Deletion of a mycobacterial gene encoding a reductase leads to an altered cell wall containing β-oxo-mycolic acid analogues, and the accumulation of long-chain ketones related to mycolic acids

Colonies of wild type (mc²155), mutant (∆MSMEG4722) and complemented (∆MSMEG4722-C)
strains on TSB-agar (-) or TSB-agar + 0.05% Tween-80 (+). Colony growth shown in upper row
was obtained by inoculating 5μL of a broth culture on the agar plate while lower row shows
pictures of a single isolated colony of each strain. Scale bar =1mm. (C) Growth curve of wild
type (mc²155), mutant (∆MSMEG4722) and complemented (∆MSMEG4722-C) strains in TSB.

Figure 3 TLC analysis of mycolic acid methyl esters (MAMEs)

(A) TLC analysis of MAMEs extracted from mc²155, ∆MSMEG4722, ∆MSMEG4722-C and
∆MSMEG4722-CRv strains. The rapidly migrating species observed in ∆MSMEG4722 are
indicated by a question mark. FAMEs; fatty acid methyl esters. (B) 2D Ag⁺-TLC of MAMEs
extracted from mc²155, ∆MSMEG4722, ∆MSMEG4722-C. UFAMEs, unsaturated fatty acid
methyl esters. (C) TLC analysis of MAMEs extracted from cells pre-treated with NaBH4.

Figure 4 2D-TLC analysis of apolar lipids extracted from mc²155, ∆MSMEG4722 and
∆MSMEG4722-C strains

Panel (A), direction 1, chloroform:methanol:water, 100:14:0.8 (v/v); direction 2,
chloroform:acetone:methanol:water, 50:60:2.5:3 (v/v). Panel (B), direction 1, petroleum
ether:acetone, 98:2 (v/v, thrice); direction 2, toluene:acetone, 98:2 (v/v). Panel (C), direction 1,
chloroform:methanol, 96:4 (v/v); direction 2, toluene:acetone, 80:20 (v/v). TMM, trehalose
monomycolate; TDM, trehalose dimycolate; GMM, glucose monomycolate; FA, fatty acids;
MA, mycolic acids. Lipid-Y is indicated by an arrow and question mark in Panel (B).

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