Capsular exopolysaccharide biosynthesis gene of Propionibacterium freudenreichii subsp. shermanii

International Journal of Food Microbiology 125 (2008) 252–258

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International Journal of Food Microbiology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / i j f o o d m i c r o

Capsular exopolysaccharide biosynthesis gene of Propionibacterium freudenreichii subsp. shermanii Stéphanie-Marie Deutsch a,b,d,⁎, Hélène Falentin a,b, Marguerite Dols-Lafargue c, Gisèle LaPointe d, Denis Roy d a

INRA, UMR1253, 35000 Rennes, France Agrocampus Rennes, UMR1253, 35000 Rennes, France UMR1219 Œnologie, Université Victor Segalen Bordeaux2 / INRA ISVV, 351 Cours de la Libération, 33405 Talence, France d Institut des Nutraceutiques et des Aliments fonctionnels, Université Laval, Québec, Canada G1V 0A6 b c

a r t i c l e

i n f o

Article history: Received 4 February 2008 Received in revised form 16 April 2008 Accepted 16 April 2008

a b s t r a c t In the dairy industry, exopolysaccharides (EPS) contribute to improving the texture and viscosity of cheese and yoghurt and also receive increasing attention because of their beneficial properties for health. For lactic acid bacteria, the production of EPS is well studied. However, for dairy propionibacteria the biosynthesis of EPS is poorly documented. A polysaccharide synthase-encoding gene was identified in the genome of Propionibacterium freudenreichii subsp. shermanii TL 34 (CIP 103027). This gene best aligns with Tts, the polysaccharide synthase gene of Streptococcus pneumoniae type 37 that is responsible for the production of a β-glucan capsular polysaccharide. PCR amplification showed the presence of an internal fragment of this gene in twelve strains of P. freudenreichii subsp. shermanii with a ropy phenotype in YEL+ medium. The gene sequence is highly conserved, as less than 1% of nucleotides differed among the 10 strains containing the complete gtf gene. The same primers failed to detect the gene in Propionibacterium acidipropionici strain TL 47, which is known to excrete exopolysaccharides in milk. The presence of (1→3, 1→2)-β-D-glucan capsule was demonstrated for 7 out of 12 strains by agglutination with a S. pneumoniae-type 37-specific antiserum. The presence of mRNA corresponding to the gene was detected by RT-PCR in three strains at both exponential and stationary growth phases. This work represents the first identification of a polysaccharide synthase gene of P. freudenreichii, and further studies will be undertaken to elucidate the role of capsular EPS. © 2008 Elsevier B.V. All rights reserved.

1. Introduction Propionibacterium freudenreichii is a Gram positive species widely used in the production of Swiss-type cheeses, because of its important role in the production of flavour compounds. This species has been used for the production of B12 vitamin (Quesada-Chanto et al., 1994), and propionate is used as a biopreservative in certain food industries (bread, silage) for inhibiting rope formation and mould growth. Besides these applications, P. freudenreichii is also studied for its probiotic capacities. Indeed, dairy propionibacteria have received GRAS status, they survive in the digestive tract (Bouglé et al., 1999; Jan et al., 2002), and depending on the strain, they can stimulate the growth of bifidobacteria in humans (Bouglé et al., 1999; Hojo et al., 2002; Satomi et al., 1999) and inhibit the growth of pathogenic microorganisms (Lyon et al., 1993). In addition, immunomodulatory effects have been shown in rodents (Kirjavainen et al., 1999; Pérez Chaia et al., 1995). Exopolysaccharides are classified according to their composition into homopolysaccharides, which are composed of one type of monosac-

