Analysis of a Bacteroides conjugative transposon using a novel "targeted capture" model system

Large conjugative transposons (CTn's) are widespread among Bacteroides spp. and they are responsible for the high rates of Bacteroides tetracycline resistance, which is mediated by the tetQ gene. These elements are self-transmissible and conjugation can be induced up to 1000-fold by the addition of tetracycline to cultures prior to mating. In addition to self-transfer, the Bacteroides CTn's, such as CTn341, are able to mobilize unlinked genetic elements such as plasmids and mobilizable transposons in a tetracycline-inducible manner. To study the molecular properties of these unique elements, a vector was designed to capture CTn's for analysis in heterologous hosts. This plasmid, pFD670, consisted of the low-copy vector pWSK29, the RK2 oriT, an ermF gene, and a tetQ gene fragment containing the N-terminus and promoter. The vector was transferred into Bacteroides recipients containing CTn341 where it integrated into the tetQ gene by homologous recombination. This integrated construct then was transferred back into an Escherichia coli host where it replicated as a plasmid, pFD699, about 56 kb in size. Further analysis showed that pFD699 could be transferred into Bacteroides hosts where it displayed the same tetracycline-inducible properties as the native CTn341. The captured element appeared to utilize a circular intermediate in both transfer and transposition, and integration into the chromosome seemed to be random. Hybridization studies with a range of Bacteroides CTn's encoding tetracycline resistance revealed a great deal of homology between most of the CTn's but there was much variation seen in the restriction patterns of these elements, suggesting great diversity among this group.