Structural Characterization of the Exocellular Polysaccharides ...

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 1997, p. 3512–3518 0099-2240/97/$04.0010 Copyright © 1997, American Society for Microbiology

Vol. 63, No. 9

Structural Characterization of the Exocellular Polysaccharides Produced by Streptococcus thermophilus SFi39 and SFi12 ´ RO ˆ ME LEMOINE,1 FRE ´ DE ´ RIC CHIRAT,1 JEAN-MICHEL WIERUSZESKI,1 JE ´ RARD STRECKER,1 NICOLE FAVRE,2 AND JEAN-RICHARD NEESER2* GE Universite´ des Sciences et Techniques de Lille, F-59655 Villeneuve d’Ascq, France,1 and Nestle´ Research Center, Vers-chez-les-Blanc, CH-1000 Lausanne 26, Switzerland2 Received 5 February 1997/Accepted 9 June 1997

We investigated the structures of the exopolysaccharides (EPSs) produced by Streptococcus thermophilus SFi39 and SFi12. Both polymers were found to have molecular masses of greater than 2 3 106 Da. The SFi39 EPS consisted of D-glucose and D-galactose in a molar ratio of 1:1, whereas the SFi12 EPS was composed of D-galactose, L-rhamnose, and D-glucose in a molar ratio of 3:2:1. Methylation analysis of and nuclear magnetic resonance spectra recorded from the native polysaccharide, as well as oligosaccharides released by partial acid hydrolysis, allowed the complete structural determination of the SFi39 EPS, which consists of the following tetrasaccharide repeating unit: b-D-Galp1 2 6 33)-a-D-Glcp-(133)-b-D-Glcp-(133)-b-D-Galf-(13

Similar spectra recorded only from the native polysaccharide were sufficient to allow the structural determination of the SFi12 EPS, which consists of the following hexasaccharide repeating unit: b-D-Galp1 2 4 32)-a-L-Rhap-(132)-a-D-Galp-(133)-a-D-Glcp-(133)-a-D-Galp-(133)-a-L-Rhap-(13

This study shows that the texturizing properties of different S. thermophilus ropy strains are based on the production of EPSs exhibiting chemical similarities but structural differences. mophilus strains in our collection, we realized that there exists a large structural diversity among these EPSs. In the present report, we describe the characterization of two more S. thermophilus EPSs, produced by our strains SFi39 and SFi12.

There have been prior studies on bacterial exopolysaccharides (EPSs), mainly prompted by their exciting functional properties and their use as food thickeners (14). In recent years, several publications reporting the structures of EPSs obtained from lactic acid bacteria have appeared (1, 3, 4, 6, 11, 12, 16, 19–23, 26, 29, 30). Some of these EPSs, in addition to having the ability to improve the texture of fermented products, exhibit advantageous biological properties, such as immunostimulation and antitumor and antiulcer activities (15, 18). In 1989-1990, Doco et al. (3, 4) reported the first structural characterization of an EPS produced by three Streptococcus thermophilus strains, which was found to be composed of Dgalactose, D-glucose, and 2-acetamido-2-deoxy-D-galactose in a molar ratio of 2:1:1. This EPS was found to consist of the following tetrasaccharide repeating unit:

