Lactobacillus helveticus glycosyltransferases: from genes to carbohydrate synthesis

Laure Jolly, John Newell, Ida Porcelli, Sébastien J.F. Vincent1 and Francesca Stingele2

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.


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Bioactive carbohydrates are crucial in mediating essential biological processes, and their biosynthesis is an essential aspect to develop for a global view of their biological functions. Lactic acid bacteria display an array of diverse and complex carbohydrates and, therefore, are of particular interest. Here we present the identification of a novel exocellular polysaccharide structure and the corresponding gene cluster from Lactobacillus helveticus NCC2745. The development of a glycosyltransferase-specific enzymatic assay allowed the assignment of sugar specificities, which as a general approach will for the future permit a faster and more direct characterization of glycosyltransferase specificities. A model of the biosynthesis of the repeating unit is proposed. EpsE is a phosphoglucosyltransferase initiating the repeating unit biosynthesis by linking a glucose residue to a membrane-associated lipophilic acceptor. EpsF elongates the carbohydrate chain by forming an {alpha}(1,3)-Glcp linkage onto the first Glcp, whereas EpsG adds a backbone {alpha}(1,6)-Galp onto {alpha}-Glcp and EpsH attaches a {alpha}(1,6)-Glcp branch onto the first glucose residue. Finally, EpsI would add a ß(1,6)-Galp linkage onto {alpha}-Glcp terminating the sidechain and EpsJ would terminate the synthesis of the polysaccharides’ repeating unit by forming a ß(1,3)-Galp linkage onto {alpha}-Galp.

Key words: enzymatic synthesis of oligosaccharides/eps gene cluster/exopolysaccharide biosynthesis/glycosyltransferases/NMR


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Carbohydrates play essential biological roles in bacterial recognition processes, immune system, and cellular growth. Specific polysaccharides have been shown to be essential in biofilm formation (Costerton et al., 1999Go), others to have prebiotic effect (Roberfroid, 2001Go) and some to mediate host–pathogen interactions (Casadevall and Pirofski, 2000Go), such as Streptococcus pneumoniae capsular polysaccharides that are key virulence factors (Sutherland, 1998Go). Specific carbohydrates have been linked to specific stimulations of the immune system in T-cell activation (Oda et al., 1983Go; Nakajima et al., 1995Go). Carbohydrate activity relating to ulcer (Nagaoka et al., 1994Go) and apoptosis and cancer (Hughes and Rowland, 2001Go) have been described.

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, 2001Go; Plante et al., 2001Go; Burkhart et al., 2001Go). The second approach is based on the use of enzymes, either in chemoenzymatic synthesis, which already has shown successes in vitro (Palcic, 1999Go; Sears and Wong, 2001Go), 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., 1998Go; DeFrees et al., 2000Go).

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, 2000Go; DeFrees et al., 2000Go). 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, 2001Go; Jiang et al., 2001Go; Jolly and Stingele, 2001Go). 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., 2001Go).

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.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
eps genetic analysis
The genetics of bacterial EPS production has been described for a few lactic acid bacteria strains (Stingele et al., 1996Go; van Kranenburg et al., 1997Go; Lamothe, 2000Go; Germond et al., 2001Go) and shown to be linked to eps gene clusters encoding putative GTFs (Jolly and Stingele, 2001Go). To identify the eps locus in L. helveticus NCC2745, degenerated oligonucleotides were designed on the basis of conserved regions found between homologs of the highly conserved phospho-GalT EpsE from S. thermophilus Sfi6. Polymerase chain reaction amplification with genomic DNA from L. helveticus NCC2745 produced a 120-bp DNA fragment that was used as a probe to screen a {lambda}-ZAP genomic library of L. helveticus NCC2745. Thus a 14-kb region encoding 11 open reading frames (ORFs) oriented in the same direction was identified (Figure 1). The 1042 bp upstream of the eps cluster are identical to parts of the L. helveticus cryptic plasmid pLH3. pLH3 contains an ORF oriented in the opposite direction to the gene cluster and encodes a 186-amino-acid protein with high similarity to the transposase of the insertion element IS982 of Lactococcus lactis B40 (van Kranenburg et al., 1997Go). Downstream of epsO, an ORF sharing no homology with ORFs from other eps genes was detected. Detailed ORF sequence analysis with the hydrophobic cluster analysis tool indicated the presence of six putative UDP-dependent GTFs (Campbell et al., 1997Go; Jolly and Stingele, 2001Go; Henrissat and Coutinho, 2001Go) falling into family 4 of retaining {alpha}-GTF (epsF, epsG, and epsH) and family 2 of inverting ß-GTF (epsI and epsJ). On the basis of similarities, five further ORFs were identified in the cluster and predicted to encode the following: epsK, a polymerase; epsL, a glycogenin homologous protein ({alpha}-GTF); epsM, a UDP-galactopyranoside mutase; epsN, a flippase analog to Wxz (Feldman et al., 1999Go); and epsO a putative transport protein involved in exporting the repeating unit toward the external side of the membrane.



