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



delipidated cells and the whole cell pellets were subjected to alkaline hydrolysis using 5 %
aqueous tetrabutylammonium hydroxide (TBAH) at 100°C overnight, followed by the addition
of 4 ml of CH2Cl2, 500 μl of CH3I, 2 ml of water, followed by mixing for 30 min. The upper
aqueous phase was discarded following centrifugation and the lower organic phase washed thrice
with water and evaporated to dryness. The resulting fatty acid methyl esters (FAMEs) and
MAMEs were dissolved in diethyl ether, insoluble residues were removed by centrifugation and
the ether solution evaporated to dryness and re-dissolved in 200 μl of CH2Cl2. Equivalent
volumes of the resulting solution of FAMEs and MAMEs was subjected to thin-layer
chromatography (TLC) using silica gel plates (5735 silica gel 60F
254; Merck, Darmstadt,
Germany), developed in petroleum ether-acetone (95:5). Autoradiograms were produced by
overnight exposure of Kodak X-Omat AR film to the plates to reveal [14C]-labelled FAMEs and
MAMEs. Argentation-TLC was performed as above after saturation of TLC plates with 10%
aqueous silver nitrate solution and prior activation at 100°C for 1 h. Lipid-Y was purified by
preparative silica gel TLC, using petroleum ether:ethyl acetate (98:2, v/v) and detection by
spraying with ethanolic Rhodamine 6G to visualize the lipid under a 366 nm U.V. lamp. The
area containing Lipid-Y was removed and extracted from the silica gel, using diethyl ether. The
extracted sample was then resolved on a second TLC plate in toluene:acetone (95:5, v/v) and
purified as above. Matrix-Assisted Laser Desorption Ionisation-Time of Flight/Mass
Spectroscopy (MALDI-TOF/MS) of all samples was done using the Voyager DE-STR MALDI-
TOF instrument (PerSeptive Biosystems, Framingham, MA). Nuclear Magnetic Resonance
(NMR) spectra for Lipid-Y were recorded in CDCl
3 on a Bruker DRX500 operating at 500.13
MHz for 1H-NMR and 125.77 MHz for 13C-NMR.

19



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