Nestlé Research Center, Vers-chez-les-Blanc, CH-1000 Lausanne 26, Switzerland
Received on November 13, 2001; revised on January 16, 2002; accepted on January 17, 2002.
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Abstract |
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Key words: enzymatic synthesis of oligosaccharides/eps gene cluster/exopolysaccharide biosynthesis/glycosyltransferases/NMR
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Introduction |
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Ongoing research efforts aim at developing efficient synthesis of bioactive oligosaccharides to influence and modulate these important processes. This can be achieved using different approaches. The first is chemical synthesis, and it requires a complete control of the stereoselectivity. Recent synthetic strategies have shown increasing success (Bertozzi and Kiessling, 2001; Plante et al., 2001
; Burkhart et al., 2001
). The second approach is based on the use of enzymes, either in chemoenzymatic synthesis, which already has shown successes in vitro (Palcic, 1999
; Sears and Wong, 2001
), or in the context of in vivo carbohydrate bioengineering, where the approach is to design recombinant bacterial systems for targeted carbohydrate synthesis (Koizumi et al., 1998
; DeFrees et al., 2000
).
Glycosyltransferases (GTFs) are key carbohydrate-interacting enzymes involved in the synthesis of complex carbohydrate structures, which offer the advantages of high regio- and stereospecificities compared to a chemical approach, as well as the potential availability of many different glycosidic linkages. One major limitation is the limited number of characterized enzymes available. To address this issue, some reports have appeared detailing acceptor specificities of GTFs but mostly on mammalian fucosyl and sialyl transferases (Wymer and Tooze, 2000; DeFrees et al., 2000
). The identification of putative GTF genes involved in bacterial exocellular polysaccharide (EPS), capsular polysaccharides, and lipopolysaccharide biosynthesis is rapidly increasing as a result of research efforts in the field of genomics (Wang and Reeves, 2001
; Jiang et al., 2001
; Jolly and Stingele, 2001
). The diversity of EPS structures described so far is large and provides the basis for a rapid identification of a wide variety of novel enzymes for the catalysis of specific reactions. All GTFs from lactic acid bacteria represent an array of putative specificities, which altogether could be defined as a "toolbox" for the production of tailored carbohydrates. However, the characterization of the specificity of the putative GTFs toward its two reactants (the activated nucleotide sugar and the acceptor) is required. This task is made difficult due to the specific individual properties of each GTF, which does not permit the use of one general biochemical assay (Guan et al., 2001
).
This study presents several aspects of EPS production by Lactobacillus helveticus strain NCC2745: cloning of putative genes, determination of the EPS structure, characterization of enzymatic function using biochemical assays, and the in vitro enzymatic biosynthesis of two polyprenol-linked trisaccharides. This lead to the establishment of an integrated EPS biosynthesis model for L. helveticus strain NCC2745.
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Results |
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The 1D 1H nuclear magnetic resonance (NMR) spectra of L. helveticus NCC2745 EPS (Figure 2b) and the anomeric region of the 1H-13C heterologus single quantum correlation (HSQC) spectrum (Figure 2c) showed six anomeric resonances within the repeating unit. Ring forms (pyranose or furanose) and anomeric configurations were deduced from H-1 chemical shifts and one-bond C-1, H-1 scalar couplings obtained by direct measurement from the cross-correlated dipoledipole relaxation spectra (Vincent and Zwahlen, 2000).
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Purification of Eps proteins
Proteins were expressed in Escherichia coli BL21-SI cells harboring either the pLJLHHi plasmid or the pLJLHGi plasmid under the control of the tightly regulated T7 promoter inducible with NaCl. Although a majority of the gluthatione-S-transferase (GST)-Eps proteins formed inclusion bodies, a significant amount of soluble protein could be recovered from the supernatant. GST-Eps proteins could be purified in one step using an affinity chromatography matrix with 0.1% 3[(3-chloramidopropyl)dimethyl ammonio]-1-propane-sulfate (CHAPS). The isolated GST-Eps proteins were pure on Coomassie Bluestained sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDSPAGE) gel (Figure 3).
