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Title:Excision of CTnDOT
Author(s):Keeton, Carolyn
Director of Research:Gardner, Jeffrey F.
Doctoral Committee Chair(s):Gardner, Jeffrey F.
Doctoral Committee Member(s):Shisler, Joanna L.; Slauch, James M.; Olsen, Gary J.
Department / Program:Microbiology
Discipline:Microbiology
Degree Granting Institution:University of Illinois at Urbana-Champaign
Degree:Ph.D.
Genre:Dissertation
Subject(s):CTnDOT
Xis2c
Xis2d
Exc
IntDOT
recombination directionality factors (RDFs)
integrative conjugative elements (ICEs)
conjugative transposons
Abstract:Bacteroides spp. compose about 25-30% of the culturable microflora of the human colon and have acquired genes for resistance to tetracycline (tetQ) and erythromycin (ermF) due to conjugative transposons such as CTnDOT. Excision from the chromosome is the first step during transfer of a conjugative transposon (CTn) to a recipient. We previously showed that excision of CTnDOT is more complex than the excision of lambdoid phages and CTns such as Tn916. Excision of CTnDOT in vivo utilizes four CTnDOT-encoded proteins: IntDOT, Xis2c, Xis2d, and Exc as well as a host factor. We previously developed an in vitro excision reaction where the recombination sites, attL and attR, were located on different plasmids. The reaction was inefficient and did not require Exc, suggesting that the reaction conditions did not mimic in vivo conditions. Here we report the development of an intramolecular excision reaction where the attL and attR sites are located on the same DNA molecule. We found that Exc stimulates the reaction 3 to 5-fold. The efficiency of the excision reaction was also dependent on the distance between the attL and attR sites and on the sequences of the overlap regions between the sites of strand exchange. Substrates with identical overlap sequences recombined more efficiently than substrates with heterologous overlap sequences. This was surprising because the integration reaction is not sensitive to heterology in the overlap regions. We identified the binding sites of the excision proteins on the attR site during excision. CTnDOT 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. Xis2c and Xis2d are required for excision, while the Exc protein stimulates excision in vitro. 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.
Issue Date:2013-05-24
URI:http://hdl.handle.net/2142/44427
Rights Information:Copyright 2013 Carolyn Keeton
Date Available in IDEALS:2013-05-24
2015-05-24
Date Deposited:2013-05


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