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

mycolate analogues in their envelopes, strongly suggests that an exchange of a native β-hydroxy

group for an unnatural β-oxo unit is permitted. However, as discussed later, the ∆MSMEG4722
mutant cells were more permeable to lipophilic antibiotics and colony morphology was affected.
Further studies will be needed to clarify these intriguing observations.

Additionally, replacement of TMM and TDM with derivatives with slightly altered TLC
mobility in ∆MSMEG4722 (Figure 4A) suggested that the α-alkyl, β-oxo mycolate precursors
were also esterified to trehalose. Indeed, we obtained similar results for MAME analysis from
extracts of whole cells (which contain wall bound and trehalose bound mycolates) as well as
delipidated cells (which contain only wall bound mycolates) demonstrating that mycolic acids in
both the mAGP complex and in TMM/TDM were replaced by the α-alkyl, β-oxo fatty acid
precursors.

While the loss of the reductase was expected to generate precursors of mycolic acids, it
was surprising that the α-alkyl, β-oxo fatty acids were associated with the cell wall. These data
suggested that mycobacterial components involved in the processing, transport and subsequent
transfer of mycolic acids to the cell wall (including the hypothetical mycolyl transferases I and II
and the proteins of the Antigen 85 complex; [(Takayama et al., 2005)]) were probably able to do
the same with the α-alkyl, β-oxo fatty acid intermediates. In contrast, Lea-Smith et al (Lea-
Smith et al., 2007) reported reduced levels of AG-mycolylation in the C. glutamicum reductase
mutant (∆NCgl2385). Corynomycolate derivatives released from mutant cell wall were distinct
from wild type corynomycolates and showed a ~80% reduction in abundance. However, the
extraction method used in this previous study involved acid-methanolyis. In light of our findings
it is likely that rather than a reduction in mycolylation, the ∆NCgl2385 mutant contained equally

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