| Fibrobacter succinogenes S85, a rod-shaped Gram-negative, strictly anaerobic bacterium, is one of the major cellulolytic bacteria in the rumen. Its fibrolytic activity against plant materials is similar to that of rumen fungi and exceeds that of most other cellulolytic microorganisms. Despite its high cellulolytic activity, the mechanism by which it digests cellulose has been elusive, but evidence obtained by other researchers has indicated that adhesion of the bacterium to cellulose is a prerequisite to degradation of the substrate by cell associated enzymes. Thus this research was performed for the purpose of; (i) investigation of the genetics underpinning the degradation of plant materials by F. succinogenes S85, (ii) identification of adhesin(s) on the cell surface of the bacterium, and (iii) characterization of enzymes involved in cellulose biodegradation.; The genome of F. succinogenes S85 was sequenced at The Institute for Genomic Research and a consortium including myself was involved in annotation of the genome. My focus included glycosyl hydrolase, carbohydrate esterase, and pectate lyase genes. The F. succinogenes S85 genome contains genes putatively coding for 33 cellulases, 24 xylanases, 14 carbohydrate esterases, and 7 pectate lyases. Notable features with respect to genes coding for putative fibrolytic enzymes are that (i) the S85 genome possesses a greater number of glycosyl hydrolase, carbohydrate esterase, and pectate lyase genes than any other cellulolytic bacteria, (ii) many hemicellulase genes are clustered together, (iii) many of the encoded glycosyl hydrolase proteins contain a basic terminal domain, and (iv) few cellulase but many hemicellulase enzymes contain carbohydrate-binding modules (CBM).; Through proteomic analyses using the Isotope Coded Affinity Tagged labeling technique and 1- and 2-dimensional gel electrophoresis in conjunction with mass spectrometry, previously unknown cellulases were shown to be induced in S85 cells grown on cellulose and these included Cel5B, Cel5H, Cel8B, Cel9H, Cel9I, and Cel45C, making it a total of at least thirteen cellulases synthesized by F. succinogenes S85 grown on cellulose; the cellulases Cel10A (chloride-stimulated cellobiosidase) and Cel5H contain one and two CBMs, respectively. Despite the absence of known CBMs, Cel9H and Cel9I had an affinity for crystalline cellulose, suggestive of a novel CBM. Thirteen proteins were induced by growth of F. succinogenes grown on cellulose and of these, seven proteins were annotated as either hypothetical proteins and lipoproteins.; Through the use of two F. succinogenes mutants that lacked the capability to bind to crystalline cellulose and exhibited differential binding to amorphous cellulose, novel candidate non-catalytic cellulose binding proteins (CBP-4, -7, -10, -11, and -12) from the outer membrane of cells were identified, suggesting that these five CBPs have roles in adhesion of S85 cells to amorphous cellulose. Unique features of the CBPs are exemplified by CBP-4 and CBP-12, the former contained a CBM belonging to family 2 and nine consecutive tetratricopeptide repeats and the latter contained a family 30 CBM. Furthermore, four CBPs bound to crystalline cellulose were detected only in outer membranes of F. succinogenes S85 grown on cellulose, suggesting that the CBPs can be involved in binding of F. succinogenes S85 to cellulose in a late phase of adhesion of cells to the substrate rather than in an initial phase. One of the candidate proteins binding to crystalline cellulose was a pilin that was missing from the mutants.; In conclusion, cellulose biodegradation by F. succinogenes appears to occur by a novel mechanism that is very different from both the cellulosomal complex and the non-cellulosomal cellulase systems of other cellulolytic bacteria and fungi. This research has provided the basis for determination of the mechanism of cellulose digestion. |
