Identification of genes required for excision of CTnDOT, a Bacteroides conjugative transposon.

Abstract

Integrated self-transmissible elements called conjugative transposons have been found in many different bacteria, but little is known about how they excise from the chromosome to form the circular intermediate, which is then transferred by conjugation. We have now identified a gene, exc, which is required for the excision of the Bacteroides conjugative transposon, CTnDOT. The int gene of CTnDOT is a member of the lambda integrase family of recombinases, a family that also contains the integrase of the Gram-positive conjugative transposon Tn916. The exc gene was located 15 kbp from the int gene, which is located at one end of the 65 kbp element. The exc gene, together with the regulatory genes, rteA, rteB and rteC, were necessary to excise a miniature form of CTnDOT that contained only the ends of the element and the int gene. Another open reading frame (ORF) in the same operon and upstream of exc, orf3, was not essential for excision and had no significant amino acid sequence similarity to any proteins in the databases. The deduced amino acid sequence of the CTnDOT Exc protein has significant similarity to topoisomerases. A small ORF (orf2) that could encode a small, basic protein comparable with lambda and Tn916 excision proteins (Xis) was located immediately downstream of the CTnDOT int gene. Although Xis proteins are required for excision of lambda and Tn916, orf2 had no effect on excision of the element. Excision of the CTnDOT mini-element was not affected by the site in which it was integrated, another difference from Tn916. Our results demonstrate that the Bacteroides CTnDOT excision system is tightly regulated and appears to be different from that of any other known integrated transmissible element, including those of some Bacteroides mobilizable transposons that are mobilized by CTnDOT.