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Incubating purified GST-EpsF with other synthetic acceptor composed of a monosaccharide linked to a long hydrophobic aliphatic chain, 2-(octadecylthio)-ethyl -CH2CH2SC18H37 (OTE)-ß-Glc (C18H37SCH2CH2-ß-Glc), and labeled UDP-[14C]Glc as donor yielded a disaccharide product, OTE-ß-Glc-Glc, that could be detected and identified by liquid chromatographymass spectrometry (LC-MS). Hence, in agreement with the C35-P acceptor experiment, EpsF acted as a GlcT.
Known acceptors (p-aminophenyl [p-AP], p-nitrophenyl [p-NP], and 6-[fluorescein-5-carboxamido]-hexanoic acid succimidyl ester [Fchase]) previously used for GTF and glycosidase activity assays were further tested (Wakarchuk et al., 1996; Kolkman et al., 1997
; Palcic, 1999
), but no disaccharide formation could be detected with purified GST-EpsF and either labeled UDP-[14C]Glc or labeled UDP-[14C]Gal. The natural substrate analog, C35-P therefore proved to be the most adequate acceptor to assay the activity of GTFs.
According to the EPS structure (Figure 2a), two glycosidic linkages are possible for the attachment of Glc onto the ß-Glc-acceptor: either (1,3) or
(1,6). None of the five other GST-Eps added either Glc or Gal onto the labeled C35-P-P-ß-[14C]Glc acceptor. These results together with the EPS structure (Figure 2a) indicate that EpsF is the second enzyme involved in the biosynthesis of the EPS repeating unit, adding either a Glc in a
(1,3) or a
(1,6) linkage onto the initial ß-Glc.
To discriminate between the two possible activities, the disaccharide attached to the lipid carrier produced by the action of GST-EpsF was released by mild hydrolysis and analyzed by high-performance liquid chromatography (HPLC). As standards, both nigerose (-D-Glcp-(1
3)-D-Glcp) and isomaltose (
-D-Glcp-(1
6)-D-Glcp) were used. The EpsF product both had the same retention time as and comigrated with nigerose when it was coinjected with this standard disaccharide (Figure 5). This experiment allowed the determination of not only the sugar specificity but also the regio- and stereospecificities of EpsF, which is an
(1,3)-GlcT.
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EpsI and EpsJ activities
Sequence comparison using hydrophobic cluster analysis suggested that EpsI and EpsJ are ß-GTF. However, neither purified GST-EpsI nor GST-EpsJ showed any activity with any combination of acceptor or donor substrates tested (Table III). On the other hand, GST-EpsI, unlike GST-EpsJ, was shown to hydrolyze UDP-Gal (data not shown), which indicates specific affinity of EpsI for Gal substrate.
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Discussion |
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EpsE transferred a phospho-Glc onto a prenylphosphate carrier showing that epsE encoded a phospho-GlcT initiating the biosynthesis of the repeating unit. EpsF is the second enzyme involved in the biosynthesis of the EPS repeating unit, adding a Glc in a (1,3) linkage onto the initial ß-Glc. GST-EpsG and GST-EpsH carried out both
-GlcT and
-GalT activities and were both specific for the C35-P-P-ß-[14C]Glc-(3
1)-
-Glc acceptor. EpsI, not EpsJ, was shown to have a specific affinity for a Gal substrate.
Repeating unit biosynthesis
The first enzyme initiating the biosynthesis of the polysaccharide repeating unit was shown to be EpsE, a phospho-GlcT capable of transferring a phospho-Glc onto a C35-prenylphosphate carrier. For the second enzyme EpsF, the sugar-, regio- and stereo-specificities were determined unequivocally showing that EpsF was an (1,3)-GlcT.
