Wang L, Slayden R A, Barry C E, Liu J
Department of Medical Genetics and Microbiology, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
J Biol Chem. 2000 Mar 10;275(10):7224-9. doi: 10.1074/jbc.275.10.7224.
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-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.
最近分离出了一株在分枝菌酸生物合成方面存在缺陷的耻垢分枝杆菌突变株（Liu, J., 和 Nikaido, H. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 4011 - 4016）。该突变株无法合成全长的分枝菌酸，并积累了一系列长链β-羟基分枝菌酸。在这项工作中，我们对分枝菌酸的定位以及突变株的细胞壁结构进行了详细表征。薄层色谱显示，用氯仿/甲醇和十二烷基硫酸钠提取后剩余的不溶性细胞壁基质仍含有大部分总分枝菌酸。对不溶性细胞壁基质进行史密斯降解产生的片段进行基质辅助激光解吸/电离和电喷雾电离质谱分析表明，分枝菌酸在末端阿拉伯呋喃糖基残基的5-OH位置与阿拉伯半乳聚糖共价连接。通过核磁共振分析，突变株中的阿拉伯半乳聚糖似乎是正常的。对有机相脂质的分析表明，突变株细胞壁含有一些可提取的脂质，但缺乏糖肽脂和脂寡糖。突变株细胞壁的差示扫描量热法未能显示完整分枝杆菌细胞壁典型的大型协同热转变。透射电子显微镜显示，突变株细胞壁具有异常的超微结构（没有与不对称分枝菌酸脂质层相关的电子透明区）。综上所述，这些结果证明了分枝菌酸对分枝杆菌细胞壁结构和功能完整性的重要性。突变株细胞壁中缺乏高度组织化的脂质结构域解释了该突变株的药物敏感和温度敏感表型。
