Liu J, Nikaido H
Department of Medical Genetics and Microbiology, University of Toronto, Toronto, Ontario, Canada M5S 1A8.
Proc Natl Acad Sci U S A. 1999 Mar 30;96(7):4011-6. doi: 10.1073/pnas.96.7.4011.
Mycolic acids are a major constituent of the mycobacterial cell wall, and they form an effective permeability barrier to protect mycobacteria from antimicrobial agents. Although the chemical structures of mycolic acids are well established, little is known on their biosynthesis. We have isolated a mycolate-deficient mutant strain of Mycobacterium smegmatis mc2-155 by chemical mutagenesis followed by screening for increased sensitivity to novobiocin. This mutant also was hypersensitive to other hydrophobic compounds such as crystal violet, rifampicin, and erythromycin. Entry of hydrophobic probes into mutant cells occurred much more rapidly than that into the wild-type cells. HPLC and TLC analysis of fatty acid composition after saponification showed that the mutant failed to synthesize full-length mycolic acids. Instead, it accumulated a series of long-chain fatty acids, which were not detected in the wild-type strain. Analysis by 1H NMR, electrospray and electron impact mass spectroscopy, and permanganate cleavage of double bonds showed that these compounds corresponded to the incomplete meromycolate chain of mycolic acids, except for the presence of a beta-hydroxyl group. This direct identification of meromycolates as precursors of mycolic acids provides a strong support for the previously proposed pathway for mycolic acid biosynthesis involving the separate synthesis of meromycolate chain and the alpha-branch of mycolic acids, followed by the joining of these two branches.
分枝菌酸是分枝杆菌细胞壁的主要成分，它们形成了一个有效的渗透屏障，以保护分枝杆菌免受抗菌剂的侵害。尽管分枝菌酸的化学结构已得到充分确定，但其生物合成过程却鲜为人知。我们通过化学诱变，随后筛选对新生霉素敏感性增加的菌株，分离出了耻垢分枝杆菌mc2-155的一个分枝菌酸缺陷突变株。该突变株对其他疏水化合物如结晶紫、利福平和红霉素也高度敏感。疏水探针进入突变细胞的速度比进入野生型细胞的速度快得多。皂化后脂肪酸组成的HPLC和TLC分析表明，该突变株无法合成全长的分枝菌酸。相反，它积累了一系列长链脂肪酸，这些脂肪酸在野生型菌株中未被检测到。通过1H NMR、电喷雾和电子轰击质谱分析以及双键的高锰酸盐裂解表明，除了存在β-羟基外，这些化合物对应于分枝菌酸不完整的分枝菌酸链。将分枝菌酸直接鉴定为分枝菌酸的前体，为先前提出的分枝菌酸生物合成途径提供了有力支持，该途径涉及分枝菌酸链和分枝菌酸α-分支分别合成，随后这两个分支连接起来。