From the results presented, the only two possibilities for EpsG and EpsH were either a (1,6)-GalT elongating the backbone by using the 3-linked
-Glc as acceptor or a branching
(1,6)-GlcT using the (internal) ß-Glc as acceptor. Because GST-EpsG showed higher
-GalT than
-GlcT activity (Table III), EpsG is proposed to be an
(1,6)-GalT while, by exclusion, EpsH is most likely responsible for the branching of the EPS repeating unit with a
(1,6)-GlcT activity. Moreover, the fact that GST-EpsH exhibited the lowest GTF activity (Table III) could have resulted from the requirement of the correct "anchoring" trisaccharide
-D-Galp-(1
6)-
-D-Glcp-(1
3)-D-Glcp for recognition of the precursor instead of the available disaccharide
-D-Glcp-(1
3)-D-Glcp, an effect shown to be important in vivo for S. thermophilus Sfi6 EPS synthesis (Stingele et al., 1999
). According to the EPS structure (Figure 2a), the necessary acceptors for the two putative ßGTFs, EpsI and EpsJ, would be either a
(1,6)-Glc or a
(1,6)-Gal resulting from the action of EpsG and EpsH. Unfortunately, the tetrasaccharide precursor could not be synthesized in sufficient amounts to test the activities of either EpsG or EpsH. Due to the observed affinity of EpsI for UDP-Gal, we concluded that EpsI could be a ß(1,6)-GalT acting on the branched
(1,6)-Glc and that therefore EpsJ would finish the backbone by adding the terminal ß(1,6)-GalT and be the last enzyme to react before the translocation. Transfer of the eps gene cluster from S. thermophilus Sfi6 to the nonEPS-producing host L. lactis MG1363 yielded the production of an EPS with no side chain, different from the original EPS. It was therefore concluded that for the proper translocation of EPS the intact EPS backbone was needed, whereas the side chain could be missing without perturbing EPS biosynthesis (Stingele et al., 1999
). All the activity assays carried out with purified GST-EPS proteins enabled us to design a model for the biosynthesis of the repeating unit. These results suggest that the order of enzyme reaction for the biosynthesis of the repeating unit is EpsE, EpsF, EpsG and EpsH, and EpsI and EpsJ, following the order of the eps genes present in the Eps cluster.
In this report we present a comprehensive study, including the identification of the gene cluster, the elucidation of the EPS structure and the characterization of the key GTFs, of the EPS synthesis in L. helveticus NCC2745. The identified EPS structure represents a novel structure for the L. helveticus species. GTFs from lactic acid bacteria were cloned, then heterologously expressed and purified. GTF substrates were characterized using a synthesized acceptor mimicking the natural substrate. For the second enzyme, the stereo- and regiospecificities were also demonstrated. Furthermore, an example of in vitro polyprenol-linked oligosaccharide synthesis based on purified GTFs was shown, with two trisaccharide products suggested to be -D-Galp-(1
6)-
-D-Glcp-(1
3)-ß-Glcp and
-D-Glcp-(1
6)-[
-D-Glcp-(1
3)-]-D-Glcp (Figure 4, lanes 11 and 12, respectively).
Several lactic acid bacteria enzymes were identified that could be targeted as tools for both in vitro chemoenzymatic and in vivo engineering of oligosaccharide and polysaccharides, effectively creating a GTF toolbox. The enzymes listed in Table IV were chosen from lactic acid bacteria strains for which genes had been described and whose EPSs had been characterized allowing a preliminary classification of these enzymes. Several of these enzymes will need to be characterized further before they can be effectively used.
