Tuberculosis, caused by Mycobacterium tuberculosis, remains a leading infectious disease despite the availability of chemotherapy and BCG vaccine. The commonly used avirulent M. tuberculosis strain H37Ra was derived from virulent strain H37 in 1935 but the basis of virulence attenuation has remained obscure despite numerous studies. We determined the complete genomic sequence of H37Ra ATCC25177 and compared that with its virulent counterpart H37Rv and a clinical isolate CDC1551. The H37Ra genome is highly similar to that of H37Rv with respect to gene content and order but is 8,445 bp larger as a result of 53 insertions and 21 deletions in H37Ra relative to H37Rv. Variations in repetitive sequences such as IS6110 and PE/PPE/PE-PGRS family genes are responsible for most of the gross genetic changes. A total of 198 single nucleotide variations (SNVs) that are different between H37Ra and H37Rv were identified, yet 119 of them are identical between H37Ra and CDC1551 and 3 are due to H37Rv strain variation, leaving only 76 H37Ra-specific SNVs that affect only 32 genes. The biological impact of missense mutations in protein coding sequences was analyzed in silico while nucleotide variations in potential promoter regions of several important genes were verified by quantitative RT-PCR. Mutations affecting transcription factors and/or global metabolic regulations related to in vitro survival under aging stress, and mutations affecting cell envelope, primary metabolism, in vivo growth as well as variations in the PE/PPE/PE-PGRS family genes, may underlie the basis of virulence attenuation. These findings have implications not only for improved understanding of pathogenesis of M. tuberculosis but also for development of new vaccines and new therapeutic agents.

MazG nucleoside triphosphate pyrophosphohydrolase (NTP-PPaseMycobacterium tuberculosis H37Ra is an avirulent relative of the typical laboratory strain H37Rv, both of which are derived from the parent strain H37 that was originally isolated from a 19-year-old patient with chronic pulmonary tuberculosis by Edward R. Baldwin in 1905 (1). Our systematic comparison of the complete genomic sequences of H37Rv and H37Ra identified multiple H37Ra-specific single nucleotide polymorphisms (SNPs) affecting about 30 genes including the virulence determinants phoP, nrp, and pks12 (2), consistent with the widely accepted notion (3-7) that the virulence attenuation of H37Ra is due to multiple mutations. One of these mutations, the A219E substitution located in a highly conserved sequence of H37Ra MazG, prompted the current study aiming to reveal the function of the corresponding prototype enzyme in M. tuberculosis (MtMazG) and compare its role with that of