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ARS Home » Midwest Area » Ames, Iowa » National Animal Disease Center » Infectious Bacterial Diseases Research » Research » Publications at this Location » Publication #342959

Research Project: Characterization of Antigens, Virulence Markers, and Host Immunity in the Pathogenesis of Johne’s Disease

Location: Infectious Bacterial Diseases Research

Title: Transposon mutagenesis in Mycobacterium avium subspecies paratuberculosis

item Bannantine, John
item ZINNIEL, DENISE K - University Of Nebraska
item BARLETTA, RAUL - University Of Nebraska

Submitted to: Methods in Molecular Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/3/2019
Publication Date: 6/14/2019
Citation: Bannantine, J.P., Zinniel, D., Barletta, R.G. 2019. Transposon mutagenesis in Mycobacterium avium subspecies paratuberculosis. Methods in Molecular Biology. 2016/117-125.

Interpretive Summary: Genetic tools are important to study pathogens that cause disease. For Mycobacterium avium subspecies paratuberculosis, the bacterium that causes Johne's disease, these tools have been underdeveloped due to the slow growth, unique culture requirements, and especially, the unique and nearly impenetrable cell wall structure. However, our group has overcome these limitations and assembled and published on the construction and use of a mutant library for this bacterium. The mutations are created by insertion of a DNA element, called a transposon, into genes of the bacterium, rendering them nonfunctional. This advance has allowed us to determine which genes are essential to the bacterium and which can be dispensed or compensated for. In this communication, we provide the details of how transposon mutagenesis was conducted in Mycobacterium avium subspecies paratuberculosis with the hope that other investigators working in Johne's disease will use this tool to enhance their investigations.

Technical 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, MycoMarT7, 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 de-mystify the procedure and encourage additional investigators to incorporate transposon mutagenesis in their studies on Johne’s disease.