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Fig. 1. Molecular organization of the eps cluster from L. helveticus NCC2475. Phospho-GTF (black vertically striped arrow), three {alpha}-GTF (black arrows), two ß-GTF (white arrows), polymerization enzyme (gray striped arrow), protein homolog to glycogenin (black horizontally striped arrow), mutase (gray vertically striped arrow), two enzymes involved in the export (gray striped arrow) and an insertion element at the beginning of the cluster (IS, gray arrow). Numbers indicate the start position of the genes.

 
Structural analysis of the EPS
The EPS produced by L. helveticus NCC2745 was composed of glucose and galactose in a molar ratio of 1:1. Although most other EPS from L. helveticus strains seem to be composed of glucose and galactose, the molar ratio between the two vary from 5:2 (in L. helveticus Lb161; (Staaf et al., 2000Go) to 1:2 (in L. helveticus VCCN 2091; Staaf et al., 1996Go), and L. helveticus TY1-2 also reported the additional presence of GlcpNAc (Yamamoto et al., 1994Go).

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 dipole–dipole relaxation spectra (Vincent and Zwahlen, 2000Go).



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Fig. 2. (a) Structure of L. helveticus NCC2745 EPS. (b) 1D 1H NMR spectra. (c) 1H-13C HSQC of L. helveticus NCC2745 EPS. The anomeric resonances are identified by the letters A to F associated with the monosaccharide unit (see Table I).

 
Several polysaccharide NMR experiments were recorded on L. helveticus NCC2745 EPS at 67°C. The 1H and 13C NMR assignments were collected in Table I. The 1H NMR assignment of L. helveticus NCC2745 EPS started from the anomeric (H-1) resonances of all residues A to F (assigned by decreasing 1H anomeric resonance) in the total correlation spectroscopy (TOCSY) spectra yielding connectivities H-2,3,4 for all six residues. Overlap of anomeric (E[H-1] and F[H-1] at 4.53 ppm) and nonanomeric proton resonances made the use of H-2,3,4,5 TOCSY traces, intramonosaccharide strong nuclear Overhauser effect spectroscopy (NOESY) cross-peaks, and complete assignment of the 1H-13C HSQC spectrum necessary. Resonances of aglyconic carbon atoms involved in a glycosidic linkage were determined from the 13C chemical shifts by identifying differences (> 4 ppm) in comparison to monosaccharide methyl glycoside references.


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Table I. 1H and 13C NMR chemical shifts of the L. helveticus NCC2745 EPS
 
The sequence of the monosaccharide residues was deduced from the results of the chemical analysis, from the 13C NMR assignments and from the presence of cross-peaks in 1H-13C cross-correlated dipole–dipole relaxation spectra (Vincent and Zwahlen, 2000Go) and 1H-13C heteronuclear multiple bonds correlation (HMBC) spectra, as well as NOESY spectra. Structurally important cross-peaks are summarized in Table II.


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Table II. NMR structural information available from cross-correlated dipole–dipole relaxation, HMBC, and NOESY
 
The structure of the repeating unit of the L. helveticus NCC2745 EPS was ->3)-ß-D-Galp-(1->3)-{alpha}-D-Galp-(1->6)-{alpha}-D-Glcp-(1->3)-[ß-D-Galp-(1->6)-{alpha}-D-Glcp-(1->6)-]-ß-D-Glcp-(1-> (Figure 2a).

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 Blue–stained sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) gel (Figure 3).



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Fig. 3. SDS–PAGE of purified GST-Eps proteins from L. helveticus NCC2475. Proteins were visualized with Coomassie blue staining. Lanes 1–5 purified GST-EpsF to purified GST-EpsJ. The expected molecular weights with GST are: epsF 69.9 kD, epsG 69.7 kD, epsH 73.3 kD, epsI 67.3 kD and epsJ 67.2 kD.