MATERIALS AND METHODS Bacterial strains and fermentation conditions. S. thermophilus SFi39 and SFi12 are both ropy strains of the Nestle´ strain collection. The growth medium consisted of skim milk powder, reconstituted at 10% and heat treated (115°C, 35 min) for sterilization (9 parts), plus an amino acid mixture (per liter, 495 mg of Ala, 343 mg of Arg, 682 mg of Asp, 59 mg of Cys, 1,229 mg of Glu, 759 mg of Gly, 153 mg of His, 215 mg of Iso, 470 mg of Leu, 565 mg of Lys, 122 mg of Met, 255 mg of Phe, 436 mg of Pro, 68 mg of Ser, 170 mg of Thr, 61 mg of Try, and 304 mg of Val) (one part), adjusted to pH 5.0 with 1 M NaOH and filtered for sterilization. The fermentations were carried out in a 1-liter-scale fermentor for 24 h at 40°C with a 1% inoculum. The pH was maintained at 5.5 by using 2 N NaOH and a stirring rate of 60 rpm. Extraction of the polysaccharides. The removal of proteins and bacteria from the spent fermented cultures was achieved by the addition of an equal volume of a solution of trichloroacetic acid (40%) followed by centrifugation (17,000 3 g, 20 min). Then, the same volume of acetone was added to the supernatant fraction to precipitate the EPSs, which were finally collected by centrifugation (17,000 3 g, 20 min). Such precipitated EPS fractions were dissolved in distilled water, and the pH was adjusted to 7.0 with a sodium hydroxide solution. After dialysis against distilled water (16 h), insoluble material was removed by ultracentrifugation (110,000 3 g, 1 h) and the EPSs were lyophilized. The total neutral sugar contents of these crude dehydrated EPSs were determined by the phenol-sulfuric acid method (5). Sizes of the exopolysaccharides. To confirm the purity and estimate the molecular weights of the polysaccharides, gel filtration chromatography was conducted with a fast protein liquid chromatography (FPLC) system (Pharmacia) containing a Superose 6 column (1.0 by 30 cm). Samples (200 ml), each containing 200 to 400 mg of dehydrated polysaccharide, were applied onto the column, and the polysaccharides were eluted with 50 mM phosphate buffer, pH 7.2, at the rate of 0.5 ml/min. Fractions of 1.0 ml were collected, and the total neutral sugar content in each fraction was determined by the phenol-sulfuric acid method (5). Monosaccharide compositions. Monosaccharide compositions were determined by gas-liquid chromatography (GLC) of O-methyloxime acetate deriva-

33)-b-D-Galp-(133)-b-D-Glcp-(133)-a-D-GalpNAc-(13 6 1 a-D-Galp1

When we first started analyzing our Nestle´ strain collection for texturizing S. thermophilus strains, we isolated two strains (S. thermophilus SFi6 and SFi20) secreting an EPS with a structure identical to the one depicted above (unpublished results). The genes responsible for the synthesis of the SFi6 EPS were identified and characterized (24). While we were looking for new polysaccharides secreted by other S. ther-

* Corresponding author. Mailing address: Nestle´ Research Center, Vers-chez-les-Blanc, CH-1000 Lausanne 26, Switzerland. Phone: (41 21) 785 86 98. Fax: (41 21) 785 89 25. E-mail: jean-richard.neeser @chlsnr.nestrd.ch. 3512

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S. THERMOPHILUS EXOCELLULAR POLYSACCHARIDE STRUCTURES

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FIG. 1. 2-D COSY spectrum (400 MHz) of the native SFi39 polysaccharide, recorded in D2O at 333 K. (A) Complete spectrum between d5.4 and d3.4 ppm. (B) Expanded spectrum between d4.1 and d3.4 ppm. tives obtained after acid hydrolysis of the polysaccharides (1 h, 125°C) in a 4 N trifluoroacetic acid (TFA) solution (17). Independently, polysaccharide samples (0.1 mg) were methanolyzed (methanolic 0.5 N HCl, 80°C, 24 h), N-acetylated, and trimethylsilylated. These N-acetylated trimethylsilylated methyl glycosides were analyzed with a Varian 3400 gas chromatograph (temperature program, 120 to 240°C at 2°C/min) on a BP1 fused-silica capillary column (25 by 0.32 mm) (13). The absolute configurations of the monosaccharides were also determined by GLC, using the trimethylsilylated N-reacetylated (2)-2-butyl glycoside derivatives (9, 10). Linkage analysis. The polysaccharides or oligosaccharides derived from acid hydrolysis were permethylated according to the method of Ciucanu and Kerek (2). Permethylated products were then subjected either to methanolysis (see above) or to acid hydrolysis (4 N TFA, 4 h, 100°C) followed by reduction with NaBD4. The partially methylated and acetylated (pyridine-acetic anhydride [1:2], overnight at room temperature) methylglycosides and alditol acetates were identified by GLC against reference compounds, as well as by GLC-mass spectrometry in the electron impact mode with a Nermag R10-10S mass spectrometer using an electron energy of 70 eV and an ionizing current of 0.2 mA (13).

