Figure 3.

Lipid compositions of MVs

(A) Trehalose dicorynomycolic acids (TDCMs) were detected from MVs using LC/MS. The structure of each TDCM was determined based on the results of GC/MS and LC/MS/MS analyses shown in Figures S12 and S14.

(B) Intensities of TDCM and phospholipids (PLs) in TLC analysis were compared using ImageJ. Each lipid was extracted from mycomembrane (MM), inner membrane (IM), and MV of Corynebacterium glutamicum, and then separated by TLC as described under Methods. All values indicated by the bars represent the mean value ± SD for three experiments.

(C) Mycomembrane-specific lipids (MMSLs) and inner membrane-specific lipids (IMSLs) of C. glutamicum cells under various growth conditions were identified by LC/MS analyses. In these analyses, we defined MMSLs and IMSLs as the lipids that were detected in either the mycomembrane (1-butanol extract) or the inner membrane extract (total of chloroform/methanol and chloroform/methanol/water extracts) of C. glutamicum cell under each of the designated culture conditions. Detailed information of these analyses is shown in Methods and Figures S15–S18. Denominator of each fraction indicates total number of specific lipids in the mycomembrane or the inner membrane extract of C. glutamicum cells under different culture condition, and the numerator of each fraction indicates total number of those MMSLs or IMSLs detected in MVs.

(D) Lipid compositions of mycomembrane, inner membrane, and MVs were analyzed. Mycomembranes and inner membranes were extracted separately from cells using 1-butanol and chloroform/methanol solutions, respectively. Lipids were quantified using TLC and standard lipids. All values indicated by the bars represent the mean value ± SD for three experiments.

(E) Cardiolipins were visualized using acridine orange 10-nonyl bromide. White arrowheads indicate the localization of cardiolipins. Scale bars, 2 μm.