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Trimethoprim resistance mediated by the Staphylococcus aureus multi-resistance plasmid pSK1 is encoded by a structure with characteristics of a composite transposon which we have designated Tn4003. Nucleotide sequence analysis of Tn4003 revealed it to be 4717 bp in length and to contain three copies of the insertion element IS257 (789-790 bp), the outside two of which are flanked by directly repeated 8-bp target sequences. IS257 has imperfect terminal inverted repeats of 27-28 bp and encodes for a putative transposase with two potential alpha-helix-turn-alpha-helix DNA recognition motifs. IS257 shares sequence similarities with members of the IS15 family of insertion sequences from Gram-negative bacteria and with ISS1 from Streptococcus lactis. The central region of the transposon contains the dfrA gene that specifies the S1 dihydrofolate reductase (DHFR) responsible for trimethoprim resistance. The S1 enzyme shows sequence homology with type I and V trimethoprim-resistant DHFRs from Gram-negative bacteria and with chromosomally encoded DHFRs from Gram-positive and Gram-negative bacteria. 5' to dfrA is a thymidylate synthetase gene, designated thyE.

DNA sequences corresponding to the 4.7-kb gentamicin, tobramycin and kanamycin resistance (GmrTmrKmr) transposon Tn4001 have been detected on a series of nine structurally-related plasmids that mediate this phenotype in Australian isolates of Staphylococcus aureus. Tn4001 sequences have also been demonstrated on the chromosomes of GmrTmrKmr isolates that do not possess these plasmids, and the exhibited diversity of chromosomal sites occupied by this element implies that Tn4001 has transposed to the chromosome on numerous occasions in vivo. These results suggest that the rapid emergence of nosocomial GmrTmrKmr S. aureus in the early 1980s may have been the result of the transposition of Tn4001 from a chromosomal site to a readily disseminated plasmid.