TABLE 1. 1H chemical shifts of the native polysaccharide from SFi39 Chemical shift (d) in residue Proton

C (33-aD-Glcp)

B [33(6)b-D-Glcp]

A (33-bD-Galf)

D (D-bGalp)

H-1 H-2 H-3 H-4 H-5 H-6 H-69

5.373 3.741 3.911 3.548 4.070 3.880 3.818

4.700 3.491 3.725 3.777 3.702 4.234 3.936

5.358 4.414 4.359 4.310 4.014 3.770 3.714

4.886 3.628 3.695 3.989 3.729 3.850 3.820

Partial acid hydrolysis. Polysaccharide samples (10 mg) were hydrolyzed in 4 ml of a 0.2 N TFA solution for 1 h at 100°C. The degree of polysaccharide hydrolysis and the obtainment of low-mass oligosaccharides were followed by thin-layer chromatography on silica gel 60 F254 aluminum sheets (Merck) developed in a butanol-water-acetic acid (2:1:1.5) mixture, with the sugars being detected with an orcinol-sulfuric acid solution. To recover the hydrolyzed polysaccharides, TFA was removed by vacuum evaporation and lyophilization. Direct fractionation of the oligosaccharide mixtures was performed by high-pressure anion-exchange pulse amperometric detection chromatography (HPAE-PAD), as described below, followed by reduction with NaBD4. Periodate oxidation. A 10-mg polysaccharide sample was dissolved in 10 ml of sodium acetate buffer, pH 3.9. Sodium metaperiodate was added to a final concentration of 0.05 M, and the solution was maintained in the dark for 7 days at 4°C. Then, the excess of periodate was reacted with 2 ml of ethylene glycol for 2 h at room temperature, and the mixture was dialyzed against double-distilled water for 48 h and lyophilized. The oxidized polysaccharide was reduced with NaBH4 (16 h), and the excess of NaBH4 was reacted with a Dowex 50x8 (H1) resin; this was followed by vacuum coevaporation with methanol to remove boric acid. The oxidized and reduced polysaccharide was finally subjected to mild acid hydrolysis in 0.5 N TFA for 1 h at 90°C (Smith degradation). After removal of the TFA by vacuum drying, the resulting oligosaccharides were fractionated by HPAE-PAD chromatography and then reduced with NaBD4. HPAE-PAD chromatography. Fractionation of oligosaccharides obtained by acid hydrolysis of either native or periodate-oxidized polysaccharides was carried out with the Dionex HPAE-PAD system, consisting of a Dionex Bio-LC quaternary gradient module, a PAD 2 detector, and a Carbopac PA-1 pellicular anionexchange column (250 by 9 mm). The elution program was as follows: 100% eluant A (0.1 N NaOH) for 5 min and then 75% eluant A–25% eluant B (0.1 N NaOH containing 1 N CH3COONa) for 60 min, with a flow rate of 3 ml/min. The eluted fractions were immediately neutralized with 1 N acetic acid and lyophilized. The fractions were successively desalted on a column (6 by 1 cm) of Dowex 50x8 (H1) resin and on a column of Fractogel HW40F (55 by 2 cm; Merck) with water as the eluent. NMR spectroscopy. The samples were repeatedly exchanged in D2O (99.9%) with intermediate lyophilizations and then finally dissolved in D2O (99.95%).

