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Journal of Bacteriology, May 2000, p. 2445-2452, Vol. 182, No. 9
0021-9193/00/$04.00+0

Efficient Targeted Mutagenesis in Borrelia burgdorferi

James L. Bono,* Abdallah F. Elias, John J. Kupko III, Brian Stevenson,dagger Kit Tilly, and Patricia Rosa

Laboratory of Human Bacterial Pathogenesis, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Disease, National Institutes of Health, Hamilton, Montana 59840

Received 25 October 1999/Accepted 16 February 2000

Genetic studies in Borrelia burgdorferi have been hindered by the lack of a nonborrelial selectable marker. Currently, the only selectable marker is gyrBr, a mutated form of the chromosomal gyrB gene that encodes the B subunit of DNA gyrase and confers resistance to the antibiotic coumermycin A1. The utility of the coumermycin-resistant gyrBr gene for targeted gene disruption is limited by a high frequency of recombination with the endogenous gyrB gene. A kanamycin resistance gene (kan) was introduced into B. burgdorferi, and its use as a selectable marker was explored in an effort to improve the genetic manipulation of this pathogen. B. burgdorferi transformants with the kan gene expressed from its native promoter were susceptible to kanamycin. In striking contrast, transformants with the kan gene expressed from either the B. burgdorferi flaB or flgB promoter were resistant to high levels of kanamycin. The kanamycin resistance marker allows efficient direct selection of mutants in B. burgdorferi and hence is a significant improvement in the ability to construct isogenic mutant strains in this pathogen.

* Corresponding author. Mailing address: Laboratory of Human Bacterial Pathogenesis, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Disease, National Institutes of Health, 903 South 4th St., Hamilton, MT 59840. Phone: (406) 363-9301. Fax: (406) 363-9204. E-mail: jbono{at}niaid.nih.gov.

dagger Present address: Department of Microbiology and Immunology, University of Kentucky College of Medicine, MS415 UKMC, Lexington, KY 40536.

Journal of Bacteriology, May 2000, p. 2445-2452, Vol. 182, No. 9
0021-9193/00/$04.00+0


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