcp-(1 (9, 16). A plasmid-located eps gene cluster
including 14 coordinately transcribed genes with the order
epsRXABCDEFGHIJKL has been implicated in the biosynthesis of
this EPS. A single gene disruption and heterologous expression of the
epsD gene have been used to demonstrate that it is essential
for the synthesis of the repeating unit and encodes a GTF transferring
glucose from UDP-glucose to a lipid carrier (16). Because of
its homology to GumD from Xanthomonas campestris, we assume
that EpsD, like GumD, catalyzes the transfer of glucosyl-1-phosphate
from UDP-glucose to undecaprenyl phosphate (4). To determine
the function and substrate specificities of other NIZO B40 GTF gene
products and to establish the order of assembly of the backbone of the
EPS repeating unit, which could not be determined by our previous results, we expressed the relevant GTF genes in E. coli.
Fragments containing the epsD, epsDE,
epsDEF, and epsDEFG genes (Fig.
1) were cloned under control of the
isopropyl--D-thiogalactopyranoside (IPTG)-inducible
lac promoter in pUC19 (18) and introduced
into E. coli DH5 (3), which has no background
GTF activity, as was shown previously (16). The fragment
containing epsDE was generated by PCR with the primers
5'-CCGCGCGGATCCGGGTATAGATGATTATC-3' and
5'-CCGCGCCGAATTCTTGAAACGCCCTTGCTATCTC-3' by
using the BamHI and EcoRI sites of the primers
(underlined) for cloning. Since the backbone of the EPS repeating unit
is known to contain glucose and galactose, permeabilized cells of
IPTG-induced (0.1 ng ml
1) E. coli harboring
these plasmids were incubated with UDP-[14C]glucose and
UDP-[14C]galactose. The lipid fraction was extracted,
subjected to complete and mild acid hydrolysis, and analyzed by TLC and
autoradiography as described previously (16) to detect the
14C-labelled monosaccharides (complete acid hydrolysis) and
oligosaccharides (mild acid hydrolysis), respectively (Fig.
2). Expression of epsDE showed
the same sugar incorporation as expression of epsD alone. In
contrast, expression of epsDEF resulted in the production of a lipid-linked oligosaccharide with the same mobility on TLC as cellobiose, which is the
-D-Glcp-(14)--D-Glcp
part of the repeating unit. Expression of epsDEFG resulted
in the production of lipid-linked oligosaccharides containing glucose
and galactose. Mild acid hydrolysis of these products yielded two
oligosaccharides with lower mobility on TLC than that of cellobiose
that were retrieved from the TLC plate and subjected to complete acid
hydrolysis, followed by a second TLC analysis. The oligosaccharide with
the higher mobility consisted of glucose and galactose with a ratio of
approximately 2:1, as judged from the intensity of the spots on the
autoradiograph of the TLC, and is likely to be the
-D-Galp-(14)--D-Glcp-(14)--D-Glcp trisaccharide of the backbone of the repeating unit. The other oligosaccharide contained only 14C-labelled galactose,
indicating that EpsG may act with slightly lower affinity on another
lipid acceptor than lipid-linked cellobiose in the E. coli
membrane. These results indicate that EpsF is the second GTF, linking
glucose to lipid-linked glucose, and that EpsG is the third GTF,
linking galactose to lipid-linked cellobiose. As EpsE and EpsF are
homologous to pneumococcal Cps14F and Cps14G, respectively, we assume
that, like Cps14F and Cps14G, both lactococcal gene products act
together as one GTF and that EpsF contains the GTF activity while EpsE
has an accessory function (5).
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FIG. 1.
Physical and genetic map of the eps gene
cluster of plasmid pNZ4000. For BsaAI, EcoRI,
HincII, NdeI, ScaI, and
SstI, only sites relevant for subcloning are indicated. The
plasmids used for heterologous and homologous expression of the
eps genes are listed. Plasmids pNZ4060 through pNZ4065 are
pUC19 derivatives carrying the indicated fragments under control of the
lac promoter. Plasmids pNZ4070, pNZ4071, and pNZ4072 are
pNZ8020 derivatives carrying the indicated fragments under control of
the nisA promoter.
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FIG. 2.
TLC of 14C-labelled intermediates isolated
from the lipid fraction of permeabilized E. coli cells. The
positions of the standard sugars glucose (Glc), galactose (Gal), and
cellobiose (Glc-Glc), as well as those of the predicted trisaccharide
(Glc-Glc-Gal) and the unknown galactose-containing molecule (?-Gal),
are indicated on the right. Additional products of incomplete mild
hydrolysis, putative sugar phosphates (A) and putative lipid-linked
sugars (B), are indicated on the left. Lanes: 1 and 2, E. coli carrying pNZ4060; 3 and 4, E. coli carrying
pNZ4061; 5 and 6, E. coli carrying pNZ4062; 7 and 8, E. coli carrying pNZ4063. Complete (C) and mild (M) acid
hydrolysis treatments are indicated.
