USE OF A MODIFIED BACTEROIDES-PREVOTELLA SHUTTLE VECTOR TO TRANSFER ARECONSTRUCTED BETA-1,4-D-ENDOGLUCANASE GENE INTO BACTEROIDES-UNIFORMIS AND PREVOTELLA-RUMINICOLA B(1)4

A carboxymethyl cellulase (CMCase) gene from Prevotella ruminicola B(1 )4 was reconstructed by adding a cellulose binding domain from a Therm omonospora fusca cellulase and was conjugally transferred from Escheri chia coli to Bacteroides uniformis 0061 by using a chloramphenicol and tetracycline resistance shuttle vector (pTC-COW), pTC-COW was specifi cally constructed to facilitate conjugal transfer of vectors from B. u niformis donors to P. ruminicola recipients, B, uniformis transconjuga nts containing CMCase constructs cloned into pTC-COW expressed Cm-r, b ut they did not produce the reconstructed CMCase until a xylanase prom oter from P. ruminicola 23 was added upstream of the CMCase (pTC-XRCMC ), The xylanase promoter allowed the B. uniformis transconjugants to p roduce large amounts of the reconstructed CMCase, which was present on the outside surface of the cells, Although the reconstructed CMCase a lone did not allow B. uniformis to grow on acid-swollen cellulose, rap id growth was observed when two exocellulases were added to the cultur e supernatant. tinder these conditions, the reconstructed CMCase permi tted faster growth than the wild-type CMCase. The frequency of transfe r of pTC-XRCMC from B. uniformis to P, ruminicola B(1)4 was increased 100-fold when strictly anaerobic conditions, nitrocellulose filters (c ell immobilization), and more stringent selections were employed. Alth ough the P. ruminicola B(1)4 (pTC-XRCMC) transconjugants expressed Tc- r and had DNA that hybridized with a probe to the shuttle vector, thes e transconjugants did not produce detectable levels of the reconstruct ed CMCase even when xylan was the carbon source. On the basis of these results, it appears that hot all of the promoters recognized by B, un iformis and P. ruminicola 23 are functional in P. ruminicola B(1)4, Ho wever, the results with B. uniformis suggest that the introduction of a P. ruminicola B(1)4 promoter should allow expression of the reconstr ucted CMCase in P. ruminicola B(1)4.