Use of a modified Bacteroides-Prevotella shuttle vector to transfer a reconstructed β-1,4-D-endoglucanase gene into Bacteroides uniformis and Prevotella ruminicola B14

Abstract

A carboxymethyl cellulase (CMCase) gene from Prevotella ruminicola B14 was reconstructed by adding a cellulose binding domain from a Thermomonospora fusca cellulase and was conjugally transferred from Escherichia coli to Bacteroides uniformis 0061 by using a chloramphenicol and tetracycline resistance shuttle vector (pTC-COW), pTC-COW was specifically constructed to facilitate conjugal transfer of vectors from B. uniformis donors to P. ruminicola recipients. B. uniformis transconjugants containing CMCase constructs cloned into pTC-COW expressed Cm(r), 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 transconjugants to produce large amounts of the reconstructed CMCase, which was present on the outside surface of the cells. 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. Under these conditions, the reconstructed CMCase permitted faster growth than the wild-type CMCase. The frequency of transfer of pTC-XRCMC from B. uniformis to P. ruminicola B14 was increased 100-fold when strictly anaerobic conditions, nitrocelluose filters (cell immobilization), and more stringent selections were employed. Although the P. ruminicola B14 (pTC-XRCMC) transconjugants expressed Tc(r) and 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 the carbon source. On the basis of these results, it appears that not all of the promoters recognized by B. uniformis and 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.