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Microbiology 154 (2008), 3084-3094; DOI  10.1099/mic.0.2008/017418-0
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Microbiology 154 (2008), 3084-3094; DOI  10.1099/mic.0.2008/017418-0
© 2008 Society for General Microbiology

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

Stephen M. Kwong, Ricky Lim, Rebecca J. LeBard{dagger}, Ronald A. Skurray and Neville Firth

School of Biological Sciences, University of Sydney, New South Wales 2006, Australia

Correspondence
Neville Firth
nfirth{at}bio.usyd.edu.au

Multidrug-resistant staphylococci often harbour plasmids that carry genes conferring resistance to several antimicrobial compounds. Many of these multiresistance plasmids appear to utilize a related theta-type replication system for which multiresistance plasmid pSK1 serves as a prototype. Essential pSK1 replication elements were identified by cloning segments of the replication region and testing the resulting plasmids for replication proficiency. An iterated region within rep and a DNA segment of up to 68 bp upstream of the rep promoter were both found to be essential for origin activity. The Rep protein was overexpressed as a 6xHis-tagged C-terminal fusion protein and was shown to bind in vitro to four Rep boxes located within the rep coding region. Inactivation of a divergently oriented promoter upstream of rep, designated PrnaI, resulted in an elevated plasmid copy number. Comparative analyses suggest that the replication systems of many staphylococcal multiresistance plasmids share a similar genetic organization and utilize an antisense-RNA-mediated regulatory mechanism for copy number control.

Abbreviations: CAT, chloramphenicol acetyltransferase; EMSA, electrophoretic mobility shift assay; TSP, transcriptional start point

{dagger}Present address: School of Biotechnology and Biomolecular Sciences, University of New South Wales, NSW 2052, Australia.






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