Figure 5. Each of CarD’s functional domains is important for resistance to clinically relevant antibiotics.

A–C. Survival of M. smegmatis strains during the disk zone of inhibition assays. Five hundred microliters of log-phase M. smegmatis strains expressing CarD^WT, CarD^R25E, CarD^R47E, CarD^W85A, CarD^K90A, CarD^K125A, or CarD^K125E were plated on LB agar, and a disk spotted with 5μl of 1 mg ml⁻¹ ciprofloxacin (A), 100 mg ml⁻¹ rifampicin (B), or 200 mg ml⁻¹ streptomycin (C) was placed in the middle of the bacterial lawn. A representative experiment is shown above the x-axis. The radius of the zone of inhibition for each replicate and the mean ± SEM for each set of replicates is graphed, with each sample represented by a black circle.

D. Survival of M. smegmatis strains during transient ciprofloxacin treatment. Log-phase cultures of the M. smegmatis strains expressing CarD^WT, CarD^R25E, CarD^R47E, CarD^W85A, CarD^K90A, CarD^K125A, or CarD^K125E growing in LB were treated for 2 hr with 10 μg ml⁻¹ ciprofloxacin before the dilutions were plated to determine the surviving CFU. Graphed is the ratio of CFUs in treated cultures to that in untreated cultures for each replicate and the mean ± SEM for each set of replicates, with each sample represented by a black circle.

For all panels, the significance of differences between mutant strains and WT were determined as described in Figure 2.