Through examination of one of the fundamental in vitro characteristics of Mycobacterium bovis - its requirement for pyruvate in glycerol medium - we have revealed a lesion in central metabolism that has profound implications for in vivo growth and nutrition. Not only is M. bovis unable to use glycerol as a sole carbon source but the lack of a functioning pyruvate kinase (PK) means that carbohydrates cannot be used to generate energy. This disruption in sugar catabolism is caused by a single nucleotide polymorphism in pykA, the gene which encodes PK, that substitutes glutamic acid residue 220 with an aspartic acid residue. Substitution of this highly conserved amino acid residue renders PK inactive and thus blocks the ATP generating roles of glycolysis and the pentose phosphate pathway. This mutation was found to occur in other members of the M. tuberculosis complex, namely M. microti and M. africanum. With carbohydrates unable to act as carbon sources, the importance of lipids and gluconeogenesis for growth in vivo becomes apparent. Complementation of M. bovis with the pykA gene from M. tuberculosis H37Rv restored growth on glycerol. Additionally, the presence of a functioning PK caused the colony morphology of the complemented strain to change from the characteristic dysgonic growth of M. bovis to eugonic growth, an appearance normally associated with M. tuberculosis. We also suggest that the glycerol-soaked potato slices used for the derivation of the M. bovis bacillus Calmette and Guérin (BCG) vaccine strain selected for an M. bovis PK+ mutant, a finding that explains the alteration in colony morphology noted during the derivation of BCG. In summary, the disruption of a key step in glycolysis divides the M. tuberculosis complex into two groups with distinct carbon source utilization.