SUPPLEMENTARY FIGURES

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1 SUPPLEMENTARY FIGURES Fig. S1: Effect of ISO- and TAC-treatments on the biosynthesis of FAS-II elongation products in M. tb H37Ra. LC/MS chromatograms showing a decrease in products with elemental compositions (deduced from the exact masses of the [M-1] - at and ) consistent with C34 monounsaturated and C52 diunsaturated meromycolate precursors in the ISO- and TAC-treated cells. Drug treatments and sample processing were as described in Fig. 3B. C34:1 untreated ISO-treated TAC-treated untreated C52:2 ISO-treated TAC-treated Counts versus acquisition time (min)

2 Fig. S2: GC/MS total ion chromatograms of M. tb H37Ra untreated or treated with ISO or TAC. Drug treatments and sample processing were as described in Fig. 3B. The percent areas of the fatty acids to the total areas of the GC/MS chromatograms in the untreated and ISO- or TAC-treated cells are shown in the table below. GC/MS chromatograms show a 62 and 81% increase in C26 fatty acid in the ISO and TAC-treated samples relative to the untreated control, respectively, probably reflecting the inhibition of mycolic acid synthesis in the drug-treated cells and, thus, the build-up of the α-alkyl C26 fatty acid branch originating from FAS-I (7). The percent areas represented by oleic and tuberculostearic acids show a 13.4 and 17.7% decrease in the ISO- and TAC-treated cells, respectively. C18:1 untreated C18:0 C22:0 C16:0 C19:0 C20:0 C24:0 C26:0 ISO-treated TAC-treated Retention time (min) Percent area of the fatty acids to the total area of the chromatogram Control ISO treated TAC treated Palmitic acid methyl ester (C16:0) Oleic acid methyl ester (C18:1) Stearic acid methyl ester (C18:0) Tuberculostearic acid methyl ester (C19:0) Eicosanoic acid methyl ester (C20:0) Behenic acid methyl ester (C22:0) Tetracosanoic acid methyl ester (C24:0) Hexacosanoic acid methyl ester (C26:0)

3 Fig. S3: Mycolic acid profile and fatty acid composition of a hadc transposon mutant of M. tb CDC1551. Mycolic acid composition - α, methoxy- and keto- MA methyl esters were prepared from the same amount of whole wild-type (wt) and transposon mutant (mut) cells, analyzed vol. to vol. by TLC in the solvent system [n-hexanes:ethyl acetate] (95:5, 3 developments) and revealed by spraying with cupric sulfate (10% in a 8% phosphoric acid solution) and heating. A decrease in methoxy-mycolates is visible in the mutant strain. The position of insertion of the transposon is at bp 1 of hadc in the mutant strain [BEI Resources mutant NR-18624]. α methoxy keto wt mut Fatty acid composition - Fatty acids were extracted from the same hadc transposon mutant of M. tb CDC1551 and the WT parent strain and processed for LC/MS and GC/MS analysis as described under Experimental Procedures. The percent areas of the fatty acids of interest to the total areas of the LC/MS or GC/MS chromatograms in the mutant cells (red bars) was compared to that of the corresponding fatty acids in the WT parent cells (blue bars) and values expressed as fold change (increase or decrease) over the WT parent values arbitrarily set to 1 for each fatty acid species. Note the build-up of 3-hydroxy -C18 up to -C30 fatty acids (as determined by GC/MS) and the overall decrease in downstream FAS-II elongation products identified as di-unsaturated C43 to C54 fatty acids (as determined by LC/MS) in the transposon mutant. 3-OH C18-C30 fatty acids Fold change over the WT cells Fold change over the WT cells Di-unsaturated C43-C54 fatty acids

4 Fig. S4: Effect of ISO treatment on the synthesis of mycolic acids by the M. tb spontaneous ISOresistant mutants ISO-R10, ISO-R1 and ISO-R12. ISO-R10, ISO-R1 and ISO-R12 were grown on 7H11-OADC agar plates in the presence of various concentrations of ISO. The bacteria were then harvested, delipidated and the MA methyl esters prepared from delipidated cells were analyzed by TLC in the solvent system [n-hexanes:ethyl acetate] (95:5, 3 developments) and revealed by spraying with cupric sulfate (10% in a 8% phosphoric acid solution) and heating. ISO-R1 ISO-R10 ISO-R12 α methoxy keto [ISO] µg ml H37Rv WT No ISO

5 Fig. S5: Restoration of oxygenated MA synthesis upon complementation of ISO-R2 and ISO-R10 with a WT copy of mmaa4 from M. tb H37Rv expressed from pcg76-mmaa4. MA methyl esters were prepared from delipidated cells, analyzed by TLC in the solvent system [nhexanes:ethyl acetate] (95:5, 3 developments) and revealed by spraying with cupric sulfate (10% in a 8% phosphoric acid solution) and heating. 1, M. tb H37Rv/pCG76; 2, M. tb H37Rv/pCG76-mmaA4; 3, ISO- R2/pCG76; 4, ISO-R2/pCG76-mmaA4; 5, ISO-R10/pCG76; 6, ISO-R10/pCG76-mmaA4.

6 Fig. S6: Metabolism of ISO and TAC by purified EthA and MmaA4 in vitro. A recombinant His-tagged forms of EthA was purified from M. smegmatis mc as described previously (11). MmaA4 was purified from and E. coli BL21 DE3 plyss/pet15b-hma as described by Boissier et al. [2006; J. Biol. Chem. 281(7), ]. All enzymatic reactions were performed in pentaplicates and the reaction mixtures contained 100 mm KCl, 0.2 mg ml -1 bovine serum albumin, 2 mm NADPH, 2 mm SAM, 10 µg ml -1 of TAC or ISO in DMSO (final concentration, 1 %), purified recombinant enzyme(s) (32 µg of EthA protein or 200 µg of MmaA4 protein) and 50 mm Tris HCl ph 7.5 in a final volume of 2 ml. Reactions run for 1 h at 37 C and were stopped by the addition of 2.4 ml of ethyl acetate. The organic and water phases were partitioned, the organic phase pentaplicates combined, dried under nitrogen and finally analyzed by LC-MS. The LC/MS analysis of ISO and TAC metabolites was carried out on the same Agilent LC/MS as described for the fatty acids with the following changes. The ESI/APCI source was in the positive mode. A Zorbax 2.1 x 50 mm SB-C µm column was used. Solvent A was 10 mm trifluoroacetic acid in water and Solvent B was 10 mm trifluoroacetic acid in acetonitrile. The solvent program was 10% solvent B in solvent A for 2 min, followed by a linear gradient to 90% solvent B over the next 10 min followed by a 10 min wash at 90% solvent B. The flow rate was 0.32 ml min -1. Table: In vitro activation of ISO and TAC by EthA, MmaA4 or EthA+MmaA4. The metabolites of each drug were analyzed by LC/MS (see chromatograms on next page) and their percent area relative to total metabolites (including ISO and TAC) were determined. The metabolites of ISO and TAC were as described in refs 11 and 12. [M+H] of ISO metabolites Sample (ISO) ISO ISO + EthA ISO + MmaA ISO + EthA + MmaA [M+H] of TAC metabolites Sample (TAC) TAC TAC + EthA TAC + MmaA TAC + EthA + MmaA

7 LC/MS total ion chromatograms of in vitro ISO and TAC activation showing the metabolites of both drugs. ISO ISO ISO + EthA ISO + MmaA4 ISO + EthA + MmaA4 purified EthA protein purified MmaA4 protein TAC TAC TAC + EthA TAC metabolites TAC + MmaA4 TAC + EthA + MmaA4

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