The Staphylococcus aureus strains used in this study were derived from a USA300 strain isolated from a skin and soft tissue infection in a detainee from the Los Angeles County Jail (LAC) (35 (link)). S. aureus strain LAC contains two plasmids, p01 (a 3.1-kb cryptic plasmid) and p03 (a 27-kb plasmid conferring resistance to erythromycin) (36 (link)). Both p01 and p03 were removed from LAC for two reasons. First, curing of these two plasmids eliminated potential plasmid incompatibility concerns with pFA545 and pBursa (see below), and second, curing ensured that the bursa aurealis transposon did not insert preferentially into plasmid DNA. For removal of p03, strain LAC was grown overnight in Tryptic soy broth (TSB) with shaking at 37°C without antibiotics. The next day, serial dilutions were made and the cells were plated on Trypic soy agar (TSA) plates without any antibiotics. Colonies arising after overnight incubation at 37°C were then replica plated onto plates with and without erythromycin (5 µg/ml). Those colonies unable to grow on erythromycin were chosen, and the loss of the plasmid was confirmed by performing plasmid analysis (Promega, Madison, WI). After confirmation that no major genomic rearrangements had occurred by pulsed-field gel electrophoresis (PFGE) (37 (link)), one colony, designated LAC-13C, was selected for subsequent manipulation.
We next cured p01 from LAC-13C utilizing a plasmid incompatibility strategy. First, a chimeric plasmid in which pCL84 (conferring tetracycline resistance) (38 (link)) and p01 were ligated together at their unique EcoRI sites was created. This plasmid, designated pJE06, was then electroporated into Escherichia coli DH5α, purified, and subsequently electroporated into S. aureus RN4220 (39 (link)). pJE06 was then transduced from RN4220 using bacteriophage ϕ11 (40 (link)) into LAC-13C using methods as described by McNamara (41 ). Plasmid preparations of the resulting tetracycline-resistant transductants were screened for the loss of p01 due to plasmid incompatibility (42 (link)). The resulting strains were then cured of pJE06 by following the process described above for removal of pUSA03 except that the colonies were screened for tetracycline sensitivity. Multiple tetracycline-susceptible derivatives were examined by PFGE to ensure that no chromosomal rearrangements were detected; one strain was selected for subsequent use and designated JE2.
We next cured p01 from LAC-13C utilizing a plasmid incompatibility strategy. First, a chimeric plasmid in which pCL84 (conferring tetracycline resistance) (38 (link)) and p01 were ligated together at their unique EcoRI sites was created. This plasmid, designated pJE06, was then electroporated into Escherichia coli DH5α, purified, and subsequently electroporated into S. aureus RN4220 (39 (link)). pJE06 was then transduced from RN4220 using bacteriophage ϕ11 (40 (link)) into LAC-13C using methods as described by McNamara (41 ). Plasmid preparations of the resulting tetracycline-resistant transductants were screened for the loss of p01 due to plasmid incompatibility (42 (link)). The resulting strains were then cured of pJE06 by following the process described above for removal of pUSA03 except that the colonies were screened for tetracycline sensitivity. Multiple tetracycline-susceptible derivatives were examined by PFGE to ensure that no chromosomal rearrangements were detected; one strain was selected for subsequent use and designated JE2.
