Rf value (Rf 0.147 and Rf 0.39) than parental TMM and TDM (Rf 0.117 and Rf 0.36) in
direction 1. This was likely due to esterification of the α-alkyl, β-oxo mycolic acid precursors
rather than mature mycolic acids to trehalose in the mutant strain as was observed in the C.
glutamicum mutant (Lea-Smith et al., 2007). Furthermore, the mutant strain showed
accumulation of a non-polar species accompanied by a total loss of free mycolic acids (Figure
4B, C). This lipid species, referred to as Lipid-Y, did not stain with Dittmer-Lester reagent or
with α-napthol-sulfuric acid indicating the absence of phosphate groups and carbohydrates (data
not shown). Following purification by preparative TLC, Lipid-Y was characterized by MALDI-
TOF/MS, and NMR (Figure 5). Three species of m/z 907.6, 935.6 and 963.6 were observed in
the mass spectra (Figure 5A) indicating a difference of m/z 28 between each species. Further,
1H-NMR and 13C-NMR revealed a signal characteristic of -CH2- groups (1.3 ppm and 30 ppm,
respectively) and indicated the presence of alkyl chains differing (CH2)2 units. Additionally, 1H-
NMR and 13C-NMR provided evidence for the presence of cis and/or trans double bonds (1, 2, 3
, Figure 5B-D) with latter possessing an adjacent methyl branch, (5, Figure 5B-D) and a keto
group (14, Figure 5C-D) suggesting that Lipid-Y was a mixture of cis and trans-unsaturated long
chain ketones (Figure 5D). Based on the masses obtained by MALDI-TOF/MS, these
unsaturated, branched-ketones were of chain lengths C62, C64 or C66. Since the accumulation of
Lipid-Y was accompanied by a loss of mycolates (and no detectable free α-alkyl, β-oxo
mycolate precursors), it seemed likely that the ketones that comprised Lipid-Y were derived
from free β-oxo precursors. Free α-alkyl, β-oxo mycolate precursors could undergo
decarboxylation to form ketones. If this was the case, then the oxo group would be situated
between alkyl chains that originate from a meroacid on one side and the α-branch on the other.
Using Electron Impact-Mass Spectroscopy (EI-MS) we were able to confirm the presence of a
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