The integration and excision mechanisms of the Bacteroides conjugative transposon CTnDOT

Bacteroides spp. are Gram-negative, anaerobic bacteria that are predominant species in the human colon. One of the major reasons for the spread of antibiotic resistance genes among different Bacteroides spp. is due to the transposition of Bacteroides conjugative transposons (CTns). In this thesis, I have taken both genetic and biochemical approaches to investigate the integration and excision mechanisms of a typical Bacteroides conjugative transposon CTnDOT.; To study integration, I constructed a mini integration element after identification of a gene (intDOT) which has homology at amino acid level to members of Tyrosine Recombinase family. The results suggest that the integration step requires the intDOT, the attachment site (attDOT) and possibly a host factor. Integration of this miniature form of CTnDOT was not regulated by tetracycline. Excision of CTnDOT and formation of the circular intermediate were detected by PCR. Sequence analysis of the PCR products revealed that excision and integration of CTnDOT involve a coupling sequence-type mechanism.; Using the integrated mini-element as an excision indicator strain, I constructed a mini-excision system and confirmed that a gene called orf4, together with the regulatory genes, rteA-rteB-rteC , were necessary for excision of the integrated mini-element containing intDOT and the ends of CTnDOT. It was shown that orf3, the open reading frame upstream of orf4, is not essential for excision. My results suggest that the excision mechanism of CTnDOT appears to be different from that of any other known integrated transmissible element.; A critical requirement for studying transposition is the availability of a defined in vitro transposition system. Using the partial-purified CTnDOT Int protein, an in vitro integration system was developed and a host factor(s) from Bacteroides cell extract was shown to be important for integration. The CTnDOT Int protein catalyzes the in vitro integration reactions by a coupling sequence mechanism similar to the in vivo reaction. Purified E. coli integration host factor (IHF) can substitute for the Bacteroides host factor(s) and significantly stimulates in vitro integration.