Total mycolic acid methyl ester fractions were isolated from 24 representatives of Mycobacterium tuberculosis, Mycobacterium bovis (including BCG), Mycobacterium microti, Mycobacterium kansasii and Mycobacterium avium. The total mycolate functional group composition was estimated from 1 H-NMR spectra. Mycolates were separated into α-mycolates, methoxymycolates and ketomycolates and each class was further separated by argentation chromatography into mycolates with no double bonds, with one trans-double bond and with one cis-double bond. Mass spectrometry revealed the mycolate chain lengths and 1 H-NMR the cis-and trans-double bond and cyclopropane ring content. The same species had similar mycolate profiles ; the major type of each class had cis-or trans-cyclopropane rings and lacked double bonds. Minor proportions of possible unsaturated precursors of the cyclopropane mycolates were commonly encountered. Among unusual α-mycolates, many strains had tricyclopropyl components with chains extended by 6 to 8 carbons.Significantly, M. tuberculosis (Canetti) and M. avium had α-mycolates with a trans-double bond and cyclopropane ring, whose chain lengths suggested a relationship to possible precursors of oxygenated mycolates. The methoxyand ketomycolates from a majority of M. tuberculosis strains had minor amounts of components with additional cyclopropane rings, some of whose chains were also extended by 6 to 8 carbons. These latter mycolates were major components in the attenuated M. tuberculosis H37Ra strain, whose mycolate profile was distinct from those of other strains of M. tuberculosis.Keywords : mycobacterial mycolates, cyclopropane rings, double bonds, stereochemistry, tuberculosis INTRODUCTIONMycobacterial mycolic acids, whose general structures are indicated in Fig. 1, are high-molecular-mass 2-alkyl branched, 3-hydroxy fatty acids, which are characteristic components of the cell envelope of mycobacteria.Although they are present in the extractable lipids, mostly as trehalose dimycolates, the major portion is covalently bound in the cell envelope esterified to the 5-hydroxy groups of arabinofuranosyl residues to form the terminal [5-mycoloyl-β-Araf-(1 2)-5-mycoloyl-α-Araf-(1 )] units of a major arabinogalactan polysaccharide (McNeil et al., 1991 galactan is, in turn, attached to the shape-forming peptidoglycan. Schematic models of mycobacterial envelope structure have been proposed to show possible arrangements for the mycolic acids and other lipid components (McNeil & Brennan, 1991 ;Minnikin, 1991 ;Paul & Beveridge, 1994). According to the currently accepted structural model (Brennan & Nikaido, 1995 ;Daffe! & Draper, 1998 ;Draper, 1998), the long hydrocarbon chains of the mycolic acids are arranged in an orderly fashion, in parallel, with the methyl ends towards the outside surface. This arrangement results in the electrontransparent layer of the cell envelope, the presence of which is revealed by electron micrographs of ultra-thin sections of mycobacterial cells (Paul & Beveridge, 1992 in the major ' merom...

Mycobacterial α-, methoxy-and keto-mycolic acid methyl esters were separated by argentation chromatography into mycolates with no double bond, with one trans double bond or with one cis double bond. Meromycolic acids were prepared from each methyl mycolate fraction by pyrolysis, followed by silver oxide oxidation, and analysed by high-energy collision-induced dissociation/fast atom bombardment MS to reveal the exact locations of the functional groups within the meromycolate chain. The locations of cis and trans double bonds, cis and trans cyclopropane rings, methoxy and keto groups, and methyl branches within the meromycolate chain were determined from their characteristic fragment ion profiles, and the structures of the meromycolic acids, including those with three functional groups extracted from Mycobacterium tuberculosis H37Ra, Mycobacterium bovis BCG and Mycobacterium microti, were established. Meromycolic acids with one cis double bond were structurally closely related to those with one cis cyclopropane ring, whereas the meromycolic acids with one trans cyclopropane ring were closely related to the corresponding meromycolic acids with one cis cyclopropane ring. A close relationship between methoxy-and keto-meromycolic acids was also implied. The relationship between the meromycolic acids with a trans double bond and the other meromycolic acids was not clearly revealed, and they did not appear to be immediate substrates for trans cyclopropanation.

Phase diagrams of the Langmuir monolayer of dicyclopropyl alpha mycolic acid (alpha-MA), cyclopropyl methoxy mycolic acid (MeO-MA), and cyclopropyl ketomycolic acids (Keto-MA) from Mycobacterium tuberculosis were obtained by thermodynamic analysis of the surface pressure (pi) vs. average molecular area (A) isotherms at temperatures in the range of 10-46 degrees C. The Langmuir monolayers of MAs were shown to exhibit various phases depending on the temperature (T) and the pi values. In the Langmuir monolayer of Keto-MA, the carbonyl group in the meromycolate chain apparently touches the water surface to give the molecule a W-shape in all the temperatures and surface pressures studied. Keto-MA formed a rigid solid condensed film, with four hydrocarbon chains packing together, not observed in the others. In contrast, the monolayer films of alpha-and MeO-MAs having no such highly hydrophilic intra-chain groups in the meromycolate chain were mostly in liquid condensed phase. This novel insight into the packing of mycolic acids opens up new avenues for the study of the role of mycolic acids in the mycobacterial cell envelopes and pathogenic processes.

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Phase diagrams of Langmuir monolayers of oxygenated mycolic acids, i.e. methoxy mycolic acid (MeO-MA), ketomycolic acid (Keto-MA), and artificially obtained deoxo-mycolic acid (deoxo-MA) from Mycobacterium bovis BCG were obtained by thermodynamic analysis of the surface pressure (pi) vs. average molecular area (A) isotherms. At lower temperatures and lower surface pressures, both Keto- and MeO-MAs formed rigid condensed monolayers where each MA molecule was considered to be in a 4-chain form, in which the three carbon chain segments due to bending of the 3-hydroxy aliphatic carboxylate chain and the 2-side chain were in compact parallel arrangement. At higher temperatures and surface pressures, MeO-MA and deoxo-MA tended to take stretched-out conformations in which the 3-hydroxy aliphatic carboxylate chain was more or less in an extended form, but Keto-MA retained the original 4-chain structure. The thickness measurement of the monolayers in situ by ellipsometry at different pi values and temperatures supported the above conclusions derived from the phase diagrams. The enthalpy changes associated with the phase transitions of MeO-MA and deoxo-MA implied that the MeO-MA needed larger energy to change from a compact conformation to an extended one, possibly and partly due to the dehydration of the methoxy group from water surface involved. Molecular dynamics studies of MA models derived from Monte Carlo calculations were also performed, which confirmed the conformational behavior of MAs suggested by the thermodynamic studies on the Langmuir monolayers.