Search

Mycolic acids, the hallmark of mycobacteria and related bacteria, are major and specific components of their cell envelope and essential for the mycobacterial survival. Mycobacteria contain structurally related long-chain lipids, but the metabolic relationships between these various classes of compounds remain obscure. To address this question a series of C(35) to C(54) nonhydroxylated fatty acids (mycobacteric acids), ketones, and alcohols structurally related to the C(70-80) dicyclopropanated or diethylenic mycolic acids were characterized in three mycobacterial strains and their structures compared. The relationships between these long-chain acids and mycolic acids were established by following the in vivo traffic of (14)C labeled alpha-mycolic acids purified from the same mycobacterial species. The labeling was exclusively found in mycobacteric acids. The mechanism of this degradation was established by incorporation of (18)O(2) into long-chain lipids and shown to consist in the rupture of mycolic acids between carbon 3 and 4 by a Baeyer-Villiger-like reaction. We also demonstrated that mycobacteric acids occur exclusively in the triacylglycerol (TAG) fraction where one molecule of these acids esterifies one of the three hydroxyl groups of glycerol. Altogether, these data suggest that these compounds represent a pathway of metabolic energy that would be used by mycobacteria in particular circumstances.

Because of their structures, phleic acids (general formula: CH3-(CH2)m-(CH=CH-CH2-CH2)n-CO2H; main component: m = 14, n = 5) cannot be synthesized by the same kinds of enzymatic systems as other natural polyunsaturated fatty acids. By using specifically labelled 14C compounds, we have tested the ability of different molecules to be incorporated in the phleate skeletons by Mycobacterium phlei. The localisation of radioactive carbon atoms has been studied by chemical degradation of labelled phleates, isolation and purification of the degradation products, and determination of their specific radioactivity. When M. phlei cells are incubated with labelled acetate, the unsaturated and saturated parts of the molecules of phleic acids are unequally labelled. The radioactivity of succinate monoester on the one hand and fatty acids (mixture of myristic and palmitic acids) on the other hand, measured after oxidative degradation of phleate esters, shows a constant ratio under definite conditions. Whether [1-14C]acetate or [2-14C]acetate is used for incubation, the same ratio is observed. Therefore acetate is the precursor of the unsaturated part as well as of the saturated part of the phleate molecules. By using labelled fatty acid esters, it has been found that palmitic acid is the precursor of phleates with m = 14, while myristic acid is the precursor of phleates with m = 12. Stearic and eicosanoic acids are not incorporated without degradation. The hypothesis of a condensation of a saturated fatty acid with a preformed polyunsaturated molecule was examined. Search for such a molecule in the lipids of M. phlei gives negative results. Pentaunsaturated phleate arising from palmitate is more abundant than pentaunsaturated phleate arising from myristate, while the reverse is true for hexaunsaturated phleates. These observations make very unlikely such an hypothesis. An elongation process fits well with the observed facts provided that this process involves elongation by two acetate units simultaneously, making elongation by four carbon atoms at a time. Such a requirement would be easily satisfied if two molecules of acetate are condensed together before their utilization in the elongation process. In such a hypothetical process, crotonate would be the most probable substrate of the elongation reaction.

Synchronous cultures of Mycobacterium aurum were used to prove a close relationship between cellular division and active synthesis of mycolic acids (characteristic long-chain 3-hydroxyacids, branched at position 2), confirming previous proposals. Mycolic acid biosynthesis was studied in two species (Mycobacterium phlei and M. aurum) each producing three types of mycolic acids: di-unsatured mycolates, oxomycolates and wax-ester mycolates (ester of dicarboxymycolic acid and 2-icosanol or 2-octadecanol). It was shown that unsaturated mycolates and oxomycolic acids were not directly related, whereas a metabolic filiation was confirmed between oxomycolate and wax ester mycolate: the latter derived from the former by a Baeyer-Villiger oxidation step, as has been proposed on the basis of structural considerations. By observing the labelling of the different mycolate pools in the cell, i.e. the organic-solvent-extractable fraction (essentially containing esters of trehalose and of glycerol) and the cell residue (assumed to be the cell-wall polymers), it was clear that oxomycolates and unsaturated mycolates appeared first in the extractable lipids, then in the wall-linked mycolates while wax-ester mycolates appeared first as wall-linked derivatives. Thus, it is proposed that mycolates could follow separate routes involving differently located enzymes to reach their complex forms either in extractable lipids or in the wall-linked arabino-galactan.

The biosynthesis of mycolic acids was studied in whole cells of Mycobacterium fortuitum. At first the structures of the main mycolates produced by the used strain were established as diunsaturated and epoxymycolates. By using [1-14C]acetate as a radiotracer of the lipid synthesis, it was observed that the turnover of the mycolates during the exponential phase of growth of M. fortuitum is fast enough to make very difficult the identification of their precursors. If the growth of the bacterial cells is stopped or highly diminished, by the removal of a large part of their nutritional medium, mycolate synthesis, in contrast to the synthesis of other fatty acids, is stopped as shown by incubation of the concentrated bacterial culture with [1-14C]acetate. After removal of aliquots of the sedimented bacteria at intervals, during several hours, mycolate synthesis resumes when the cell concentration becomes lighter. In these conditions the sequence of radiolabeling of mycolates and of their potential precursors (tetracosanoate and meromycolates) can be observed. In spite of their low accumulation, tetracosanoate and meromycolates were isolated and purified and their specific radioactivity, after different incubation times, could be measured. The results are in agreement with the hypothesis that meromycolates are condensed with tetracosanoate to produce mycolates. However, because of the large differences of isotopic dilution of these two precursors inside the mycolate molecule, this hypothesis, generally taken as evidence, has to be modified. A hypothetical pathway of the mycolate synthesis is proposed, taking into account all these observations.