Replication of Staphylococcal Multiresistance Plasmids

Abstract

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...

Shuttle Vectors Derived from pN315 for Study of Essential Genes in Staphylococcus aureus

“…9) From the previous studies, SAP001 protein is estimated to be Rep protein for plasmid replication initiation enabling the replicon to replicate via theta mode and SAP031 protein is Par protein for stable maintenance of the plasmid. 10,11,18,19) However, it is unknown if plasmid shuffling occurs quickly or requires a longer time-period when a DNA region for replication and partition of the plasmid is subcloned in order to construct shuttle vectors.…”

“…Among them, pN315 is a 24653 bp plasmid harbored in multi-drug resistant S. aureus N315 strain 9) and belongs to pSK1-type replicon being presumed to replicate via the theta mode. 10,11) In this study, we constructed a set of pN315-based vectors and used the plasmid shuffling assay to study the representative example of the mraY gene and as expected, found that mraY is required for cell growth or viability in S. aureus. Our initial findings suggest that the pN315-based plasmids are suitable for identification of genes essential for staphylococcal cell growth.…”

Analysis of the pSK1 replicon, a prototype from the staphylococcal multiresistance plasmid family

“…DNA sequence analyses have shown that representative replicons from each of these three staphylococcal multiresistance plasmid groups (pI9789 : : Tn552, pSK1 and pSK41, respectively) are, in fact related, and the plasmids actually utilize evolutionarily common replication systems (Firth et al, 2000). pSK1 and examples of the b-lactamase/heavymetal-resistance plasmids belong to the same incompatibility group, whereas pSK41 has been shown to be compatible with these plasmids (Firth et al, 2000).…”

“…Hence, many large theta-replicating plasmids confer resistance to more than one antimicrobial compound and are collectively termed multiresistance plasmids. Staphylococcal multiresistance plasmids have been broadly divided into three groups based on resistance phenotypes and conjugative ability; (i) the b-lactamase/heavy-metalresistance plasmids, (ii) pSK1-like multiresistance plasmids; and (iii) conjugative pSK41-like multiresistance plasmids (Paulsen et al, 1997;Firth et al, 2000). DNA sequence analyses have shown that representative replicons from each of these three staphylococcal multiresistance plasmid groups (pI9789 : : Tn552, pSK1 and pSK41, respectively) are, in fact related, and the plasmids actually utilize evolutionarily common replication systems (Firth et al, 2000).…”

“…Staphylococcal multiresistance plasmids have been broadly divided into three groups based on resistance phenotypes and conjugative ability; (i) the b-lactamase/heavy-metalresistance plasmids, (ii) pSK1-like multiresistance plasmids; and (iii) conjugative pSK41-like multiresistance plasmids (Paulsen et al, 1997;Firth et al, 2000). DNA sequence analyses have shown that representative replicons from each of these three staphylococcal multiresistance plasmid groups (pI9789 : : Tn552, pSK1 and pSK41, respectively) are, in fact related, and the plasmids actually utilize evolutionarily common replication systems (Firth et al, 2000). pSK1 and examples of the b-lactamase/heavymetal-resistance plasmids belong to the same incompatibility group, whereas pSK41 has been shown to be compatible with these plasmids (Firth et al, 2000).…”