esters (MAMEs) (Dobson et al., 1985) which are analysed by TLC. Following base-treatment
and derivatisation, [14C]- labelled extracts obtained from strains were analyzed by TLC. While
α, a´ and epoxy MAMEs were present in the parental mc2155 strain, all three species were
missing in the extract from the ∆MSMEG4722 strain (Figure 3A). Instead, the mutant strain
showed the accumulation of a product(s) with a higher retardation factor (Rf) migrating above the
methyl esters of fatty acids (FAMEs). When extracts from the mutant strain were analysed by
2D-Ag+-argentation TLC, the rapidly migrating species resolved into multiple subspecies in the
second, Ag+-containing dimension indicating the presence of multiple species differing in
degrees of unsaturation (Figure 3B). An identical result was obtained for extracts from
delipidated cells which only contain cell-wall bound mycolates (Figure 3A) indicating that the
observed changes in mycolate profiles applied to both total and specifically cell wall bound
mycolates.
Mycolic acid biosynthesis was restored in the mutant strain following complementation
with not only MSMEG4722, but also Rv2509, indicating that Rv2509 was a functional
homologue of MSMEG4722 in M. tuberculosis (strains ∆MSMEG4722-C and ∆MSMEG4722-
CRv respectively, Figure 3A).
The ∆MSMEG4722 mutant accumulates precursors of mycolic acids in the cell wall
The complete absence of α, α' and epoxy MAMEs and appearance of new, rapidly migrating
species in extracts of the ∆MSMEG4722 mutant suggested that these new species may either be
precursors of mycolates or decomposition products of precursors generated as a result of the
extraction procedure. The latter seemed more likely as base treatment of an unreduced α-alkyl,