FIG 6.
Reduced Spa sensitivity in M. tuberculosis (mc27000) expressing C− ribosomes. (A) qRT-PCR-based analysis of the expression of two genes of the c− operon (encoding S18C− and L28C−) in high- (1 mM ZnSO4) and low-zinc (without any zinc supplement) cultures of mc27000 and an isogenic Δc− strain transformed with either pYL60 (expressing c− operon from a constitutive promoter) or an empty vector control (pYL3). Growth inhibitory activity (B) and bactericidal activity (C) of Spa against an mc27000:Δc− strain transformed with either pYL60 or empty vector control (pYL3). Growth inhibition was determined by resazurin assay as described for Fig. 2B. For measuring bactericidal activity of Spa, high-zinc saturated cultures of the indicated strains were inoculated in Sauton’s medium with 100 µg/ml of Spa and incubated for the indicated time periods prior to plating dilutions and enumerating colonies. Data in both panels represent means ± SDs from three biologically independent experiments. *, P < 0.05; ****, P < 0.0001 by t test. (D) Translation activities of 70S ribosomes from the indicated strains in the presence of Spa. 70S C+ and C− ribosomes were purified from high- and low-zinc pellicle cultures of mc27000, while 70S C− ribosomes without S14C− were purified from low-zinc pellicle cultures of an isogenic Δc− strain transformed with pYL97 (a plasmid expressing c− operon without S14c− from the native promoter). IC50 values were calculated as described for Fig. 1B. The values in parentheses indicate 95% confidence intervals determined from the plots of corresponding colors. Data represent two biologically independent experiments.