RESUMO
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 beta-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 theta-mode 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 theta-mode replication system.
Assuntos
Replicação do DNA , DNA Bacteriano/biossíntese , Proteínas de Ligação a DNA , Resistência Microbiana a Medicamentos/genética , Resistência a Múltiplos Medicamentos/genética , Plasmídeos/genética , Replicon , Staphylococcus aureus/genética , Sequência de Aminoácidos , Sequência de Bases , Conjugação Genética/genética , DNA Helicases/classificação , DNA Helicases/genética , Dados de Sequência Molecular , Fatores R/genética , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Transativadores/classificação , Transativadores/genética , Resistência a Trimetoprima/genética , Resistência beta-Lactâmica/genéticaRESUMO
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 beta-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.
Assuntos
Evolução Molecular , Fatores R , Staphylococcus/genética , Sequência de Aminoácidos , Sequência de Bases , Conjugação Genética , DNA Bacteriano , Resistência Microbiana a Medicamentos/genética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Filogenia , Deleção de Sequência , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido NucleicoRESUMO
The Staphylococcus epidermidis plasmid pSK639 is the prototype of a newly described family of small plasmids identified in clinical staphylococcal isolates. pSK639 is 8 kb in length and possesses a composite structure consisting of an IS257-flanked segment mediating trimethoprim resistance (Tpr), and regions responsible for replication and mobilization of the plasmid. Comparative sequence analysis suggests that a pSK639-like plasmid may represent a progenitor of previously identified staphylococcal Tpr determinants related to the transposon-like structure, Tn4003. In contrast to the small staphylococcal plasmids characterized to date that all utilize a rolling circle mode of replication, the replication region of pSK639 was found to contain features typical of an iteron-controlled theta-mode replicon. pSK639 is the first small plasmid of this type to be identified in the staphylococci.