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Materials and methods |
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DNA manipulations
E. coli plasmid DNA was isolated using the QIAprep 8 Turbo Miniprep Kit (Qiagen GmbH, Hilden, Germany) (Sambrook et al., 1989). L. helveticus chromosomal DNA was purified as previously described for Streptococcus thermophilus (Slos et al., 1991
). Restriction nuclease digestions, ligations, agarose gel electrophoresis, and Southern blots were done according to established protocols (Sambrook et al., 1989
). DNA sequencing was done using the Thermo Sequenase Cycle Sequencing Kit (Pharmacia, Peapack, NJ). Reactions were carried out using a LI-COR DNA Sequencer Model 4000L (LI-COR, Lincoln, NE), and sequences were analyzed using GCG software, version 8 (Genetic Computer Group, University of Wisconson, Madison).
To identify the eps gene cluster of L. helveticus NCC2745, a -ZAP (Stratagene, La Jolla, CA) genomic library was constructed. The NCC2745 genomic DNA was partially digested with the restriction enzyme Sau3A and separated on agarose gel. The DNA fragments ranging from 6 to 10 kb were extracted from the gel using the QIAex extraction kit (Qiagen) and ligated into a
-ZAP Express Vector after ligation of cos-ends and subsequent digestion with BamHI. This construct was transformed into E. coli XL1-Blue MRF- cells on NZY plates and incubated at 37°C overnight. Library screening was performed following a standard protocol (Stratagene).
Plasmid construction
Plasmids suitable for the overproduction of both His-tagged and GST-tagged Epsi (E < i < J) proteins, pLJHLHi and pLJGLHi respectively, were constructed by inserting each L. helveticus epsi ORF into the GatewayTM cloning system (Invitrogen AG, Basel, Switzerland). Expression vectors carrying either a 6xHis tag or a GST at the N-terminal end were used. Plasmid vectors were sequenced to verify the integrity of the epsi ORF (GATC Biotech AG, Taegerwilen, Switzerland).
EPS expression and purification
EPS from a 1-L culture of L. helveticus NCC2745, grown at 40°C in 10% MSK supplemented with a mixture of amino acids, was isolated and purified as previously described (Stingele et al., 1996).
EPS chemical analysis
EPS were hydrolyzed by mild hydrolysis (500 µl of purified EPS solution at 1 g/L mixed to a final volume of 500 µl at a concentration of 4 N trifluoroacetic acid and heated for 1 h at 125°C) before analysis of monosaccharides by high-pH anion exchange chromatographypulsed amperometric detector (PAD) chromatographic separation (1 ml/min, 15 mM NaOH for 25 min on a Pa1 column 4 x 250 mm, Dionex DX-500 system, Dionex, Sunnyvale, CA). External standards of galactosamine, glucosamine, galactose, and glucose (2.5 µg each) were used for quantification.
NMR spectroscopy
All experiments were recorded on 5 mg lyophilized EPS dissolved in 500 µl of 99.96 atom% 2H2O on a Bruker AVANCE 600 MHz spectrometer. Chemical shifts were given in ppm by reference to the -anomeric signal of external [13C-1]-D-glucose (
H-1 5.15 for H-1 and
C-1 92.90 for C-1). Phase-sensitive 2D experiments were recorded: TOCSY with mixing times between 15 ms and 85 ms, NOESY with mixing times between 25 ms and 300 ms, gradient sensitivity-enhanced 1H-13C HSQC and cross-correlated dipoledipole relaxation experiment (Vincent and Zwahlen, 2000
) for determining glycosidic linkages with constant-time durations of 1040 ms. A magnitude mode gradient-filtered 1H-13C HMBC was recorded with a J-evolution time of 40 ms. The following number of complex points were acquired (F1, F2): 256 x 2048 (NOESY and HMBC), 512 x 2048 (TOCSY and HSQC), or 100 to 256 x 2048 (cross-correlated dipoledipole relaxation), with averaging over 16 scans (TOCSY), 64 scans (HSQC and NOESY), or 256 scans (cross-correlated dipoledipole relaxation and HMBC). Spectral widths of 3600 Hz in the 1H dimensions and of 3780 Hz in the 13C dimensions were used. A 90° shifted square sine bell was used in all cases with zero-filling once. All data were processed using Bruker XWINNMR 2.6 software.
