Genetic variation and evolutionary origin of the direct repeat locus of Mycobacterium tuberculosis complex bacteria

The direct repeat region in Mycobacter tuberculosis complex strains is comp osed of multiple direct variant repeats (DVRs), each of which is composed o f a 36-bp direct repeat (DR) plus a nonrepetitive spacer sequence of simila r size. It has been shown previously that clinical isolates show extensive polymorphism in the DR region by the variable presence of DVRs, and this po lymorphism has been used in the epidemiology of tuberculosis. In an attempt to better understand the evolutionary scenario leading to polymorphic DR l oci and to improve strain differentiation by spoligotyping, we characterize d and compared the DNA sequences of the complete DR region and its flanking DNA of M. tuberculosis complex strains. We identified 94 different spacer sequences among 26 M. tuberculosis complex strains. No sequence homology wa s found between any of these spacers and M. tuberculosis DNA outside of the DR region or with any other known bacterial sequence. Although strains dif fered extensively in the presence or absence of DVRs, the order of the spac ers in the DR locus was found to be well conserved. The data strongly sugge st that the polymorphism in clinical isolates is the result of successive d eletions of single discrete DVRs or of multiple contiguous DVRs from a prim ordial DR region containing many more DVRs than seen in present day isolate s and that virtually no scrambling of DVRs took place during evolution. Bec ause the majority of the novel spacer sequences identified in this study we re confined to isolates of the rare Mycobacterium canettii taxon, the use o f the novel spacers in spoligotyping led only to a slight improvement of st rain differentiation by spoligotyping.