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Transposon Mutagenesis in Mycobacterium avium Subspecies Paratuberculosis

  • John P. BannantineEmail author
  • Denise K. Zinniel
  • Raúl G. Barletta
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2016)

Abstract

While transposon mutagenesis has been developed for Mycobacterium avium subspecies paratuberculosis (Map), relatively few laboratories have adopted this important genetic tool to examine gene function and essentiality. Here we describe the construction of a Map transposon library using the Himar1 mariner transposon, but concepts can also be applied to the Tn5367 transposon, which has also been used by our group. Delivery of the transposon is by a temperature-sensitive phagemid, ϕMycoMarT7, and plating transductants requires patience and specialized media due to length of incubation required to observe colonies. Several transposon mutants obtained from these libraries have been tested in vaccine and pathogenesis studies. By providing the following detailed protocol herein, we expect to demystify the procedure and encourage additional investigators to incorporate transposon mutagenesis in their studies on Johne’s disease.

Key words

Transposon mutagenesis Mycobacterium Johne’s disease 

Notes

Acknowledgments

This work was supported by the USDA-Agricultural Research Service and the USDA-National Institute of Food and Agriculture grant award 2013-67015.

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • John P. Bannantine
    • 1
    Email author
  • Denise K. Zinniel
    • 2
  • Raúl G. Barletta
    • 2
  1. 1.USDA-ARS-National Animal Disease CenterAmesUSA
  2. 2.School of Veterinary Medicine and Biomedical SciencesUniversity of NebraskaLincolnUSA

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