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APPL. ENVIRON. MICROBIOL. TABLE 3. Methylation analysis (methylglycoside and itol acetate derivatives) of the native SFi39 EPS; of OS-I, derived from the EPS hydrolyzed after periodate oxidation (itol acetate derivatives); and of OS-III and -IV, derived from partial acid hydrolysis of the native EPS (itol acetate derivatives) Molar ratio for:

Derivativea SFi39 EPS

1,2,4,5-Ara 1,2,4,5,6-Gal 2,3,4,6-Gal 2,3,4,6-Glc 2,5,6-Galf 2,4,6-Glc 2,3,4-Glc 2,4-Glc

OS-I

OS-III

OS-IV

0.7 1.0

1.1 1.0 0.7

b

0.3 1.0 1.0 1.0 1.3

0.9 1.6c

0.9

1.0

a

The numbers 1 to 6 refer to the hydroxyl positions which carry a methyl group and which were not previously engaged in a glycosidic linkage. Other, unstated hydroxyl groups are acetylated. b Due to the high volatility of this derivative, the ratio was lower than expected. c This value was higher than expected, since this derivative is less volatile than those with residues carrying more methyl groups. ms; 256W by 2K FID data matrices were acquired, which were zero filled prior to Fourier transform, to obtain a 1K by 2K spectral data matrix; a sine-bell squared filter function was used in both dimensions. The 2-D 13C-1H COSY experiments were performed with simultaneous suppression of 1H homonuclear couplings by the use of the standard Bruker pulse program XHCORRD. Refocusing delays were adjusted to an average 1JC,H coupling constant of 150 Hz. 1H and 13C 90° pulse widths were 10.6 and 6 ms, respectively. The relaxation delay was 0.8 s. A 128W by 4K FID data matrix was acquired, which was zero filled prior to Fourier transform, to obtain a 512W by 4K spectral data matrix. An exponential function (LB 5 1 Hz) for 13C subspectra and a sine-bell filter function for 1H spectra were applied to enhance the signalto-noise ratio. The 2-D rotating-frame Overhauser effect spectroscopy (ROESY) spectra of the native polysaccharide from S. thermophilus SFi12 were recorded with standard Bruker programs for D2O solutions at 333 K and a mixing time of 400 ms. FIG. 2. 1H-13C heteronuclear correlation spectrum of the native SFi39 polysaccharide, recorded in D2O at 333 K. The 400-MHz 1H nuclear magnetic resonance (NMR) experiments were performed with a Bruker AM-400 wide-bore spectrometer equipped with a 5-mmdiameter 1H-13C dual-probe head, operating in the pulsed Fourier transform mode and controlled by an Aspect 3000 computer. All spectra were obtained at a probe temperature of 333 K. For one-dimensional (1-D) spectra, a 90° pulse of 10.6 ms and 1-s recycle delay were used. The chemical shifts are given relative to the signal of the methyl group of acetone (d 2.225 for 1H and d 31.55 for 13C). 2-D homonuclear correlation spectroscopy (COSY) 45 and COSY with simple, double, and triple relay transfers were performed by the use of the standard Bruker pulse program library or the programs given by B. Perly (Centre d’Etudes Atomiques, Saclay, France). For all relayed coherence transfer experiments, refocusing delays of 35 ms were chosen and the relaxation delay was 2 s. In all of these experiments, the spectral width was 1,840 Hz and the 1H 90° pulse was 10.6

TABLE 2. 13C chemical shifts of the native polysaccharide from SFi39 Chemical shift (d) in residuea Carbon

C-1 C-2 C-3 C-4 C-5 C-6

C (33-aD-Glcp)

B [33(6)-bD-Glcp]

A (33-bD-Galf)

D (D-bGalp)

100.0 72.50 81.09 69.07 72.84 61.62

102.99 72.72 83.61 70.82 73.70 69.54

109.37 80.70 85.39 83.20 71.40 63.97

104.42 71.84 73.81 69.70 76.03 61.94

a Values in boldface type indicate deshielded carbon resonances used for structure determination (see text).

