Integration and excision of a Bacteroides conjugative transposon, CTnDOT

Bacteroides conjugative transposons (CTns) are thought to transfer by first excising themselves from the chromosome to form a nonreplicating circle, w hich is then transferred by conjugation to a recipient. Earlier studies sho wed that transfer of most Bacteroides CTns is stimulated by tetracycline, b ut it was not known which step in transfer is regulated. We have cloned and sequenced both ends of the Bacteroides CTn, CTnDOT, and have used this inf ormation to examine excision and integration events. A segment of DNA that contains the joined ends of CTnDOT and an adjacent open reading frame (ORF) , intDOT, was necessary and sufficient for integration into the Bacteroides chromosome. Integration of this miniature form of the CTn was not regulate d by tetracycline. Excision of CTnDOT and formation of the circular interme diate were detected by PCR, using primers designed from the end sequences. Sequence analysis of the PCR products revealed that excision and integratio n involve a 5-bp coupling sequence-type mechanism possibly similar to that used by CTn Tn916, a CTn found originally in enterococci. PCR analysis also demonstrated that excision is a tetracycline-regulated step in transfer. T he integrated minielement containing intDOT and the ends of CTnDOT did not excise, nor did a larger minielement that also contained an ORF located imm ediately dowmstream of intDOT designated orf2. Thus, excision involves othe r genes besides intDOT and orf2. Both intDOT and orf2 were disrupted by sin gle-crossover insertions. Analysis of the disruption mutants showed that in tDOT was essential for excision but orf2 was not. Despite its proximity to the integrase gene, orf2 appears not to be essential for excision.