Fig 6.
Weakening the interaction between CarD and the RNAP and depleting CarD increases sensitivity to streptomycin. (A and B) Survival of the M. smegmatis strains during the disk zone of inhibition assays with streptomycin. Five hundred microliters of the log-phase M. smegmatis ΔcarD attB∷tet-carD strains expressing CarDWT or CarDR25E was plated on LB agar, and a disk spotted with 5 μl of 200-mg/ml streptomycin was placed in the middle of the bacterial lawn. (A) Representative experiment. (B) The radius of the zone of inhibition as the mean ± SEM of data from three biological replicates for each strain. (C and D) Survival of M. smegmatis strains during transient streptomycin treatment. The log-phase M. smegmatis ΔcarD attB∷tet-carD strains expressing CarDWT or CarDR25E growing in LB were treated for 2 h with 10 μg/ml streptomycin before the dilutions were plated to determine the surviving CFU. M. smegmatis Tet-CarD and control strains were grown in LB without ATc for 13 h to deplete carD transcript levels in Tet-CarD and treated for 2 h with 10 μg/ml streptomycin, and dilutions were plated on LB plus ATc to determine the surviving CFU. (C) Representative experiment. (D) Survival as the mean ratio of CFU in treated cultures to that in untreated cultures ± SEM, with each sample represented by a black circle. (E) Western blot analysis of protein lysates from M. smegmatis Tet-CarD (lanes 2, 3, 5, 6, and 8) and control (lanes 1, 4, and 7) strain replicates grown in LB without ATc for 13 h to confirm the depletion of CarD in Tet-CarD samples. The RNAP β subunit was detected in each sample as a loading control. The significance levels for all panels were determined as described for Fig. 3.