Characterisation of cellulose-binding proteins that are involved in the adhesion mechanism of Fibrobacter intestinalis DR7.
Cellulose-binding proteins (CBP) isolated from cell envelopes of the cellulolytic bacterium Fibrobacter intestinalis strain DR7 were studied in order to investigate the adhesion mechanism. The proteins were examined for their reaction with antibodies that specifically block bacterial adhesion, response to glycosylation staining and monosaccharide composition. To this end, the effect of some monosaccharides (CBP components) on blocking of DR7 adhesion to cellulose was determined. Previous study had shown the occurrence of 16 CBP in the outer membrane and periplasm of DR7, of which 6 had endoglucanase activity (Miron and Forsberg 1998). Data from the present study show that most of the 16 CBP of DR7, except for the 38-, 90- and 180-kDa proteins, are glycosylated. Rabbit antibodies that specifically block DR7 adhesion were prepared by affinity preabsorption of antiserum against wild-type DR7 with bacterial cells of its adherence-defective mutant (DR7-M). The preabsorbed antibodies reacted positively in Western blotting with glycosylated CBP of 225, 200, 150, 70, 45 and < 38 kDa from the DR7 outer membrane, and reacted weakly with CBP of DR7-M. Modification of glycosidic residues attached to the CBP of DR7 by periodate oxidation prevented any reaction with the preabsorbed antibodies. Monosaccharide analysis by HPLC of isolated CBP from the outer membrane and periplasm of DR7 cells, showed that galactosamine, glucosamine, galacturonic acid, and glucuronic acid were the predominant monosaccharide components of CBP that can block the adhesion of DR7 cells to cellulose. It is suggested that some glycosylated residues of CBP may have a predominant role in the adhesion of DR7 to cellulose.
Miron
J
J
Metabolic Unit, Volcani Center, Bet Dagan, Israel. jmiron@actcom.co.il
Forsberg
C W
CW
D000818:Animals; D001422:Bacterial Adhesion / Q000502:physiology*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D015153:Blotting, Western; D002482:Cellulose / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D006089:Gram-Negative Anaerobic Bacteria / Q000378:metabolism / Q000502:physiology*; D009005:Monosaccharides / Q000032:analysis; D011817:Rabbits
D001426:Bacterial Proteins; D009005:Monosaccharides; D002482:Cellulose