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Isolation of Bacterial Type IV Machine Subassemblies

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Book cover Bacterial Cell Surfaces

Part of the book series: Methods in Molecular Biology ((MIMB,volume 966))

Abstract

The bacterial type IV secretion systems (T4SSs) deliver DNA and protein substrates to bacterial and eukaryotic target cells generally by a mechanism requiring direct contact between donor and target cells. Recent advances in defining the architectures of T4SSs have been made through isolation of machine subassemblies for further biochemical and ultrastructural analysis. Here, we describe a protocol for isolation and characterization of VirB protein complexes from the paradigmatic VirB/VirD4 T4SS of Agrobacterium tumefaciens. This protocol can be adapted for isolation of T4SS subassemblies from other gram-negative bacteria as well as gram-positive bacteria. The biological importance of isolated T4SS subcomplexes can be assessed by assaying for copurification of trapped or cross-linked substrates. This can be achieved with a modified form of the chromatin immunoprecipitation (ChIP) assay termed transfer DNA immunoprecipitation (TrIP). Here, a TrIP protocol is described for recovery of formaldehyde-cross-linked DNA substrate–channel subunit complexes from cells employing T4SSs for conjugative DNA transfer.

Mayukh K. Sarkar and Seyyed I. Husnain contributed equally to this work.

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Acknowledgments

We thank members of the Waksman, Galan, and Baron laboratories for sharing ideas regarding biochemical enrichments of T4SS subassemblies. We thank Drs. Sayyed Shah and Eric Cascales for initiating studies in this laboratory aimed at isolation of T4SS subassemblies and cross-linked DNA substrate–channel subunit complexes. We thank other members of the Christie laboratory for valuable discussions and technical expertise. This work was supported by NIH grant GM48746 to PJC.

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Authors and Affiliations

  1. Department of Microbiology and Molecular Genetics, University of Texas Medical School at Houston, Houston, TX, USA

    Mayukh K. Sarkar, Simon J. Jakubowski & Peter J. Christie

  2. Department of Biochemistry, University of Leicester, Leicester, UK

    Seyyed I. Husnain

Authors
  1. Mayukh K. Sarkar
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  2. Seyyed I. Husnain
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  3. Simon J. Jakubowski
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  4. Peter J. Christie
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Correspondence to Peter J. Christie .

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  1. , Dept. of Biology and Biochemistry, University of Houston, Calhoun Road 4800, Houston, 77004, Texas, USA

    Anne H. Delcour

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Sarkar, M.K., Husnain, S.I., Jakubowski, S.J., Christie, P.J. (2013). Isolation of Bacterial Type IV Machine Subassemblies. In: Delcour, A. (eds) Bacterial Cell Surfaces. Methods in Molecular Biology, vol 966. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-245-2_12

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  • DOI: https://doi.org/10.1007/978-1-62703-245-2_12

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-244-5

  • Online ISBN: 978-1-62703-245-2

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