 
EpsE activity
To test the activity of the epsE gene product, permeabilized E. coli BL21SI(pLJHLHE) cells were induced to overproduce 6xHis-EpsE. These cells showed incorporation of labeled [14C]Glc when incubated with UDP-[14C]Glc, whereas no incorporation was found when the incubation was performed with UDP-[14C]Gal. Similarly, in vitro testing of the C35-P acceptor and crude extracts also showed incorporation of [14C]Glc but not [14C]Gal. Incorporation of [14C]Glc was found only in presence of the acceptor C35-P and crude extracts from induced E. coli BL21SI(pLJHLHE) cells (data not shown). The labeled product as seen on thin-layer chromatography (TLC) was subjected to mild acid hydrolysis and the resulting sugar was a Glc, showing that one Glc had been transferred onto the lipid carrier (Figure 4). Similarly to E. coli Rfe (Rush et al., 1997Go), 6xHis-EpsE transferred a phospho-Glc onto a prenylphosphate carrier showing that epsE encoded a phospho-glucosyltransferase (phospho-GlcT) initiating the biosynthesis of the repeating unit.



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Fig. 4. TLC analysis of (a) purification of C35-P-P-ß-Glc by reversed-phase chromatography. Lane 1: reaction mixture of EpsE assay with C35-P and labeled UDP-[14C]Glc. Lanes 2–4: elution with 10%, 50%, and 100% acetonitrile. (b) Activity assays with purified GST-EpsF, labeled C35-P-P-ß-[14C]Glc, and UDP-Glc. Lane 5: control without enzyme. Lanes 6 and 7: reactions at room temperature at t = 8 h and t = overnight. Lane 8: control without UDP-Glc. Lane 9: control without MgCl2. (c) Activity assays with labeled precursor. Lane 10: labeled C35-P-P-ß-[14C]Glc-(3<-1)-{alpha}-[14C]Glc alone. Lane 11: reaction of labeled C35-P-P-ß-[14C]Glc-(3<-1)-{alpha}-[14C]Glc with purified GST-EpsG and UDP-Gal. Lane 12: reaction of labeled C35-P-P-ß-[14C]Glc-(3<-1)-{alpha}-[14C]Glc with purified GST-EpsH and UDP-Glc. m, d, t1 t2, u, and g designate monosaccharide, disaccharide, first trisaccharide, second trisaccharide, free UDP-[14C]Glc, and free [14C]Glc, respectively.

 
EpsF activity
To identify the enzymatic activity of the subsequent biosynthesis step, a natural receptor analog, C35-P-P-ß-[14C]Glc, was produced by incubating EpsE with C35-P and UDP-[14C]Glc and purified by reversed-phase chromatography (Figure 4, lanes 1–4). Subsequently, purified GST-EpsF was used in a reaction with labeled C35-P-P-ß-[14C]Glc and either UDP-Glc or UDP-Gal. A new labeled compound could be detected on TLC plates only in the presence of UDP-Glc (Figure 4, lane 6). Mild acid hydrolysis revealed that the sugar moiety carried by C35-P was a disaccharide. This indicates that EpsF had a GlcT activity. This activity was shown to be MgCl2-dependent (Figure 4, lane 9), because no disaccharide product was detected either in the absence of magnesium ions or in the presence of manganese ions. UDP-sugar dependent GTFs are known to require the presence of a divalent metal cation for binding of the donor sugar by GTFs (Unligil and Rini, 2000Go). The yield of the reaction was low and corresponded to a specific activity of approximately 0.2 nmol/min/mg.

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 chromatography–mass 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., 1996Go; Kolkman et al., 1997Go; Palcic, 1999Go), 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 {alpha}(1,3) or {alpha}(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 {alpha}(1,3) or a {alpha}(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 ({alpha}-D-Glcp-(1->3)-D-Glcp) and isomaltose ({alpha}-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 {alpha}(1,3)-GlcT.



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Fig. 5. HPLC analysis. (a) 25 ng of glucose (1) (b) 100 ng of isomaltose (2) and nigerose (3). (c) Hydrolysate of the EpsF product C35-P-P-ß-[14C]Glc-(3<-1)-{alpha}-Glc. Only glucose (1) and nigerose (3) could be detected.

