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Protocols for Tn-seq Analyses in the Group A Streptococcus

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Group A Streptococcus

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2136))

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Abstract

Transposon-sequencing (Tn-seq) has revolutionized forward-genetic analyses to study genotype–phenotype associations and interrogate bacterial cell physiology. The Tn-seq approach allows the en masse monitoring of highly complex mutant libraries, leveraging massive parallel DNA sequencing as a means to characterize the composition of these mutant pools on a genome-scale with unprecedented nucleotide-level high resolution. In this chapter, we present step-by-step protocols for Tn-seq analyses in the human pathogen Streptococcus pyogenes (Group A Streptococcus or GAS) using the mariner-based Krmit transposon. We detail how to generate highly complex Krmit mutant libraries in GAS and the en masse production of Krmit insertion tags for Illumina sequencing of the transposon–genome junctions for Tn-seq analyses. Most of the protocols presented here were developed and implemented using the S. pyogenes M1T1 serotype clinical isolate 5448, but they have been successfully applied to multiple GAS serotypes as well as other pathogenic Streptococci.

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Acknowledgments

Material has been reviewed by the Walter Reed Army Institute of Research. There is no objection to its presentation and/or publication. The opinions and assertions contained herein are the private views of the author and are not to be construed as official or as reflecting true views of the Department of the Army or the Department of Defense. This work was supported by grants (AI047928 and AI094773) from the National Institute of Allergy and Infectious Diseases (NIAID) at the National Institutes of Health (NIH).

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Authors and Affiliations

  1. Wound Infections Department, Bacterial Diseases Branch, Walter Reed Army Institute for Research, Silver Spring, MD, USA

    Yoann Le Breton

  2. Department of Cell Biology and Molecular Genetics and Maryland Pathogen Research Institute, University of Maryland, College Park, College Park, MD, USA

    Ashton T. Belew & Kevin S. McIver

  3. Center for Bioinformatics and Computational Biology, University of Maryland, College Park, College Park, MD, USA

    Ashton T. Belew

Authors
  1. Yoann Le Breton
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  2. Ashton T. Belew
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  3. Kevin S. McIver
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Corresponding author

Correspondence to Yoann Le Breton .

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Editors and Affiliations

  1. School of Medical Sciences, The University of Auckland, Auckland, New Zealand

    Thomas Proft

  2. School of Medical Sciences, The University of Auckland, Auckland, New Zealand

    Jacelyn M. S. Loh

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Le Breton, Y., Belew, A.T., McIver, K.S. (2020). Protocols for Tn-seq Analyses in the Group A Streptococcus. In: Proft, T., Loh, J. (eds) Group A Streptococcus. Methods in Molecular Biology, vol 2136. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0467-0_4

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  • DOI: https://doi.org/10.1007/978-1-0716-0467-0_4

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0466-3

  • Online ISBN: 978-1-0716-0467-0

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