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Oligonucleoticle-tnediated genetic transformation of Borrelia burgdorferi

Division of Biological Sciences, University of Montana, Missoula, Montana 59812, USA
Bacterial Pathogenesis Section, Rocky Mountain Laboratories Microscopy Branch, National Institute of Allergy and Infectious Diseases, Hamilton, Montana 59840, USA

³Author for correspondence: Scott Samuels. Tel: + 1 406 243 6145. Fax: + 1 406 243 4184. e-mail: Samuels@selway.umt.edu

ABSTRACT

We have used short oligonucleotides to genetically transform the Lyme disease spirochaete Borrelia burgdorferi. The oligonucleotides are derived from the sequence of an Arg-133 to He mutant gyrB (chromosomal) gene that confers resistance to the antibiotic coumermycin A₁. Oligonucleotides were about 10000-fold less efficient at transformation, on a molar basis, than longer PCR-generated substrates. All of the transformants tested contained the predicted site-directed silent mutation in their gyrB genes. Antisense oligonucleotides were more efficient at transformation than either sense or double-stranded oligonucleotides. This is the first demonstration of oligonucleotides used to introduce site-directed mutations directly into the genome of a bacterium.

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