The genetic manipulation of clinical Staphylococcus 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, using the ability of PacBio sequencing to detect adenine methylation, 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. These strains, called the IMXXB series mimic the type I adenine methylation profiles ofS. aureus clonal complexes 1, 8, 30, and ST93 and enable direct, high-efficiency transformation and streamlined genetic manipulation of major S. aureus lineages. 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.