The publication of the complete genome sequence for Mycobacterium tuberculosis H37Rv in 1998 has had a great impact on the research community. Nonetheless, it is suspected that genetic differences have arisen in stocks of H37Rv that are maintained in different laboratories. In order to assess the consistency of the genome sequences among H37Rv strains in use and the extent to which they have diverged from the original strain sequenced, we carried out whole-genome sequencing on six strains of H37Rv from different laboratories. Polymorphisms at 73 sites were observed, which were shared among the lab strains, though 72 of these were also shared with H37Ra and are likely to be due to sequencing errors in the original H37Rv reference sequence. An updated H37Rv genome sequence should be valuable to the tuberculosis research community as well as the broader microbial research community. In addition, several polymorphisms unique to individual strains and several shared polymorphisms were identified and shown to be consistent with the known provenance of these strains. Aside from nucleotide substitutions and insertion/deletions, multiple IS6110 transposition events were observed, supporting the theory that they play a significant role in plasticity of the M. tuberculosis genome. This genome-wide catalog of genetic differences can help explain any phenotypic differences that might be found, including a frameshift mutation in the mycocerosic acid synthase gene which causes two of the strains to be deficient in biosynthesis of the surface glycolipid phthiocerol dimycocerosate (PDIM). The resequencing of these six lab strains represents a fortuitous "in vitro evolution" experiment that demonstrates how the M. tuberculosis genome continues to evolve even in a controlled environment.Publication of the whole genome sequence of the H37Rv strain of Mycobacterium tuberculosis by Stewart Cole and colleagues in 1998 provided a breakthrough in tuberculosis (TB) research (8), leading to insights into the biology, metabolism, and evolution of this infectious pathogen. Large protein families related to fatty acid and polyketide biosynthesis, regulation (e.g., sigma factors and two-component sensor systems), drug efflux pumps and transporters, and the PE_PGRS proteins (a large duplicated family unique to the M. tuberculosis group of mycobacteria) were identified. In addition, transposons, prophage-like elements, and other repetitive and/or mobile genetic elements were identified (18). This genomic information has played an essential role in interpreting gene expression studies, modeling persistence, and identifying essential proteins as putative targets for drug discovery. However, to date the functions of only half of the genes (1,756/ 4,066) have been determined or predicted, and the rest remain annotated as "hypothetical proteins" (6).The H37Rv strain was initially selected for sequencing because it is a widely used laboratory strain that has retained its virulence. H37Rv was initially derived from a clinical isolate, H37, obtained fr...

Mycobacterium tuberculosis enoyl-acyl-ACP reductase (InhA) has been demonstrated to be the primary target of isoniazid (INH).adduct. An Escherichia coli cell-based system was designed that allowed coexpression of both M. tuberculosis katG and dfrA genes in the presence of INH. The DHFR protein isolated from the experimental sample was not found bound with any INH-NADP adduct by enzyme inhibition assay and mass spectroscopic analysis. We also used whole-genome sequencing to determine whether polymorphisms in dfrA could be detected in six INHresistant clinical isolates known to lack mutations in inhA and katG, but no such mutations were found. The dfrA overexpression experiments, together with the biochemical and sequencing studies, conclusively demonstrate that DHFR is not a target relevant to the antitubercular activity of INH.