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

abundant α-alkyl, β-oxo corynomycolate precursors esterified to the AG which were not
detected by GC and MS because of decomposition resulting from acid-methanolysis.

Incorporation of the α-alkyl, β-oxo fatty acid precursors, instead of mycolic acids in the
cell wall had a significant change in the characteristics of the cell wall of M. smegmatis
rendering the mutant strain more susceptible to lipophilic antibiotics due to an increased
permeability. The presence on a β-oxo rather than a β-hydroxyl group also affected the colony
morphology of the mutant strain, presumably due to changes in the hydrophobicity of the outer
surface of the bacterial cells.

A key distinction between the reductase mutants of C. glutamicum and M. smegmatis was
the accumulation of an unusual lipid, Lipid-Y, in the latter. MS and NMR analysis of purified
Lipid-Y revealed it to be a mixture of unsaturated, branched ketones. Similar ketones were
detected in strains of Mycobacterium tuberculosis (tuberculenone) and Corynebacterium
diptheriae (Asselineau, 1954; Pudles and Lederer, 1954). The total number of carbons (C60±C3)
in the mono unsaturated M. tuberculosis ketone, tuberculenone is similar to those of Lipid-Y
(C62-C66) in the MSMEG4722 mutant. However, tuberculenone was characterised before the
advent of mass spectrometry (Asselineau, 1954) so precise comparisons are not meaningful. It
has been suggested that ketones like tuberculenone are derived from decarboxylation of the α-
alkyl, β-oxo fatty acid intermediates of mycolic acids (Asselineau, 1966). In addition to cell wall
bound and glycolipid associated mycolates, mycobacteria also contain free mycolic acids and
would be expected to contain some transient, unreduced mycolic acid intermediates at any given
time. It is likely that tuberculenone is derived from these intermediates. In the ∆MSMEG4722
mutant however no free mycolic acids were detected (Figure 4C). Instead, an accumulation of

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