Separation of outer and cytoplasmic membranes of Fibrobacter succinogenes and membrane and glycogen granule locations of glycanases and cellobiase

Abstract

The outer membrane (OM) of Fibrobacter succinogenes was isolated by a combination of salt, sucrose, and water washes from whole cells grown on either glucose or cellulose. The cytoplasmic membrane (CM) was isolated from OM-depleted cells after disruption with a French press. The OM and membrane vesicles isolated from the extracellular culture fluid of cellulose-grown cells had a higher density, much lower succinate dehydrogenase activity, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein profiles different from those of the CM. The OM from both glucose-and cellulose-grown cells and the extracellular membrane vesicles from cellulose-grown cultures exhibited higher endoglucanase, xylanase, and acetylesterase activities than the CM and other cell fractions. Endoglucanase 2 was absent from the isolated OM fractions of glucose-and cellulose-grown cells and from the extracellular membrane vesicles of cellulose-grown cells but was present in the CM and intracellular glycogen granule fractions, while endoglucanase 3 was enriched in the OM. Cellobiosidase was located primarily in the periplasm as previously reported, while cellobiase was mainly present in the glycogen granule fraction of glucose-grown cells and in a nongranular glycogen and CM complex in cellulose-grown cells. The cellobiase was not eluted from glycogen granules by cellobiose, maltose, and maltotriose nor from either the granules or the cell membranes by nondenaturing detergents but was eluted from both glycogen granules and cell membranes by high concentrations of salts. The eluted cellobiase rebound almost quantitatively when diluted and mixed with purified glycogen granules but exhibited a low affinity for Avicel cellulose. Thus, we have documented a method for isolation of OM from F. succinogenes, identified the OM origin of the extracellular membrane vesicles, and located glycanases and cellobiase in membrane and glycogen fractions.Fibrobacter succinogenes S85 is one of the major cellulolytic bacteria in the rumen of animals receiving low-quality forage rations (44). It extensively degrades cellulose after attachment to the insoluble substrate (39), which indicates that the cellulase enzymes are functioning at the cell surface during cellulose digestion. Nevertheless, the mechanism by which the bacterium degrades cellulose is still unknown (18,44). To elucidate the physiological mechanism of cellulose degradation by the bacterium, three endoglucanases, endoglucanase 1 (EGI), EG2 (46), and EG3 (49), a chloride-stimulated cellobiosidase (31), and a cellodextrinase (28, 29) were purified and characterized. EG I was released from cells during growth, and

Fiber-Degrading Systems of Different Strains of the Genus Fibrobacter

“…This is in agreement with a previous study that showed major endoglucanase and xylanase activities of strain S85 associated with membrane vesicles in the ECCF of cellulose-grown cultures (16).…”

Cel9D, an Atypical 1,4-β- d -Glucan Glucohydrolase from Fibrobacter succinogenes : Characteristics, Catalytic Residues, and Synergistic Interactions with Other Cellulases

“…Since Cel9D cannot hydrolyze cellobiose, it can be presumed that ␤-glucosidases (EC 3.2.1.21) would exert a synergistic effect with Cel9D. Indeed, cellobiase activity has been detected in F. succinogenes (7) and two GH3 ␤-glucosidases were identified in the genome of F. succinogenes (21).…”

Outer Membrane Proteins ofFibrobacter succinogeneswith Potential Roles in Adhesion to Cellulose and in Cellulose Digestion

“…Abbreviations: NS, nonsedimentable extracellular culture fluid; ExOM, extracellular outer membrane; OM, outer membrane; PP, periplasmic fraction. ExOM and PP were prepared as previously described (13). The arrowhead indicates a proteolytic degradation product.…”