⁎ Corresponding author. INRA, Agrocampus Rennes, Unité Mixte de Recherche 1253, Science et Technologie du lait et de l'Œuf, 65 rue de Saint-Brieuc, 35042 Rennes Cédex, France. Tel.: +33 2 23 48 53 34; fax: +33 2 23 48 53 50. E-mail address: [email protected] (S.-M. Deutsch). 0168-1605/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2008.04.006

charide and heteropolysaccharides, which are composed of repeating units of several monosaccharides (3 to 8). According to their location, they can be excreted exopolysaccharides (secreted in the surrounding environment) or capsular exopolysaccharides (tightly associated with the cell wall) (Laws et al., 2001; Monsan et al., 2001; Welman and Maddox, 2003). A very large biodiversity of EPS have been studied for lactic acid bacteria (LAB), and according to their composition, molecular mass, type of glycosidic linkage and structure, the properties of the polymers are highly variable (de Vuyst et al., 2001). The genes coding for EPS synthesis have been largely studied for LAB heteropolysaccharides, and the proteins involved are encoded by operons often consisting of more than 10 genes (Laws et al., 2001). The synthesis of homopolysaccharides has been described mostly for fructans and glucans and most often is carried out by glycansucrase enzymes using sucrose as glycosyl donor (Monsan et al., 2001). The applications of EPS are highly dependent on their properties (capsular or excreted, composition, size, sugar content) (Dabour et al., 2005; Faber et al.,1998). In the dairy industry, EPS contribute to improving the texture and viscosity of yoghurt and low-fat cheeses (Cerning, 1990; de Vuyst and Degeest, 1999). In low-fat products, EPS-producing LAB are used in situ to increase the moisture content, thus contributing to correct the poor texture (Dabour et al., 2006; Low et al., 1998). EPS also are receiving increasing attention because of their beneficial properties for

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health. Indeed, for some of them, immunomodulatory properties have been described (Chabot et al., 2001; Makino et al., 2006) as well as cholesterol lowering activities (Nakajima et al., 1992). The production of EPS is poorly documented for dairy propionibacteria. The data dealing with EPS-producing propionibacteria show a strain-dependent production, influenced by the medium composition as well as by the fermentation conditions (Crow, 1988; Gorret, 2001a,b; Racine et al.,1991; Skogen et al., 1974). Recently, the primary structure of an EPS produced by P. freudenreichii subsp. shermanii strain JS has been determined, showing the production of [→3)[β-D-Glcp-(1→2)]-β-DGlcp-(1→] homopolysaccharide (Nordmark et al., 2005). Based on this observation, it can be hypothesized that β-D-glucan synthase could be involved in (1, 3) β-D-glucan synthesis in P. freudenreichii. The aim of our study was the detection and the molecular characterization of the gtf gene, which is carried by the chromosome and encodes a putative β-Dglucan synthase. 2. Materials and methods 2.1. Bacterial strains and growth conditions The strains of P. freudenreichii subsp. shermanii were obtained from the TL collection from Institut National de la Recherche Agronomique (Rennes, France). All the strains were routinely cultured in YEL broth (Malik et al., 1968) at 30 °C under microaerophilic conditions. Growth was monitored spectrophotometrically at 650 nm (A650). When required, a specific growth medium was used, called YEL+, with the same composition as YEL but with 2.4% of lactic acid instead of 1% in YEL. Strain TL 47 (DSM 4900) belongs to the Propionibacterium acidipropionici species, and was cultivated under the same conditions. 2.2. Rapid screening of EPS-producing strains of P. freudenreichii subsp. shermanii

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2.5. DNA sequencing of the gtf genes Both DNA strands of PCR products were sequenced by the sequencing service of Ouest-Genopole (Roscoff, France) on an ABIPrism 3130 xl apparatus (Applied Biosystems, Courtaboeuf, France) using ppsAllF2 and ppsAllR2N primers. The gtf gene sequences have been deposited in EMBL, the accession numbers are indicated at the end of the section “Materials and methods”. The similarity analyses were performed by BLAST at NCBI (http://www.ncbi.nlm.nih.gov/ blast). The topology of the predicted protein products was performed using the TMpred program, available at http://www.ch.embnet.org/ software/TMPRED_form.html. 2.6. RNA isolation and manipulation for detection of the gtf transcript The strains of P. freudenreichii subsp. shermanii were grown on YEL or YEL+ medium until the A650 was between 0.6 and 0.8 (exponential growth phase) or 2 (stationary growth phase). Then cells from 5 ml of the culture were harvested (8000 ×g, 10 min, 4 °C). The pellet was suspended in 200 µl of lysis buffer (50 mM Tris–HCl, 1 mM EDTA, pH 8.0) containing 20 mg/l lysozyme and 50 U/ml mutanolysin and incubated 15 min at 37 °C. RNA extraction was performed using the RNeasy mini kit (Qiagen), according to the instructions of the manufacturer. At the end of extraction, RNA was suspended in 50 µl of RNase free water. Two µl of Riboblock (Fermentas life sciences, Vilnius, Lithuania) was added and the RNA was treated with DNase (DNA-free, Ambion, Cambridgeshire, United Kingdom), according to the instructions of the supplier. RNA was stored at −80 °C. Detection of mRNA was performed with the one-step RT-PCR kit (Qiagen), according to the instructions of the supplier using ppsF2 and ppsR1 primers. The positive control was performed with the two primers V3P3 and V3P2, targeting 16S rRNA (Parayre et al., 2007). 2.7. Immunological detection of capsular polysaccharide

