The purpose of this study is to investigate the molecular basis of transposition and fertility of the conjugative transposon Tn916 from Enterococcus faecalis. Tn916 encodes resistance to tetracycline and is the prototype of a large family of nonplasmid elements believed to be a major factor in the dissemination of antibiotic resistance in Gram positive bacteria. The proposed structural and genetic studies should reveal details of a transposition process believed to be significantly different from that of most other known systems; and the hypothesis that movement of Tn9]6 is an excision/insertion event involving a circular intermediate will be examined. More specifically we plan to: 1) Sequence junction regions of a number of Tn916 inserts and determine the extent to which the degree of homology between the two regions relates to the rate of transposition; 2) determine if new DNA at junction regions is brought in by the transposon during insertion; 3) attempt to isolate a circular transposition intermediate in Escherichia coli and determine the nucleotide sequence where the ends are joined; 4) determine the complete nucleotide sequence of Tn9l6; 5) develop an efficient Tn9l6 transposition system (assay) in E. coli that can be uscd for a variety of genctic studies in this host; 6) gonetically analyze the transposition of Tn9]6 in E. coli with emphasis on characterizing controlling aspects of the excision phenomenon; 7) analyze ,Tn9l6-cncoded proteins and RNA; 8) examine the effcct of various host determinants on the excision/transposition of Tn9l6 in E. coll; 9) determinc the degrce, if any, to whicb a rcsident Tn9]6 derivative confers immunity to another transposon (i.e. a distinguishable derivative of Tn9l6) coming into the enterococcal host.
