A point mutation in the mma3 gene is responsible for impaired methoxymycolic acid production in Mycobacterium bovis BCG strains obtained after 1927

BCG vaccines are substrains of Mycobacterium bovis derived by attenuation i n vitro. After the original attenuation (1908 to 1921), BCG strains were ma intained by serial propagation in different BCG laboratories (1921 to 1961) . As a result, various BCG substrains developed which are now known to diff er 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 a nd since recent work bras shown that the mma3 gene is responsible for O-met hylation of hydroxymycolate precursors to form methoxymycolic acids, we ana lyzed 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 vitr o but that those obtained from the Pasteur Institute in 1931 and later all failed to synthesize methoxymycolates, and furthermore, the mma3 sequence o f the latter strains differs from that of Mycobacterium tuberculosis H37Rv by a point mutation at bp 293, Site-specific introduction of this guanine-t o-adenine mutation into wild-type mma3 (resulting in the replacement of gly cine 98 with aspartic acid) eliminated the ability of this enzyme to produc e O-methylated mycolic acids when the mutant was cloned in tandem with mma4 into Mycobacterium smegmatis. These findings indicate that a point mutatio n in mma3 occurred between 1927 and 1931, and that this mutant population b ecame the dominant clone of BCG at the Pasteur Institute.