The EPS-producing ability of P. freudenreichii subsp. shermanii strains was evaluated by visual observation of cultures. The strains were grown in YEL or YEL+ broth, for 24, 48 and 168 h at 30 °C. After incubation, tubes of culture were mixed, and the presence of slime or ropiness was checked visually. 2.3. Purification of P. freudenreichii genomic DNA Bacterial cultures were grown on YEL to an A650 of 1. Cells were harvested from 2 ml of each culture (8000 × g, 10 min, 4 °C), suspended in lysis buffer (lysozyme 20 mg/ml, mutanolysin 50 U/ml in 20 mM Tris–HCl (pH 8), 2 mM EDTA and 1% (v/v) Triton X 100), and incubated for 1 h at 37 °C. After lysis of the cells, the DNA was extracted with the DNeasy tissue kit (QIAGEN, Courtabœuf, France), according to the instructions of the manufacturer.

Agglutination tests were performed with Streptococcus pneumoniae-type 37-specific antisera, obtained from the Statens Serum Institut (Hillerød, Denmark). The cells of P. freudenreichii were grown on YEL, or YEL + to exponential or stationary growth phase. The assays were performed as previously described (Walling et al., 2005). 2.8. Accession numbers The 10 gtf sequences of P. freudenreichii subsp. shermanii have been deposited at EMBL (http://www.ebi.ac.uk/embl) with the following accession numbers: AM850119 for TL 20; AM850120 for TL 34; AM850121 for TL 143; AM850122 for TL 162; AM850123 for TL 176; AM850128 for TL 216; AM850124 for TL 234; AM850125 for TL 503; AM850126 for TL 1348; AM850127 for TL 1378.

2.4. PCR detection of the gtf gene 3. Results Primers ppsF2 (5′-CAGTCGGTGAAGAACCGCTACG-3′) and ppsR1 (5′-CGGCAGGGCATAGGTGAACAAC-3′) targeting an internal 342 bp fragment of gtf were used for amplification with total genomic DNA from P. freudenreichii as template. The PCR mixture (50 µl) contained: Taq polymerase buffer (10 mM Tris–HCl [pH 9.0], 50 mM KCl, 1.5 mM MgCl2), 200 µM of dNTPs, 1 µM of each primer, 2.5 U Taq polymerase (Q-BIOgene, Illkirch, France) and 1 µl of template DNA (equivalent to 10–20 ng). Cycling conditions were: 94 °C for 2 min followed by 30 cycles of 95 °C, 30 s; 60 °C, 30 s; 72 °C, 30 s; and ending with 72 °C, 10 min. For amplification of the complete gtf gene, primers ppsAllF2 (5′CAAGGGCACTGGCGACCAGGCATGAC-3′) and ppsAllR2 N (5′CCTTCAGGCCACTGGTGACGCTGCG-3′) were used. The cycling conditions were 94 °C, 2 min, followed by 30 cycles of 95 °C, 40 s; 64 °C, 30 s; 72 °C, 90 s and ending with 72 °C, 10 min.

