The Butyrivibrio fibrisolvens tet(W) gene is carried on the novel conjugative transposon TnB1230, which contains duplicated nitroreductase coding sequences

Abstract

The Butyrivibrio fibrisolvens tet(W) gene is located on the conjugative transposon TnB1230. TnB1230 encodes transfer proteins with 48 to 67% identity to some of those encoded by Tn1549. tet(W) is flanked by directly repeated sequences with significant homology to oxygen-insensitive nitroreductases. The 340 nucleotides upstream of tet(W) are strongly conserved and are required for tetracycline resistance.

FIG. 1.

Diagram showing the genetic organization of 12 kb of the sequenced region of TnB1230. The arrows indicate the location and direction of transcription of each ORF. The positions of the DRs (DR1 and DR2) are indicated by hatched boxes. The number of amino acids (aa) encoded by each ORF is shown, and the DNA G+C content is also indicated. The sequence identity to ORFs encoded by Tn1549 is indicated as appropriate. This figure is not drawn to scale.

FIG. 2.

The CLUSTALW program was used to align sequences upstream of tet(W) genes from B. fibrisolvens 1.230 (GenBank accession number AJ222769), M. multiacidus P208 (GenBank accession number AY603069), and Clostridium sp. strain K10 (GenBank accession number AY601650) with tet(M) from E. faecalis (GenBank accession number M85225) and tet(O) from Campylobacter jejuni (GenBank accession number M18896). Solid arrows indicate the inverted repeats predicted to form secondary stem-loop structures. The DRs present only in M. multiacidus P208 are indicated by dashed arrows above the sequence. The putative poly(U) attenuation terminator (TTTTT), ribosome binding sites (GGAXG), and the ATG start codon are boxed. Nucleotides conserved in all five sequences are underscored with asterisks. The positions of the upstream primers used to amplify full-length (PCRB1) or truncated [tet(W)FF] sequences for expression cloning (indicated by [1] and [2], respectively) are shown by dashed overlining. The sequences in tet(W) and tet(M) encoding the putative leader peptides are shown in bold.