Role of phosphorolytic cleavage in cellobiose and cellodextrin metabolism by the ruminal bacterium Prevotella ruminicola

Abstract

In bacteria, cellobiose and cellodextrins are usually degraded by either hydrolytic or phosphorolytic cleavage. Prevotella ruminicola B 1 4 is a noncellulolytic ruminal bacterium which has the ability to utilize the products of cellulose degradation. In this organism, cellobiose hydrolytic cleavage activity was threefold greater than phosphorolytic cleavage activity (113 versus 34 nmol/min/mg of protein), as measured by an enzymatic assay. Cellobiose phosphorylase activity (measured as the release of P i) was found in cellobiose-, mannose-, xylose-, lactose-, and cellodextrin-grown cells (>92 nmol of P i /min/mg of protein), but the activity was reduced by more than 74% for cells grown on fructose, L-arabinose, sucrose, maltose, or glucose. A small amount of cellodextrin phosphorylase activity (19 nmol/min/mg of protein) was also detected, and both phosphorylase activities were located in the cytoplasm. Degradation involving phosphorolytic cleavage conserves more metabolic energy than simple hydrolysis, and such degradation is consistent with substrate-limiting conditions such as those often found in the rumen. MATERIALS AND METHODS Organisms and cell growth. P. ruminicola B 1 4 was obtained from J. B. Russell, Cornell University, Ithaca, N.Y. The organism was cultured in an anaerobic medium containing the following (per liter): 292 mg of K 2 HPO 4 , 240 mg of KH 2 PO 4 , 480 mg of Na 2 SO 4 , 480 mg of NaCl, 100 mg of MgSO 4 ⅐ 7H 2 O, 64 mg of CaCl 2 ⅐ H 2 O, 600 mg of cysteine, 4 g of Na 2 CO 3 , 1 g of Trypticase (Becton Dickinson Microbiological Systems, Cockeysville, Md.), 500 mg of yeast extract, 100 mg of resazurin, 22.1 mM acetate, 6.0 mM propionate, 2.4 mM butyrate, 0.68 mM valerate, 0.68 mM isovalerate, 0.81 mM isobutyrate, and 0.66 mM 2-methylbutyrate. The medium was adjusted to pH 6.7 with NaOH and prepared and maintained under a carbon dioxide atmosphere. Carbohydrates were added as separate solutions, and cultures were grown at 39ЊC. Cell fractionation. Cells were harvested during logarithmic growth by centrifugation (15,000 ϫ g for 10 min at 4ЊC) and washed twice with either 50 mM PIPES [piperazine-N,NЈ-bis(2-ethanesulfonic acid) (pH 6.8)] for cellobiose phosphorylase and ␤-glucosidase assays or 50 mM Tris-maleate-NaOH (pH 6.8) for cellodextrin phosphorylase assays. Washed cells were passed through a French pressure cell (1,120 kg/cm 2) two times, and debris and whole cells were removed by centrifugation (15,000 ϫ g for 10 min at 4ЊC). Portions of the cell extracts were kept at 4ЊC for assay the same day. The remaining extract was centrifuged (140,000 ϫ g for 30 min at 4ЊC), yielding cytosol (supernatant) and membrane (pellet) fractions. The membrane pellets were washed with the appropriate buffer. Phosphoglucomutase activity was used as a cytosoplasmic marker and assayed as previously described (12). ATPase activity served as a membrane marker and was assayed as described by Kinoshita et al. (11). Cellobiose degradation by crude extracts. The total cellobiose cleavage activity was determined in an...

Alternative pathways of glucose transport in Prevotella bryantii B14

“…the phosphotransferase system (PTS) [1]. Prevotella bryantii B 1 4 is a strictly anaerobic ruminal bacterium that lacks PTS activity [2], but glucose consumption slowed the rate of cellobiose utilization [3] and repressed L-glucosidase [4] and L-glucanase synthesis [5]. Mannose, a sugar that provides a lower growth rate than glucose, did not repress any of these activities, but the nature of this regulation was not de¢ned [5].…”

Purification and Properties of a Cellobiose Phosphorylase (CepA) and a Cellodextrin Phosphorylase (CepB) from the Cellulolytic Thermophile Clostridium Stercorarium

“…A cellodextrin phosphorylase has been isolated only from C. therniocellum (Sheth and Alexander, 1969;Arai et al, 1994). Furthermore, cellobiose and cellodextrin phosphorylase activities have been detected in Fomrs unnosus (Hiittermann and Volger, 1973), several species of Cellulomonus (Schimz et al, 1983), and the ruminal bacteria Ruminococcus fluvefuciens (Ayers, 1958 ;Helaszek and White, 1991), Ruminococcus ulbus (Thurston et al, 1993), Fibrobacter sucrinogenes (Wells et al, 1995) and Prevotella ruminicolu (Lou et al, 1996). To date, however, no primary structures of cellobiose or cellodextrin phosphorylases are known.…”

Glycogen Formation by the Ruminal Bacterium Prevotella ruminicola

“…B. Russell, Cornell University, Ithaca, N.Y. The bacterium was cultured anaerobically at 39ЊC in a semidefined medium (14). Acetate, propionate, and butyrate were omitted from the medium used in continuous culture.…”

“…Enzyme assays. Crude extracts were prepared as described previously (14). The activities of maltose and sucrose phosphorylase were determined spectrophotometrically by monitoring NADPH formation at 340 nm.…”

“…The reaction mixture contained 50 mM PIPES [piperazine-N,NЈ-bis(2-ethanesulfonic acid); pH 6.8], 33 mM sodium-potassium-phosphate, 3 mM MgCl 2 , 1 mM NADP, 8 U of phosphoglucomutase, 5 U of glucose-6-phosphate dehydrogenase, a 20 mM concentration of the respective disaccharide, and aliquots of crude extracts containing approximately 120 g of protein. Maltase and sucrase activities were measured by an NADPH-linked assay for determining ␤-glucosidase (14) except that maltose or sucrose was substituted for cellobiose. Phosphoglucomutase activities were determined as described by Kotze (12).…”