3.1. Identification of EPS-producing strains of P. freudenreichii subsp. shermanii Sixty strains of P. freudenreichii subsp. shermanii, including the type strain TL 34, were tested for their slime or ropiness-producing ability. After 24 h of growth, no slime or ropiness was visually detected, whatever the growth medium for all the strains. After 48 h of incubation, twelve strains presented a ropy phenotype in the YEL+ medium, suggesting production of EPS. The presence of ropiness was mainly observed when cells were incubated in YEL+, suggesting that the production was dependent on the concentration of lactic acid in the medium. Ropiness was accentuated after 196 h of incubation.

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Fig. 1. Hydrophobicity plots of Gtf from Propionibacterium freudenreichii subsp. shermanii TL 34 (A), Tts from Streptococcus pneumoniae type 37 (B) and Gtf from Pediococcus parvulus (C), indicating the predicted transmembrane domains and their orientation (performed with the TMpred program available at www.ch.embnet.org/software/TMPRED_form.html). The accession numbers are: AAZ73237 for Gtf of P. parvulus, CAB51329 for Tts of S. pneumoniae, and AM850120 for Gtf of P. freudenreichii subsp. shermanii.

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Fig. 2. Multiple alignment of selected regions of the Gtf protein of Propionibacterium freudenreichii subsp. shermanii with those of other putative glycosyltransferases. The proteins aligned with Gtf are: Tts of Streptococcus pneumoniae (CAB51329), Dps of Pediococcus damnosus (ABB51206), and a putative glucosyltransferase of Arabidopsis thaliana (AAD15482). Position of the amino acids in the motif D,D,D,QXXRW previously identified in glycosyltransferase family 2 proteins are indicated in grey. Amino acid residues identical in all sequences are denoted by an asterisk and conserved amino acids are indicated with dots.

3.2. Identification of a polysaccharide synthase-encoding gene in the genome of P. freudenreichii subsp. shermanii strain TL 34 The analysis of the genome of the sequenced type strain P. freudenreichii subsp. shermanii TL 34 revealed the presence on the chromosome of a gene potentially coding for exopolysaccharide (EPS) biosynthesis, named gtf. The gtf gene contains 1536 nucleotides, with a predicted protein product Gtf of 511 amino acids. The predicted molecular weight of Gtf is 58,222 Da, and its calculated pl is 9.76. The predicted protein product of the gtf gene best aligned with Tts (accession number CAB51329), a polysaccharide synthase gene of S. pneumoniae type 37, with 52% identity and 70% similarity over a 451-amino-acid overlap. Gtf also showed similarity with the putative glucan synthase Dps of Pediococcus damnosus strain IEOB 8801 (now reclassified as Pediococcus parvulus) (accession number ABB51206), the Gtf of P. parvulus strain 2.6 (accession number AAZ73237) and the Gtf of Oenococcus oeni (accession number AAY87028) with 32% identity and 51% similarity over a 506amino-acid overlap. No promoter sequence was found in the region upstream of the ATG codon, even considering the specific promoter sequences identified for P. freudenreichii (Piao et al., 2004). 3.3. Gtf amino acid sequence analysis Topology prediction indicates that Gtf is a membrane-bound protein, with 6 highly hydrophobic transmembrane (TM) segments (Fig. 1). The amino acid positions for the predicted transmembrane sequences are aa 10–27 (in–out), aa 38–56 (out–in), aa 343–359 (in–out), aa 370–392 (out– in), aa 395–415 (in–out), and aa 454–487 (out–in). A potential signal