RESULTS FPLC and analysis of the compositions of the polysaccharides. After 24 h of fermentation, 1-liter samples of the spent media from S. thermophilus SFi39 and SFi12 cultures were both subjected to the extraction procedure, yielding 350 mg of polysaccharide from SFi39 and 105 mg of EPS from SFi12. The elution and the purity of these polysaccharide samples were analyzed by FPLC on a column of Superose 6 (data not shown). Both polysaccharides were eluted at the exclusion limit (greater than 2 3 106 Da). GLC analysis of the O-methyloxime acetate sugar derivatives (after acid hydrolysis) indicated the presence of galactose and glucose in a molar ratio of 1:1 for the SFi39 EPS and of galactose, rhamnose, and glucose in a molar ratio of 3:2:1 for the SFi12 EPS. NMR spectroscopy of the EPS from S. thermophilus SFi39. For clarity in the presentation of the NMR data, the numbering of the sugar residues (in boldfaced capital letters) and protons (in arabic numerals) of each residue deduced from the assignment procedure is shown here in advance: D b-D-Galp1 2 6 33)-a-D-Glcp-(133)-b-D-Glcp-(133)-b-D-Galf-(13 C B A

GLC analysis of the trimethylsilylated methyl glycosides and (2)-2-butyl glycosides confirmed the presence of D-galactose and D-glucose in a molar ratio of 1:1. The 1H NMR spectrum of the native SFi39 EPS (Fig. 1) shows the presence of four anomeric protons with H-1 signals

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FIG. 3. 2-D COSY spectrum (400 MHz) of the native SFi12 polysaccharide, recorded in D2O at 333 K. (A) Complete spectrum between d5.6 and d3.4 ppm. (B) Partial spectrum of a selected high field resonance domain.

at d 5.358 (J1,2 5 1.6 Hz) (residue A), d 4.700 (J1,2 5 8 Hz) (residue B), d 5.373 (J1,2 5 4.3 Hz) (residue C), and d 4.886 (J1,2 5 8 Hz) (residue D), indicative of a tetrasaccharide repeating unit. The set of vicinal coupling constants depicted on the 1H COSY spectrum allowed the identification of the monosaccharides. Because the magnitudes of the J3,4 and J4,5 vicinal coupling constants were lower than 4 Hz, residue D was identified as a b-Gal. The attributions of a-Glc for residue C

and b-Glc for residue B were deduced from the fact that the vicinal coupling constants J3,4 and J4,5 were high (close to 8 Hz). Based on the sugar composition (Gal/Glc, 1:1) and on the characterization in the native polysaccharide of a 1,3,4-tri-Oacetyl, 2,5,6-tri-O-methyl hexose among the methyl ether derivatives (see Table 3), we concluded that the A residue was a hexofuranose. Its 3J1,2 coupling constant, which is lower than 2 Hz, and the H-2 (d 4.414) and H-3 (d 4.359) chemical shifts

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LEMOINE ET AL. TABLE 4. 1H chemical shifts of the native polysaccharide from SFi12 Chemical shift (d) in residue

Proton

E (32aL-Rhap)

D (32-aD-Galp)

C (33-aD-Glcp)

B (33-aD-Galp)

A [33(4)-aL-Rhap]

F (b-DGalp1)

