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DNA exchange and insertional inactivation in spirochetes
1Laboratory of Human Bacterial Pathogenesis, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rocky Mountain Laboratories, Hamilton, Montana, USA.
Journal of Molecular Microbiology and Biotechnology
|November 15, 2000
Summary
Gene inactivation in spirochetes, particularly Borrelia burgdorferi, is advancing. Optimal electrotransformation requires specific bacterial growth phases, DNA amounts, and field strengths for successful genetic manipulation.
Area of Science:
- Microbiology
- Genetics
- Pathogen Research
Background:
- Spirochetes are significant pathogens with complex life cycles, yet their genetic manipulation remains underdeveloped.
- Gene inactivation has been demonstrated in key spirochete species, including Borrelia burgdorferi, Brachyspira hyodysenteriae, and Treponema denticola.
Purpose of the Study:
- To review existing methods for gene inactivation and DNA exchange in spirochetes.
- To focus on the genetic manipulation of Borrelia burgdorferi, the causative agent of Lyme disease.
- To identify factors influencing electrotransformation efficiency in Borrelia burgdorferi.
Main Methods:
- Review of published literature on spirochete gene inactivation and DNA exchange techniques.
- Analysis of factors affecting electrotransformation in Borrelia burgdorferi, including bacterial growth phase, DNA quantity, and electrical field strength.
Main Results:
- Optimal electrotransformation of Borrelia burgdorferi is achieved using log-phase bacteria, substantial DNA concentrations (up to 50 microg), and high field strengths (12.5–37.5 kV/cm).
- Infectious Borrelia burgdorferi isolates exhibit transformation frequencies 100-fold lower than high-passage, non-infectious strains.
- Bacterial surface characteristics, often linked to infectivity, present challenges for efficient electroporation.
Conclusions:
- Established protocols can facilitate gene inactivation and DNA exchange in spirochetes, with a focus on Borrelia burgdorferi.
- Understanding and overcoming barriers related to bacterial surface properties is crucial for improving transformation efficiency in infectious strains.