title: Expression of SCCmec cassette chromosome recombinases in methicillin-resistant Staphylococcus aureus and Staphylococcus epidermidis.
abstract: OBJECTIVE
  Methicillin resistance in staphylococci is mediated by the mecA gene, which is carried on the staphylococcal cassette chromosome mec (SCCmec). SCCmec is responsible for vertical and horizontal transfer of methicillin resistance. Horizontal transfer implies first SCCmec excision from the chromosome. Site-specific excision is catalysed by the Ccr recombinases, which are encoded by ccrAB genes located on the cassette. The aim of this study is to determine the promoter activity of ccrAB genes in individual cells of methicillin-resistant Staphylococcus aureus (N315, COL and MW2) and Staphylococcus epidermidis (RP62A). One mutant cured of its SCCmec (N315EX) was also used. Exposure to various stresses was included in the study.
  
  
  METHODS
  For each strain, translational promoter-green fluorescent protein (gfp) fusions were used to assess the levels of ccr promoter activity in individual cells. Analyses were performed using epifluorescence microscopy and flow cytometry.
  
  
  RESULTS
  ccr promoter activity was observed in only a small percentage of cell populations. This 'bistable' phenotype was strain dependent (GFP was expressed in N315 and RP62A, but not in COL and MW2) and growth dependent (GFP-expressing cells decreased from approximately 3% to 1% between logarithmic and stationary growth phases). The ccr promoter of strain N315 displayed normal promoter activity when expressed in SCCmec-negative N315EX. Likewise, the ccr promoter of strain COL (which was inactive in COL) showed normal N315-like activity when transformed into N315 and N315EX.
  
  
  CONCLUSIONS
  SCCmec excision operates through bistability, favouring a small fraction of cells to 'sacrifice' their genomic islands for transfer, while the rest of the population remains intact. Determinants responsible for the activity of the ccr promoter were not located on SCCmec, but were elsewhere on the genome. Thus, the staphylococcal chromosome plays a key role in determining SCCmec stability and transferability.
authors.affiliation: Department of Fundamental Microbiology, University of Lausanne, Lausanne, Switzerland. milos.stojanov@unil.ch
authors.forename: M
authors.initials: M
authors.lastname: Stojanov
authors.forename: O
authors.initials: O
authors.lastname: Sakwinska
authors.forename: P
authors.initials: P
authors.lastname: Moreillon
mesh_terms: D019677:Artificial Gene Fusion; D005434:Flow Cytometry; D020869:Gene Expression Profiling; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D055624:Methicillin-Resistant Staphylococcus aureus / Q000201:enzymology* / Q000235:genetics; D008856:Microscopy, Fluorescence; D011401:Promoter Regions, Genetic; D045522:Recombinases / Q000096:biosynthesis*; D011995:Recombination, Genetic; D013212:Staphylococcus epidermidis / Q000201:enzymology* / Q000235:genetics
chemical_list: D045522:Recombinases; D049452:Green Fluorescent Proteins