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Journal of Bacteriology, September 2002, p. 4829-4837, Vol. 184, No. 17
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.17.4829-4837.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Development of an In Vitro Integration Assay for the Bacteroides Conjugative Transposon CTnDOT

Qi Cheng, Neil Wesslund, Nadja B. Shoemaker, Abigail A. Salyers, and Jeffrey F. Gardner*

Department of Microbiology, University of Illinois, Urbana, Illinois 61801

Received 13 March 2002/ Accepted 3 June 2002

Integrated self-transmissible elements called conjugative transposons (CTns) are responsible for the transfer of antibiotic resistance genes in many different species of bacteria. One of the best characterized of these newly recognized elements is the Bacteroides CTn, CTnDOT. CTnDOT is thought to have a circular transfer intermediate that transfers to and integrates into the genome of the recipient cell. Previous investigations of the mechanism of CTnDOT integration have been hindered by the lack of an in vitro system for checking this model of integration and determining whether the CTnDOT integrase alone was sufficient to catalyze the integration reaction or whether host factors might be involved. We report here the development of an in vitro system in which a plasmid containing the joined ends of CTnDOT integrates into a plasmid carrying a CTnDOT target site. To develop this in vitro system, a His-tagged version of the integrase gene of CTnDOT was cloned and shown to be active in vivo. The protein produced by this construct was partially purified and then added to a reaction mixture that contained the joined ends of the circular form of CTnDOT and a plasmid carrying one of the CTnDOT target sites. Integration was demonstrated by using a fairly simple mixture of components, but integration was stimulated by a Bacteroides extract or by purified Escherichia coli integration host factor. The results of this study demonstrate both that the circular form of CTnDOT is the form that integrates into the target site and that host factors are involved in the integration process.

* Corresponding author. Mailing address: Department of Microbiology, 601 S. Goodwin Ave., University of Illinois, Urbana, IL 61801. Phone: (217) 333-7287. Fax: (217) 244-6697. E-mail: jeffgard{at}uiuc.edu.

Journal of Bacteriology, September 2002, p. 4829-4837, Vol. 184, No. 17
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.17.4829-4837.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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