Genetic Variation and Evolutionary Origin of the Direct Repeat Locus of Mycobacterium tuberculosis Complex Bacteria

Abstract

The direct repeat region in Mycobacterium tuberculosis complex strains is composed of multiple direct variant repeats (DVRs), each of which is composed of a 36-bp direct repeat (DR) plus a nonrepetitive spacer sequence of similar size. It has been shown previously that clinical isolates show extensive polymorphism in the DR region by the variable presence of DVRs, and this polymorphism has been used in the epidemiology of tuberculosis. In an attempt to better understand the evolutionary scenario leading to polymorphic DR loci and to improve strain differentiation by spoligotyping, we characterized 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 was found between any of these spacers and M. tuberculosis DNA outside of the DR region or with any other known bacterial sequence. Although strains differed extensively in the presence or absence of DVRs, the order of the spacers in the DR locus was found to be well conserved. The data strongly suggest that the polymorphism in clinical isolates is the result of successive deletions of single discrete DVRs or of multiple contiguous DVRs from a primordial DR region containing many more DVRs than seen in present day isolates and that virtually no scrambling of DVRs took place during evolution. Because the majority of the novel spacer sequences identified in this study were confined to isolates of the rare Mycobacterium canettii taxon, the use of the novel spacers in spoligotyping led only to a slight improvement of strain differentiation by spoligotyping.

Diversity, evolution, and therapeutic applications of small RNAs in prokaryotic and eukaryotic immune systems

“…The duplication of spacers and repeats within the CRISPR locus has been documented in the cases of methanoarchaea and mycobacteria [127]. An alignment of the CRISPR loci of three S. solfataricus strains revealed that in addition to some deletion events and different spacers being incorporated into the locus after the strains deviated from one another, there was one implied instance of recombination.…”

“…It is likely that deletion in the middle or downstream sections of the CRISPR locus do not impact the immune defense of the cell as much as an upstream deletion would, because these older spacers are not as relevant to the current or recent invaders of the cell [37,91]. Van Embden and company discovered that the CRISPR locus, also known as the DR (direct repeat) region, in strains of M. tuberculosis showed multiple deletions -but no genetic shuffling -between strains [127].…”

Identifying Mycobacterium tuberculosis complex strain families using spoligotypes

“…It has been hypothesized that the DR locus evolves by deletion of a single or multiple contiguous DVRs, whereas insertion of DVRs is very unlikely (van Embden et al, 2000;Aranaz et al, 2004). We have incorporated this knowledge into our algorithm by introducing "Hidden Parents" into the model.…”

“…The order of the spacers was found to be well conserved (van Embden et al, 2000). Currently, 94 different spacer sequences were identified of which 43 are used for MTC strain differentiation (van Embden et al, 2000). Spoligotyping differentiates isolates of MTC strains by determining the absence or presence of the 43 defined spacer sequences.…”

“…The region comprising the repeat plus the adjacent spacer has been termed the direct variable repeat (DVR) (Groenen et al, 1993). The order of the spacers was found to be well conserved (van Embden et al, 2000). Currently, 94 different spacer sequences were identified of which 43 are used for MTC strain differentiation (van Embden et al, 2000).…”