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

ABSTRACTMycobacterium microtiis a member of the Mycobacterium tuberculosiscomplex that causes tuberculosis in voles. Most strains of M. microtiare 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. microtibut present in human pathogen M. tuberculosisor Mycobacterium boviswere searched for. A minimal set of 50 bacterial artificial chromosome (BAC) clones that covers almost all of the genome of M. microtiOV254 was constructed, and individual BACs were compared to the corresponding BACs from M. bovisAF2122/97 and M. tuberculosisH37Rv. Comparison of pulsed-field gel-separated DNA digests of BAC clones led to the identification of 10 regions of difference (RD) between M. microtiOV254 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. microtistrains. 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 (plcAto -C), was missing from only the vole isolates and was present in M. microtistrains isolated from humans. Apart from RD1micand RD5micother M. microti-specific deleted regions have been identified (MiD1 to MiD3). Deletion of MiD1 has removed parts of the direct repeat region in M. microtiand thus contributes to the characteristic spoligotype of M. microtistrains.