FIG 2.

Individual Mce4 proteins, Mam proteins, and OmamA and OmamB proteins are required for M. smegmatis to grow on and take up cholesterol. (A to E) Growth of 10⁴ CFU of M. smegmatis strains with cholesterol as the sole carbon source was measured over time, using resazurin reduction as a fluorescent readout of metabolic activity. Cholesterol growth curves measured by resazurin reduction by Δmce4A and complemented strains (A), Δmce4E, Δmce4E^polar, and complemented strains (B), Δmam4A^polar and complemented strains (C), ΔomamA, ΔomamB, and complemented strains (D), and ΔomamCD and ΔomamEF mutant M. smegmatis strains (E) are depicted. (F and G) Cholesterol uptake was measured by incubating M. smegmatis strains with [4-¹⁴C]cholesterol for 3 h and measuring the cell-associated radioactivity using a scintillation counter. Measurements of cholesterol uptake were normalized as a percentage of the value of WT M. smegmatis, which was set to 100%. All strains were complemented with multicopy (mc) or single-copy (sc) plasmids as indicated. Mutant strains contain empty vectors (EV) as indicated. Error bars represent standard deviations. *, P < 0.001, compared to WT results. Results are representative of at least three independent experiments.