GTF crude extracts
E. coli BL21-SI cells were transformed with pLJLHHi and pLJLHGi plasmids and grown at 37°C in Luria-Bertani NaCl free with ampicillin (50 ml). At an optical density at 600 nm of 0.75, cells were induced with 0.3 M NaCl for 2 h at 37°C. Cells were then harvested by centrifugation for 15 min and washed once with 50 mM Tris buffer at pH 8 (buffer A). After centrifugation, cells were resuspended in 0.75 ml buffer A containing a cocktail of protease inhibitors (Complete, Roche Mannheim, Germany), 0.3 mM MgCl2 and 1 mM dithiothreitol. The cells were broken by sonication, and the crude extract was subjected to ultracentrifugation at 100,000 x g for 20 min at 4°C. In some cases, to optimize the solubilization of GTFs, one of the following detergents was added (0.1% CHAPS, 1% Triton X-100, 0.5% -dodecyl-ß-D-maltocide [DDM], or 1%
-octyl-ß-D-glycopyranoside) and incubated for 2h at 4°C.
GTFs purification
GTFs tagged with 6xHis were purified by affinity chromatography in a one-step purification under native conditions according to the manufacturers instructions (Qiagen): binding of the supernatant with Ni2+-nitriloacetate agarose overnight at 4°C, washing with phosphate buffered saline (PBS) buffer 50 mM, pH 7.5, containing 5 mM, 25 mM, and 50 mM imidazole, then elution with 100 mM imidazole in PBS. GTF tagged with GST were purified in batch using the affinity matrix glutathione sepharose 4B (Pharmacia). Soluble enzymes were first bound to the resin overnight at 4°C on a rotary shaker and washed with PBS. Bound proteins were eluted with 10 mM reduced glutathione in buffer A supplemented with 0.3 mM MgCl2 and 1 mM dithiothreitol.
Synthesis of acceptors
Synthesis of Fchase-ßGlc and Fchase-ßGal were performed as described by Wakarchuk et al. (1996) using either UDP-Glc or UDP-Gal (Sigma) and Fchase (Molecular Probes, Eugene, OR).
For synthesizing C35-P acceptors, 1 mg heptaprenylphosphate C35-P (Larodan Fine Chemicals AB, Malmö, Sweden) was dissolved in 1 ml 1% Triton-X100, and 25 µl of the solution were incubated for 3 h at 10°C with 0.5 mM UDP-Glc or UDP-Gal, 92.5 kBq UDP-[14C]Glc or UDP-[14C]Gal, 15 mg of crude extracts from recombinant phospho-GTF EpsE (as described previously) in buffer A supplemented with 10 mM MgCl2, 1 mM ethylenediamine tetra-acetic acid, and 0.1 mM dithiothreitol. The reaction was stopped by the addition of 2 ml chloroform-methanol (2:1). The unincorporated labeled sugars were removed by extracting the organic phase using three times with 0.4 ml extraction buffer (15 ml chloroform, 250 ml methanol, 1.83 g KCl, 235 ml H2O). The lower phase was vacuum-dried and resuspended in 10% acetonitrile. The C35-P-P-sugar products were purified on a Sep Pak column and eluted with 10%, 50%, and 100% acetonitrile. The fractions containing the labeled acceptors were vacuum-dried, resuspended in Triton X-100 1%, and loaded on TLC. The pure acceptors were then analyzed by mild acid hydrolysis.
