Fig 8.

Loss of crgA and cwsA causes severe defects in cell wall synthesis. (A) ¹⁴C-labeled GlcNAc incorporation in M. smegmatis WT, ΔcwsA, ΔcrgA, and DKO strains. Exponential cultures were grown with ¹⁴C-labeled GlcNAc as described in Materials and Methods. At 1 min, 2 h, 4 h, and 6 h after addition of the label, 0.5 ml of culture was filtered through a 0.2-μm-pore-size nitrocellulose filter and washed, the filters were air dried, and radioactivity was measured in a liquid scintillation counter. Data shown are normalized to the A₆₀₀ at each given point. Means ± standard deviations from 3 independent experiments are shown. (B) Cell wall turnover in M. smegmatis strains from panel A. Cells were labeled with [¹⁴C]GlcNAc for 6 h as described for panel A and pelleted. The labeled cells were resuspended in medium without label and grown at 37°C with shaking. At 0, 3, 4, 5, 6, 8, 10, and 12 h following growth in label-free medium, 0.5 ml of culture was pelleted, boiled in 4.0% SDS for 30 min, and passed through a 0.22-μm-pore-size filter. The filters were processed as described in Materials and Methods, and the retained radioactivity was measured in a liquid scintillation counter. Percent PG turnover at each time point was calculated with respect to that at time zero. Marker labels are as described for panel A. Means ± standard deviations from 3 independent experiments are shown. Statistical analysis of data using paired Student's t test indicated significant differences between WT and the ΔcrgA strain (P = 0.003), WT and DKO (P = 0.005), and WT and the ΔcwsA strain (P = 0.011). One-way ANOVA followed by Tukey's multiple-comparison test revealed significant differences between the ΔcrgA and ΔcwsA strains (P = 0.0035), the ΔcrgA and DKO strains (P = 0.0058), and the ΔcwsA and DKO strains (P = 0.0249). (C) Autolysis of M. smegmatis strains. Various M. smegmatis strains (as described for panel A) were grown to an A₆₀₀ of 0.5, and cells were pelleted, resuspended in 50 mM glycine, pH 8.0, containing 0.05% Tween 20, and incubated with shaking at 37°C. At time points of 0, 1, 2, 4, 6, 8, 10, 12, 14, and 16 h, aliquots of cells were removed and the A₆₀₀ was measured. The percentage of the initial optical density (OD) retained was calculated with respect to that at time zero. Data shown are means ± standard deviations from 3 independent experiments. Paired Student's t test showed significant differences in autolysis between the WT and ΔcwsA strains (P = 0.0078), the WT and DKO strains (P = 0.0028), the ΔcwsA and DKO strains (P = 0.0026), and the ΔcrgA and DKO strains (P = 0.0014) but not between the WT and the ΔcwsA strains (P = 0.063). (D) Lysozyme sensitivity. Fluorescein-labeled cell walls were prepared from WT and mutant M. smegmatis strains as described in the text and incubated with 1 mg/ml lysozyme in a buffer containing 50 mM Tris and 1 mM EDTA for 4 h. Reactions were stopped by addition of an equal volume of 4 M LiCl and spun, and the fluorescent label released in the supernatant was quantitated in a Jasco FP6500 fluorimeter. Reactions were done in triplicate, and average percent hydrolysis is shown.