Identification and Molecular Characterization of the Chromosomal Exopolysaccharide Biosynthesis Gene Cluster from Lactococcus lactis subsp. cremoris SMQ-461

Abstract

The exopolysaccharide (EPS) capsule-forming strain SMQ-461 of Lactococcus lactis subsp. cremoris, isolated from raw milk, produces EPS with an apparent molecular mass of >1.6 ؋ 10 6 Da. The EPS biosynthetic genes are located on the chromosome in a 13.2-kb region consisting of 15 open reading frames. This region is flanked by three IS1077-related tnp genes (L. lactis) at the 5 end and orfY, along with an IS981-related tnp gene, at the 3 end. The eps genes are organized in specific regions involved in regulation, chain length determination, biosynthesis of the repeat unit, polymerization, and export. Three (epsGIK) of the six predicted glycosyltransferase gene products showed low amino acid similarity with known glycosyltransferases. The structure of the repeat unit could thus be different from those known to date for Lactococcus. Reverse transcription-PCR analysis revealed that the eps locus is transcribed as a single mRNA. The function of the eps gene cluster was confirmed by disrupting the priming glycosyltransferase gene (epsD) in Lactococcus cremoris SMQ-461, generating non-EPS-producing reversible mutants. This is the first report of a chromosomal location for EPS genetic elements in Lactococcus cremoris, with novel glycosyltransferases not encountered before in lactic acid bacteria.Lactic acid bacteria (LAB) are widely used in fermented dairy products, mainly for lactic acid formation but also for the production of minor flavor and preservation components. Some LAB are also able to produce exopolysaccharides (EPS), which are either excreted in the growth medium as slime (ropy form) or remain attached to the bacterial cell wall forming capsular EPS (6, 32). In the dairy industry, EPS-producing LAB, including the genera Streptococcus, Lactobacillus, and Lactococcus, are used in situ to improve the textural characteristics of fermented dairy products, especially low-fat yoghurt and cheese. LAB are foodgrade bacteria that can produce a wide variety of structurally different EPS with potential uses for new applications, for example, in replacement of polysaccharides such as gellan, pullulan, xanthan, and bacterial alginates that are presently produced by non-food-grade bacteria (12,25,48).EPS-producing LAB, including strains of Lactococcus spp., have been shown to express at least two distinct phenotypic forms of EPS, either ropy and/or capsular forms (32). Moreover, they produce EPS with considerable diversity in structure and composition (14,54,55,58,64). This diversity in EPS composition indicates that LAB contain a vast pool of glycosyltransferases with a wide range of sugar and linkage specificities. EPS-producing lactococci in particular have received growing attention in recent years, especially for the analysis of genes encoding EPS biosynthesis. The first lactococcal eps locus identified was that of Lactococcus lactis subsp. cremoris NIZO B40; it comprises 14 plasmid-encoded genes (56). Since then, partial sequences of eps gene clusters have been identified in L. lactis subsp. cremoris NIZO B891 and NIZ...

Complete Genome Sequence of the Prototype Lactic Acid Bacterium Lactococcus lactis subsp. cremoris MG1363

“…Although 28 putative glycosyltransferases are encoded by the L. lactis MG1363 genome, we could not identify a gene cluster with the typical genetic organization of a lactococcal extracellular polysaccharide operon (21). The glycosyltransferases are more likely to be involved in the decoration of the lactococcal cell wall with cell wall polysaccharides (WPS).…”

Cell Surface of Lactococcus lactis Is Covered by a Protective Polysaccharide Pellicle

“…The plasmid-encoded genes responsible for EPS production in the dairy strains of L. lactis were characterized, and their organization was found to be similar to that of CPS biosynthesis gene clusters of Gram-positive pathogens (6,7). The presence of chromosomally encoded cell surface-associated polysaccharides was suggested for some lactococcal strains (8). Nevertheless, the ability of PS to form an outer layer was never demonstrated in this species.…”

Sugar source modulates exopolysaccharide biosynthesis in Bifidobacterium longum subsp. longum CRC 002

“…agitation, at a controlled pH of 6.0 by automatic addition of 4 M NH 4 OH and under anaerobic conditions by continuously bubbling CO 2 gas. The EPS was extracted following the method of Cerning et al (1994) and further purified as previously described (Dabour & LaPointe, 2005). Monosaccharide analysis was performed by GLC as described previously (Van Calsteren et al, 2002).…”