Distribution of C₂₂-, C₂₄- and C₂₆-α-Unit-Containing Mycolic Acid Homologues in Mycobacteria

Abstract

There are three mycolic acid homologues with C₂₂-, C₂₄- and C₂₆-α-units in Mycobacterium. In order to reveal the composition and distribution of these homologues in each subclass and molecular species of mycolic acids and to compare them with the composition of constitutive non-polar fatty acids (free and ound forms), we have separated non-polar fatty acids and each subclass of mycolic acids from 21 mycobacterial species by thin-layer chromatography, and analyzed non-polar fatty acid methyl esters by gas chromatography (GC) and the cleavage products of methyl mycolate by pyrolysis GC. We further performed mass chromatographic analysis of trimethylsilyl (TMS) ether derivatives of mycolic acid methyl esters by monitoring [B-29]⁺ ions (loss of CHO from the α-branched-chain structure of mycolic acids) of m/z 426, 454 and 482 which are attributed to C₂₂-, C₂₄- and C₂₆-α-units of TMS ether derivatives of methyl mycolates, respectively, (Kaneda, K. et al, J. Clin. Microbiol. 24: 1060-1070, 1986). By pyrolysis GC, C_22:0, C_24:0 and C_26:0 fatty acid methyl esters generated by the C₂-C₃ cleavage of C₂₂-, C₂₄- and C₂₆-α-unit-containing mycolic acid methyl esters, respectively, were detected. Their proportion was almost the same among subclasses of mycolic acids in every Mycobacterium and also similar to the proportion of constitutive non-polar C_22:0, C_24:0 and C_26:0 fatty acids. By mass chromatography, the composition and distribution of C₂₂- and C₂₄- α-unit-containing homologues were revealed to be similar between α- and α'-mycolic acids in every Mycobacterium. We further analyzed in detail M. vaccae and demonstrated that the mass chromatogram of C₂₂-α-unit-containing homologue was analogous in shape to that of the C₂₄-α-unit-containing one, with the latter mass chromatogram being up-shifted from the former by two carbon numbers, in every subclass of α-, α'-, keto and dicarboxy mycolic acids. The present study suggests that the compositions of three homologues of both mycolic acids and constitutive non-polar fatty acids, which are characteristic to each mycobacterial species, may reflect the proportion of the amount of free C_22:0, C_24:0 and C_26:0 fatty acids synthesized in the cell. It is further demonstrated that intermolecular condensation of two fatty acids which become α- and β-units of mycolic acids will occur independently of the carbon chain length or kinds of polar moieties of fatty acids.