FIG. 1.

Effect of Rv3779 overexpression on the incorporation of [¹⁴C]Man from GDP-[¹⁴C]Man into mannolipids by cell extracts from M. smegmatis. (A) TLC analysis of an in vitro cell-free assay using GDP-[¹⁴C]Man and crude cell lysates from mc²155/pVV16 and mc²155/pVV16-Rv3779. Lysates were incubated with GDP-[¹⁴C]Man at 37°C for the indicated periods of time. The synthesized mannolipids were extracted with CHCl₃-CH₃OH (2:1, vol/vol), and a 10% aliquot of each sample was analyzed by TLC followed by autoradiography. The TLC plate was developed in CHCl₃-CH₃OH-H₂O-NH₄OH (65:25:4:0.5). Lanes: C, mc²155/pVV16; O, mc²155/pVV16-Rv3779. (B and C) Incorporation of [¹⁴C]Man from GDP-[¹⁴C]Man into exogenous decaprenyl-phosphate (C₅₀)-P using membrane extracts from mc²155/pVV16 and mc²155/pVV16-Rv3779. (B) TLC analysis of the first 15 min of the in vitro cell-free assays using 0.5 mM of (C₅₀)-P. (C) Quantification of the Man incorporated by the control (open symbols) and overexpressor (filled symbols) into C₅₀-P-Man over time. The concentrations of (C₅₀)-P used in the assays were 0 (circles), 0.005 (rectangles), and 0.05 (diamonds) mM. The inset shows a closeup of results of the assays using 0 and 0.005 mM (C₅₀)-P.