GTF enzymatic assays
A GTF assay using permeabilized cells was performed as described by Kolkman et al. (1996). Purified recombinant GST-GTF fusion proteins were also tested in vitro for enzyme activity. In a 5 µl reaction volume of buffer A supplemented with 10 mM MgCl2 and 0.1 mM dithiothreitol, various amount of acceptor substrates (15 mM) were tested with 0.5 mM UDP-Glc or UDP-Gal as donor substrates, which were supplemented with 100 Bq of UDP-[14C]Glc or UDP-[14C]Gal (Pharmacia). The substrates were: pAP-ßGal, or pAP-ßGlc, or pNP-
Gal, or pNP-
Glc, or pNP-
GlcNAC (Sigma), or OTE-ßGlc and OTE-ßGal (Glycorex AB, Sölvegatan, Sweden). For the activity assays using labeled prenylphosphate acceptors C35-P-P-ß-[14C]Glc or C35-P-P-ß-[14C]Gal, unlabeled nucleotide sugars were used. The reaction mixture was incubated at room temperature for up to 8 h, loaded on a 20 x 20 cm TLC silica gel 60 plate (Merck) and developed in ethylacetate-methanol-water-acetic acid (7:2:1:0.1). The TLC plates were analyzed using a phosphoimager (Fujifilm Bas 1800 II, Kanagava, Japan) equipped with Raytest software (Raytest Isotopenmessgerate GmbH, Straubenhardt, Germany).
HPLC analysis of oligosaccharides
Isomaltose and nigerose (Sigma) were analyzed by means of high-pH anion exchange chromatographyPAD using a Dionex DX-500 system equipped with a PA1 column. The elution of the disaccharides was achieved using a gradient starting from 60 mM sodium hydroxide and 25 mM sodium acetate to 75 mM sodium hydroxide and 25 mM sodium acetate after 20 min, at a flow rate of 1ml/min.
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Acknowledgments |
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Abbreviations |
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Footnotes |
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2 Present address: Neurim Pharmaceuticals SA, 18 Rte de Genève, CH-1280 Nyon, Switzerland
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References |
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---|
Burkhart, F., Zhang, Z., Wacowich-Sgarbi, S., and Wong, C.-H. (2001) Synthesis of the globo H hexasaccharide using the programmable reactivity-based one-pot strategy. Angew. Chem. Int. Ed., 40, 12741277.[CrossRef][ISI]
Campbell, J.A., Davies, G.J., Bulone, V., and Henrissat, B. (1997) A classification of nucleotide-diphospho-sugar glycosyltransferases based on amino acid sequence similarities. Biochem. J., 326, 929939.[ISI][Medline]
Casadevall, A. and Pirofski, L.A. (2000) Host-pathogen interactions: basic concepts of microbial commensalism, colonization, infection, and disease. Infect. Immun., 68, 65116518.
Costerton, J.W., Stewart, P.S., and Greenberg, E.P. (1999) Bacterial biofilms: a common cause of persistent infections. Science, 284, 13181822.
DeFrees, S., Bayer, R. J., and Ratcliffe, M. (2000) Enzymatic synthesis of oligosaccharides. Neose Technologies, US Patent No. 6, 030, 815.
Editorial (1999) Policy on papers contributors: Nature is encouraging authors of papers to say who did what. Nature, 399, 393.[CrossRef][Medline]
Feldman, M.F., Marolda, C.L., Monteiro, M.A., Perry, M.B., Parodi, A.J., and Valvano, M.A. (1999) The activity of a putative polyisoprenol-linked sugar translocase (Wzx) involved in Escherichia coli O antigen assembly is independent of the chemical structure of the O repeat. J. Biol. Chem., 274, 3512935138.
Germond, J.-E., Delley, M., DAmico, N., and Vincent, S.J.F. (2001) Heterologuous expression and characterization of the exopolysaccharide from Streptococcus thernophilus Sfi39. Eur. J. Biochem., 268, 51495156.
Guan, S., Clarke, A.J., and Whitfield, C. (2001) Functional analysis of the galactosyltransferases required for biosynthesis of D-galactan I, a component of the lipopolysaccharide O1 antigen of Klebsiella pneumoniae. J. Bacteriol., 183, 33183327.