H-1 H-2 H-3 H-4 H-5 H-6 H-69

5.282 4.098 3.931 3.514 3.858 1.332

5.497 4.000 4.097 4.073 4.313 3.79 3.79

5.159 3.766 4.019 3.721 4.069 3.84 3.84

5.306 4.078 4.171 4.234 4.262 3.81 3.81

5.057 4.388 4.181 4.019 3.892 1.369

4.687 3.552 3.697 3.966 3.686 3.85 3.80

that appear at a relatively low field are all characteristic of a b-anomeric galactosyl conformation (30). Finally, the two-step relayed COSY spectra (Fig. 1A and B) allowed the complete assignment of the proton resonances (Table 1). After these values were reported on the 1H-13C heteronuclear correlation spectrum (Fig. 2), the 24 13C atom resonances were fully assigned (Table 2). These values clearly show the deshielding of C-3 for a-Glcp (C), C-3 for b-Galf (A), and C-3 and C-6 for b-Glc (B), whereas the 13C resonances of b-Gal (D) were specific for a nonreducing monosaccharide unit. Therefore, the polysaccharide was determined to be composed of a terminal bGalp, a 3-linked a-Glcp, a 3-linked bGlcp, and a 3-linked bGalf. To achieve the complete elucidation of the repeating unit sequence, methylation analysis of the native polysaccharide and of three oligosaccharides derived from it was performed (Table 3). Oligosaccharide I (OS-I) was obtained after partial acid hydrolysis of the periodate-oxidized EPS, whereas oligo-

saccharides III and IV were obtained after partial acid hydrolysis of the native EPS. Methylation analysis of OS-III revealed a terminal Gal, a 6-linked Glc, and a 3-linked Gal-ol that furnish the sequence Gal136Glc133Gal-ol. The occurrence of terminal Gal and Glc residues in the methylation data of OSIV, together with a 3,6-linked Glc and a 3-linked Gal-ol, indicated a tetrasaccharide, for which the following sequence may be proposed by combination with the analytical results for OS-III: Gal1 2 6 Glc(133)Glc(133)Gal-ol

The structure of the repeating unit was finally deduced from the analysis of OS-I derived from the periodate-oxidized polysaccharide that led, after methylation analysis, to a terminal Glc, a 3-linked Glc, and a 3-linked Ara-ol, this last pentose originating from the C-5–C-6-oxidized galactofuranose. Then, the Smith degradation produced a trisaccharidic unit by hydrolysis of the acid-sensitive glycosidic bonds involving the terminal branched galactose and arabinofuranose, the structure of which is Glc133Glc133Ara-ol. By combining the methylation and NMR data described above, the primary structure of the repeating unit was formulated: Galp1 2 6 33)-Glcp-(133)-Glc-(133)-Galf-(13

Finally, the whole set of analytical results collected here permitted the complete assignment of the tetrasaccharide unit depicted at the beginning of this section. NMR spectroscopy of the EPS from S. thermophilus SFi12. Again, for clarity in the presentation of the NMR data, the

FIG. 4. HMQC 1H-13C spectrum of the native SFi12 polysaccharide, recorded in D2O at 333 K. (A) Partial spectrum of the low field resonance domain. (B) Partial spectrum of the high field resonance domain.


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TABLE 5. 13C chemical shifts of the native polysaccharide from SFi12 Chemical shift (d) in residuea Carbon E (32a-L- D (32-a- C (33-a- B (33-a- A [33(4)-a- F (b-DD-Galp) D-Glcp) D-Galp) L-Rhap] Rhap) Galp1)

C-1 C-2 C-3 C-4 C-5 C-6

100.9 79.7 70.8 73.2 70.1 17.6

98.7 75.0 71.0 70.6 71.7 61.9

96.5 70.9 79.5 70.9 72.5 61.2

94.0 67.6 73.8 69.2 71.7 61.9

102.8 66.1 75.7 76.4 69.0 18.0

103.7 71.9 74.0 69.7 76.0 62.1

a Values in boldface type indicate deshielded carbon resonances used for structure determination (see text).

numbering of the sugar residues (in boldfaced capital letters) and protons (in arabic numerals) of each residue deduced from the assignment procedure is shown in advance: F b-D-Galp1 2 4 32)-a-L-Rhap-(132)-a-D-Galp(133)-a-D-Glcp(133)-a-D-Galp(133)-a-L-Rhap(13 E D C B A

