Behr M A, Schroeder B G, Brinkman J N, Slayden R A, Barry C E
McGill University Health Centre, Montreal, Canada.
J Bacteriol. 2000 Jun;182(12):3394-9. doi: 10.1128/JB.182.12.3394-3399.2000.
BCG vaccines are substrains of Mycobacterium bovis derived by attenuation in vitro. After the original attenuation (1908 to 1921), BCG strains were maintained by serial propagation in different BCG laboratories (1921 to 1961). As a result, various BCG substrains developed which are now known to differ in a number of genetic and phenotypic properties. However, to date, none of these differences has permitted a direct phenotype-genotype link. Since BCG strains differ in their abilities to synthesize methoxymycolic acids and since recent work has shown that the mma3 gene is responsible for O-methylation of hydroxymycolate precursors to form methoxymycolic acids, we analyzed methoxymycolate production and mma3 gene sequences for a genetically defined collection of BCG strains. We found that BCG strains obtained from the Pasteur Institute in 1927 and earlier produced methoxymycolates in vitro but that those obtained from the Pasteur Institute in 1931 and later all failed to synthesize methoxymycolates, and furthermore, the mma3 sequence of the latter strains differs from that of Mycobacterium tuberculosis H37Rv by a point mutation at bp 293. Site-specific introduction of this guanine-to-adenine mutation into wild-type mma3 (resulting in the replacement of glycine 98 with aspartic acid) eliminated the ability of this enzyme to produce O-methylated mycolic acids when the mutant was cloned in tandem with mma4 into Mycobacterium smegmatis. These findings indicate that a point mutation in mma3 occurred between 1927 and 1931, and that this mutant population became the dominant clone of BCG at the Pasteur Institute.
卡介苗是通过体外减毒获得的牛分枝杆菌亚菌株。在最初减毒（1908年至1921年）后，卡介苗菌株在不同的卡介苗实验室通过连续传代进行保存（1921年至1961年）。结果，产生了各种卡介苗亚菌株，现在已知它们在许多遗传和表型特性上存在差异。然而，迄今为止，这些差异中没有一个能够建立直接的表型-基因型联系。由于卡介苗菌株在合成甲氧基分枝菌酸的能力上存在差异，并且由于最近的研究表明mma3基因负责将羟基分枝菌酸前体进行O-甲基化以形成甲氧基分枝菌酸，我们分析了一组基因明确的卡介苗菌株的甲氧基分枝菌酸产生情况和mma3基因序列。我们发现，1927年及更早从巴斯德研究所获得的卡介苗菌株在体外能产生甲氧基分枝菌酸，但1931年及以后从巴斯德研究所获得的菌株均无法合成甲氧基分枝菌酸，此外，后一类菌株的mma3序列与结核分枝杆菌H37Rv的序列在第293位碱基处存在一个点突变。当将该鸟嘌呤到腺嘌呤的突变位点特异性引入野生型mma3（导致第98位甘氨酸被天冬氨酸取代），并将该突变体与mma4串联克隆到耻垢分枝杆菌中时，该酶产生O-甲基化分枝菌酸的能力丧失。这些发现表明，mma3在1927年至1931年间发生了一个点突变，并且这个突变群体成为了巴斯德研究所卡介苗的优势克隆。
