Replication of staphylococcal multiresistance plasmids

Based on structural and functional properties, three groups of large staphy lococcal multiresistance plasmids have been recognized, viz., the pSK1 fami ly, pSK41-like conjugative plasmids, and beta-lactamase-heavy-metal resista nce plasmids. Here we describe an analysis of the replication functions of a representative of each of these plasmid groups. The replication initiatio n genes from the Staphylococcus aureus plasmids pSK1, pSK41, and pI9789::Tn 552 were found to be related to each other and to the Staphylococcus xylosu s plasmid pSX267 and are also related to rep genes of several plasmids from other gram-positive genera. Nucleotide sequence similarity between pSK1 an d pI9789::Tn552 extended beyond their rep genes, encompassing upstream dive rgently transcribed genes, orf245 and orf256, respectively. Our analyses re vealed that genes encoding proteins related to the deduced orf245 product a re variously represented, in several types of organization, on plasmids pos sessing six seemingly evolutionarily distinct types of replication initiati on genes and including both theta-mode and rolling-circle replicons. Constr uction of minireplicons and subsequent functional analysis demonstrated tha t orf245 is required for the segregational stability of the pSK1 replicon. In contrast, no gene equivalent to orf245 is evident on the conjugative pla smid pSK41, and a minireplicon encoding only the pSK41 rep gene was found t o exhibit a segregational stability approaching that of the parent plasmid. Significantly, the results described establish that many of the large mult iresistance plasmids that have been identified in clinical staphylococci, w hich were formerly presumed to be unrelated, actually utilize an evolutiona rily related thetamode replication system.