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

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.

Functional Characterization of a Mucus-Specific LPXTG Surface Adhesin from Probiotic Lactobacillus rhamnosus GG

“…This not only indicated that the MBF antiserum was functional but also provided visual evidence for the surface localization of the MBF homolog in this related L. rhamnosus strain. Since these two genetically similar L. rhamnosus strains have been reported to differ in the composition of their EPS layers (21,23), we suspected that the longer galactose-rich EPS repeating units found in L. rhamnosus GG, but not in L. rhamnosus LC705, might be the reason for the observed difference in surface-localized MBF protein.…”

“…As these results would imply, MBF proteins are responsible for mediating a low level of mucosal adhesion in L. rhamnosus GG and LC705 cells. Consequently, whereas MBF likely represents one of the key mucus adhesins on the cell surface of the less adhesive L. rhamnosus LC705 strain, this surface-localized (23), which was recently reported to wield a shielding effect against host protective factors (24), WT L. rhamnosus GG cells are apparently able to mediate mucosal adhesion through the surface-localized MBF. This might reflect the need for a closely interwoven association between the L. rhamnosus GG cell surface and the mucosal layer so that MBF proteins can bind optimally to mucus-related molecules.…”

“…The L. rhamnosus GG knockout derivative strain, an inactivated exopolysaccharide (EPS) welE (CMPG5351; ⌬welE::Tc r ) mutant, was used for demonstrating MBF cell surface localization by electron microscopic means. Details about its construction have been described previously (23).…”

“…To test this possibility, we examined the uncovered surface of an inactivated EPS knockout mutant of L. rhamnosus GG (CMPG5351), which was used previously for establishing the EPS layer as a potential shield of certain surface adhesins (23). As shown in Fig.…”

Comparative genomic analysis of Lactobacillus rhamnosus GG reveals pili containing a human- mucus binding protein

“…Other cell surface components putatively involved in hostcell interactions (29,30) and potentially implicated in promoting beneficial health effects are the cell envelope-bound or -secreted EPSs. The EPS biosynthetic gene cluster (LCILS3) in the genome of strain LC705 (Fig.…”