Cell wall structure of a mutant of Mycobacterium smegmatis defective in the biosynthesis of mycolic acids

A mutant strain of Mycobacterium smegmatis defective in the biosynthesis of mycolic acids was recently isolated (Liu, J., and Nikaido, H. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 4011-4016), This mutant failed to synthesize full-length mycolic acids and accumulated a series of long chain beta-hydr oxymeromycolates. In this work, we provide a detailed characterization of t he localization of meromycolates and of the cell wall structure of the muta nt. Thin layer chromatography showed that the insoluble cell wall matrix re maining after extraction with chloroform/methanol and SDS still contained a large portion of the total meromycolates. Matrix-assisted laser desorption /ionization and electrospray ionization mass spectroscopy analysis of fragm ents arising from Smith degradation of the insoluble cell wall matrix revea led that the meromycolates were covalently attached to arabinogalactan at t he 5-OH positions of the terminal arabinofuranosyl residues. The arabinogal actan appeared to be normal in the mutant strain, as analyzed by NMR, Analy sis of organic phase lipids showed that the mutant cell wall contained some of the extractable lipids but lacked glycopeptidolipids and lipooligosacch arides. Differential scanning calorimetry of the mutant cell wall failed to show the large cooperative thermal transitions typical of intact mycobacte rial cell walls. Transmission electron microscopy showed that the mutant ce ll wall had an abnormal ultrastructure (without the electron-transparent zo ne associated with the asymmetric mycolate lipid layer). Taken together, th ese results demonstrate the importance of mycolic acids for the structural and functional integrity of the mycobacterial cell mall. The lack of highly organized lipid domains in the mutant cell wall explains the drug-sensitiv e and temperature-sensitive phenotypes of the mutant.