Development and use of cloning systems for Bacteroides fragilis: Cloning of a plasmid-encoded clindamycin resistance determinant

Abstract

Chimeric plasmids able to replicate in Bacteroides fragilis or in B. fragilis and Escherichia coli were constructed and used as molecular cloning vectors. The 2.7-kilobase pair (kb) cryptic Bacteroides plasmid pBI143 and the E. coli cloning vector pUC19 were the two replicons used for these constructions. Selection of the plasmid vectors in B. fragilis was made possible by ligation to a restriction fragment bearing the clindamycin resistance (Cc(r)) determinant from a Bacteroides R plasmid, pBF4; Cc(r) was not expressed in E. coli. The chimeric plasmids ranged from 5.3 to 7.3 kb in size and contained at least 10 unique restriction enzyme recognition sites suitable for cloning. Transformation of B. fragilis with the chimeric plasmids was dependent upon the source of the DNA; generally 105 transformants μg-1 of DNA were recovered when plasmid purified from B. fragilis was used. When the source of DNA was E. coli, there was a 1,000-fold decrease in the number of transformants obtained. Two of the shuttle plasmids not containing the pBF4 Cc(r) determinant were used in an analysis of the transposon-like structure encoding Cc(r) in the R plasmid pBI136. This gene encoding Cc(r) was located on a 0.85-kb EcoRI-HaeII fragment and cloned nonselectively in E. coli. Recombinants containing the gene inserted in both orientations at the unique ClaI site within the pB1143 portion of the shuttle plasmids could transform B. fragilis to clindamycin resistance. These results together with previous structural data show that the gene encoding Cc(r) lies directly adjacent to one of the repeated sequences of the pBI136 transposon-like structure.