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Mycolic acids are major and specific lipid components of the cell envelope of mycobacteria that include the causative agents of tuberculosis and leprosy, Mycobacterium tuberculosis and Mycobacterium leprae, respectively. Subtle structural variations that are known to be crucial for both their virulence and the permeability of their cell envelope occur in mycolic acids. Among these are the introduction of cyclopropyl groups and methyl branches by mycolic acid S-adenosylmethionine-dependent methyltransferases (MA-MTs). While the functions of seven of the M. tuberculosis MA-MTs have been either established or strongly presumed nothing is known of the roles of the remaining umaA gene product and those of M. smegmatis MA-MTs. Mutants of the M. tuberculosis umaA gene and its putative M. smegmatis orthologue, MSMEG0913, were created. The lipid extracts of the resulting mutants were analyzed in detail using a combination of analytical techniques such as matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and proton nuclear magnetic resonance spectroscopy, and chemical degradation methods. The M. smegmatis mutants no longer synthesized subtypes of mycolates containing a methyl branch adjacent to either trans cyclopropyl group or trans double bond at the "proximal" position of both alpha- and epoxy-mycolates. Complementation with MSMEG0913, but not with umaA, fully restored the wild-type phenotype in M. smegmatis. Consistently, no modification was observed in the structures of mycolic acids produced by the M. tuberculosis umaA mutant. These data proved that despite their synteny and high similarity umaA and MSMEG0913 are not functionally orthologous.

The surface-enhanced Raman scattering (SERS) technique for Fourier transform Raman spectrometry is employed to reveal the chemical structure of biological aliphatic compounds consisting of folded, long aliphatic chains. The structural analysis is performed via the measurements of the accordion-vibration modes generated in the ordered, long aliphatic chain. The SERS spectra after subtraction of a background spectrum give segment lengths that are almost perfectly consistent with the chemical structures studied by mass spectrometry. The agreement of the SERS results with those of mass spectrometry suggests the positions of kinks in the long hydrocarbon chain. The combination technique of SERS and mass spectrometry is useful to discuss the structure of folded, long biological lipids., (Copyright 2004 Wiley Periodicals, Inc.)

Three kinds of mycolic acids (MAs) (alpha-, keto and methoxy-MAs) extracted from several species of mycobacteria were used to prepare monolayer films on water, and the surface pressure-area (pi-A) isotherms of the monolayers have been compared, so that the monolayer characteristics of the MAs as in cell walls would be revealed, since the monolayer molecular aggregation is related to drug permeability via the molecular packing. It was expected that the limiting molecular areas of the isotherms would be changed only a little, which reflects the minor difference in chemical structure and conformation of the mycobacteria. Nevertheless, the results are largely different from the expectation, and two greatly different patterns of the limiting molecular area have been observed. In a new model for elucidation of the results, two parts in an MA molecule are separately considered, and both contributions to the molecular unfolding by the monolayer compression have been suggested. This model is found to be useful to totally understand the isotherm behaviors of MAs. The relationship between monolayer properties and chemical structures for MAs has been summarized for the first time.

A method using Selective Ion Monitoring (SIM) gas chromatography-mass spectrometry is described for analysis of mycolic acids which reveals a hitherto unrecognised chemical structural diversity among these in some members of the Mycolata. The structural interpretation of mass spectral data of mycolic acids from Rhodococcus spp and Gordonia [corrected] spp using SIM is discussed.

An important class of fatty acids contained in the cell envelopes of Mycobacterium organisms is the group of high-molecular-weight, long-chain, alpha-branched, beta-hydroxylated mycolic acids. By using standard saponification techniques and derivatization of the acids to their p-bromophenacyl esters, it is possible to differentiate them by high-performance liquid chromatography. Mycolic acid chromatograms of 63 clinical isolates of Mycobacterium gordonae were compared with conventional biochemical methods for identification. The data show two distinct pattern types for this species, only one of which has been elaborated in the literature by using this protocol. Laboratory workers who intend to use this method as a clinical tool need to be aware that these two pattern types exist.

The effect of growth temperature on mycolic acid composition in eight strains of Mycobacterium smegmatis was investigated by gas chromatography/mass spectrometry. A change in growth temperature from 45 to 20 degrees C caused a shift in the subclass and molecular species composition of mycolic acids. The relative amount of alpha'-mycolic acids to alpha-mycolic acids decreased, and that of hydroxy mycolic acids increased at lower temperatures. Moreover, the proportion of shorter-chain species of alpha-mycolic acids increased, and those of longer-chain species of alpha-mycolic and hydroxy mycolic acids decreased. This observation seems to be due to the changes of the chain length of meromycolates because the alpha-alkyl chain unit of mycolic acids was not affected. The ratio of odd to even carbon-numbered alpha-mycolates decreased as the growth temperature was lowered. In contrast, the molecular species composition of alpha'-mycolic acid was not influenced by the growth temperature.