Henrissat, B. and Coutinho, P. (2001) CAZyCarbohydrate-Active enZYmes. Available online at http://afmb.cnrs-mrs.fr/~cazy/CAZY/db.html.
Hughes, R. and Rowland, I.R. (2001) Stimulation of apoptosis by two prebiotic chicory fructans in the rat colon. Carcinogenesis, 22, 4347.
Jiang, S.M., Wang, L., and Reeves, P.R. (2001) Molecular characterization of Streptococcus pneumoniae type 4, 6B, 8, and 18C capsular polysaccharide gene clusters. Infect. Immun., 69, 12441255.
Jolly, L. and Stingele, F. (2001) Molecular organization and functionality of exopolysaccharide gene clusters from lactic acid bacteria. Int. Dairy J., 11, 733745.[CrossRef][ISI]
Koizumi, S., Endo, T., Tabata, K., and Ozaki, A. (1998) Large-scale production of UDP-galactose and globotriose by coupling metabolically engineered bacteria. Nat. Biotech., 16, 847850.[ISI]
Kolkman, M.A., Morrison, D.A., van der Zeijst, B.A., and Nuijten, P.J. (1996) The capsule polysaccharide synthesis locus of Streptococcus pneumoniae serotype 14: identification of the glycosyl transferase gene cps14E. J. Bacteriol., 178, 37363741.[Abstract]
Kolkman, M.A., van der Zeijst, B.A., and Nuijten, P.J. (1997) Functional analysis of glycosyltransferases encoded by the capsular polysaccharide biosynthesis locus of Streptococcus pneumoniae serotype 14. J. Biol. Chem., 272, 1950219508.
Lamothe, G. T. (2000) Molecular characterisation of exopolysaccharide biosynthesis by Lactobacillus delbrueckii subsp. bulgaricus. Université de Lausanne, Lausanne, Switzerland.
Nagaoka, M., Hashimoto, S., Watanabe, T., Yokokura, T., and Mori, Y. (1994) Anti-ulcer effects of lactic acid bacteria and their cell wall polysaccharides. Biol. Pharm. Bull., 17, 10121017.[ISI][Medline]
Nakajima, H., Toba, T., and Toyoda, S. (1995) Enhancement of antigen-specific antibody production by extracellular slime products from slime-forming Lactococcus lactis subspecies cremoris SBT 0495 in mice. Int. J. Food Microbiol., 25, 153158.[CrossRef][ISI][Medline]
Oda, M., Hasegawa, H., Komatsu, S., Kambe, M., and Tsuchiya, F. (1983) Anti-tumor polysaccharides from Lactobacillus species. Agric. Biol. Chem., 47, 16231625.[ISI]
Palcic, M.M. (1999) Biocatalytic synthesis of oligosaccharides. Curr. Opin. Biotechnol., 10, 616624.[CrossRef][ISI][Medline]
Plante, O.J., Palmacci, E.R., and Seeberger, P.H. (2001) Automated solid-phase synthesis of oligosaccharides. Science, 291, 15231527.
Roberfroid, M.B. (2001) Prebiotics: preferential substrates for specific germs? Am. J. Clin. Nutr., 73, 406S-409S.
Rush, J.S., Rick, P.D., and Waechter, C.J. (1997) Polyisoprenyl phosphate specificity of UDP-GlcNAc:undecaprenyl phosphate N-acetylglucosaminyl 1-P transferase from E. coli. Glycobiology, 7, 315322.[Abstract]
Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular cloning: a laboratory manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
Sears, P. and Wong, C.-H. (2001) Toward automated synthesis of oligosaccharides and glycoproteins. Science, 291, 23442350.