 
EpsG and EpsH activities
To test the activity of EpsG and EpsH the C35-P-P-ß-[14C]Glc-(3<-1)-{alpha}-Glc acceptor were produced by incubating EpsE and EpsF in the presence of C35-P and labeled UDP-[14C]Glc and subsequent reversed-phase chromatography purification. Both purified GST-EpsG and GST-EpsH were assayed with the C35-P-P-ß-[14C]Glc-(3<-1)-{alpha}-Glc. Both were able to add UDP-Glc and UDP-Gal to this acceptor in independent and separate reaction mixtures (i.e., one enzyme, one acceptor, one donor). Mild acid hydrolysis revealed that all four products contained a trisaccharide moiety. According to hydrophobic cluster analysis, both enzymes were predicted to be {alpha}-GTFs. Hence, GTF-EpsG and GST-EpsH carried out both {alpha}-GlcT and {alpha}-GalT activities and were both specific for the C35-P-P-ß-[14C]Glc-(3<-1)-{alpha}-Glc acceptor.

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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Table III. GTF affinity for different acceptor and donor sugars determined with purified GST-GTF fusion proteins
 

    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
The overall structure of other eps cluster (Figure 1) was found to be similar to other described lactic acid bacteria (Jolly and Stingele, 2001Go), however the first gene present in the NCC2745 eps cluster is epsE, a gene coding for a putative phospho-GTF initiating the biosynthesis of the repeating unit of the EPS, with the first gene in other clusters encoding a putative transcriptional regulatory protein and proteins involved in the polymerization and chain length determination of the EPS. The structure of the repeating unit of the L. helveticus NCC2745 EPS was found to be ->3)-ß-D-Galp-(1->3)-{alpha}-D-Galp-(1->6)-{alpha}-D-Glcp-(1->3)-[ß-D-Galp-(1->6)-{alpha}-D-Glcp-(1->6)-]-ß-D-Glcp-(1-> (Figure 2a).

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 {alpha}(1,3) linkage onto the initial ß-Glc. GST-EpsG and GST-EpsH carried out both {alpha}-GlcT and {alpha}-GalT activities and were both specific for the C35-P-P-ß-[14C]Glc-(3<-1)-{alpha}-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 {alpha}(1,3)-GlcT.

From the results presented, the only two possibilities for EpsG and EpsH were either a {alpha}(1,6)-GalT elongating the backbone by using the 3-linked {alpha}-Glc as acceptor or a branching {alpha}(1,6)-GlcT using the (internal) ß-Glc as acceptor. Because GST-EpsG showed higher {alpha}-GalT than {alpha}-GlcT activity (Table III), EpsG is proposed to be an {alpha}(1,6)-GalT while, by exclusion, EpsH is most likely responsible for the branching of the EPS repeating unit with a {alpha}(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 {alpha}-D-Galp-(1->6)-{alpha}-D-Glcp-(1->3)-D-Glcp for recognition of the precursor instead of the available disaccharide {alpha}-D-Glcp-(1->3)-D-Glcp, an effect shown to be important in vivo for S. thermophilus Sfi6 EPS synthesis (Stingele et al., 1999Go). According to the EPS structure (Figure 2a), the necessary acceptors for the two putative ßGTFs, EpsI and EpsJ, would be either a {alpha}(1,6)-Glc or a {alpha}(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 {alpha}(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 non–EPS-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., 1999Go). 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 {alpha}-D-Galp-(1->6)-{alpha}-D-Glcp-(1->3)-ß-Glcp and {alpha}-D-Glcp-(1->6)-[{alpha}-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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Table IV. Lactic acid bacteria GTF toolbox for glycosidic linkage synthesis
 
Today 17 different types of glycosidic linkages can be selectively created using GTFs from lactic acid bacteria that are involved in EPS biosynthesis, 6 of which were studied in the present work. Synthesis of biologically important structures like human milk oligosaccharide lacto-N-neotetraose could be obtained using three GTFs from S. macedonicus Sc136 and LgtB from Neisseria meningitidis (Table IV). This concept can be transferred to in vivo and in vitro carbohydrate biosynthesis using recombinant microorganisms expressing heterologous GTFs. Two studies have already shown that such a heterologous carbohydrate expression is feasible (Stingele et al., 1999Go; Germond et al., 2001Go).


