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Efficient targeted mutagenesis in Borrelia burgdorferi
J L Bono1, A F Elias, J J Kupko
1Laboratory of Human Bacterial Pathogenesis, National Institute of Allergy and Infectious Disease, National Institutes of Health, Hamilton, Montana 59840, USA. jbono@niaid.nih.gov
Journal of Bacteriology
|April 13, 2000
Summary
A new kanamycin resistance gene offers improved genetic manipulation for Borrelia burgdorferi (Lyme disease bacteria). This selectable marker facilitates efficient creation of mutant strains, overcoming limitations of previous methods.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- Genetic studies of Borrelia burgdorferi are challenging due to limited selectable markers.
- The existing gyrB(r) marker for coumermycin A(1) resistance has high recombination rates with the endogenous gene, hindering targeted gene disruption.
Purpose of the Study:
- To introduce and evaluate a kanamycin resistance gene (kan) as a novel selectable marker for Borrelia burgdorferi.
- To improve genetic manipulation techniques and facilitate the construction of isogenic mutant strains in this pathogen.
Main Methods:
- A kanamycin resistance gene (kan) was introduced into Borrelia burgdorferi.
- The kan gene was expressed using its native promoter and also the Borrelia burgdorferi flaB or flgB promoters.
- Transformants were selected on kanamycin-containing media.
Main Results:
- Borrelia burgdorferi transformants expressing kan from its native promoter were susceptible to kanamycin.
- Transformants expressing kan from the flaB or flgB promoters exhibited resistance to high kanamycin concentrations.
- The kan gene proved effective for direct selection of mutants.
Conclusions:
- The kanamycin resistance gene serves as a robust and efficient selectable marker for Borrelia burgdorferi.
- This marker significantly enhances the ability to create isogenic mutants, advancing genetic studies of the pathogen.