Unraveling the function of glycosyltransferases in Streptococcus thermophilus Sfi6

Streptococcus thermophilus Sfi6 produces a texturizing exopolysaccharide (E PS) consisting of a -->3) [alpha-D-Galp-(1--> 6)-beta-D-Glcp-(1 --> 3)-alph a-D-GalpNAc-(1 --> 3)-beta-D-Galp-(1 --> repeating unit. We previously iden tified and analyzed a 14.5-kb gene cluster from S, thermophilus Sfi6 consis ting of 13 genes responsible for its EPS production. Within this gene clust er, we found a central region of genes (epsE, epsF, epsG, and epsI) that sh owed similarity to glycosyltransferases In this study, we investigated the sugar specificity of these enzymes. EpsE catalyzes the first step in the bi osynthesis of the EPS repeating unit. It exhibits phosphogalactosyltransfer ase activity and transfers galactose onto the lipophilic carrier. The secon d step is fulfilled by EpsG1 which transfers an alpha-N-acetylgalactosamine onto the first beta-galactoside. The activity of EpsF was determined by ch aracterizing the EPS produced by an S, thermophilus epsF deletion mutant. T his EPS consisted of the monosaccharides Gal, Glc, and GalNAc in an approxi mately equimolar ratio, thus suggesting that epsF codes for the branching g alactosyltransferase. epsI probably codes for the beta-1,3-glucosyltransfer ase, since it is the only glycosyltransferase to which no gene has been ass igned and it exhibits similarity to other beta-glycosyltransferases. EpsE s hows the conserved features of phosphoglycosyltransferases, whereas EpsF an d EpsG exhibit the primary structure of alpha-glycosyltransferases, belongi ng to glycosyltransferase family 4, whose members are conserved in all majo r phylogenetic lineages, including the Archaea and Eukaryota.