Fig. 6: Growth inhibition experiments in MRSA validate mechanistic findings.

a View of the inward-occluded NorA cryo-EM structure at pH 5.0 displaying Glu222 and Asp307 and residues within 5 Å from these two residues. b Norfloxacin minimum inhibitory concentration (MIC) values were measured in an MRSA strain where the native norA gene was disrupted by a transposon insertion (MRSA^ΔnorA). Wild-type NorA and indicated mutants were encoded on a hemin-inducible plasmid. The number of independent MIC experiments (five for empty vector ΔnorA, two for other variants) is represented by the black circles overlaid on the bar graph for each sample. Data are presented as mean values ± s.d. across independent experiments. The upper limit of MIC detection was set at 256 μg/mL. Statistical significance was assessed using an unpaired, two-tailed t-test for each single-site mutant in comparison to MRSA^ΔnorA + NorA. P values were labeled on each bar. c Immunoblotting results performed on MRSA cell lysates following the induction of MRSA^ΔnorA or MRSA^ΔnorA transformed with a hemin-inducible plasmid carrying wild-type NorA and NorA mutants. The immunoblots probed for the Myc tag on the C-terminus of NorA and SrtA (control protein in S. aureus). Similar results were observed in two independent experiments. d Ethidium efflux assay results for MRSA^ΔnorA transformed with plasmids corresponding to empty vector (ΔnorA), wild-type NorA, E222Q, and D307N. The graph displays the normalized ethidium fluorescence (F/F0) over time, starting from the addition of glucose at time zero. Solid and shaded lines in the same color depict the mean values ± s.d. from two independent experiments with a total of four technical replicates. e A plot of the normalized fluorescence value (F/F0) at 1200 s from the data in d. The number of technical replicates from two independent experiments is represented by the black circles overlaid on the bar graph for each sample. Data are presented as mean values ± s.d. from four technical replicates. Statistical significance was evaluated using an unpaired, two-tailed t-test for individual single-site mutants compared to MRSA^ΔnorA + NorA. P values were labeled on each bar.