General DNA manipulations were performed using standard procedures. Plasmid constructs used in this study were generated by cloning PCR products obtained with the oligonucleotide primers indicated in table S8. To introduce restriction sites in strains NCTC8325 and N315, we used plasmids pMAD and pBT2_bgaB and the oligonucleotides listed in table S8. pJP1501 and pJP1502 were constructed by a two-piece overlap assembly PCR. The whole fragments were subsequently cloned into the pMAD and pBT2_bgaB vectors, and the plasmids were transformed by electroporation into S. aureus RN4220 and transduced into NCTC8325 and N315 strains. Allelic replacements were carried out by a two-step procedure as follows: First, the plasmids were integrated into the chromosome by homologous recombination under nonpermissive conditions (44°C) and subsequent growth of the cointegrates at permissive temperature (30°C) followed by a second homologous recombination under nonpermissive conditions (44°C), resulting in their resolution. Erythromycin (for pMAD)– and chloramphenicol (for pBT2_bgaB)–sensitive white colonies, which no longer contained the pMAD or pBT2_bgaB plasmid, were tested by PCR. To differentiate between wild-type (wt) and mutant strains, we performed PCRs with oligonucleotides indicated in table S8 and digestion with restriction enzymes specific for Eco RI in NCTC8325s strain or Hind III for N315s isolates. All mutations were confirmed by DNA sequencing.

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