Submitted to: Applied and Environmental Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/29/1995
Publication Date: N/A
Interpretive Summary: Ruminant animals lose their capacity to digest cellulose when the diets are high in starch. Starch causes a decrease in ruminal pH, and the cellulolytic bacteria cannot tolerate even modest decline in pH. We have undertaken a project of convert an acid-resistant bacterium into one that can digest cellulose at low pH. This project entials the conversion of a endoglucanase into a true cellulase by that addition of cellulose binding domain. This manuscript describes the cellular location and occurence of this enzyme in other strains of ruminal bacteria. If overall project is successful, we will be able to re-inoculate the rumen with a pH resistant cellulolytic bacterium and increase the rate of cellulose digestion in animals that have low ruminal pH.
Technical Abstract: Prevotella ruminicola B14, TC-1, TF1-3 and TS1-5 all produced immunologically crossreacting 88 kDa and 82 kDa CMCases. P. rumincola 23, 118B, 20-63 and 20-78 had much less CMCase activity and no cross-reaction with the B14 CMCase antiserum. Fibrobacter succinogenes S85 and Selenomonas ruminantium HD4 and D produced smaller CMCases and none of these enzymes cross-reacted with the B14 CMCase antiserum. Addition of the B14 CMCase antiserum to whole cells of P. ruminicola B14 inhibited CMCase activity and agglutinated the cells, indicating that the active site of the CMCase was located on the outside surface of the cells. Purified 88 kDa B14 CMcase was able to bind to P. ruminicola cells when added to the supernatant, but a fully active, truncated form of the CMCase, which lacked the N-terminal domain of the 88 kDa B14 CMCase, was not able to bind to P. ruminicola. P. ruminicola B14 cells growing exponentially on cellobiose did not release CMCase into the culture supernatant and contained approximately 85% of the CMCase that could experimentally bound to B14 cells lacking CMCase. CMCase could only be detected in the culture supernatant when P. ruminicola B14 cultures entered stationary phase and the cells started to lyse. Based on the observation that when cultures entered stationary phase the 88 kDa CMCase decreased and the 82 kDa CMCase increased proportionally, it appears that the 82 kDa protein may be a proteolytic degradation product of the 88 kDa CMCase.