To test the substrate specificities of the epsDEFG gene
products in L. lactis, fragments of pNZ4000 carrying
epsD, epsDEF, or epsDEFG were cloned
under control of the nisA promoter of pNZ8020, which, when
introduced into L. lactis NZ3900 (1), allows the use of the NICE (nisin-controlled expression system) (2, 7). Cultures were induced with 0.1 ng of nisin A ml1 at an
optical density at 600 nm of 0.5, and cells were harvested 2 h
after induction. After lysozyme treatment, permeabilized cells were
prepared as described previously (16). Incubation of
(uninduced) permeabilized, plasmid-free L. lactis NZ3900
cells with UDP-[14C]glucose or
UDP-[14C]galactose resulted in a high level of
incorporation of [14C]glucose in the lipid fraction by an
unknown glucosyltransferase activity that may be involved in the
biosynthesis of other cell surface polysaccharides (Fig.
3). Mild acid hydrolysis of the extracted
lipid fractions yielded five species of labelled saccharides (Fig. 3):
one with the same mobility on TLC as glucose, one migrating slightly
slower than cellobiose, and three with higher mobility than glucose
(the latter three are not shown in Fig. 3). This glucosyltransferase
activity was not restricted to L. lactis subsp. cremoris MG1363 derivative NZ3900 but was also observed for
L. lactis subsp. lactis IL1403 and L. lactis subsp. lactis biovar diacetylactis
BU2-60 (data not shown). The background incorporation prevented
detection of the activity of EpsD and EpsF, as no additional effect of
expression of the epsD or epsDEF genes was
observed (data not shown). However, expression of
epsDEFG resulted in the formation of a new lipid-linked
oligosaccharide. Its complete acid hydrolysis yielded an additional
product with the same mobility as galactose. Its hydrolysis by mild
acid resulted in a product with the same mobility as the putative
trisaccharide detected in E. coli expressing
epsDEFG (Fig. 3). The latter product was retrieved from the
TLC plate and subjected to complete acid hydrolysis and a second TLC
analysis. The oligosaccharide consisted of glucose and galactose,
identical to the glucose-and-galactose-containing oligosaccharide found
in E. coli expressing epsDEFG. Therefore, we
conclude that the functions of the epsDEFG genes in E. coli and L. lactis are identical.
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); 2 and 5, L. lactis carrying pNZ4072 (L1); 3 and 6, E. coli carrying pNZ4063 (Ec); 7, mixture of mild
acid hydrolysates of L. lactis carrying pNZ4072 and E. coli carrying pNZ4063 (mix). Complete (C) and mild (M)
acid hydrolysis treatments are indicated.
It is likely that the subsequent steps of the repeating unit synthesis include the coupling of the side chain sugars rhamnose and galactose-phosphate to the galactose of the backbone. Possible candidates for these activities are EpsH, which is homologous to several GTFs, and EpsJ, which is homologous to a CDP-glycerol:poly(glycerophosphate) glycerophosphotransferase of Bacillus subtilis designated TagH (RodC) (11, 16). To test the function of epsH, a fragment containing epsDEFGH (Fig. 1) was cloned under control of the lac promoter in pUC19. Incubation of permeabilized E. coli cells expressing epsDEFGH with UDP-[14C]glucose and UDP-[14C]galactose resulted in the same products as those of cells expressing epsDEFG, indicating that EpsH is either not a galactosyltransferase, inactive in this assay, or not expressed. EpsH may be the rhamnosyltransferase, which could not be tested, as its substrate, dTDP-rhamnose, is unstable. The epsJ gene was cloned downstream of epsDEFG in pNZ4063 (Fig. 1). The resulting plasmid, pNZ4065, was readily obtained. Incubation of permeabilized E. coli cells expressing epsDEFGJ resulted in complete loss of incorporation of 14C-labelled sugars from the lipid fraction and no radioactivity on TLC. This strongly indicates that the epsJ gene is active and may encode either an enzyme linking galactosyl phosphate to galactose, after which E. coli enzymes can release the oligosaccharide from the membrane, or an enzyme releasing the trisaccharide backbone from the lipid carrier. Presently, we cannot distinguish between these two alternatives.
In conclusion, our report describes the heterologous and homologous expression of the lactococcal eps genes encoding the GTFs involved in the assembly of the EPS repeating unit. EpsD, EpsE, and EpsF, and EpsG link glucose-1-phosphate to a lipid carrier (presumably undecaprenyl phosphate), glucose to lipid-linked glucose, and galactose to lipid-linked cellobiose, respectively. The epsJ gene product is active and likely to be a galactosyl phosphotransferase or an enzyme releasing the trisaccharide backbone from the lipid carrier. Furthermore, to the best of our knowledge, this is the first report describing controlled homologous expression of GTF genes in gram-positive bacteria.
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ACKNOWLEDGMENTS |
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This work was partly supported by EC research grants 1116/92 1.6 and BIOT-CT96-0498.
We thank Roland Siezen and Ingeborg Boels for critically reading the manuscript.
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FOOTNOTES |
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* Corresponding author. Mailing address: Microbial Ingredients Section, NIZO Food Research, Kernhemseweg 2, 6718 ZB, Ede, The Netherlands. Phone: 31 318 659511. Fax: 31 318 650400. E-mail: kranenbu{at}nizo.nl.
Present address: Laboratory of Microbiology, Centre for
Neurosciences of Coimbra, University of Coimbra, 3049 Coimbra Codex, Portugal.
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Journal of Bacteriology, January 1999, p. 338-340, Vol. 181, No. 1
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