J Bacteriol 181(20): 6354-6360

Unraveling the Function of Glycosyltransferases in <em>Streptococcus thermophilus</em> Sfi6

Nestlé Research Center, Nestec Ltd., Vers-chez-les-Blanc, 1000 Lausanne 26, Switzerland

^{Corresponding author. Mailing address: Nestlé Research Center, Nestec Ltd., P.O. Box 44, Vers-chez-les-Blanc, CH-1000 Lausanne 26, Switzerland. Phone: 41-21-7858923. Fax: 41-21-7858925. E-mail:}moc.eltsen.sldr@elegnits.acsecnarf.

Received 1999 May 3; Accepted 1999 Jul 26.

Abstract

Streptococcus thermophilus Sfi6 produces a texturizing exopolysaccharide (EPS) consisting of a →3)[α-d-Galp-(1→6)]-β-d-Glcp-(1→3)-α-d-GalpNAc-(1→3)-β-d-Galp-(1→ repeating unit. We previously identified and analyzed a 14.5-kb gene cluster from S. thermophilus Sfi6 consisting of 13 genes responsible for its EPS production. Within this gene cluster, we found a central region of genes (epsE, epsF, epsG, and epsI) that showed similarity to glycosyltransferases. In this study, we investigated the sugar specificity of these enzymes. EpsE catalyzes the first step in the biosynthesis of the EPS repeating unit. It exhibits phosphogalactosyltransferase activity and transfers galactose onto the lipophilic carrier. The second step is fulfilled by EpsG, which transfers an α-N-acetylgalactosamine onto the first β-galactoside. The activity of EpsF was determined by characterizing the EPS produced by an S. thermophilus epsF deletion mutant. This EPS consisted of the monosaccharides Gal, Glc, and GalNAc in an approximately equimolar ratio, thus suggesting that epsF codes for the branching galactosyltransferase. epsI probably codes for the β-1,3-glucosyltransferase, since it is the only glycosyltransferase to which no gene has been assigned and it exhibits similarity to other β-glycosyltransferases. EpsE shows the conserved features of phosphoglycosyltransferases, whereas EpsF and EpsG exhibit the primary structure of α-glycosyltransferases, belonging to glycosyltransferase family 4, whose members are conserved in all major phylogenetic lineages, including the Archaea and Eukaryota.

Abstract

Extracellular polysaccharides have a wide array of functions in bacteria and can play roles as varied as protection from desiccation or improvement of adherence, pathogenesis, or symbiosis (19, 20, 31, 32). They may take the form of capsules attached to the cell membrane or be secreted extracellularly. More commonly, in gram-negative bacteria they are present in the form of the O antigen of the lipopolysaccharide, and in gram-positive bacteria they occur as cell wall polysaccharides. Besides their biological role, polysaccharides are also of industrial interest because of their rheological properties. Exopolysaccharides (EPS) produced by lactic acid bacteria have in particular attracted the attention of the food industry because of their GRAS (generally regarded as safe) status. This has resulted in the elucidation of a large number of varied EPS structures from gram-positive Streptococcus (3, 5, 12), Lactococcus (7, 17), and Lactobacillus (8, 21–24, 26, 28, 37) strains.

While investigation of polysaccharide gene clusters in gram-negative bacteria began over 20 years ago (31, 36), research on those of gram-positive microorganisms has advanced only lately. Recent reports include the characterization of genes involved in polysaccharide biosynthesis from the pathogens Staphylococcus aureus and Streptococcus pneumoniae (11, 13, 16) and from the food microorganisms Streptococcus thermophilus and Lactococcus lactis (27, 33). The general organization of these clusters seems to be conserved: a central region with similarity to glycosyltransferase genes is flanked by two regions exhibiting similarity to genes involved in polymerization and export, and a putative regulatory region can be found at the beginning of each cluster. Even though this organization is not always conserved among genes involved in O-antigen synthesis, their homology points to similar biosynthetic pathways (10, 36). The repeating unit is first assembled by the sequential transfer of sugar residues onto a lipophilic carrier by specific glycosyltransferases. Unlike the other glycosyltransferases, the first glycosyltransferase does not catalyze a glycosidic linkage but transfers a sugar-1-phosphate onto a lipophilic anchor, such as undecaprenylphosphate. Subsequently, the completed repeating unit is exported and polymerized. In the case of cell surface polysaccharides, it is anchored to a cell envelope component while secreted polysaccharides are released.

Of the enzymes required for the biosynthesis of EPS, we were especially interested in the glycosyltransferases, because their sugar specificities determine the nature of the polysaccharide. Understanding and being able to predict their function are prerequisites for their use in carbohydrate bioengineering. However, even though a large number of polysaccharide gene clusters have been sequenced, only a very limited number of glycosyltransferases have been biochemically characterized (11, 34). Little is known about their sugar acceptor/donor specificities and their active centers.

Here we present a functional analysis of the S. thermophilus Sfi6 glycosyltransferase gene region, comprising the epsE-epsF-epsG-epsH-epsI genes, which are part of the previously characterized eps gene cluster (27, 29). Except for epsH, which shows homology to genes encoding O-acetyltransferases (2), all genes located in this region exhibit homology to glycosyltransferases involved in polysaccharide biosynthesis. By using a combination of biochemical and genetic approaches, we were able to shed light on the sugar specificity of the glycosyltransferases.

ACKNOWLEDGMENTS

We thank M. Kolkman for helpful advice on setting up the glycosyltransferase assays and B. Henrissat for assistance in interpreting the hydrophobic cluster analysis data for glycosyltransferases.

ACKNOWLEDGMENTS

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