Among enterococci and streptococci, two clinically important groups of bacteria, conjugative transposons are ubiquitous and are probably as much responsible for the dissemination of multiple antibiotic resistance as transferable plasmids. The purpose of this study is to investigate the molecular basis of conjugative transposition of the Enterococcus faecalis tetracycline-resistance transposon Tn916. Our recent determination of the 18 kb nucleotide sequence of this element should greatly facilitate our efforts to reveal details of a transposition process believed to be significantly different from other conjugative systems. Our studies to date have allowed the formulation of a hypothesis that describes how conjugative transposition may be regulated, and parts of the proposed work are designed to test that model. More specifically we will: 1. Utilize our recently developed transactivation system to perform a genetic complementation analysis of numerous previously-generated Tn916:: Tn5 derivatives whose Tn5 insertions can now be precisely located; 2. Test our hypothesis that a determinant, traA, in the left arm of the transposon encodes a key positive regulator of the overall process of conjugative transposition; 3. Develop a system whereby transposition can be controlled through direct induction of traA or xis-Tn/int-Tn; 4. Analyze transcriptional and translation products appearing at various times following induced circularization; 5. Attempt to identify determinants for two hypothetical products, TraO and TraP, proposed to act in regulating the extent to which intermediate circular DNA can integrate into targets within the donor; 6. Determine possible functional roles for two putative products, ORF3 and ORF4, which are encoded within and in- frame with int-Tn; 7. Determine the minimum substrate size for excision and integration; 8. Characterize the transfer origin oriT; 9. Attempt to identify and characterize a Tn916-intermediate relaxosome.