peptide was found for Gtf, with the cleavage site between amino acids 30 and 31. The central part of the protein is predicted to be cytoplasmic because it is more hydrophilic. Using COG analysis, Gtf might be a member of group 1215, glycosyltransferases involved in cell wall biogenesis (Tatusov et al., 2000). According to the CAZy database (http://www.cazy. org), Gtf belongs to family 2 (GT2) (Campbell et al., 1997). Gtf exhibits significant similarities with regions shown to be conserved among GT2 members. The conserved motif found in Gtf (D1,D2,D3,QXXRW) (Saxena et al., 1995; Stasinopoulos et al., 1999) has been identified as characteristic of the GT2 enzymes (Fig. 2). The three Asp residues are located in the cytosolic part of the protein in positions 124, 177 and 266. 3.4. PCR detection and sequencing of the gtf gene According to the nucleotide sequence of gtf detected in the type strain TL 34, the primers ppsF1 and ppsR2 targeting the central part of the gene were designed to detect gtf in other strains of the species, and in P. acidipropionici strain TL 47. The PCR reaction with these primers was positive for all 12 strains of P. freudenreichii subsp. shermanii tested, and negative for strain TL 47 (Fig. 3). The size of the amplicon was 342 bp, as expected. The complete gtf gene was amplified with primers ppsAllF2 and ppsAllR2N for 10 out of the 12 ropy strains of P. freudenreichii subsp. shermanii, and the PCR products were sequenced. For strains TL 1323 and TL 1337, no amplicon was obtained with primers ppsAllF2 and ppsAllR2N, suggesting that the sequence of gtf was divergent in the region of these primers. The predicted protein products are identical for strains TL 20 and TL 1378, for strains TL 34, TL 143 and TL 162 and for strains TL 176 and TL 234. Within the 10 sequences, 7 amino acids differed from one sequence to another (Table 1). Among the divergent amino acids, for those in positions 83, 196, 366 and 394, the substitution

Table 1 Divergent amino acids in the deduced protein sequence of Gtf from the different strains of Propionibacterium freudenreichii subsp. shermanii Amino acid position

Strain TL 20

TL 34

TL 176

TL 1378

TL 143

TL 234

TL 216

TL 503

TL 1348

F Y K Q S F R

F C K Q G F R

S Y E R G F R

TL 162

Fig. 3. PCR detection of the gtf gene in the genome of Propionibacterium freudenreichii subsp. shermanii. Agarose gel analysis of PCR reactions performed with total DNA as template and ppsF1 and ppsR2 as primers. Strain names are indicated above each lane. The boxed strain belongs to the Propionibacterium acidipropionici species. “M” indicates DNA molecular size marker (100 bp DNA Ladder, Invitrogen).

50 68 83 196 258 366 394

F Y K Q G F R

S Y E R G F K

S Y E R G L K

The names of the strains are indicated in the first line and the positions of the divergent amino acids are indicated in the first column.

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was strongly conservative whereas for the amino acid in position 258 the substitution was weakly conservative. According to the topology prediction, the amino acids in positions 83, 196 and 258 are located in the cytosolic part of the protein, amino acid in position 366 is located in the external part of the protein, and the amino acid in position 394 is located in the fourth TM segment. The substitutions in positions 50 (located in the first N-terminal TM segment) and 68 (located in the cytosolic part) were not conservative. The hydrophobicity profiles of the 10 proteins are identical (data not shown). 3.5. Immunological detection of capsular EPS The 12 strains of P. freudenreichii subsp. shermanii were tested for agglutination in the presence of the S. pneumoniae serotype 37 antibody (Table 2), which was raised against the β-glucan capsule of S. pneumoniae. The capsular strain of P. damnosus IEOB8801 was used as positive control, and its plasmid cured variant IEOB0206 was used as negative control (Walling et al., 2005). Seven strains of P. freudenreichii subsp. shermanii were agglutinated in the presence of the antiserum. For strains TL 34, TL 143, TL 162 and TL 234, the same positive results were obtained with cells grown on YEL or YEL+, and at both exponential and stationary phases. For strains TL 20, TL 216, TL 1337, TL 1348 and TL 1378, no agglutination was observed whatever the medium and the growth phase. For strains TL 1323, TL 503 and TL 176, agglutination was dependent on the growth phase and/or the medium. In YEL+ medium, no agglutination was observed during the exponential growth phase for these three strains. No agglutination was observed with P. acidipropionici strain TL 47, which was also negative by PCR for the gtf gene.

Fig. 4. Transcription analysis by one-step RT-PCR of gtf gene from Propionibacterium freudenreichii subsp. shermanii TL 34. Reactions were carried out using total RNA as template, and gtf targeting primers ppsF1 and ppsR2, or 16s rRNA targeting primers V3P3 and V3P2. Lane 1: ppsF1 and ppsR2; lane 2: V3P3 and V3P2; lane 3: same as lane 1, but without the reverse transcription step; lane 4: genomic DNA as template, ppsF1 and ppsR2 primers; lane 5: negative control with water as template; lane 6: molecular mass marker (100 bp DNA Ladder, Invitrogen).

