title: Complete Bypass of Restriction Systems for Major Staphylococcus aureus Lineages.
abstract: Staphylococcus aureus is a prominent global nosocomial and community-acquired bacterial pathogen. A strong restriction barrier presents a major hurdle for the introduction of recombinant DNA into clinical isolates of S. aureus. Here, we describe the construction and characterization of the IMXXB series of Escherichia coli strains that mimic the type I adenine methylation profiles of S. aureus clonal complexes 1, 8, 30, and ST93. The IMXXB strains enable direct, high-efficiency transformation and streamlined genetic manipulation of major S. aureus lineages.
  
  
  OBJECTIVE
  The genetic manipulation of clinical S. aureus isolates has been hampered due to the presence of restriction modification barriers that detect and subsequently degrade inappropriately methylated DNA. Current methods allow the introduction of plasmid DNA into a limited subset of S. aureus strains at high efficiency after passage of plasmid DNA through the restriction-negative, modification-proficient strain RN4220. Here, we have constructed and validated a suite of E. coli strains that mimic the adenine methylation profiles of different clonal complexes and show high-efficiency plasmid DNA transfer. The ability to bypass RN4220 will reduce the cost and time involved for plasmid transfer into S. aureus. The IMXXB series of E. coli strains should expedite the process of mutant construction in diverse genetic backgrounds and allow the application of new techniques to the genetic manipulation of S. aureus.
authors.affiliation: imonk@unimelb.edu.au.
authors.forename: Ian R
authors.initials: IR
authors.lastname: Monk
authors.affiliation: Department of Microbiology and Immunology, University of Melbourne at the Doherty Institute for Infection and Immunity, Melbourne, Australia.
authors.forename: Jai J
authors.initials: JJ
authors.lastname: Tree
authors.forename: Benjamin P
authors.initials: BP
authors.lastname: Howden
authors.affiliation: Department of Microbiology and Immunology, University of Melbourne at the Doherty Institute for Infection and Immunity, Melbourne, Australia.
authors.forename: Timothy P
authors.initials: TP
authors.lastname: Stinear
authors.affiliation: Moyne Institute of Preventative Medicine, Department of Microbiology, School of Genetics and Microbiology, Trinity College Dublin, Dublin, Ireland.
authors.forename: Timothy J
authors.initials: TJ
authors.lastname: Foster
mesh_terms: D019175:DNA Methylation; D015280:DNA Restriction-Modification Enzymes / Q000235:genetics / Q000378:metabolism; D004269:DNA, Bacterial / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D009154:Mutation; D010957:Plasmids; D013211:Staphylococcus aureus / Q000235:genetics*; D014169:Transformation, Bacterial
chemical_list: D015280:DNA Restriction-Modification Enzymes; D004269:DNA, Bacterial