OR32

Although it is often thought that the progenitor of the Mycobacterium tuberculosis complex (MTBC) arose from soil, its environmental origin remains elusive. In order to search for this ancestral source, we exploited unique combined features of CRISPR-Cas systems and M. canettii TB clinical strains. As prokaryotic CRISPR-Cas systems provide adaptive immunity to foreign invasion by storing specific spacer sequences excised from viral genomes and other parasitic genetic elements, CRISPR spacers represent a molecular memory of prior microbial ecology. In contrast with the MTBC, M. canettii strains are recombinogenic, show diverse CRISPR-Cas systems, and represent evolutionarily early branching lineages of tubercle bacilli, with an unknown putative environmental reservoir in East Africa. Therefore, we identified and comprehensively analyzed CRISPR-cas loci and spacer repertoires in all previously known and newly sequenced genomes of M. canettii, and in representative genomes of all known human- and animal-associated lineages. On top of CRISPR-Cas Type III-A (shared with the MTBC), I-C and I-E previously known, Type I-D and I-U systems were newly identified. Among multiple other events of horizontal evolution among M. canettii strains, phylogenetic reconstruction and CRISPR-cas structural analysis indicated the involvement of an environmental mycobacterium-like ancestor in past horizontal exchanges of the CRISPR-cas locus from a particular strain subgroup. Moreover, we found multiple CRISPR spacers in M. canettii and some MTBC spacers that match sequences of environmental mycobacteriophages or prophages integrated in genomes of free-living (myco)bacteria. The distribution of the environmental sources of these organisms indicates a non-telluric ancestral origin for the MTBC