We report the identification and characterization of the eps gene cluster 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 consists of the following tetrasaccharide repeating unit: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 from subclones of a genomic library of Sfi6, revealed a 15.25-kb region encoding 15 open reading frames. EPS expression in the non-EPS-producing 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 genes epsA to epsM was capable of directing EPS synthesis and secretion in 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 Streptococcus 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, and E and the genes involved in exopolysaccharide I (EPSI) synthesis in Rhizobium meliloti for epsC to epsJ and the genes involved in lipopolysaccharide synthesis in members of the Enterobacteriaceae, and finally for epsK and lipB of Neisseria meningitidis. Genes (epsJ, epsL, and epsM) 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.

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Lactobacillus delbrueckiisubsp. bulgaricus produces exopolysaccharides (EPSs), which play a role in the rheological properties of fermented food products. Lb. bulgaricus Lfi5 produces a high-molecular-weight EPS composed of galactose, glucose, and rhamnose in the molar ratio 5:1:1. An 18-kb DNA region containing 14 genes, designated epsA to epsN, was isolated by genomic DNA library screening and inverted PCR. The predicted gene products are homologous to proteins involved in the biosynthesis of other bacterial polysaccharides and the genetic organization was found to be similar to that of other eps clusters from lactic acid bacteria. Transcriptional analysis revealed that the 14 eps genes are co-ordinately expressed and transcribed as a single mRNA of 15-16 kb. The transcription start site of the promoter was mapped upstream of the first gene, epsA. Genes encoding glycosyltranferases were further studied by heterologous expression and functional assays. We showed that the epsE gene product is a phospho-glucosyltransferase initiating the biosynthesis of EPS. Heterologous expression of epsE in a Lactococcus lactis epsDmutant restored EPS production, demonstrating its role and importance in EPS biosynthesis. Functional assays of other glycosyltransferases allowed their sugar specificity to be elucidated and an overall biosynthetic pathway for EPS synthesis by Lb. bulgaricus to be proposed.

The aim of the present study was to select bacterial strains with potential properties as oral probiotics, namely for the prevention of dental caries. We examined 23 dairy microorganisms, out of which we identified two Streptococcus thermophilus and two Lactcoccus lactis strains that were able to adhere to saliva-coated hydroxyapatite beads to the same extent as Streptococcus sobrinus OMZ176. Two of them, Strep. thermophilus NCC1561 and Lactoc. lactis ssp. lactis NCC2211, were further successfully incorporated into a biofilm mimicking the dental plaque. Furthermore, they could grow in such a biofilm together with five strains of oral bacterial species, representative of supragingival plaque. In this system, Lactoc. lactis NCC2211 was able to modulate the growth of the oral bacteria, and in particular to diminish the colonization of Streptococcus oralis OMZ607, Veillonella dispar OMZ493, Actinomyces naeslundii OMZ745 and of the cariogenic Strep. sobrinus OMZ176. These findings encourage further research with selected non-pathogenic dairy bacterial strains with the aim to decrease the cariogenic potential of dental plaque.

Zwitterionic polysaccharides (Zps) from pathogenic bacteria, such as Bacteroides fragilis, are virulence factors responsible for abscess formation associated with intra-abdominal sepsis. The underlying cellular mechanism for abscess formation requires T cell activation. Conversely, abscess formation can be prevented by prophylactic s.c. injection of purified Zps alone, a process also dependent on T cells. Hence, the modulatory role of T cells in abscess formation was investigated. We show that Zps interact directly with T cells with fast association/dissociation kinetics. Vβ repertoire analysis using RT-PCR demonstrates that Zps have broad Vβ usage. Zps-specific hybridomas responded to a variety of other Zps, but not to a nonzwitterionic polysaccharide, indicating cross-reactivity between different Zps. Furthermore, Zps-reactive T cell hybridomas could effectively transfer protection against abscess formation. Analysis of the proliferative capacity of T cells recovered from Zps-treated animals revealed that these T cells are anergic to subsequent stimulation by the different Zps or to alloantigens in an MLR. This anergic response was relieved by addition of IL-2. Taken together, the data show that this class of polysaccharides interacts directly with T cells in a nonbiased manner to elicit an IL-2-dependent anergic response that confers protection against abscess formation.