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



findings with molecular sizes detected in all strains corresponding to those of α, α', epoxy
MAMEs and to the reduced, hydroxylated products of epoxy MAMEs (Table 2). Similar results
were obtained when the analyses were performed on delipidated cells indicating that the
α-alkyl,
β-oxo fatty acyl precursors of mycolates from M. smegmatis were esterified to the AG in the cell
wall (Figure 3C). However, in this case only a single MAME-I hydroxylated component was
produced on NaBH
4-reduction of the delipidated cells (Figure 3C).

The NaBH4-reduction of the β-oxo group, in a mycolate precursor, would result in two
diastereoisomers of MAMEs which would migrate differently on TLC (Minnikin and Polgar,
1966). MAME-II, therefore, was likely to be a mycolate
β-epimer and, on MALDI-TOF/MS,
the molecular size was found to be identical to that of
α-MAMES. Indeed, purified MAME-II
co-migrated with a known standard for α-MAME
β-epimer (data not shown). These results
showed that the
MSMEG4722 mutant, due to loss of mycolyl reductase function, failed to make
mature mycolic acids and instead synthesized the
α-alkyl, β-oxo fatty acyl precursors of α, a´
and epoxy mycolates that were transported and subsequently esterified to the reducing termini of
the AG-complex.

Lipid analysis revealed accumulation of a non-polar lipid in the MSMEG4722 mutant

Polar and non-polar lipids labelled with [14C]-acetate were extracted from mc2155,
MSMEG4722 and MSMEG4722-C strains and analyzed by 2D-TLC (Dobson et al., 1985).
Interestingly, the lipids corresponding to TMM and TDM in mc2155 were replaced by two lipids
with slightly altered mobilities in the mutant strain (Figure 4A). Both lipids had a slightly higher



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