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. 2009 Jun;75(11):3554-63.
doi: 10.1128/AEM.02919-08. Epub 2009 Apr 3.

Identification of a Gene Cluster for the Biosynthesis of a Long, Galactose-Rich Exopolysaccharide in Lactobacillus rhamnosus GG and Functional Analysis of the Priming Glycosyltransferase

Sarah Lebeer  1 , Tine L A Verhoeven, Grégory Francius, Geert Schoofs, Ivo Lambrichts, Yves Dufrêne, Jos Vanderleyden, Sigrid C J De Keersmaecker
Affiliations

Identification of a Gene Cluster for the Biosynthesis of a Long, Galactose-Rich Exopolysaccharide in Lactobacillus rhamnosus GG and Functional Analysis of the Priming Glycosyltransferase

Sarah Lebeer et al. Appl Environ Microbiol. 2009 Jun.

Abstract

Cell surface polysaccharides have an established role as virulence factors in human bacterial pathogens. Less documented are the biosynthesis and biological functions of surface polysaccharides in beneficial bacteria. We identified a gene cluster that encodes the enzymes and regulatory and transporter proteins for the different steps in the biosynthesis of extracellular polysaccharides (EPS) of the well-documented probiotic strain Lactobacillus rhamnosus GG. Subsequent mutation of the welE gene, encoding the priming glycosyltransferase within this cluster, and comparative phenotypic analyses of wild-type versus mutant strains confirmed the specific function of this gene cluster in the biosynthesis of high-molecular-weight, galactose-rich heteropolymeric EPS molecules. The phenotypic analyses included monomer composition determination, estimation of the polymer length of the isolated EPS molecules, and single-molecule force spectroscopy of the surface polysaccharides. Further characterization of the welE mutant also showed that deprivation of these long, galactose-rich EPS molecules results in an increased adherence and biofilm formation capacity of L. rhamnosus GG, possibly because of less shielding of adhesins such as fimbria-like structures.

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Figures

FIG. 1.
FIG. 1.
(A) Organization of the EPS gene cluster of L. rhamnosus GG. The organization of the EPS gene cluster of L. rhamnosus GG is compared by BLASTx analysis with that of the EPS gene cluster of the phylogenetically related strain L. rhamnosus ATCC 9595 (43). Genes encoding similar functions in EPS biosynthesis have a similar gray scaling code. Genes indicated in dark gray encode proteins putatively involved in the regulation of EPS production and polymerization. wzx and wzy (light gray) encode the putative polysaccharide transporter and polymerase, respectively. Genes indicated in white encode the putative glycosyltransferases, with wzm in L. rhamnosus ATCC 9595 encoding a putative pyruvyltransferase. Long stripes indicate genes encoding the proteins involved in the biosynthesis of the dTDP-rhamnose precursor. The lightest gray indicates the glf gene, encoding the UDP-galactopyranose mutase for the biosynthesis of the sugar nucleotide precursor for galactofuranose present in the EPS molecules of L. rhamnosus GG. The triangles indicate insertion sequence elements (IS). The insertion sequence of the L. rhamnosus GG EPS gene cluster is indicated with orf1 present on ISLrh2 in Table 2. Finally, genes sharing high nucleotide identity are linked by light gray boxes. Cutoff values for the displayed degrees of similarity are >88% identity. (B) Schematic representation of the putative steps in EPS biosynthesis by L. rhamnosus GG that are encoded within the EPS gene cluster. First, a phosphogalactosyl residue is transferred from an activated nucleotide sugar to the undecaprenyl phosphate (UndP)-lipid carrier on the cytoplasmic face of the membrane. This step is catalyzed by the membrane-associated priming glycosyltransferase WelE. Subsequently, unique glycosyltransferases WelF to -J add the remaining sugars in a sugar and glycosidic linkage-dependent manner, of which the exact order remains to be determined (indicated by brackets). The substrates for these glycosyltransferases are sugar nucleotides, which are available from existing cellular pools (e.g., UDP-galactose, UDP-N-acetylglucosamine) or synthesized by EPS-specific enzymes encoded within the EPS gene cluster (e.g., UDP-galactofuranose by the glf gene). Once an EPS subunit is completed, it needs to be translocated across the cytoplasmic membrane by a Wzx flippase, followed by linkage of the repeating units into long polysaccharides by a specific Wzy polymerase. A phosphorylation complex including a Wze autophosphorylating tyrosine kinase and a Wzb phosphotyrosine protein phosphatase is thought to be involved in the regulation of EPS biosynthesis. This figure is partly based on the model for Streptococcus pneumoniae (5), with modifications based on reference and the gene cluster and phenotypic analyses presented here.
FIG. 2.
FIG. 2.
Mutation of the welE gene in L. rhamnosus GG reduces the level of CW-PS. (A) TEM analysis of L. rhamnosus GG wild-type and welE mutant CMPG5351 strains grown in AOAC medium. For wild-type L. rhamnosus GG, the cell-bound EPS layer is indicated. While this layer is absent in the welE mutant CMPG5351, some patches of presumably polysaccharide accumulations could still be detected (black arrow). Additionally, fimbria-like appendages appear to become exposed in welE mutant cells (white arrow). (B) CW-PS were extracted from cell pellets from wild-type L. rhamnosus GG (WT), welE mutant CMPG5351, and the complemented strain CMPG5354. Error bars indicate standard deviations.
FIG. 3.
FIG. 3.
Mutation of the welE gene in L. rhamnosus GG specifically attenuates the high-molecular-mass, galactose-rich EPS molecules. (A) Analysis of the sugar monomer compositions of the CW-PS extracts of wild-type L. rhamnosus GG (WT), welE mutant CMPG5351, and the complemented strain CMPG5354. The data are expressed in relative amounts, taking the total amount of detected monomeric sugars as 100%. (B) Polyacrylamide gel electrophoresis of the CW-PS extract of wild-type L. rhamnosus GG, welE mutant CMPG5351, and complemented strain CMPG5354. Equal amounts of material were loaded. The upper band corresponds to polysaccharides with a polymer size of >1.4 × 106 Da based on size exclusion chromatography (data not shown). (C) Detection of individual galactose-rich polysaccharides on wild-type L. rhamnosus GG and welE mutant CMPG5351 by SMFS with PA-1 lectin-functionalized tips. Histograms (n = 1,024) of adhesion forces (left) and rupture distances (right) are shown.
FIG. 4.
FIG. 4.
Biofilm formation and adhesion to mucus and Caco-2 cells by wild-type L. rhamnosus GG (WT), the EPS welE mutant CMPG5351, and complemented strain CMPG5354. (A) Biofilm formation (in AOAC medium) is expressed relative to the amount formed by wild-type L. rhamnosus GG. (B) The adhesion capacity to mucus is expressed relative to wild-type L. rhamnosus GG (set as 100%), of which ca. 22% of the added cells adhered. (C) The adhesion capacity to Caco-2 cells is expressed relative to wild-type L. rhamnosus GG, of which ca. 4% of the added cells adhered. Error bars indicate standard deviations.
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