Fig. 4

Metabolic incorporation of 3-azido pHB occurs only in live M. marinum with an intact PGL biosynthetic pathway. (A) Mycobacteria that lack the PGL biosynthetic pathway were treated with various concentrations of 3-azido pHB and analyzed by flow cytometry. Flow cytometry data are averages of three independent replicates. Statistical analysis was performed using an ordinary two-way ANOVA, followed by Šídák’s multiple comparisons test. Significance is represented, where ****p < 0.0001 and ns (not significant) for p > 0.05. (B) Lipid extracts of PGL-deficient M. marinum strains treated with 3-azido pHB, stained with AF647-DBCO, and analyzed by TLC. Crude lipid extracts (100 μg) or AF647-DBCO (20 μg) were loaded onto a silica gel 60 TLC plate, which was then developed with 4:6 methanol/chloroform. TLC was visualized using a ChemiDoc MP imaging system with a 700 nm wavelength. (C) Competition experiment using various concentrations of pHB added to M. marinum treated with 750 μM 3-azido pHB. Flow cytometry data are averages of three independent replicates. Relative MFI is determined by normalizing against DMSO control. Statistical analysis was performed using a two-way ANOVA followed by a Dunnett’s multiple comparisons test. Significance is represented by ****p < 0.0001 and ns (not significant) for p > 0.05. (D) M. marinum were heat killed (80 °C, 30 min), treated with 3-azido pHB for 18 h, stained with AF647-DBCO, and analyzed by flow cytometry. Statistical analysis was performed using a two-way ANOVA followed by a Dunnett’s multiple comparisons test. Significance is represented by p < 0.001 and ns (not significant) for p > 0.05.