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

α-alkyl, β-oxo fatty acid intermediates would be expected to occur. Free acids of such
intermediates would then undergo decarboxylation to form the ketones that comprise Lipid-Y.
Using EI-MS we were able to confirm that this was indeed the case.

Environmental mycobacteria are known alter cell wall mycolate composition in response
to growth substrates resulting in a more hydrophobic wall when grown in the presence of
hydrophobic substrates (Wick et al., 2002). It is not clear whether mycolate reduction is
regulated by environmental factors but our studies herein have shown that loss of the β-hydroxy
mycolate-motif reduction alters cell wall hydrophobicity. Whether this has an effect in vivo (in
the case of M. tuberculosis) remains to be studied.

In conclusion, our results clearly demonstrate that MSMEG4722 is the reductase
involved in generation of the mycolic acid motif in M. smegmatis. The loss of this function is not
lethal, allowing cell wall incorporation of β-oxo mycolate analogues but affecting the growth
characteristics of the bacterium.

SIGNIFICANCE

Mycolic acid biosynthesis is essential for mycobacterial survival and many antituberculosis
drugs like isoniazid, ethionamide and thiolactomycin target enzymes of this exclusive pathway
(Banerjee et al., 1994; Kremer et al., 2000). Interestingly, MSMEG4722, which catalyses the
final step in mycolic acid biosynthesis in M. smegmatis, is non-essential and α-alkyl, β-oxo
mycolate precursors are attached to arabinogalactan and trehalose. However, we have
demonstrated that loss of function does cause major changes in the cell wall of M. smegmatis,

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