IS6110-mediated deletions of wild-type chromosomes of Mycobacterium tuberculosis

Abstract

The ipl locus is a site for the preferential insertion of IS6110 and has been identified as an insertion sequence, IS1547, in its own right. Various deletions around the ipl locus of clinical isolates of Mycobacterium tuberculosis were identified, and these deletions ranged in length from several hundred base pairs up to several kilobase pairs. The most obvious feature shared by these deletions was the presence of an IS6110 copy at the deletion sites, which suggested two possible mechanisms for their occurrence, IS6110 transposition and homologous recombination. To clarify the mechanism, an investigation was conducted; the results suggest that although deletion transpositionally mediated by IS6110 was a possibility, homologous recombination was a more likely one. The implications of such chromosomal rearrangements for the evolution of M. tuberculosis, for IS6110-mediated mutagenesis, and for the development of genetic tools are discussed. The deletion of genomic DNA in isolates of M. tuberculosis has previously been noted at only a few sites. This study examined the deletional loss of genetic material at a new site and suggests that such losses may occur elsewhere too and may be more prevalent than was previously thought. Distinct from the study of laboratory-induced mutations, the detailed analysis of clinical isolates, in combination with knowledge of their evolutionary relationships to each other, gives us the opportunity to study mutational diversity in isolates that have survived in the human host and therefore offers a different perspective on the importance of particular genetic markers in pathogenesis.

FIG. 1

PCR products from the hr-gr-lpdh::IS1547 loci in different isolates. Lanes 1 to 3, products of long PCR with primers P3 and P8 from isolate 9110 (hr-gr-lpdh::IS1547), isolate 41909 (hr-gr-lpdh::IS1547::IS6110), and isolate 9013, respectively; lanes 4 to 6, products of conventional PCR with the long PCR products from isolate 9013 as templates with primer pairs INS2 and INS3 (lane 4), P3 and INS1 (lane 5), and INS4 and P8 (lane 6).

FIG. 2

Schematic illustration of locus hr-gr-lpdh::IS1547 of strains 9110, 9013, and 9504. The DNA sequences of the inverted repeats of the IS6110 copy in strain 9013 and their adjacent DNA sequences are detailed. The positions of the primers used in this study are illustrated with arrows.

FIG. 3

Autoradiograph of Southern blot of PvuII-digested chromosomal DNA of M. tuberculosis 41909 with intact IS1547 copies (lane 1) and isolate 9013 (lane 2) probed with the DIG-labelled IS1547 probe. In comparison with isolate 41909, isolate 9013 has lost one hybridized band due to the deletion of the DNA segment of IS1547 where the probe would hybridize.

FIG. 4

Dendrogram of the 19 isolates and their IS6110 RFLP profiles with a scale of Dice coefficients. Two distinct subclusters (A and B) appear in the dendrogram.

FIG. 5

Models of the formation of the structures observed in isolates 9013 and 9504. Among the three possible mechanisms (IS6110 transposition, site-specific recombination, and homologous recombination) that may be responsible for the generation of the structures, homologous recombination is the most likely one and is detailed.

FIG. 6

(A) Schematic illustration of strain H37Rv of M. tuberculosis in comparison with clinical isolate F4 of M. tuberculosis, showing strain H37Rv’s deletion. (B) PCR fragments from these two strains (lane 1, H37Rv, and lane 2, F4) with primers Tf and P6. The fragment in isolate F4 (i.e., segment of b-c-d) was confirmed to contain no IS6110 element by DNA sequencing (data not shown).