Abstract
(Englisch)
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The eps Gene Cluster from Streptococcus thermophilus Sfi39: Characterization, stability of EPS production and heterologous expression in Lactococcus lactis.
Streptococcus thermophilus Sfi39 is a ropy strain of lactic acid bacteria (LAB). The high-molecular-weight exopolysaccharide (EPS) produced is composed of glucose and galactose in a molar ratio of 1:1 and has the following tetrasaccharide repeating unit: ® 3[b-D-Galp (1-6) -b-D-Glcp-(1®3)-b-D-Galf-(1®3)-a-D-Glcp-(1®(J. Lemoine et al., Appl. Environ. Microbiol., 63(1997) 3512-3518).
The genetic locus involved in the production of the EPS was isolated by PCR using specific primers designed from the sequence of the S. thermophilus Sfi6 gene cluster (Stingele et al., J. of Bactedol., 178 (1996) 1680-1690). A genomic region of 20kb was isolated, encoding 14 open reading frames. Homology searches of the predicted proteins on the Swiss-Prot database revealed that the first 5 genes are identical to epsA to epsE of other S. thermophilus strains. These genes are involved in regulation (epsA), chain-length determination (epsB to epsD), glycosyitransferase (epsE to epsH) and transmembrane transport (epsl and epsJ) functions. The epsE is the glycosyltransferase responsible for the anchoring of the first monosaccharide to the lipophilic carrier. Moderate homologies (30-45% identity) were detected between epsF, epsG, epsH and genes involved in capsular polysaccharide synthesis in Streptococcus. pneumoniae (epsF) and glycosyltranferases from S. thermophilus, S. pneumoniae, Klebsiella pneumoniae (epsG, epsH). The next genes, epsl epsJ showsed homologies to enzymes involved in the transport of the repeating unit in S. thermophilus and Lactococcus lactis. The last gene involved in EPS synthesis is a galactopyranose mutase (epsK).
Three IS elements were found inserted at the extremity of the eps cluster and between epsE and epsF. The same IS elements were found in L. lactis (IS905, IS892) and S. pneumoniae (IS1167). The stability of EPS production was tested by screening for spontaneous non-ropy mutants. Several mutants were isolated: in one case an IS element (IS905) was found inserted in the middle of epsF.
To asses the implication of the gene cluster in EPS synthesis, epsE and epsG were disrupted by simple integration. In both cases, the ropy character was lost. The eps gene cluster was cloned and transferred into a non-EPS-producing heterologuous host, L lactis MG1363, and an EPS was produced. This EPS was shown by means of nuclear magnetic resonance (NMR) spectroscopy analysis to have the same structure as the EPS produced by Sfi39, thereby demonstrating the function of the eps gene cluster.
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