FIGURE 5.

The rates of TDM and mycolyl arabinogalactan biosynthesis in the M. smegmatis mmpL11 mutant are similar to those in the wild type. A, logarithmic phase cultures of wild-type M. smegmatis (mc²155/pVV16), the mmpL11 mutant (GP02/pVV16), and the complemented mutant (GP02/pVV16mmpL11) were pulsed with [¹⁴C]acetate for 5, 15, 30, and 60 min to label mycolic acids. A. To examine TDM levels, lipids were extracted and resolved by TLC in chloroform/methanol/ammonium hydroxide (80:20:2, v/v/v). A representative image is shown. Densitometry was performed to determine the intensity of the TDM spots, and the mean ± S.D. of three independent experiments was plotted over time. B, MAMEs of mycolyl arabinogalactan (mAG) were isolated from wild-type M. smegmatis (mc²155/pVV16) and the mmpL11 mutant (GP02/pVV16) and then resolved by TLC in petroleum ether/diethyl ether (9:1, v/v; 5 times). α, α′, and epoxy refer to the three mycolic acid forms present in M. smegmatis. A representative image is shown. Densitometry was performed to determine the intensity of mycolyl arabinogalactan, and the mean ± S.D. of three independent experiments was plotted over time. C, uptake of acetate by wild-type M. smegmatis (mc²155/pVV16) and the mmpL11 mutant (GP02/pVV16) at 37 °C. Analysis of the initial time points indicated uptake rates of 25.9 and 19.4 mmol/mg/min, respectively, with correlation coefficients of 1.0.