Figure 5.

Deciphering the roles of rodA and pbpA in mycobacterial cell fitness. a–d, TEM analysis of Mtb rodA and pbpA deletion mutants in 7H9 (a and c) or Sauton's medium (b and d). Fresh cultures of Mtb, MtbΔr, MtbΔp, and MtbΔrp were seeded at an initial A₆₀₀ of 0.1 and grown for 6 days at 37 °C at 100 rpm in 7H9 or Sauton's medium followed by fixation. Fixed cells were processed for TEM, and cell wall architecture was observed at 200 kV, ×50,000 in 7H9 (a) and Sauton's medium (b). c and d, quantification of cell wall thickness in MtbΔr, MtbΔp, MtbΔrp strains (n = 6–7) grown in 7H9 medium (mean cell wall thickness: Mtb, MtbΔr, and MtbΔp, 17 nm; MtbΔrp, 20.2 nm) (c) or Sauton's medium (mean cell wall thickness: Mtb, 18 nm; MtbΔr, 15.9 nm; MtbΔp, 28.7 nm; MtbΔrp, 30.6 nm) (d). Cell wall thickness was measured independently using SmartTiff software and analyzed using GraphPad Prism version 6, and statistical analysis was performed using a two-way ANOVA test. ****, p < 0.0001; ***, p < 0.001. Red bars, cell wall thickness. e and f, hypoxia and persisters analysis for Mtb and Msm deletion mutants. e, bacterial survival analysis of day 42 posthypoxia (Wayne model) for Mtb, MtbΔr, and MtbΔp. Results were plotted using GraphPad prism version 6. Error bar, S.D. Statistical analysis was performed using a two-tailed test. *, p < 0.05. Similar results were obtained in two independent experiments performed in duplicates. f, persisters analysis of Msm, MsmΔr, and MsmΔp. Cultures were grown until exponential phase and treated with 10 μg/ml isoniazid for 48 h, and cfu obtained before and after isoniazid treatment were plotted. Statistical analysis was performed using a two-tailed test. **, p < 0.005. Error bars, S.D. Similar results were obtained in two independent experiments performed in triplicates.