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Figure S1. (A) Genetic contexts of four cmpL genes encoded on Corynebacterium glutamicum chromosome. (B) Localization and orientation of cmpL1 and cmpL4 genes expressed under the IPTG-inducible Ptac promoter in LY115 and LY116.

Figure S2. Protein composition of membranes isolated from LY112 (Δcmpl1,2,3,4 [pML1-Tc]) and LY113 (Δcmpl1,2,3,4 [pML4-Tc]) in the presence and absence of IPTG. Membrane fractions were isolated by ultracentrifugation and analyzed by 12% SDS-PAGE. Overproduced CmpL1 and CmpL4 can be seen in unboiled samples (marked with asterisk).

Figure S3. Composition of lipids extracted from Corynebacterium glutamicum mutants. (A) Cells were grown in the minimal medium and noncovalently associated lipids extracted with CMW mixture. TLC was carried out with C:M:W 30:8:1 as the mobile phase and lipids were stained with 5% phosphomolybdic solution. (B) Mutant cells were grown in minimal medium. Lipids were extracted either with CMW (left panel) or RMS (right) mixtures, separated on TLC plates with C:M:W 30:8:1 as the mobile phase and stained with anthrone. (C). Glycolipid composition of LY114 and LY115 knock-in mutant strains. Lipids were extracted from cells grown in minimal medium with CMW and analyzed as in (B).

Figure S4. (A) LC/MS chromatogram of Lipid X. Component I which barely stuck to the reversed phase LC column was not further analyzed and may or may not be part of the Lipid X TLC spot. Component III is like component II except it is based on a C-34 fully saturated corynomycolate. (B) The mass spectrum of component II consistent with acetylated C-32 fully saturated corynomycolate (m/z = 537.4886) whose structure is shown on the right) and monounsaturated C-34 corynomycolate (m/z = 563.5049).

Figure S5. The structure of the methyl ester trimethylsilyl ether of C-32 corynomycolate as determined by GC/MS after formation of the methyl ester and trimethylsilylation of the alcohol. The m/z 207 ion (asterisk) is from column bleed. M-15 is 567.6. The original LC/MS analysis also had a complex early eluting peak containing ions at m/z 485.3854, 489.3359, and 521.3622 and the GC/MS had many components in addition to the corynomycolates. These possible other components have not been investigated.

Table S1. Strains and plasmids used in this study.

Table S2. Antibiotic susceptibility of Corynebacterium glutamicum cells lacking cmpLs.

Table S3. Antibiotic susceptibility of Corynebacterium glutamicum mutant cells overproducing various CmpL proteins.

Table S4. Primers used in this study.

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