Molecular Evolutionary History of Tubercle Bacilli Assessed by Study of the Polymorphic Nucleotide within the Nitrate Reductase ( narGHJI ) Operon Promoter

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Abstract
A well-characterized collection of Mycobacterium tuberculosis complex (MTC) isolates, representing all known subspecies as well as some relevant genotypic families of M. tuberculosis , was analyzed for the newly discovered narGHJI −215 C-to-T promoter single-nucleotide polymorphism (SNP). This point mutation has been shown in earlier studies to be responsible for the differential nitrate reductase activity of M. tuberculosis versus M. bovis . As previously defined by the presence or the absence of the TbD1 genetic locus, the group included both the “modern” W-Beijing, Haarlem, and Central-Asian1 (CAS1) families as well as the “ancestral” East-African-Indian (EAI) clade. Interestingly, among “modern” M. tuberculosis isolates, those previously classified as Principal Genetic Group 1 (PGG1) organisms by katG 463 -gyrA 95 polymorphism analysis did not present the two-banded narGHJI restriction fragment length polymorphism analysis of PCR products pattern common to the other PGG1 MTC members, including the “ancestral” M. tuberculosis isolates. Instead, they showed a one-banded pattern, aligning them with other evolutionarily recent M. tuberculosis isolates of the PGG2 and PGG3 groups, such as Haarlem, Latin-American and Mediterranean (LAM), and X families. The presence of a nitrate reductase producer phenotype in “ Mycobacterium canettii” and some “ancestral” M. tuberculosis isolates, despite a two-band −215C genotype, argues in favor of an alternate mechanism to explain the differential nitrate reductase activity of certain PGG1 subspecies of the MTC. Overall, these findings may help to establish the precise evolutionary history of important genotype families such as W-Beijing and suggest that the −215T genotype may have contributed the virulence, spread, and evolutionary success of “modern” M. tuberculosis strains compared to the remaining MTC organisms.

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