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1 Supporting Information Bansal-Mutalik and Nikaido /pnas Relative lipid release (% ) Relative lipid release (%) Extraction time (minutes) Step 1 Step 2 Step 3 Number of extraction stage (c) 1 (d) 1 R ela tiv e lipid relea se (% ) Water content of RMS (% v/v) Relative lipid release (%) AOT concentration (mm) Fig. S1. Optimization of RMS extraction for maximum lipid release from C. glutamicum. Error bars represent SDs. (A) Effect of time: Separate aliquots of wet cells corresponding to similar dry weights were extracted with 25mM AOT in heptane (containing 0.45% (vol/vol) water) for varying times; Values expressed as % lipid release relative to that in maximum time. (B) Effect of number of extraction cycles: An aliquot of wet cell of known dry weight was subjected to multiple extractions of 15 min each under conditions similar to A; Values are expressed as % lipid release relative to that in first extraction cycle. (C) Effect of % (vol/vol) of water in the RMS: Separate aliquots of wet cells corresponding to similar dry weights were extracted with RMS of 25mM AOT in heptane containing varying amounts of water, for 15 min each; Values expressed as % lipid release relative to that with maximum water content. (D) Effect of AOT concentration. Separate aliquots of wet cells corresponding to similar dry weights were extracted with varying concentrations of AOT in heptane (containing water at molar concentrations 10 times higher than that of AOT) for 15 min each. Values are expressed as % lipid release relative to that with maximum AOT concentration. 1of6

2 Relative lipid release (% ) Extraction time (minutes) Relative lipid release (%) Step 1 Step 2 Step 3 Number of extraction stage Fig. S2. Optimization of extraction for maximum lipid release from C. glutamicum. Error bars represent SDs. (A) Effect of time. Separate aliquots of wet cells corresponding to similar dry weights were extracted with for varying times; Values expressed as % lipid release relative to that in maximum time. (B) Effect of number of extraction cycles. An aliquot of wet cells of known dry weight was subjected to multiple extractions of 15 min each. Values are expressed as % lipid release relative to that in first extraction cycle. Top C 12 -C 20 FAMEs Mycolols PI Rf~0.24 TLC origin Primary alcohols PI Top Fig. S3. (A) TLC profile of extract of C. glutamicum using (30:8:1) as the developing solvent. Individual lipid s from this TLC profile were subjected to transmethylation using 15% aqueous tetrabutyl ammonium hydroxide. (B) This was followed by TLC of the products with petroleum-ether:diethyl ether (85:15). FAME, fatty acid methyl ester; cmame, corynomycolic acid methyl ester. 2of6

3 C 12 -C 20 fatty acid methyl esters Top region TLC origin Primary alcohols (R 1 -CH 2 OH) Top region Fig. S4. (A) TLC profile of RMS extract of C. glutamicum using (30:8:1) as the developing solvent. Individual lipid s from this TLC plate were subjected to transmethylation using 15% aqueous tetrabutyl ammonium hydroxide. (B) This was followed by TLC of the products with petroleum-ether:diethyl ether (85:15). cmame, corynomycolic acid methyl ester. Fig. S5. ESI-MS profile of the major glycolipid seen in the and RM extract, identified as (isolated from the TLC plate of extracts of intact cells). 3of6

4 (d) (c) RMS Std Std TP OA RMS std TP Std OA Std Fig. S6. Identification of lipids in the TLC profiles of and RMS extracts using various 1D-TLC developing systems and lipid standards. (A) TLC with chloroform:methanol (9:1) for identification of the OM-specific glycolipid of modest polarity, visualized by anthrone reagent. (B D) TLC for identification of triacylglycerols and FFAs with petroleum ether:diethyl ether (9:1) (B), petroleum ether:diethyl ether (7:3) (C), and hexane-diethyl ether-acetic acid (70:30:1) (D). Visualization was done for all lipid groups by spraying with phosphomolybdotungstate reagent (B and C) or by phosporimaging of radioactive lipids (D). Samples:, extract; RMS, RMS extract. Standards: std TP, tripalmitin (Sigma-Aldrich); std OA, oleic acid (Sigma-Aldrich); std, radiolabeled mycolic acid isolated from Mycobacterium smegmatis mc DAGs DAGs UI 1 UI 1 UI 2 UI 2 FFA PI+PIMs PIMs PI+PIMs DAG (c) DAG FF A FFA PIMs PI+PIMs (d) PI (e) (f) Fig. S7. 2D-TLC profiles of lipids from C. glutamicum using (30:8:1) in the first direction and hexane-diethyl ether-acetic acid (70:30:1) in the second direction, followed by phosphorimaging. (A C) TLC profiles of lipids from fresh cells. (D F) TLC profiles of lipids from cells that were kept frozen for more than 1 mo. (A and D) extract of whole cells. (B and E) RMS extract of whole cells. (C and F) extract of cells pretreated with RMS. PG, phosphatidylglycerol; CL, cardiolopidin; DAGs, diacyl gycerols;, free (coryno)mycolic acids; Ul, unidentified lipid. 4 of 6

5 C12-C20 FAMEs Origin Fig. S8. Fatty acid methyl esters obtained after alkaline hydrolysis and transmethylation of delipidated (-extracted) cells. Fatty acid esters were converted into methyl esters by phase-transfer catalysis as described in Experimental Procedures. 2D-TLC was performed with petroleum ether-acetone (95:5) (three times) in the first direction and with toluene-acetone (97:3) in the second direction. cmame, corynomycolic acid methyl ester; FAME, fatty acid methyl ester. 1 2 C 12 -C 20 fatty acid methyl esters extracted extract of cell envelope cell envelope Fig. S9. TLC profiles of the fatty acid methyl esters from cell envelope obtained by alkaline hydrolysis with 15% aqueous tetrabutyl ammonium hydroxide followed by transmethylation. The samples were the cell wall after extraction, intended to remove the IM lipids as well as the extractable lipids of OM (lane 1) and the extract of the cell envelope (lane 2). TLC was done with petroleum-ether:diethyl ether (85:15). cmame, corynomycolic acid methyl ester. GC-MS quantitation of the methyl esters of C12-20 fatty acids found that the sample in lane 1 contained only 1% of the amount in the sample in lane 2. 5of6

6 60 C12:0 C14:0 C16:1 C16:0 C17:0 C18:1 C18:0 TS C20:0 C21: Total cell envelope FAs OL of OM FAs (RMS extract of intact cells) IM FAs (-extract of RMS-extracted cells) PG associated FAs ( extract of lysozyme-treated delipidated cells) Fig. S10. Relative abundance of individual fatty acids in various fractions. The figure shows the distribution, from left, in the total cell envelope, the outer leaflet (OL) of OM (RMS extract of cells), IM ( extract of cells preextracted with RMS), and peptidoglycan-embedded fraction ( reextract of lysozymetreated delipidated cells). Fatty acids were converted into methyl esters as described in Experimental Procedures, and then separated and quantitated using GC-MS. Note that mycolic acid esters are not eluted in the GC system used. TS, tuberculostearic acid. Error bars represent SDs. 6of6