Introduction of specific mutations of the gyrA, gyrB, parC, and parE genes into the Rd strain.

The cells were transformed by electroporation. The electrocompetent cells were prepared as described by Ubukata et al. (7). Specific primers (Table S6) were used to PCR amplify amplicons of approximately 3 kb; 200 ng of the DNA was then mixed with electrocompetent cells followed by electroporation. The mixtures were incubated for 24 h at 37°C under an atmosphere of 5% CO₂. The transformants were selected by spreading on sBHI agar containing 0.03 and 0.06 mg/liter moxifloxacin. The target genes in the transformants selected on the moxifloxacin-containing agar (at least three transformants for each transformation attempt) were sequenced to confirm the presence of the target mutations, and subsequently MIC values of the transformants were determined as described above. We also confirmed the contribution of the gyrB, parC, and parE mutations by introducing each mutation into gyrB, parC, and parE sequences of Rd by using site-direct mutagenesis methods as described in Table S7 and Fig. S2.