Molecular and genetic characterization of the conjugative transposon Tn916.

Tn916 is a 16.4 kb conjugative transposon encoding resistance to tetracycline (tet (M)), originally identified on the chromosome of Enterococcus faecalis DS16. Tn916 moves by an excision/insertion mechanism and exhibits a broad host-range among gram-positive bacteria. Transfer frequencies (solid surface matings) range from 10$\sp{-9}$ to 10$\sp{-5}$ per donor, depending on the particular donor isolate. Excision is believed to be the rate-limiting step for movement. When located on a multicopy plasmid in E. coli, Tn916 excises and segregates at a high frequency (e.g. $>$50% in overnight cultures without selection). Mutations near the left end of Tn916 generate excision-defective mutants which can be complemented in trans. This study represents a molecular and genetic analysis of the leftmost 40% of Tn916, a region responsible for the excision and the resistance. The 6,533-bp sequence of the left region was determined, and over ten open reading frames (ORFs) were identified. ORF1 and ORF2 were found to be essential to excision of Tn916; whereas products of ORF3 and/or ORF4, present in-frame and within ORF2, appeared to inhibit the excision process, probably by competing with the ORF2 product for specific binding sites; this may relate to the overall regulation of transposition. The tet(M) determinant exhibited significant homology with other tet(M) genes and a GTP-binding domain present in determinants of bacterial translational initiation and elongation factors. Transcription of the tet(M) gene and an extension of related transcripts well beyond the resistance determinant were found to be inducible by tetracycline when Tn916 was located on the E. faecalis chromosome. Three transcripts (0.25, 2.5, and 3.2 kb) detected by Northern hybridization were related to expression of the tet(M) gene. Both the 2.5- and 3.2-kb transcripts, including the tet(M) ORF and representing extensions of a 0.25-kb leader mRNA, were significantly increased in the presence of tetracycline. The induction of these transcripts probably involves transcriptional attenuation mechanisms.PhDOral BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/103319/1/9308460.pdfDescription of 9308460.pdf : Restricted to UM users only