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.
Samuels
D Scott
DS
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.
Garon
Claude F
CF
Bacterial Pathogenesis Section, Rocky Mountain Laboratories Microscopy Branch, National Institute of Allergy and Infectious Diseases, Hamilton, Montana 59840, USA.
D049933:Aminocoumarins; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D015748:Borrelia burgdorferi Group / Q000235:genetics*; D003374:Coumarins / Q000494:pharmacology; D027081:DNA Gyrase; D004250:DNA Topoisomerases, Type II / Q000235:genetics; D004277:DNA, Single-Stranded; D004352:Drug Resistance, Microbial; D018274:Electroporation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D009841:Oligonucleotides; D059005:Topoisomerase II Inhibitors; D014170:Transformation, Genetic
D049933:Aminocoumarins; D000900:Anti-Bacterial Agents; D003374:Coumarins; D004277:DNA, Single-Stranded; D004791:Enzyme Inhibitors; D009841:Oligonucleotides; D059005:Topoisomerase II Inhibitors; D027081:DNA Gyrase; D004250:DNA Topoisomerases, Type II; C004628:coumermycin