Host-Bacteria Interactions pp 329-345 | Cite as
Gene Inactivation in Coxiella burnetii
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Abstract
Coxiella burnetii, the agent of human Q fever, is a zoonotic bacterial pathogen with a worldwide distribution. Owing to an historic lack of methods for genetic manipulation, virulence factors deployed by this bacterium for disease pathogenesis are poorly understood. However, the recent advance of host cell-free (axenic) growth of C. burnetii has coincided with development of several new genetic technologies including site-specific and random transposon systems, shuttle vectors, and an inducible gene expression system. We have recently added two methods for targeted gene inactivation to the expanding C. burnetii genetics toolbox. Here, we describe a “loop in/loop out” gene inactivation system for C. burnetii. This procedure allows for generation of site-directed mutants in approximately 10 weeks and has been used by our laboratory to generate more than 50 individual C. burnetii mutants. The collection of C. burnetii genetic tools now allows for conventional mutation and complementation strategies to define virulence factors.
Key words
Coxiella burnetii Electroporation Genetic transformation Targeted gene inactivation Axenic media Transposon Counterselection Homologous recombinationNotes
Acknowledgement
This study was supported by the Intramural Research Program of the National Institutes of Health, National Institute of Allergy and Infectious Diseases.
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