Fig. 2

Fluorescent labeling of M. marinum upon treatment with 3-azido pHB. (A) Workflow of labeling experiments with 2- or 3-azido pHB. M. marinum was treated for 18 h in the presence of various concentrations of 2- or 3-azido pHB, followed by staining with AF647-DBCO (30 μM). Cells were then fixed and analyzed by flow cytometry. (B) Flow cytometry analysis of the labeled cells. Relative MFI is determined by normalizing against the DMSO control. Flow cytometry data are the averages of three independent replicates. Statistical analysis was performed using a two-way ANOVA followed by a Dunnett’s multiple comparisons test. Significance is represented as follows: ***p < 0.001, ****p < 0.0001, and ns (not significant) for p > 0.05. (C) Effects of bacterial growth (A600) of M. marinum treated with 2- or 3-azido pHB. The data represent three independent replicates. Statistical analysis was performed using an ordinary two-way ANOVA, followed by Šídák’s multiple comparisons test. Significance is represented by ns (not significant) for p > 0.05. (D) TLC analysis of crude lipid extracts from M. marinum cells treated with 3-azido pHB and stained with AF647-DBCO. Crude lipid extracts (50 μg) or AF647-DBCO (1 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 using the 700 nm wavelength. (E) Confocal images of M. marinum cells treated with 750 μM 3-azido pHB and stained with AF647-DBCO. DIC = differential interference contrast. Scale bar = 2 μm.