GLC analysis of the trimethylsilylated methyl glycosides and (2)-2-butyl glycosides confirmed the presence of D-galactose, L-rhamnose, and D-glucose in a molar ratio of 3:2:1. The occurrence of a hexasaccharidic repeating unit was further confirmed by the 1H and 13C NMR data from the native polysaccharide, which revealed six anomeric protons and carbons. The 3J1,2 values were measured on the two-step relayed COSY spectrum (Fig. 3A), which revealed three a- and one bhexosyl residues, together with two rhamnose units (3J1,2 ' 1 Hz). The six sugar unit residues were identified on the basis of their vicinal proton constant values with the aid of the 2-D relayed COSY spectra (one and two relays successively [Fig. 3]). These sugar units were respectively identified according to the magnitude of the 3JH,H values: residues B, D, and F were identified as galacto compounds due to the small J3,4 and J4,5 coupling constant values, whereas the larger values (;8 Hz) for J2,3, J3,4, and J4,5 exhibited by residue C indicated a gluco configuration. Resonances corresponding to H-6 of rhamnosyl residues A and E were correlated to their anomeric protons via the H-63H-53H-43H-3 and the H-33H-23H-1 connectivities depicted on the relayed COSY spectrum (Fig. 3A and B). Moreover, the a-anomericity of these rhamnosyl residues was latter confirmed by the observation of their C-5 atom resonances at 69 to ;70 ppm (see Table 5). Owing to the correlation peaks observed on the relayed COSY spectrum, a complete assignment of protons was performed (Table 4). After having reported these data on the HMQC spectrum (Fig. 4A and B), all carbon resonances were also assigned (Table 5). The low field values of some 13C resonances (in boldfaced type in Table 5) revealed the following linkage positions: a 2-substituted aRhap (E), a 2-substituted aGalp (D), a 3-substituted aGlcp (C), a 3-substituted aGalp (B), a 3,4-disubstituted aRhap (A), and a nonreducing terminal bGalp (F). Finally, the repeating unit sequence was established and the linkage positions were confirmed with the aid of ROESY correlation spectroscopy (Fig. 5). The NOEs for the corresponding transglycosidic anomeric-aglyconic proton pairs were found for the following sugar units: H-1 Gal (F)3H-4 Rha (A), H-1 Gal (B)3H-2,H-3 Rha (A), H-1 Glc (C)3H-4 Gal (B), H-1 Gal (D)3H-2,H-3,H-4 Glc (C), and H-1 Rha (E)3H-2 Gal (D).

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Based on the comparison of the HMQC and ROESY spectra (Fig. 4), interresidual connectivities F H-1–A H-4 proved that the terminal Gal F is 4-linked to the Rha A residue, the latter being 3-substituted with a Gal B residue rather than 2-substituted, since only its C-3 and C-4 were downshifted on HMQC 1H-13C spectrum (Fig. 4). Only a single correlation cross peak was observed between residues C and B, indicating a 1-3 linkage. Since only C-3 of residue C was shifted down to the low field, we concluded that there was a bond between residue D and C-3 of residue C. Similarly, the single interresidual connectivity H-1 H-2 between residues E and D demonstrated the 2-substitution of monosaccharide Gal D. Finally, based on the HMQC spectrum (Fig. 4), we concluded that Rha A was linking the 2 position of residue E. Taken together, the analytical results collected here permitted the complete elucidation of the hexasaccharide unit depicted at the beginning of this section. DISCUSSION The results presented here augment the list of the primary structures characterizing the EPSs secreted by lactic acid bacteria. Once again, the (1- and 2-D homonuclear and heteronuclear) NMR methods which have become available in the last few years were found to be extremely powerful when applied to the determination of the structures of soluble heteropolysaccharides. Ropy strains of S. thermophilus (3, 4), Lactobacillus bulgaricus (12), Lactobacillus helveticus (6, 20, 23, 29,

FIG. 5. 2-D ROESY spectrum (400 MHz) of the native SFi12 polysaccharide, recorded in D2O at 333 K with a mixing time of 400 ms.

