Complete Bypass of Restriction Systems for Major Staphylococcus aureus Lineages

T.P.S. and T.J.F. are joint senior authors. ABSTRACT Staphylococcus aureus is a prominent global nosocomial and community-acquired bacterial pathogen. A strong re-striction 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 transfor-mation and streamlined genetic manipulation of major S. aureus lineages. IMPORTANCE The genetic manipulation of clinical S. aureus isolates has been hampered due to the presence of restriction modi-fication barriers that detect and subsequently degrade inappropriately methylated DNA. Current methods allow the introduc-tion 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 ne