Conjugative transposons (CTns) are integrated self-transmissible elements. The first step in their transfer is excision from the bacterial chromosome to form a circular intermediate. A single-stranded copy of this intermediate is then transferred to a recipient cell where it integrates into the chromosome. CTns are proving to be major contributors to antibiotic resistance gene transfer. Yet little is know about their activities, particularly the process by which they excise from the chromosome. The focus of this proposal is on excision of a widely distributed CTn found in human colonic Bacteroides species, CTnDOT. CTnDOT has an unusually complex excision system that consists of an integrase (IntDOT).and 3 other proteins (Orf2c, Orf2d and Exc) that are essential for excision. Nothing is known about the mechanisms of Orf2c, Orf2d and Exc. We will purify these proteins and use a variety of biochemical techniques, ranging from gel shift analysis to footprinting, to determine how they bind DNA during the excision process and how they interact with each other and with IntDOT. Site-directed and random mutagenesis will be used to learn what amino acid residues are important for their functions. Orf2c, Orf2d and Exc also participate in the regulation of transfer genes. We will determine how these proteins contribute to control of transfer gene expression. Finally, we will characterize further three regulatory proteins, RteA, RteB and RteC, which are central regulators of CTnDOT functions including excision and transfer. Of special interest is the question of what RteA, the putative sensor protein, is sensing. The results of the proposed experiments will provide new information about the functions and regulatory machinery of a unique family of transmissible integrated elements. Information about excision might be used in the future to develop compounds that """"""""cure"""""""" certain types of bacteria of genes on CTns that confer resistance to antibiotics or encode virulence factors. Also, information about how CTn functions are regulated is already beginning to be applied to the development of strategies that prevent their spread in nature and to guide evaluation of regulatory measures in the food and pharmaceutical industry.
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