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Bacterial strains and media
E. coli cells were grown at 37°C with vigorous shaking in NZ-amine (casein hydrolysate)-yeast extract (NZY) medium for the {lambda}-ZAP genomic library screening, and a salt free Luria-Bertani medium for E. coli BL21-SI and a 2-yeast extract-tryptone (2YT) medium for E. coli DH5{alpha} were used. L. helveticus strain NCC2745 was grown at 40°C in de Man-Rogosa-Sharpe medium (MRS) broth without agitation or in 10% reconstituted skim milk powder (MSK) supplemented with an amino acid mixture corresponding to concentrations found in 1% Protease Peptone Nr 3 (Oxide, Hampshire, UK). Where required, media were supplemented with kanamycin (50 µg/ml), chloramphenicol (34 µg/ml), or ampicillin (100 µg/ml).

DNA manipulations
E. coli plasmid DNA was isolated using the QIAprep 8 Turbo Miniprep Kit (Qiagen GmbH, Hilden, Germany) (Sambrook et al., 1989Go). L. helveticus chromosomal DNA was purified as previously described for Streptococcus thermophilus (Slos et al., 1991Go). Restriction nuclease digestions, ligations, agarose gel electrophoresis, and Southern blots were done according to established protocols (Sambrook et al., 1989Go). 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 {lambda}-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 {lambda}-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., 1996Go).

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 chromatography–pulsed 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 {alpha}-anomeric signal of external [13C-1]-D-glucose ({delta}H-1 5.15 for H-1 and {delta}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 dipole–dipole relaxation experiment (Vincent and Zwahlen, 2000Go) for determining glycosidic linkages with constant-time durations of 10–40 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 dipole–dipole relaxation), with averaging over 16 scans (TOCSY), 64 scans (HSQC and NOESY), or 256 scans (cross-correlated dipole–dipole 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% {eta}-dodecyl-ß-D-maltocide [DDM], or 1% {eta}-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 manufacturer’s 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)Go 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, 1–5 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)Go. 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 (1–5 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-{alpha}Gal, or pNP-{alpha}Glc, or pNP-{alpha}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 chromatography–PAD 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.


    Acknowledgments
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
We thank Jean-Richard Neeser for continuous support, Norbert Sprenger for critical comments and discussions, Joerg Hau for the mass spectrometry analysis, and Bernard Henrissat for help with the GTF classification. According to recent proposals (White, 1997Go; Zyzik and Goldmann, 1999Go; Editorial, 1999Go), full authorship contributions are provided: L.J., analyzed data, collected data, managed data, wrote paper; J.N., analyzed data, collected data, edited paper; I.P., analyzed data, collected data; S.J.F.V., analyzed data, collected data, managed data, wrote paper; F.S., analyzed data, coordinated study, edited paper, initiated the study, secured funding. The gene sequences were deposited in GenBank under the following accession codes: epsE, CAC07462.1; epsF, CAC07463.1; epsG, CAC07464.1; epsH, CAC07465.1; epsI, CAC07466.1; epsJ, CAC07467.1; epsK, CAC07468.1; epsL, CAC07469.1; epsM, CAC07470.1; epsN0, CAC07471.1; epsO, CAC07472.1.


    Abbreviations
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
CHAPS, 3-[(3-chloramidopropyl)dimethyl ammonio]-1-propane-sulfate; EPS, exocellular polysaccharides; Fchase, 6-(fluorescein-5-carboxamido)-hexanoic acid succimidyl ester; GlcT, glucosyltransferase; GTF, glycosyltransferase; GST, glutathione-S-transferase; HPLC, high-performance liquid chromatography; HMBC, heteronuclear multiple bonds correlation; HSQC, heterologus single quantum correlation; LC-MS, liquid chromatography mass spectrometry; MSK, reconstituted skim milk powder NMR, nuclear magnetic resonance; NZ-amine (casein hydrolysate)-yeast extract; NOESY, nuclear Overhauser effect spectroscopy; ORF, open reading frame; OTE, 2-(octadecylthio)-ethyl -CH2CH2SC18H37; pAP, p-aminophenyl; pNP, p-nitrophenyl; PAD, pulsed amperometric detector; PBS, phosphate buffered saline; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; TLC, thin-layer chromatography; TOCSY, total correlation spectroscopy.


    Footnotes
 
1 To whom correspondence should be addressed Back

2 Present address: Neurim Pharmaceuticals SA, 18 Rte de Genève, CH-1280 Nyon, Switzerland Back


    References
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
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