Alain Billault

Identification of variable regions in the genomes of tubercle bacilli using bacterial artificial chromosome arrays

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.

Bacterial Artificial Chromosome-Based Comparative Genomic Analysis Identifies Mycobacterium microti as a Natural ESAT-6 Deletion Mutant

ABSTRACT Mycobacterium microti is a member of the Mycobacterium tuberculosis complex that causes tuberculosis in voles. Most strains of M. microti are harmless for humans, and some have been successfully used as live tuberculosis vaccines. In an attempt to identify putative virulence factors of the tubercle bacilli, genes that are absent from the avirulent M. microti but present in human pathogen M. tuberculosis or Mycobacterium bovis were searched for. A minimal set of 50 bacterial artificial chromosome (BAC) clones that covers almost all of the genome of M. microti OV254 was constructed, and individual BACs were compared to the corresponding BACs from M. bovis AF2122/97 and M. tuberculosis H37Rv. Comparison of pulsed-field gel-separated DNA digests of BAC clones led to the identification of 10 regions of difference (RD) between M. microti OV254 and M. tuberculosis. A 14-kb chromosomal region (RD1mic) that partly overlaps the RD1 deletion in the BCG vaccine strain was missing from the genomes of all nine tested M. microti strains. This region covers 13 genes, Rv3864 to Rv3876, in M. tuberculosis, including those encoding the potent ESAT-6 and CFP-10 antigens. In contrast, RD5mic, a region that contains three phospholipase C genes (plcA to -C), was missing from only the vole isolates and was present in M. microti strains isolated from humans. Apart from RD1mic and RD5mic other M. microti-specific deleted regions have been identified (MiD1 to MiD3). Deletion of MiD1 has removed parts of the direct repeat region in M. microti and thus contributes to the characteristic spoligotype of M. microti strains.