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J Biol Chem, Vol. 275, Issue 10, 7224-7229, March 10, 2000

Cell Wall Structure of a Mutant of Mycobacterium smegmatis Defective in the Biosynthesis of Mycolic Acids^*

Linda Wang, Richard A. Slayden^§, Clifton E. Barry III^§, and Jun Liu^¶

From the  Department of Medical Genetics and Microbiology, University of Toronto, Toronto, Ontario M5S 1A8, Canada and the ^§ Tuberculosis Research Section, Laboratory of Host Defenses, NIAID, National Institutes of Health, Rockville, Maryland 20852

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 -hydroxymeromycolates. In this work, we provide a detailed characterization of the localization of meromycolates and of the cell wall structure of the mutant. Thin layer chromatography showed that the insoluble cell wall matrix remaining 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 fragments arising from Smith degradation of the insoluble cell wall matrix revealed that the meromycolates were covalently attached to arabinogalactan at the 5-OH positions of the terminal arabinofuranosyl residues. The arabinogalactan appeared to be normal in the mutant strain, as analyzed by NMR. Analysis of organic phase lipids showed that the mutant cell wall contained some of the extractable lipids but lacked glycopeptidolipids and lipooligosaccharides. Differential scanning calorimetry of the mutant cell wall failed to show the large cooperative thermal transitions typical of intact mycobacterial cell walls. Transmission electron microscopy showed that the mutant cell wall had an abnormal ultrastructure (without the electron-transparent zone associated with the asymmetric mycolate lipid layer). Taken together, these results demonstrate the importance of mycolic acids for the structural and functional integrity of the mycobacterial cell wall. The lack of highly organized lipid domains in the mutant cell wall explains the drug-sensitive and temperature-sensitive phenotypes of the mutant.

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^* This work was supported by Medical Research Council of Canada Grant MT-15107 (to J. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Dept. of Medical Genetics and Microbiology, 4382 Medical Sciences Bldg., University of Toronto, 1 King's College Circle, Toronto, Ontario M5S 1A8, Canada. Tel.: 416-946-5067; Fax: 416-978-6885; E-mail: jun.liu@utoronto.ca.

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Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.

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