The joint of Tn916 circular intermediates is a homoduplex in Enterococcus faecalis

Tn916 is a 18-kb conjugative transposon originally identified in Enterococcus faecalis. The first step for Tn916 movement is its excision from the donor replicon with the formation of a nonreplicating covalently closed circular intermediate. Studies on formation of circular intermediates in Escherichia coli have shown that the joint between the Tn916 termini is a 6-bp heteroduplex formed by the two regions flanking the transposon before its excision (coupling sequences). In this work we studied the joint of Tn916 termini in circular intermediates formed in both E. coli and E. faecalis. Our strategy was to use direct sequencing of amplification products obtained from the joint region of single target molecules. In E. coli, 50% of circular intermediates contained a heteroduplex joint, while the remaining 50% displayed a homoduplex joint formed by one of the two coupling sequences. In E. faecalis, we could not demonstrate the presence of any heteroduplex joint. In this case 77.7% of the analyzed joints were formed by the left coupling sequence.