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United States Department of Agriculture

Agricultural Research Service

Title: Use of a New Bacteriodes/prevotella Shuttle Vector to Transfer a Reconstructed B, 1,4 D Endoglucanase Gene into Bacteroides Uniformis and Prevotella Ruminicola B14

Authors
item Gardner, Richard - CORNELL UNIVERSITY
item Russell, James
item Wilson, David - CORNELL UNIVERSITY
item Wang, Gui - UNIV. OF ILLINOIS, URBANA
item Shoemaker, Nadja - UNIV. OF ILLINOIS, URBANA

Submitted to: Applied and Environmental Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: November 4, 1995
Publication Date: N/A

Interpretive Summary: Ruminant farm animals (cattle, sheep, goats) lose their capacity to digest cellulose, a major component of many animal feeds, when the diets are high in starch. Starch causes a decrease in ruminal pH (an increase in acidity) and cellulolytic (cellulose-digesting) bacteria cannot tolerate even a modest decline in pH. We have undertaken a project to convert an acid- resistant bacterium into one that can digest cellulose at low pH. This project entails the conversion of an endoglucanase (a cellulolytic enzyme that digests soluble cellulose efficiently, but which digests the native cellulose found in plants either poorly or not at all) into a true cellulase. This is being done by the addition of a "cellulose-binding domain" to the endoglucanase, which would allow it bind to, and digest, native cellulose. This manuscript describes a procedure for transferring the reconstructed gene back into the cellulolytic ruminal bacterium. If this project is successful, we will be able to re-inoculate the rumen with an acid-resistant cellulolytic bacterium and increase the efficiency of cellulose digestion in animals that have low ruminal pH. The net result would be to increase the efficiency with which ruminant produce meat and milk.

Technical Abstract: A CMCase from Prevotella ruminicola B14 was reconstructed by adding a cellulose binding domain was transferred from Escherichia coli to Bacteroides uniformis 0061 strains using a resistance shuttle vector (pTC- COW). pTC-COW was constructed to facilitate conjugal transfer of vectors from B. uniformis to P. ruminicola recipients. B. uniformis containing CMCase constructs cloned into pTC-COW expressed Cmr, but they did not produce the reconstructed CMCase until a xylanase promoter from P. ruminicola 23 was added upstream of the CMCase (pTC-XRCMC). The xylanase promoter allowed the B. uniformis to produce large amounts of the reconstructed CMCase. Although the reconstructed CMCase alone did not allow B. uniformis to grow on acid swollen cellulose, rapid growth was observed when two exocellulases were added to the culture supernatant. The frequency of transfer of pTC-XRCMC from B. uniformis to P. ruminicola B14 was increased 100-fold when strictly anaerobic conditions, nitrocelluose filters, & more stringent selections were employed. Although the P. ruminicola B14 (pTC-XRCMC) transconjugants expressed Tc1 & had DNA that hybridized with a probe to the shuttle vector, these transconjugants did not produce detectable levels of the reconstructed CMCase even when xylan was in the carbon source. Based on these results, it appears that the promoters recognized by B. uniformis & P. ruminicola 23 are functional in P. ruminicola B14. However, the results with b. uniformis suggest that the introduction of a P. ruminicola B14 promoter should allow expression of the reconstructed CMCase in P. ruminicola B14.

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