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Mutagenesis of Vibrio fischeri and Other Marine Bacteria Using Hyperactive Mini-Tn5 Derivatives

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Microbial Transposon Mutagenesis

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2016))

Abstract

Mutagenizing bacterial genomes with selectable transposon insertions is an effective approach for identifying the genes underlying important phenotypes. Specific bacteria may require different tools and methods for effective transposon mutagenesis, and here we describe methods to mutagenize Vibrio fischeri using an engineered mini-Tn5 transposon with synthetic “mosaic” transposon ends. The transposon is delivered by conjugation on a plasmid that cannot replicate in V. fischeri and that encodes a hyperactive transposase outside the transposon itself. The chromosomal location of insertions can be readily identified by cloning and/or PCR-based methods described here. Although developed in V. fischeri, these tools and methods have proven effective in some other bacteria as well.

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Acknowledgments

The authors wish to thank Alecia Septer, Mark Mandel, Tim Miyashiro, and Karen Visick for helpful discussions, and Helen Dukes for technical assistance. This work was supported by NSF grants CAREER-MCB-0347317, IOS-0841480, IOS-1557964, IOS-0843317, and MCB-1716232.

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Authors and Affiliations

  1. Department of Microbiology, University of Georgia, Athens, GA, USA

    Julie L. Stoudenmire & Eric V. Stabb

  2. Biological Sciences Department, California Polytechnic University, San Luis Obispo, CA, USA

    Michael Black & Pat M. Fidopiastis

Authors
  1. Julie L. Stoudenmire
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  2. Michael Black
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  3. Pat M. Fidopiastis
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  4. Eric V. Stabb
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Corresponding author

Correspondence to Eric V. Stabb .

Editor information

Editors and Affiliations

  1. Department of Food Science, Center for Food Safety, University of Arkansas, Fayetteville, AR, USA

    Steven C. Ricke

  2. Department of Food Science and Technology, Oregon State University, Corvallis, OR, USA

    Si Hong Park

  3. Department of Food Science, University of Arkansas, Fayetteville, AR, USA

    Morgan L. Davis

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Stoudenmire, J.L., Black, M., Fidopiastis, P.M., Stabb, E.V. (2019). Mutagenesis of Vibrio fischeri and Other Marine Bacteria Using Hyperactive Mini-Tn5 Derivatives. In: Ricke, S., Park, S., Davis, M. (eds) Microbial Transposon Mutagenesis. Methods in Molecular Biology, vol 2016. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9570-7_9

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  • DOI: https://doi.org/10.1007/978-1-4939-9570-7_9

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9569-1

  • Online ISBN: 978-1-4939-9570-7

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