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Genetics and Cell Morphology Analyses of the Actinomyces oris srtA Mutant

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Bacterial Cell Wall Homeostasis

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1440))

Abstract

Sortase is a cysteine-transpeptidase that anchors LPXTG-containing proteins on the Gram-positive bacterial cell wall. Previously, sortase was considered to be an important factor for bacterial pathogenesis and fitness, but not cell growth. However, the Actinomyces oris sortase is essential for cell viability, due to its coupling to a glycosylation pathway. In this chapter, we describe the methods to generate conditional srtA deletion mutants and identify srtA suppressors by Tn5 transposon mutagenesis. We also provide procedures for analyzing cell morphology of this mutant by thin-section electron microscopy. These techniques can be applied for analyses of other essential genes in A. oris.

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Acknowledgments

This work was supported by the National Institute of Dental and Craniofacial Research of the NIH under award numbers F31DE024004 (to M.E.R.-R.) and DE017382 and DE025015 (to H.T.-T.).

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Authors and Affiliations

  1. Department of Microbiology & Molecular Genetics, University of Texas Health Science Center, 6431 Fannin Street, R226/MSE, Houston, TX, 77030, USA

    Chenggang Wu

  2. Department of Microbiology & Molecular Genetics, University of Texas Health Science Center, Houston, TX, USA

    Melissa Elizabeth Reardon-Robinson

  3. Department of Microbiology & Molecular Genetics, University of Texas Health Science Center, 6431 Fannin Street, R224/MSE, Houston, TX, 77030, USA

    Hung Ton-That

Authors
  1. Chenggang Wu
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  2. Melissa Elizabeth Reardon-Robinson
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  3. Hung Ton-That
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Corresponding authors

Correspondence to Chenggang Wu or Hung Ton-That .

Editor information

Editors and Affiliations

  1. Dept. of Biochem & Dept. of Bio and Med., Univ. of Cambridge andOxford Brookes Unv, Cambridge, United Kingdom

    Hee-Jeon Hong

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Wu, C., Reardon-Robinson, M.E., Ton-That, H. (2016). Genetics and Cell Morphology Analyses of the Actinomyces oris srtA Mutant. In: Hong, HJ. (eds) Bacterial Cell Wall Homeostasis. Methods in Molecular Biology, vol 1440. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3676-2_9

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  • DOI: https://doi.org/10.1007/978-1-4939-3676-2_9

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-3674-8

  • Online ISBN: 978-1-4939-3676-2

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