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

doi:10.1128/JB.182.9.2393-2401.2000

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Journal of Bacteriology, May 2000, p. 2393-2401, Vol. 182, No. 9
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

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

J. D. A. van Embden,¹^,^* T. van Gorkom,¹ K. Kremer,² R. Jansen,³ B. A. M. van der Zeijst,³ and L. M. Schouls¹

Department of Bacteriology of the Research Laboratory for Infectious Disease,¹ and Diagnostic Laboratory for Infectious Diseases and Perinatal Screening,² National Institute of Public Health and the Environment, 3720 BA Bilthoven, and Veterinary Faculty, Infectious Diseases and Immunology, Department of Bacteriology, 3508 TD Utrecht,³ The Netherlands

Received 12 November 1999/Accepted 26 January 2000

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 anonrepetitive spacer sequence of similar size. It has been shownpreviously that clinical isolates show extensive polymorphismin the DR region by the variable presence of DVRs, and this polymorphismhas been used in the epidemiology of tuberculosis. In an attemptto better understand the evolutionary scenario leading to polymorphicDR loci and to improve strain differentiation by spoligotyping,we characterized and compared the DNA sequences of the completeDR region and its flanking DNA of M. tuberculosis complex strains.We identified 94 different spacer sequences among 26 M. tuberculosiscomplex strains. No sequence homology was found between any ofthese spacers and M. tuberculosis DNA outside of the DR regionor with any other known bacterial sequence. Although strains differedextensively in the presence or absence of DVRs, the order of thespacers in the DR locus was found to be well conserved. The datastrongly suggest that the polymorphism in clinical isolates isthe result of successive deletions of single discrete DVRs orof multiple contiguous DVRs from a primordial DR region containingmany more DVRs than seen in present day isolates and that virtuallyno scrambling of DVRs took place during evolution. Because themajority of the novel spacer sequences identified in this studywere confined to isolates of the rare Mycobacterium canettii taxon,the use of the novel spacers in spoligotyping led only to a slightimprovement of strain differentiation byspoligotyping.

^* Corresponding author. Mailing address: Department of Bacteriology of the Research Laboratory for Infectious Disease, NationalInstitute of Public Health and the Environment, P.O. Box 1, 3720BA Bilthoven, The Netherlands. Phone: 31-30-2742113. Fax: 31-30-2744449.E-mail: JDA.van.Embden{at}rivm.nl.

Journal of Bacteriology, May 2000, p. 2393-2401, Vol. 182, No. 9
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

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