Abstract
Three major techniques have been employed for broad-range in vitro mutagenesis of Brucella species. Shotgun approaches capable of generating large libraries of randomly inserted transposon mutants include Tn5, mariner (Himar1), and mini-Tn5 signature-tagged mutagenesis. Allelic exchange has also been extensively employed for targeted gene deletion. In general, plasmid and transposon delivery into Brucella has relied upon electroporation; however, conjugation has also been successfully employed. Both approaches have been used to identify critical virulence determinants necessary for disease and intracellular survival of the organism. Perhaps more importantly these approaches have provided an opportunity to develop attenuated vaccine candidates of improved safety and efficacy. Future experiments are designed to identify individual functions that govern the interaction between host and agent and control intracellular trafficking and survival. Toward this goal, this chapter describes current approaches used to mutagenize Brucella spp.
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Acknowledgments
We would like to acknowledge the support of NIH (R01048496) and the WRCE (1U54AI057156-0100) for the construction of B. melitensis mutant banks and the USDA (99-35204-7550) for the development of B. abortus mutant banks. We gratefully acknowledge the contributions of Chris Allen, Renée Tsolis, Priscilla Hong, and Carol Turse without whose efforts this publication would not be possible.
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Ficht, T.A., Pei, J., Kahl-McDonagh, M. (2010). In Vitro Mutagenesis of Brucella Species. In: Braman, J. (eds) In Vitro Mutagenesis Protocols. Methods in Molecular Biology, vol 634. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-652-8_2
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DOI: https://doi.org/10.1007/978-1-60761-652-8_2
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