Slos, P., Bourquin, J.C., Lemoine, Y., and Mercenier, A. (1991) Isolation and characterization of chromosomal promoters of Streptococcus salivarius subsp. thermophilus. Appl. Environ. Microbiol., 57, 13331339.[ISI][Medline]
Staaf, M., Widmalm, G., Yang, Z., and Huttunen, E. (1996) Structural elucidation of an extracellular polysaccharide produced by Lactobacillus helveticus. Carbohydr. Res., 291, 155164.[CrossRef][ISI][Medline]
Staaf, M., Widmalm, G., Yang, Z., and Huttunen, E. (2000) Structural elucidation of the viscous exopolysaccharide produced by Lactobacillus helveticus Lb161. Carbohydr. Res., 326, 113119.[CrossRef][ISI][Medline]
Stingele, F., Neeser, J.-R., and Mollet, B. (1996) Identification and characterization of the eps (exopolysaccharide) gene cluster from Streptococcus thermophilus Sfi6. J. Bacteriol., 178, 16801690.[Abstract]
Stingele, F., Vincent, S.J.F., Faber, E.J., Newell, J.W., Kamerling, J.P., and Neeser, J.-R. (1999) Introduction of the exopolysaccharide gene cluster from Streptococcus thermophilus Sfi6 into Lactococcus lactis MG1363: production and characterization of an altered polysaccharide. Mol. Microbiol., 32, 12871295.[CrossRef][ISI][Medline]
Sutherland, I.W. (1998) Novel and established applications of microbial polysaccharides. Trends Biotech., 16, 4146.[CrossRef][ISI][Medline]
Unligil, U.M. and Rini, J.M. (2000) Glycosyltransferase structure and mechanism. Curr. Opin. Struct. Biol., 10, 510517.[CrossRef][ISI][Medline]
van Kranenburg, R., Marugg, J.D., van Swam, I.I., Willem, N.J., and de Vos, W.M. (1997) Molecular characterization of the plasmid-encoded eps gene cluster essential for exopolysaccharide biosynthesis in Lactococcus lactis. Mol. Microbiol., 24, 387397.[ISI][Medline]
van Kranenburg, R., van Swam, I.I., Marugg, J.D., Kleerebezem, M., and de Vos, W.M. (1999) Exopolysaccharide biosynthesis in Lactococcus lactis NIZO B40: functional analysis of the glycosyltransferase genes involved in synthesis of the polysaccharide backbone. J. Bacteriol., 181, 338340.
Vincent, S.J.F. and Zwahlen, C. (2000) Dipole-dipole cross-correlation at 13C natural abundance: a structural tool for polysaccharides. J. Am. Chem. Soc., 122, 83078308.[CrossRef][ISI]
Vincent, S.J.F., Faber, E.J., Neeser, J.R., Stingele, F., and Kamerling, J.P. (2001) Structure and properties of the exopolysaccharide produced by Streptococcus macedonicus Sc136. Glycobiology, 11, 131139.
Wakarchuk, W.W., Martin, A., Jennings, M.P., Moxon, E.R., and Richards, J.C. (1996) Functional relationships of the genetic locus encoding the glycosyltransferase enzymes involved in expression of the lacto-N-neotetraose terminal lipopolysaccharide structure in Neisseria meningitidis. J. Biol. Chem., 271, 1916619173.
Wang, L. and Reeves, P.R. (2001) The Escherichia coli O111 and Salmonella enterica O35 gene clusters: gene clusters encoding the same colitose-containing O-antigen are highly conserved. J. Bacteriol., 182, 52565261.
White, B. (1997) Whodunnit ? Nature, 389, 326.
Wymer, N. and Tooze, E.J. (2000) Enzyme-catalyzed synthesis of carbohydrates. Curr. Opin. Chem. Biol., 4, 110119.[CrossRef][ISI][Medline]
Yamamoto, Y., Murosaki, S., Yamauchi, R., Kato, K., and Sone, Y. (1994) Structural study on an exocellular polysaccharide produced by Lactobacillus helveticus TY1-2. Carbohydr. Res., 261, 6778.[CrossRef][ISI][Medline]
Zyzik, A. and Goldmann, T. (1999) Papers should spell out authors roles. Nature, 399, 406.