Complete nucleotide sequence and comparative analysis of pPR9, a 41.7-kilobase conjugative staphylococcal multiresistance plasmid conferring high-level mupirocin resistance

Abstract

We have sequenced the conjugative plasmid pPR9, which carries the ileS2 gene, which had contributed to the dissemination of high-level mupirocin resistance at our institution. The plasmid backbone shows extensive genetic conservation with plasmids belonging to the pSK41/pGO1 family, but comparative analyses have revealed key differences that provide important insights into the evolution of these medically important plasmids and high-level mupirocin resistance in staphylococci and highlight the role of insertion sequence IS257 in these processes.

FIG. 1.

Physical and genetic map of Staphylococcus aureus multiresistance plasmid pPR9 (GenBank entry NC_013653). The approximate positions of the putative origin of transfer (oriT), EcoRI and HindIII restriction sites, IS257 elements (IS257-L and IS257-R), and Tn552 are shown on the rim of the outer circle. The first G of the IS257-L terminal inverted repeat left (TIR_L) was designated the first nucleotide of the plasmid, and all 42 open reading frames (ORFs; tentatively named orf1 to orf42) likely to represent translated genes are numbered in relation to this site. ORFs are represented by arrows indicating the direction of transcription; tnp marks the gene for IS257 transposase. The accessory region genes are shown in gray, and the plasmid backbone genes are shown in black. All but six ORFs were found to be transcribed in the same direction (clockwise). The nucleotide sequence was verified by comparison of in silico-generated restriction maps with experimental data. Also, the numbers and sizes of HindIII and EcoRI restriction fragments that hybridized with specific probes for the ileS2, blaZ, traK, and tnp genes corresponded to those predicted from the pPR9 sequence (data not shown). orf42, interrupted by insertion of the IS257-flanked ileS2 segment, is indicated by 42′ and ′42. The deletion of the 3′ portion of orf4 is indicated as 4′.

FIG. 2.

Relationships between representative pSK41/pGO1 family plasmids. Linear physical and genetic maps of staphylococcal conjugative plasmids pGO1 (6), pSK41 (4), pPR9, pV030-8, pUSA03 (10), and pGO400 (22) are presented, with accession numbers given in parentheses; plasmid sizes are shown on the right. Shaded connections between plasmid maps illustrate that they all share a conserved basic backbone. The plasmid accessory regions are shown as a thick line in each map. The genetic loci shown are aacA-aphD (gentamicin-tobramycin-kanamycin resistance), aadD (kanamycin-neomycin-paromomycin-tobramycin resistance), ble (bleomycin resistance), dfrA (trimethoprim resistance), smr (antiseptic and disinfectant resistance), ileS2 (high-level mupirocin resistance), blaZ (penicillin resistance), ermC (macrolide, lincosamide, and streptogramin B resistance), tra (conjugative transfer functions), rep (replication initiation), res (resolvase), and oriT (origin of conjugative DNA transfer). The positions and extents of the cointegrated copy of the plasmids pUB110 and pSK639, the Tn4001-IS257 hybrid structure, and the Tn552-like transposon are indicated. Inverted and truncated copies of IS256 associated with the Tn4001 hybrid structures are represented by open boxes, whereas IS257 elements are represented as solid boxes containing an arrowhead indicating the direction of the transposase transcription and hence the element's orientation. Where known, the 8-bp sequences adjacent to each IS257 element are indicated. IS257 element designations for pGO1 and pSK41 are taken from the work of Caryl and O'Neill (6) and Berg et al. (4), respectively. The location of oriT is based on homology to pGO1, where it has been mapped precisely (7). Recognition sites for the restriction endonuclease EcoRI (E) are shown. A distance scale in kb is given below the figure.

FIG. 3.

Structural organization of the regions encompassing the ileS2 genes of plasmids pPR9, pV030-8, pUSA03, and pGO400. The respective plasmid names are shown on the left. Restriction endonuclease cleavage sites are abbreviated as follows: E, EcoRI; H, HindIII. IS257 elements (IS-L and IS-R) flanking the ileS2 gene are represented by solid boxes; the white arrows indicate the direction of IS257 transposase transcription. The ileS2 gene and the predicted ORFs upstream and downstream are represented as arrows with the arrowhead indicating their orientation. A segment of approximately 2 kb on pV030-8 has been omitted for clarity. Truncated ORFs are shown using a dotted outline. The positions of the −35 and −10 sequences for the putative promoters P_hybrid and P_out are shown above each plasmid. The spacing between the −35 and −10 promoter sequences is indicated below each putative promoter. A distance scale in kb is given below the figure.