Studies on the role of glycosylation in adherence of Fibrobacter succinogenes to cellulose

Four cellulose-binding proteins (CBPs) of 240, 220, 180, and 120 kDa molecular masses, constituting about 10% of total outer membrane (OM) proteins, were identified in Fibrobacter succinogenes strain S85, a highly cellulolytic rumen bacterium. Two of the CBPs, the 240 and 220 kDa CBPs lacked cellulase and hemicellulase activities, while the other two, a 120 kDa protein previously demonstrated to be endoglucanase CelF, and a 180 kDa protein that appears to be a xylanase are catalytic CBPs. Polyclonal antiserum specific for cellulose adhesins of F. succinogenes were produced by adsorption of antiserum raised against whole cells of the wild type bacterium with intact cells of an adhesion defective mutant Ad4. The adsorbed antiserum was used to probe cellulose-binding proteins (CBPs) from the outer membrane of F. succinogenes S85 and Ad4 by Western blotting. All four of CBPs interacted strongly with the adsorbed antiserum. Oxidation of glycosylated CBPs with periodate resulted in complete loss of antibody binding, thus demonstrating that the epitope(s) recognized are glycosylated. Monovalent Fab antibodies were prepared from the adsorbed antiserum by the treatment with papain. The Fab preparation was capable of inhibiting the binding of F. succinogenes S85 to cellulose by 95%. Fab preparations from the anti-180 kDa CBP and anti-120 kDa CBP blocked binding of S85 cells to cellulose by 70% and 35%, respectively. Monosaccharides covalently linked to the 180 kDa CBP of S85 and Ad4 was determined using a high pressure liquid chromatography (HPLC) method. These sugar residues include rhamnose, galactose, mannose, galactosamine and galacturonic acid.; Capsular polysaccharide (CP) and lipooligosaccharide-like material (designated LOS-like material for convenience) isolated from OM of S85 cells grown on cellulose accounted for 18% (w/w) of the polysaccharide in the OM of S85. Tricine SDS-PAGE analysis showed the presence of LOS-like material of about 3 kDa molecular mass. Constituent sugar analysis by high pressure anion exchange chromatography-pulsed amperometric detection (HPAEC-PAD) revealed the presence of rhamnose, galactose, glucose mannose, and, galacturonic acid in both polymers with monosaccharide with the same retention time as xylose exclusively found in the capsule that contained 79.5% of total OM monosaccharides. The predominant fatty acids present in the LOS-like material were pentadecanoic acid, anteiso pentadecanoic acid, and heptadecanoic acid. In contrast, only pentadecanoic was present in the capsule. F. succinogenes polysaccharides form amphipathic aggregates in dilute salt solutions as judged by critical aggregation concentration (CAC) measurement and elecctron microscopy. The adsorbed antiserum did not react with purified LOS-like material and CP of S85, thus precluding these structures as having a direct role in binding of cells to cellulose.; Since there is no information in the literature regarding the CP and LOS-like material of F. succinogenes we performed a structural analysis of these exo-polysaccharides. Our data showed that the LOS-like material lacks the typical lipopolysaccharide components of lipid A, 3-deoxy-D-manno-octulosonic acids, heptose and O-polysaccharide. Instead the LOS-like material and a CP contain N-(2-hydroxyethyl)-2-aminoethylphosphonic acid which possesses a stable carbon-phosphorus bond that may decrease the susceptibility of the bacterium to a variety of hydrolytic enzymes.