CIRCULARIZATION OF TN916 IS REQUIRED FOR EXPRESSION OF THE TRANSPOSON-ENCODED TRANSFER-FUNCTIONS - CHARACTERIZATION OF LONG TETRACYCLINE-INDUCIBLE TRANSCRIPTS READING THROUGH THE ATTACHMENT SITE

A detailed transcriptional analysis of the conjugative transposon Tn91 6 was carried out, which revealed that transcription of the transfer f unctions requires excision of the element and dramatically increases i n the presence of tetracycline. The key components of this regulatory system are two contiguous transposon-borne genes, orf7 and orf8, locat ed downstream from and having the same polarity of transcription as th e tetracycline resistance determinant tetM. The gene orf7 encodes a 14 0-amino-acid (aa) protein exhibiting limited homology with sigma(F) of Bacillus subtilis, whereas orf8 encodes a 76-aa peptide that does not share any sequence homology with any cognate proteins. In the presenc e of tetracycline, an attenuation mechanism enables the transcription of orf7 and orf8 from the tetM promoter. The resulting increased synth esis of ORF7 and ORF8 activates the promoter P-orf7 located upstream f rom orf7, which then directs the expression of the transfer functions in the transposon circular intermediate through long transcripts encom passing the attachment site. The apparently non-regulated promoter P-x is located upstream of the excisionase encoding gene xis could also pa rticipate in the expression of the tra genes. We also demonstrate that Tn916 carries another regulated promoter, P-orf9, which directs trans cription of a single gene, orf9, located downstream from and transcrib ed counterclockwise to tetM. This gene encodes a 117-aa putative trans criptional repressor, but the exact role of this protein in the mobili ty of Tn916, as well as the regulation of its expression, remains to b e elucidated. Our results constitute the molecular basis for the obser vation that tetracycline increased the transfer frequency of this type of element.