The clinically significant clindamycin resistance (Cln-r) plasmids were used as model systems to study genetic exchange mechanisms and gene expression in Bacteriodes fragilis. Previous work with these plasmids suggested that the Cln-r determinants were located on transposon-like structures bounded by 1.2 kb directly repeated sequences. DNA sequence analysis of the resistance gene from one of these plasmids, pBI136, showed that it started just 17 bp from the terminus of one of the repeated sequences. There were no promoter-like sequences associated with the gene and cloning experiments indicated that sequences upstream, from within the direct repeat, were required for expression of Cln-r in Bacteroides. Cloning vectors containing the Cln-r gene without its promoter sequences were constructed and used to demonstrate the presence of promoter activity in both copies of the direct repeat sequence. Transposition of the pBI136 Cln-r determinant in Bacteroides was demonstrated by cloning the entire transposon-like structure onto a shuttle plasmid vector defective for replication in Bacteroides. Following transformation with this plasmid, clindamycin resistant clones of B. fragilis were screened by Southern hybridization for the location of the resistance determinant. Results indicated that the determinant had inserted into the bacterial chromosome at random sites. Further analysis showed that in 60% of the clones tested, a second copy of the direct repeat sequence has also integrated into the chromosome but in these cases the insertion occurred at a specific site. These results indicate that the Cln-r determinant is located within a classic composite transposon, and provides preliminary evidence that the directly repeated sequences can act as insertion elements in Bacteroides spp.
