The excision proteins of CTnDOT positively regulate the transfer operon

The Bacteroides conjugative transposon, CTnDOT, is an integrated conjugative element (ICE), found in many human colonic Bacteroides spp. strains. It has a complex regulatory system for both excision from the chromosome and transfer and mobilization into a new host. It was previously shown that a cloned DNA segment encoding the xis2c, xis2d, orf3, and exc genes was required for tetracycline dependent activation of the Ptra promoter. The Xis2c and Xis2d proteins are required for excision while the Exc protein stimulates excision. We report here that neither the Orf3 nor the Exc proteins are involved in activation of the Ptra promoter. Deletion analysis and electromobility shift assays showed that the Xis2c and Xis2d proteins bind to the Ptra promoter to activate the tra operon. Thus, the recombination directionality factors of CTnDOT excision also function as activator proteins of the Ptra promoter.

► We identified the proteins responsible for activation of the transfer operon- Xis2c and Xis2d.
► Xis2c and Xis2d are involved in excision of CTnDOT.
► Xis2c and Xis2d also bind upstream of the Ptra promoter and activate expression of the transfer operon.
► The other two proteins, Exc and Orf3, in the excision operon do not activate the Ptra promoter.