FIG 2.

Ribosome remodeling is sufficient to reduce Spa sensitivity in M. smegmatis. (A) Spa sensitivity in C+ and C− ribosomes, purified from recombinant M. smegmatis strains pYL53 and pYL42 cultured in high-zinc Sauton’s medium. C+ ribosomes were purified from a high-zinc culture of a Δc− mutant harboring pYL42 (an integrative plasmid containing the wild-type c− operon), whereas C− ribosomes were purified from a high-zinc culture of a Δc− mutant harboring pYL53 (an integrative plasmid constitutively expressing the c− operon). IC₅₀ values were calculated as described for Fig. 1B. Values in parentheses denote 95% confidence intervals determined from the plots of corresponding colors. (B) Growth inhibitory activity of Spa against pYL42 and pYL53 strains in high-zinc Sauton’s medium. Approximately 100 µl of log-phase cultures of each strain from 7H9-ADCTw medium were diluted to an OD₆₀₀ of 0.025 and mixed with 100 µl 7H9-ADCTw medium containing Spa at the indicated concentrations. Cellular viability was assessed with resazurin dye after 16 h of incubation in the antibiotic containing medium. Data represent averages from three biologically independent experiments. (C) Time-dependent bactericidal activity of Spa against pYL42 and pYL53 strains in high-zinc Sauton’s medium. A saturated culture of each strain was exposed to 50 µg/ml of Spa, and the number of viable cells in the culture was determined at the indicated time points by plating dilutions on 7H10ADC medium and incubating for 3 days. A representative photograph of a plate from a 4-day time point of Spa exposure is shown on the right. Data represent means ± standard deviations (SDs) from three biologically independent experiments. ***, P < 0.001; ****, P < 0.0001 by t test.