Assessing Putative Resistance Gene Function

Putative PLM resistance genes identified in this study were introduced into a PLM-susceptible S. aureus host to assess their ability to confer resistance. DNA fragments corresponding to these genes were either generated by PCR-amplification with Phusion® High-Fidelity DNA Polymerase (NEB) using oligonucleotide primers described in Supplementary Table S1 or were obtained by synthesis (Genewiz). PCR amplicons and synthesized DNA fragments were digested with KpnI and EcoRI (NEB) to enable directional ligation into similarly-digested expression plasmid pRMC2 (24 (link)) for transformation of Escherichia coli XL10-Gold (Agilent Technologies). DNA-sequence verified constructs were then introduced into S. aureus RN4220 (25 (link)) by electroporation (12 (link)). Transformants were grown in cation-adjusted MHB at 37°C with vigorous aeration to an OD₆₂₅ of 0.6, and expression induced with anhydrotetracycline hydrochloride (ATc) (Sigma-Aldrich) at a final concentration of 100 ng/ml for 3 h. Susceptibility testing of these induced cultures was carried out as above, using MHB supplemented with ATc (100 ng/ml).