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Site-Directed Mutagenesis and Its Application in Studying the Interactions of T3S Components

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Type 3 Secretion Systems

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1531))

Abstract

Type III secretion systems are a prolific virulence determinant among Gram-negative bacteria. They are used to paralyze the host cell, which enables bacterial pathogens to establish often fatal infections—unless an effective therapeutic intervention is available. However, as a result of a catastrophic rise in infectious bacteria resistant to conventional antibiotics, these bacteria are again a leading cause of worldwide mortality. Hence, this report describes a pDM4-based site-directed mutagenesis strategy that is assisting in our foremost objective to better understand the fundamental workings of the T3SS, using Yersinia as a model pathogenic bacterium. Examples are given that clearly document how pDM4-mediated site-directed mutagenesis has been used to establish clean point mutations and in-frame deletion mutations that have been instrumental in identifying and understanding the molecular interactions between components of the Yersinia type III secretion system.

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Acknowledgments

This work was supported by Swedish Research Council grant 2014–2105 and the Medical Research Foundation of Umeå University to MSF.

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Authors and Affiliations

  1. Department of Molecular Biology, Umeå University, 6K och 6L, Sjukhusområdet, Umeå, 901 87, Sweden

    Matthew S. Francis, Ayad A. A. Amer, Debra L. Milton & Tiago R. D. Costa

  2. Umeå Centre for Microbial Research, Umeå University, 6K och 6L, Sjukhusområdet, Umeå, 901 87, Sweden

    Matthew S. Francis, Ayad A. A. Amer, Debra L. Milton & Tiago R. D. Costa

  3. Helmholtz Centre for Infection Research, Braunschweig, Germany

    Ayad A. A. Amer

  4. Department of Biological and Environmental Sciences, Troy University, Troy, AL, USA

    Debra L. Milton

  5. Institute of Structural and Molecular Biology, University College London and Birkbeck, Malet Street, London, UK

    Tiago R. D. Costa

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  1. Matthew S. Francis
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  2. Ayad A. A. Amer
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  3. Debra L. Milton
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  4. Tiago R. D. Costa
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Correspondence to Matthew S. Francis .

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Editors and Affiliations

  1. Department of Biomedical Sciences, School of Medicine and Health Sciences University of North Dakota, Grand Forks, North Dakota, USA

    Matthew L. Nilles

  2. Department of Biomedical Sciences, School of Medicine and Health Sciences University of North Dakota, Grand Forks, North Dakota, USA

    Danielle L. Jessen Condry

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Francis, M.S., Amer, A.A.A., Milton, D.L., Costa, T.R.D. (2017). Site-Directed Mutagenesis and Its Application in Studying the Interactions of T3S Components. In: Nilles, M., Condry, D. (eds) Type 3 Secretion Systems. Methods in Molecular Biology, vol 1531. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6649-3_2

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  • DOI: https://doi.org/10.1007/978-1-4939-6649-3_2

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