Submitted to: Federation of European Microbiological Societies Microbiology Letters
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/24/1997
Publication Date: N/A
Interpretive Summary: Ruminant animals (cattle, sheep, goats, etc.) lose their capacity to digest cellulose when the diets are high in starch. Starch causes a decrease in rumen pH, and the cellululose-digesting bacteria cannot tolerate even modest decline in pH. We have undertaken a project to produce an acid-resistant rumen bacterium that can digest cellulose at low pH. This project entails the gene reconstruction. The gene that we have modified i adjacent to another gene. This other gene encodes an enzyme that degrades another plant polysaccharide, mannan. We produced a mutant that does not produce either enzyme, and it appears that the two enzymes have the same regulatory element. We are now trying to isolate this regulatory element. If this project is successful, we will be able to re-inoculate the rumen with a pH resistant cellulose-degrading bacterium and increase the rate of cellulose digestion in animals that have low ruminal pH.
Technical Abstract: A mutant of Prevotella ruminicola B14 was isolated after chemical mutagenesis with ethylmethanesulfonate. The mutant, MW1, did not produce polypeptides that cross-reacted with antibody to wild type B-1,4- endoglucanase and virtually no B-1-4-endoglucanase or mannanase activity was detected. MW1 grew more slowly on cellodextrins and no growth was observed with guar gum (mannan) as an energy source. MW1 cultures that repeatedly transferred with cellobiose and mannan eventually regained the ability to utilize mannan as an energy source. The revertant (MW1R) had 40 and 25% as much B-1,4-endoglucanase and mannanase activities as wild-type B14, respectively. Western blots indicated that MW1R produced a B-1,4- endoglucanase that could crossreact with wild-type B14 enzyme. Wild-type B14 had B-1,4 endoglucanase and mannanase activities when either cell or mannan was the energy source, but MW1R had these activities only when mannan was provided.