Submitted to: Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/18/2000
Publication Date: N/A
Citation: N/A Interpretive Summary: In ruminant animals, feedstuffs are digested in the rumen prior to gastric and intestinal digestion, and the animal is dependent on fermentation products for its nutrition. Rumen bacteria that digest cellulose cannot grow at the low pH values that modern cattle diets create, but Prevotella bryantii is pH-resistant. P. ruminicola cannot digest native insoluble cellulose, but it has a b-glucanase can digest synthetic soluble cellulose When a cellulose binding domain was added to the b-glucanase, it gained the ability to digest native cellulose, but the b-glucanase was not produced if glucose was present. Our current work indicates that b-glucanase production is being regulated by the activity of glucosemannokinase, an enzyme involved in sugar metabolism. Research on P. ruminicola has the potential to improve cellulose digestion and decrease the cost of cattle production.
Technical Abstract: Prevotella bryantii B14 has a common carrier for glucose and mannose, but b-glucanase expression is only catabolite repressed by glucose. P. bryantii B14 cell extracts had ATP-dependent gluco- and mannokinase activities, and significant PEP- or GTP-dependent hexose phosphorylation was not observed. Mannose inhibited glucose phosphorylation (and vice versa), and activity gels indicated that a single protein was responsible for both activities. Glucose was phosphorylated at faster rate than mannose (Vmax 280 versus 60 nmol hexose mg protein-1 min-1, respectively), and glucose was a better substrate for the kinase (Km 0.12 versus 1.2 mM, respectively). The purified glucomannokinase (1250-fold) had a molecular weight of 68 kDa, but SDS-PAGE gels indicated that it was a dimer (34.5 kDa). The N-terminus (25 residues) had an 8 amino acid segment that was homologous to other bacterial glucokinases, and Porphyromonas gingivalis had an unidentified ORF that was closely related to this portion of the glucomannokinase (75% similarity over 25 residues). The glucomannokinase was competitively inhibited by the nonmetabolizable glucose analog, 2-deoxyglucose (2DG), and cells grown with glucose and 2DG had lower rates of glucose consumption than cells given only glucose. When the ratio of 2DG to glucose was increased, the glucose consumption rate decreased, and the b-glucanase activity increased. The 2DG-dependent glucose consumption rate was highly correlated with the 2DG-dependent glucomannokinase activity (r2=0.98), and this result suggested that glucomannokinase activity was either directly or indirectly regulating b-glucanase expression.