Oligonucleotide-mediated genetic transformation of Borrelia burgdorferi

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 Ile mutant gyrB (chromosomal) gene that confers resistance to the antibiotic coumermycin A₁. Oligonucleotides were about 10,000-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.