To extend our understanding of the mechanisms of plant cell wall degradation in the rumen, cellulosebinding proteins (CBPs) from the contents of a sheep rumen were directly isolated and identified using a metaproteomics approach. The rumen CBPs were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and some CBPs revealed endoglucanase activities toward carboxymethyl cellulose. Using mass spectrometry analyses, four CBPs were identified and annotated as known proteins from the predominant rumen cellulolytic bacterium Fibrobacter succinogenes: tetratricopeptide repeat domain protein, OmpA family protein, fibro-slime domain protein, and cellulose-binding endoglucanase F (EGF). Another CBP was identified as the cellulosomal glycosyl hydrolase family 6 exoglucanase, Cel6A, of Piromyces equi. F. succinogenes cells expressing EGF were found to be major members of the bacterial community on the surface or at the inner surface of hay stems by immunohistochemical analyses using anti-EGF antibody. The finding that four of the five CBPs isolated and identified from sheep rumen contents were from F. succinogenes indicates that F. succinogenes is significantly involved in cellulose degradation in the rumen.Efficient plant cell wall degradation depends on cooperation between microorganisms that produce fibrolytic enzymes and the ruminant, which provides an anaerobic fermentation chamber. These microorganisms include symbiotic cellulolytic bacteria, fungi, and protozoa, which are able to digest cellulosic plant materials and produce energy for the host animal. Many fibrolytic enzymes have been isolated from rumen microorganisms, and the genes encoding these enzymes have been cloned and sequenced (3,10,20,21,30,34,38,41). However, the precise mechanisms of lignocellulose degradation in the rumen microbial ecosystem are not yet fully understood. In order to extend our understanding of the mechanisms of lignocellulose degradation in the rumen, it is necessary to study fiber digestion in the rumen biochemically, genetically, and proteomically.Fibrobacter succinogenes, Ruminococcus flavefaciens, and Ruminococcus albus are considered to be the predominant cellulolytic bacteria present in the rumen (24-26, 48, 50). Transmission electron microscopy observations of plant fibers digested by rumen microbes have shown that F. succinogenesor R. flavefaciens-like bacteria are distributed over materials such as fescue and orchard grass and that sometimes these bacteria account for more than 70% of fiber-associated bacteria (1, 6). It is generally accepted that F. succinogenes contributes greatly to fiber digestion, given that this species has a potent ability to solubilize crystalline cellulose (14, 47). In contrast, when the proportions of certain species in the rumen microbial population were examined using species-specific hybridization probes, F. succinogenes and R. flavefaciens accounted for 0.1 to 6.6% and 1.3 to 2.9% of total rumen bacteria, respectively (5,26,29,47). Furthermore, in an analysis of fiber-associate...
