Comparative analysis of sequences flanking tet(W) resistance genes in multiple species of gut bacteria

Abstract

tet(W) is one of the most abundant tetracycline resistance genes found in bacteria from the mammalian gut and was first identified in the rumen anaerobe Butyrivibrio fibrisolvens 1.230, where it is highly mobile and its transfer is associated with the transposable chromosomal element TnB1230. In order to compare the genetic basis for tet(W) carriage in different bacteria, we studied sequences flanking tet(W) in representatives of seven bacterial genera originating in diverse gut environments. The sequences 657 bp upstream and 43 bp downstream of tet(W) were 96 to 100% similar in all strains examined. A common open reading frame (ORF) was identified downstream of tet(W) in five different bacteria, while another conserved ORF that flanked tet(W) in B. fibrisolvens 1.230 was also present upstream of tet(W) in a human colonic Roseburia isolate and in another rumen B. fibrisolvens isolate. In one species, Bifidobacterium longum (strain F8), a novel transposase was located within the conserved 657-bp region upstream of tet(W) and was flanked by imperfect direct repeats. Additional direct repeats 6 bp long were identified on each end of a chromosomal ORF interrupted by the insertion of the putative transposase and the tet(W) gene. This tet(W) gene was transferable at low frequencies between Bifidobacterium strains. A putative minielement carrying a copy of tet(W) was identified in B. fibrisolvens transconjugants that had acquired the tet(W) gene on TnB1230. Several different mechanisms, including mechanisms involving plasmids and conjugative transposons, appear to be involved in the horizontal transfer of tet(W) genes, but small core regions that may function as minielements are conserved.

FIG. 1.

Diagram showing the points at which the sequences upstream and downstream of tet(W) in the nine analyzed isolates diverge. The tet(W) ORF is indicated by an arrow. The thickness of the line represents the number of species for which sequence is conserved. As the sequence diverges, the line becomes thinner. This figure is not drawn to scale. *, B. longum F8 has insertion of 1,047 bp into the conserved region upstream of tet(W). **, M. multiacida P208-58 contains an insertion of a 23-bp directly repeated sequence 291 bp upstream from the tet(W) start codon.

FIG. 2.

Organization of the regions upstream and downstream of tet(W). ORFs are pattern coded and those conserved between different species are presented in the same shading. The percent identities to the closest match in the database are given above the ORFs. The rectangular boxes underneath the B. fibrisolvens diagram indicate the positions of the DRs. *, M. multiacida P208-58 contains an inserted 23-bp directly repeated sequence 291 bp upstream from the tet(W) start codon (indicated by the arrowhead). **, B. longum F8 has tandem repeats flanking the transposase inserted into the conserved core of 657 bp (represented by triangles) and duplicated 6 nucleotides flanking the insertion of the tet(W) and transposase genes (represented by circles).

FIG. 3.

Amino acid sequence of the B. longum F8 transposase. The conserved catalytic domain starts at residue P₁₃₅. Amino acids creating the DDE motif are indicated in boldface, characteristic residues upstream and/or downstream of the triad are in boldface italic, and regions N2, N3, and C1 are underlined.

FIG. 4.

Evidence for the presence of the circular minielement. ORFs are represented as solid arrows, and the locations of the primers used in PCR are shown by dotted arrows. Restriction sites are indicated, and DRs are represented by hatched boxes. (a) Organization of the tet(W) gene and its flanking regions in TnB1230 from B. fibrisolvens. (b) PCR products obtained using primers tetW_out and 3′GPtet reading outwards from the 5′ and 3′ ends of tet(W), respectively. Lane 1, 1-kb ladder (Promega, United Kingdom); lanes 2 to 7, B. fibrisolvens transconjugants Tc8 to Tc12 and Tc21, respectively; lane 8, B. fibrisolvens 1.230 (donor); lane 9, B. fibrisolvens 2221^R (recipient). (c) Organization of the PCR product shown in panel b following sequence analysis. MAFF′ and nrd′ are the truncated forms of the respective genes. (d) Diagram representing the circular form of the minielement carrying tet(W) identified in B. fibrisolvens transconjugants.

FIG. 5.

Southern blot of EcoRI-digested chromosomal DNAs from B. fibrisolvens strains hybridized to the 1-kb tet(W)-specific probe. Lanes 1 to 6, transconjugants Tc8 to Tc12 and Tc21, respectively; lane 7, Donor strain 1.230; lane 8, recipient strain 2221^R. The sizes of the bands are indicated.