Construction of new shuttle plasmid vectors for Escherichia coli‐Bacteroides transgeneric cloning

An Escherichia coli‐Bacteroides shuttle vehicle (pKBF367‐1) was constructed by combining the pBR322 derivative pKC7 (5.9 kb) with [1] a 4.6 kb cryptic plasmid from Bacteroides fragilis; and [2] the 4.2 kb EcoRI‐B fragment of the B. fragilis plasmid pBFTM10. This latter component allowed selection of clindamycin‐resistant transconjugants upon helper plasmid‐mediated transfer to a recipient strain of Bacteroides distasonis. To improve the potential of pKBF367‐1 (14.7 kb) as cloning vector, successive deletions generated derivatives of 12.8, 10.5 and 9.3 kb, which were still able to replicate in B. distasonis 419. These bifunctional vectors were successfully employed to introduce transposon Tn501 (Hgr) into B. distasonis 419, but expression of mercury resistance was not observed. This plasmid vehicles series may be useful for cloning Bacteroides genes in E. coli and studying their expression in a heterologous Bacteroides strain.