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30), Lactococcus cremoris (11, 16), Lactobacillus sake (19, 26), Lactobacillus paracasei (21), and Lactobacillus acidophilus (22) are all able to produce extracellular heteropolysaccharides, on which thickening, viscous, or slimy textures are based (7, 8, 25, 27, 28). These EPS structures are most often composed of D-galactose and D-glucose, together with other sugar residues which occur less frequently (e.g., 2-acetamido-2-deoxy sugars, rhamnose, uronic acids); these monosaccharides sometimes contain substituted phosphate or acetyl groups. Galactose may be found in the pyranose or the furanose conformation. The monosaccharides are linked by mixed a- and b-linkages in the main chain, and usually some residues are branched off. These EPSs have molecular weights of several million daltons and are made of repeating units ranging from tetra- to heptasaccharides. Besides these general features, few common structural characteristics can be observed. However, by looking more carefully at the structural analogies exhibited by the three S. thermophilus EPS structures described so far (see references 3 and 4 and both of the structures presented in Results), many similarities become apparent. Each of the three polysaccharides is made of a main backbone chain, bearing only one short side chain by a repeating unit. This side chain is always made of a galactopyranoside residue, either a-linked (3, 4) or b-linked (this study) to the main chain. Interestingly, the four S. thermophilus ropy strains of our culture collection secreting these various EPSs (namely SFi6, SFi20, SFi39, and SFi12) all yield a slimy texture rather than a thickened one when used to ferment a milk-based medium (unpublished observation). By contrast, we are currently studying another S. thermophilus ropy strain that produces a thickening texture rather than a slimy one and secretes another type of EPS, the structure of which we are in the process of establishing. Consequently, such structural studies will finally allow the establishment of a clear structure-function relationship, allowing one to predict which EPS structure will produce a specific texture (thickening, viscous, slimy, etc.) once it is secreted into a fermented product. On the other hand, we will continue our work aimed at identifying the genes responsible for EPS synthesis in S. thermophilus (24). In this regard, the fact that two new and different S. thermophilus EPS structures have now been identified will permit the characterization of two new eps gene clusters, which in turn should lead to the characterization of a complete set of bacterial glycosyltransferases. Such an effort will open the door to polysaccharide bioengineering, finally allowing the production of EPSs with more desirable properties. ACKNOWLEDGMENT We are grateful to F. Stingele for improving the manuscript and for helpful discussions. REFERENCES 1. Cerning, J. 1990. Exocellular polysaccharides produced by lactic acid bacteria. FEMS Microbiol. Rev. 87:113–130. 2. Ciucanu, I., and F. Kerek. 1984. A rapid and simple method for the permethylation of carbohydrates. Carbohydr. Res. 131:209–217. 3. Doco, T., B. Fournet, D. Carcano, P. Ramos, A. Loones, J. M. Piot, and D. Guillochon. September 1989. Polysaccharide, application comme agent ´epaississant et comme agent antitumoral. European patent 331 564. 4. Doco, T., J.-M. Wieruszeski, B. Fournet, D. Carcano, P. Ramos, and A. Loones. 1990. Structure of an exocellular polysaccharide produced by Streptococcus thermophilus. Carbohydr. Res. 198:313–321. 5. Dubois, M. A., K. A. Gilles, J. K. Hamilton, P. A. Rebers, and F. Smith. 1956. Colorimetric method for determination of sugars and related substances. Anal. Chem. 28:350–356. 6. Favre, N., J. Lemoine, and J.-R. Neeser. March 1996. Branched polysaccharide, microorganism producing it and compositions containing them. European patent 699 689.

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