Gram negative and Gram positive bacteria (Skorupska et al., 2006). This analysis did not reveal the presence of any priming-GT sequence. Moreover, the CODEHOP strategy (consensus-degenerate hybrid oligonucleotide primer) (Provencher et al., 2003) was used to attempt detection of a priming-GT in P. freudenreichii subsp. shermanii. No amplification was obtained with these primers for the 12 strains identified as ropy (data not shown). However, strain TL 47, which belongs to the P. acidipropionici species, gave a positive amplification with CODEHOP priming-GT primers.

3.6. Demonstration of gtf transcription 4. Discussion For strains TL 34, TL 1337 and TL 1378, total RNA was extracted at the exponential and stationary growth phases. After one-step reverse transcriptase-PCR (RT-PCR) using primers targeting a central region of the gtf gene, the presence of an amplicon of the expected size was detected for the three strains. The results for strain TL 34 are presented in Fig. 4. This indicates that the gtf gene is transcribed during the exponential growth phase. 3.7. Search for a priming glycosyltransferase (priming-GT) encoding gene The genome of P. freudenreichii subsp. shermanii TL 34 was examined for the presence of a priming-GT encoding gene, which is involved in the proposed mechanism for heteropolysaccharide biosynthesis in both

Table 2 Agglutination test performed with Streptococcus pneumoniae-type 37-specific antiserum against cells of Propionibacterium freudenreichii subsp. shermanii Strain

TL TL TL TL TL TL TL TL TL TL TL TL TL

20 34 47 143 162 176 216 234 503 1323 1337 1348 1378

YEL

YEL+

Exponential growth phase

Stationary phase

Exponential growth phase

Stationary phase

− + − + + + − + − − − − −

− + − + + + − + + + − − −

− + − + + − − + − − − − −

− + − + + + − + + + − − −

−: absence of agglutination; +: presence of agglutination. The underlined strain belongs to the Propionibacterium acidipropionici species.

Dairy propionibacteria have been shown to be able to produce EPS, although the biosynthesis mechanism involved is poorly documented. Recently, the structure of excreted EPS by P. freudenreichii subsp. shermanii was studied by NMR, showing a homopolysaccharide with the structure [→3)[β-D-Glcp-(1→2)]-β-D-Glcp-(1→] (Nordmark et al., 2005). To date, no data are available concerning the genes responsible for EPS biosynthesis by propionibacteria. In this work, 20% of the tested strains of P. freudenreichii subsp. shermanii, including the sequenced type strain TL 34 (Meurice et al., 2004), were identified as “ropy-producing”, suggesting the production of EPS. The chromosomal sequence of strain TL 34 encodes a gene potentially involved in EPS biosynthesis, designated gtf. The gtf gene product has high similarity with the glycosyltransferase Tts of S. pneumoniae, which is responsible for production of the type-37 capsular polysaccharide (Llull et al., 1999, 2001). The Tts glycosyltransferase has a dual specificity, synthesising both (1→3)- and (1→2)-linkages in the branched polymer (1→3, 1→2)-β-D-glucan. Gtf of P. freudenreichii subsp. shermanii also shows similarity with Gtf of P. parvulus, encoded by a plasmid-borne gene, and responsible for the secretion of β-glucan type homopolysaccharide in alcoholic beverages (Werning et al., 2006), as well as with Dps from O. oeni, encoded by a chromosomally-located gene (Walling et al., 2005; Werning et al., 2006). In addition, the genome sequence of the strain TL 34 does not contain genes potentially implicated in the biosynthesis of heteropolysaccharides. The lack of a priming-GT, which was revealed using CODEHOP primers (Provencher et al., 2003), also supports the absence of genes involved in heteropolysaccharide biosynthesis in P. freudenreichii subsp. shermanii. This CODEHOP strategy has been successfully used in several studies to detect priming-GTs (Ruas-Madiedo et al., 2007), as it is a PCR approach based on the presence of conserved motifs in the genes coding for these enzymes. Priming-GTs are involved in the first step of assembly of the repeating units of heteropolysaccharides, where the first sugar residue is linked to a lipid carrier. These priming-GTs have a key role, as

