The distribution of 20 variable regions resulting from insertion-deletion events in the genomes of the tubercle bacilli has been evaluated in a total of 100 strains of Mycobacterium tuberculosis , Mycobacterium africanum , Mycobacterium canettii , Mycobacterium microti , and Mycobacterium bovis . This approach showed that the majority of these polymorphisms did not occur independently in the different strains of the M. tuberculosis complex but, rather, resulted from ancient, irreversible genetic events in common progenitor strains. Based on the presence or absence of an M. tuberculosis specific deletion (TbD1), M. tuberculosi s strains can be divided into ancestral and “modern” strains, the latter comprising representatives of major epidemics like the Beijing, Haarlem, and African M. tuberculosis clusters. Furthermore, successive loss of DNA, reflected by region of difference 9 and other subsequent deletions, was identified for an evolutionary lineage represented by M. africanum , M. microti , and M. bovis that diverged from the progenitor of the present M. tuberculosis strains before TbD1 occurred. These findings contradict the often-presented hypothesis that M. tuberculosis, the etiological agent of human tuberculosis evolved from M. bovis , the agent of bovine disease. M. canettii and ancestral M. tuberculosis strains lack none of these deleted regions, and, therefore, seem to be direct descendants of tubercle bacilli that existed before the M. africanum→M. bovis lineage separated from the M. tuberculosis lineage. This observation suggests that the common ancestor of the tubercle bacilli resembled M. tuberculosis or M. canettii and could well have been a human pathogen already.

Mycobacterium bovisis the causative agent of tuberculosis in a range of animal species and man, with worldwide annual losses to agriculture of $3 billion. The human burden of tuberculosis caused by the bovine tubercle bacillus is still largely unknown.M. boviswas also the progenitor for theM. bovisbacillus Calmette–Guérin vaccine strain, the most widely used human vaccine. Here we describe the 4,345,492-bp genome sequence ofM. bovisAF2122/97 and its comparison with the genomes ofMycobacterium tuberculosisandMycobacterium leprae. Strikingly, the genome sequence ofM. bovisis >99.95% identical to that ofM. tuberculosis, but deletion of genetic information has led to a reduced genome size. Comparison withM. lepraereveals a number of common gene losses, suggesting the removal of functional redundancy. Cell wall components and secreted proteins show the greatest variation, indicating their potential role in host–bacillus interactions or immune evasion. Furthermore, there are no genes unique toM. bovis, implying that differential gene expression may be the key to the host tropisms of human and bovine bacilli. The genome sequence therefore offers major insight on the evolution, host preference, and pathobiology ofM. bovis.

To understand the evolution, attenuation, and variable protective efficacy of bacillus Calmette–Guérin (BCG) vaccines, Mycobacterium bovis BCG Pasteur 1173P2 has been subjected to comparative genome and transcriptome analysis. The 4,374,522-bp genome contains 3,954 protein-coding genes, 58 of which are present in two copies as a result of two independent tandem duplications, DU1 and DU2. DU1 is restricted to BCG Pasteur, although four forms of DU2 exist; DU2-I is confined to early BCG vaccines, like BCG Japan, whereas DU2-III and DU2-IV occur in the late vaccines. The glycerol-3-phosphate dehydrogenase gene, glpD2 , is one of only three genes common to all four DU2 variants, implying that BCG requires higher levels of this enzyme to grow on glycerol. Further amplification of the DU2 region is ongoing, even within vaccine preparations used to immunize humans. An evolutionary scheme for BCG vaccines was established by analyzing DU2 and other markers. Lesions in genes encoding σ-factors and pleiotropic transcriptional regulators, like PhoR and Crp, were also uncovered in various BCG strains; together with gene amplification, these affect gene expression levels, immunogenicity, and, possibly, protection against tuberculosis. Furthermore, the combined findings suggest that early BCG vaccines may even be superior to the later ones that are more widely used.

Whole‐genome comparisons of the tubercle bacilli were undertaken using ordered bacterial artificial chromosome (BAC) libraries of Mycobacterium tuberculosis and the vaccine strain, Mycobacterium bovis BCG‐Pasteur, together with the complete genome sequence of M. tuberculosis H37Rv. Restriction‐digested BAC arrays of M. tuberculosis H37Rv were used in hybridization experiments with radiolabelled M. bovis BCG genomic DNA to reveal the presence of 10 deletions (RD1–RD10) relative to M. tuberculosis. Seven of these regions, RD4–RD10, were also found to be deleted from M. bovis, with the three M. bovis BCG‐specific deletions being identical to the RD1–RD3 loci described previously. The distribution of RD4–RD10 in Mycobacterium africanum resembles that of M. tuberculosis more closely than that of M. bovis, whereas an intermediate arrangement was found in Mycobacterium microti, suggesting that the corresponding genes may affect host range and virulence of the various tubercle bacilli. Among the known products encoded by these loci are a copy of the proposed mycobacterial invasin Mce, three phospholipases, several PE, PPE and ESAT‐6 proteins, epoxide hydrolase and an insertion sequence. In a complementary approach, direct comparison of BACs uncovered a third class of deletions consisting of two M. tuberculosis H37Rv loci, RvD1 and RvD2, deleted from the genome relative to M. bovis BCG and M. bovis. These deletions affect a further seven genes, including a fourth phospholipase, plcD. In summary, the insertions and deletions described here have important implications for our understanding of the evolution of the tubercle complex.

The secreted Mycobacterium tuberculosis complex proteins CFP‐10 and ESAT‐6 have recently been shown to play an essential role in tuberculosis pathogenesis. We have determined the solution structure of the tight, 1:1 complex formed by CFP‐10 and ESAT‐6, and employed fluorescence microscopy to demonstrate specific binding of the complex to the surface of macrophage and monocyte cells. A striking feature of the complex is the long flexible arm formed by the C‐terminus of CFP‐10, which was found to be essential for binding to the surface of cells. The surface features of the CFP‐10 · ESAT‐6 complex, together with observed binding to specific host cells, strongly suggest a key signalling role for the complex, in which binding to cell surface receptors leads to modulation of host cell behaviour to the advantage of the pathogen.