The 46.4-kb nucleotide sequence of pSK41, a prototypical multiresistance plasmid from Staphylococcus aureus, has been determined, representing the first completely sequenced conjugative plasmid from a gram-positive organism. Analysis of the sequence has enabled the identification of the probable replication, maintenance, and transfer functions of the plasmid and has provided insights into the evolution of a clinically significant group of plasmids. The basis of deletions commonly associated with pSK41 family plasmids has been investigated, as has the observed insertion site specificity of Tn552-like β-lactamase transposons within them. Several of the resistance determinants carried by pSK41-like plasmids were found to be located on up to four smaller cointegrated plasmids. pSK41 and related plasmids appear to represent a consolidation of antimicrobial resistance functions, collected by a preexisting conjugative plasmid via transposon insertion and IS257-mediated cointegrative capture of other plasmids.

Based on structural and functional properties, three groups of large staphylococcal multiresistance plasmids have been recognized, viz., the pSK1 family, pSK41-like conjugative plasmids, and ␤-lactamase-heavy-metal resistance plasmids. Here we describe an analysis of the replication functions of a representative of each of these plasmid groups. The replication initiation genes from the Staphylococcus aureus plasmids pSK1, pSK41, and pI9789::Tn552 were found to be related to each other and to the Staphylococcus xylosus plasmid pSX267 and are also related to rep genes of several plasmids from other gram-positive genera. Nucleotide sequence similarity between pSK1 and pI9789::Tn552 extended beyond their rep genes, encompassing upstream divergently transcribed genes, orf245 and orf256, respectively. Our analyses revealed that genes encoding proteins related to the deduced orf245 product are variously represented, in several types of organization, on plasmids possessing six seemingly evolutionarily distinct types of replication initiation genes and including both thetamode and rolling-circle replicons. Construction of minireplicons and subsequent functional analysis demonstrated that orf245 is required for the segregational stability of the pSK1 replicon. In contrast, no gene equivalent to orf245 is evident on the conjugative plasmid pSK41, and a minireplicon encoding only the pSK41 rep gene was found to exhibit a segregational stability approaching that of the parent plasmid. Significantly, the results described establish that many of the large multiresistance plasmids that have been identified in clinical staphylococci, which were formerly presumed to be unrelated, actually utilize an evolutionarily related thetamode replication system.Clinical Staphylococcus aureus strains often harbor multiple plasmids, ranging from small rolling-circle (RC) replicating plasmids that are cryptic or encode only a single resistance determinant to larger multiresistance and conjugative plasmids (12,32,36). Three groups of multiresistance plasmids have been recognized in staphylococci. Isolates from the 1960s and 1970s were commonly found to carry multiresistance plasmids conferring resistance to penicillin and heavy metals or other inorganic ions (48). Such ␤-lactamase-heavy-metal resistance plasmids characteristically contain the ␤-lactamase-encoding transposon Tn552 or a derivative and operons mediating resistance to arsenical, cadmium, and/or mercuric ions (36). Some ␤-lactamase-heavy-metal resistance plasmids also contain Tn551, conferring resistance to macrolide-lincosamide-streptogramin type B antibiotics (33); an IS256-bounded composite aminoglycoside resistance transposon, Tn4001 (30); and/or a qacA or qacB antiseptic and disinfectant multidrug resistance determinant (30).pSK41-like conjugative multiresistance plasmids were first detected in strains isolated in the mid 1970s. Such plasmids commonly contain a Tn4001 hybrid structure (6) and IS257-flanked cointegrated copies of small plasmids, such as the aminoglycoside resist...

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