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their inactivation leads to a decrease or inhibition of EPS production (Stingele et al., 1996). The positive CODEHOP result for P. acidipropionici strain TL 47 suggests the presence of a priming-GT, which is not surprising as the synthesis of a heteropolysaccharide by this strain is well documented (Gorret et al., 2003). Our observations support the proposal that a different mechanism than that established for heteropolysaccharides is responsible for the synthesis of EPS in P. freudenreichii subsp. shermanii and suggest that the gtf gene is involved in capsular EPS production. For many homopolysaccharides, synthesis depends on the action of one single enzyme (glycansucrase), using sucrose as energy and as glycosyl donor (Korakli and Vogel, 2006; van Hijum et al., 2006). For S. pneumoniae type 37, another mechanism exists, where the synthesis is not inducible by sucrose and does not involve glycansucrase, but is due to the action of a single glycosyltransferase, Tts, without the participation of a lipid intermediate (Llull et al., 1999, 2001). Gtf presents a hydropathic profile similar to that of Tts and to that of the Gtf protein of P. parvulus. The hydropathic profile, the homology found between Gtf and Tts, together with the identification of the conserved motif D1,D2,D3,QXXRW, strongly suggests that Gtf is a membranebound glycosyltransferase, responsible for the synthesis of β-glucan. The motif D1,D2,D3,QXXRW identified as the putative catalytic site of the GT2 enzymes is divided into two domains: A, encompassing the first two Asp residues, and B, encompassing the remaining portion of the motif. These A and B domains were detected in Gtf. As for Tts of S. pneumoniae, the domain B motif is poorly conserved and the sequence RHSKW was identified. It was previously shown that processive glycosyltransferases contain both motifs A and B, whereas the B domain is absent from non-processive enzymes (Stasinopoulos et al., 1999; Saxena et al., 1995). This suggests that Gtf is a processive glycosyltransferase. Family 2 glycosyltransferases include enzymes that produce β-glucan with (1→3) linkages, such as curdlan and other β-glycans such as cellulose (Karnezis et al., 2000). The active site D1, D2,D3,QXXRW has also been described for a glucosylceramide synthase of rat cells. Site-directed mutagenesis of the glucosylceramide synthase confirmed the critical role of the motif in the active site (Marks et al., 2001). As the antiserum used to detect capsular EPS was raised against (1→3)-β-glucan, with β-(1→2) ramifications, the positive agglutination test means that a polysaccharide identical or very similar in structure is located at the surface of the cells of seven out of the 12 ropy strains. For the five agglutination-negative strains, further work is needed to determine if the quantity of polymer is too weak to permit agglutination with the antiserum or if a different polymer is produced. To our knowledge, this work represents the first identification of a polysaccharide synthase gene from P. freudenreichii. β-glucan production by lactic acid bacteria by a processive β-glycosyltransferase is rare and has been reported only for a few species: P. parvulus, O. oeni and Lactobacillus sp. It has also been reported for serotype 37 of S. pneumoniae, which is the only homopolysaccharide reported for the pneumococcus species. The physiological role of the capsule for P. freudenreichii subsp. shermanii cells requires further investigation. As P. freudenreichii subsp. shermanii has been shown to have probiotic activities, whether these activities are related to the biosynthesis of capsular EPS also needs to be studied. It would be especially interesting to investigate the immunomodulation potential of capsular P. freudenreichii, as many microbial (1→3)-β-glucans have been found to have immunomodulating activities (McIntosh et al., 2005; Zekovic and Kwiatkowski, 2005). References Bouglé, D., Roland, N., Lebeurrier, F., Arhan, P., 1999. Effect of propionibacteria supplementation on fecal bifidobacteria and segmental colonic transit time in healthy human subjects. Scandinavian Journal of Gastroenterology 34, 144–148.

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