SGM Journals
Research Article

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

Kwong, Stephen M., Lim, Ricky, LeBard, Rebecca J., Skurray, Ronald A., Firth, Neville

Microbiology 2008; 154(10):3084 · https://doi.org/10.1099/mic.0.2008/017418-0

Download PDF View at publisher PubMed

Summary auto-generated

This study characterizes the pSK1 replicon, a prototype of staphylococcal multiresistance plasmids that use theta-type replication. The researchers identified essential elements for pSK1 replication, including an iterated region within the rep gene and a 68 bp upstream segment. They determined that a promoter (PrnaI) divergently oriented from rep directs transcription of RNAI, an antisense RNA that negatively regulates plasmid copy number. Mutation of PrnaI increased plasmid copy number approximately eightfold. The Rep protein was purified and shown to bind four repeat boxes in the rep coding region. Comparative analysis revealed that pSK1 and pSK41 replicons, which serve as prototypes for non-conjugative and conjugative multiresistance plasmids respectively, share similar genetic organization and utilize antisense RNA-mediated regulation. RNAI acts as a potent incompatibility determinant, preventing stable maintenance of two pSK1 minireplicons in the same cell. The minimal replicon spans about 1,227 bp and includes the rep gene, its promoter, and essential upstream sequences.

Key findings

  • The pSK1 rep region contains four Rep protein binding sites (iterons) within the coding sequence and requires a 68 bp upstream region for autonomous replication activity
  • A divergently oriented PrnaI promoter directs synthesis of RNAI antisense RNA that negatively regulates Rep expression and plasmid copy number, with PrnaI mutation causing an 8-fold increase in copy number
  • RNAI functions as a potent incompatibility determinant, preventing stable coexistence of two pSK1 minireplicons through repression of Rep expression
  • The pSK1 and pSK41 replicons share similar genetic organization and both utilize antisense RNA-mediated copy number control, suggesting a common evolutionary origin among staphylococcal multiresistance plasmids

This summary was generated automatically from the article PDF and is not part of the original publication. Refer to the PDF for the authoritative text.

Abstract

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.