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Infection and Immunity, October 2002, p. 5568-5578, Vol. 70, No. 10
0019-9567/02/$04.00+0     DOI: 10.1128/IAI.70.10.5568-5578.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

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

Unité de Génétique Moléculaire Bactérienne, Institut Pasteur, 75724 Paris Cedex 15,¹ Molecular Engines Laboratories, 75011 Paris, France,² National Reference Center for Mycobacteria, Forschungszentrum Borstel, 23845 Borstel, Germany³

Received 26 March 2002/ Returned for modification 18 June 2002/ Accepted 5 July 2002

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 (RD1^mic) 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, RD5^mic, 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 RD1^mic and RD5^mic 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.

Editor: S. H. E. Kaufmann

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