Plasmids and primers used in this study are listed in Table 2. Minimal inhibitory concentrations (MICs) of various antibiotics were determined by microdilution as described previously (Nishi et al., 2004). Oxacillin, bacitracin and vancomycin (Sigma Sunitinib price Chemical Co. Ltd, St. Louis, MO), as well as erythromycin and ofloxacin (Wako Pure Chemical Industries Ltd., Osaka, Japan), were used. Population analysis profiles were determined by plating appropriate dilutions of an overnight culture on plates containing various concentrations of bacitracin (Nishi et al., 2004). Colonies were counted after 48 h incubation at 37 °C. All susceptibility tests were

repeated at least three times to check the reproducibility of the results. A small portion of overnight culture of S. aureus was inoculated to fresh TSB. Then, S. aureus cells were grown at 37 °C with shaking. Various concentrations (0.5, 1, 8, 16 μg mL−1) of bacitracin were added to the medium when OD 660 nm reached 0.3. After 5, 15, 30 and 60 min, the cells were

collected. Total RNA was extracted with a FastRNA Pro Blue kit (MP Biomedicals, Ohio) in accordance with the manufacturer’s protocol. One microgram of total RNA was reverse-transcribed to cDNA using a first-strand cDNA synthesis kit (Roche, click here Tokyo, Japan). Using cDNA as template DNA, quantitative PCR was performed using LightCycler system (Roche). Primers for bceR, bceA, vraD, vraF and vraR were constructed and used to determine optimal conditions for analysis of their expression. The amount

of gyrA was used as internal control. Primers for quantitative PCR are listed in Table 2. All mutants used in this study were shown in Table 1. Table 3 shows the MIC results of the mutants against various antibiotics. In MW2-derived ABC transporter mutants, the MIC of bacitracin in MM02 (ΔbceAB), MM07 (ΔbceB) and MM03 (ΔvraDE), showed two- and fourfold reductions, respectively, compared with that of the wild type, while the MIC of MM01 (ΔvraFG) showed a similar level to that of the wild type. Also, the MIC of bacitracin in a TCS mutant, MM08 (ΔbceS), was reduced fourfold compared with that of the wild type, showing a similar result with that of FK77 (ΔbceRS). In addition, two RN4220-derived filipin mutants, MM05 (ΔbceAB) and MM06 (ΔvraDE), showed increased susceptibility to bacitracin (fourfold reduction in MM05, 16-fold reduction in MM06), while another mutant MM04 (ΔvraFG) showed no change. For the complementation experiment, three complementation strains (MM09, MM10 and MM11) showed a similar susceptibility to bacitracin with that of the wild type (Table 3). MIC of oxacillin in MM02 (ΔbceAB) showed twofold reduction, while that of MM05 showed no alteration. Also, MIC of vancomycin in MM01 and MM04 (ΔvraFG) showed twofold reduction. MICs of the mutants against erythromycin and ofloxacin were similar to that of the wild type.