IDENTIFICATION AND CHARACTERIZATION OF THE EPS (EXOPOLYSACCHARIDE) GENE-CLUSTER FROM STREPTOCOCCUS-THERMOPHILUS SFI6

We report the identification and characterization of the eps gene clus ter of Streptococcus thermophilus Sfi6 required for exopolysaccharide (EPS) synthesis. This report is the first genetic work concerning EPS production in a food microorganism. The EPS secreted by this strain co nsists of the following tetrasaccharide repeating unit: 1-->6)]-beta-D -Glcp-(1-->3)-alpha-D-GalpNAc-(1-->. The genetic locus was identified by Tn916 mutagenesis in combination with a plate assay to identify Eps mutants. Sequence analysis of the gene region, which was obtained fro m subclones of a genomic library of Sfi6, revealed a 15.25-kb region e ncoding 15 open reading frames. EPS expression in the non-EPS-producin g heterologous host, Lactococcus lactis MG1363, showed that within the 15.25-kb region, a region with a size of 14.52 kb encoding the 13 gen es epsA to epsM was capable of directing EPS synthesis and secretion i n this host. Homology searches of the predicted proteins in the Swiss- Prot database revealed high homology (40 to 68% identity) for epsA, B, C, D, and E and the genes involved in capsule synthesis in Streptococ cus pneumoniae and Streptococcus agalactiae. Moderate to low homology (37 to 18% identity) was detected for epsB, D, F, and H and the genes involved in capsule synthesis in Staphylococcus aureus for epsC, D, an d E and the genes involved in expolysaccharide I (EPSI) synthesis in R hizobium meliloti for epsC to epsJ and the genes involved in lipopolys accharide synthesis in members of the Enterobacteriaceae, and finally for epsK and lipB of Neisseria meningitidis. Genes (epsJ, epsL, and ep sM) for which the predicted proteins showed little or no homology with proteins in the Swiss-Prot database were shown to be involved in EPS synthesis by single-crossover gene disruption experiments.