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Analysis of PHB Metabolism Applying Tn5 Mutagenesis in Ralstonia eutropha

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Hydrocarbon and Lipid Microbiology Protocols

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Abstract

Transposon mutagenesis presents a powerful and practicable method to generate single-gene disruption mutants of microorganisms. As naturally occurring transposons “jump” within the genome, molecular biology uses plasmid-bound transposons, which randomly disrupt genomic regions of the target organism. Obtained transposon mutants help to elucidate metabolic pathways and to identify essential genes, which are involved in syntheses or degradation of compounds or are important for other cell processes or cell structures. The best-known transposon, Tn5, codes for different antibiotic resistances as well as for a transposase mediating transposition and a transposase inhibitor protein. A notable example of applied Tn5 mutagenesis is the identification and localization of genes, which are involved in the synthesis of the industrially relevant biopolymer poly(3-hydroxybutyrate) (PHB) in Ralstonia eutropha H16. PHB is synthesized in a three-step pathway, and the key genes of R. eutropha were found to be organized as a single operon. In this chapter, the generation and analysis of Tn5-induced mutants of R. eutropha is described. This procedure starts with the transfer of the Tn5-harboring plasmid pSUP5011 into R. eutropha by conjugation, is followed by the screening of mutants defective in PHB accumulation, and is then completed by identifying genes, which have been disrupted by Tn5 by sequence analyses.

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Authors and Affiliations

  1. Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische Wilhelms-Universität, Münster, Germany

    Matthias Raberg, Daniel Heinrich & Alexander Steinbüchel

  2. Environmental Sciences Department, King Abdulaziz University, Jeddah, Saudi Arabia

    Alexander Steinbüchel

Authors
  1. Matthias Raberg
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  2. Daniel Heinrich
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  3. Alexander Steinbüchel
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Corresponding author

Correspondence to Alexander Steinbüchel .

Editor information

Editors and Affiliations

  1. School of Biological Sciences, University of Essex, Colchester, Essex, United Kingdom

    Terry J. McGenity

  2. Institute of Microbiology, Technical University Braunschweig, Braunschweig, Germany

    Kenneth N. Timmis

  3. Department of Biology, University of the Balearic Islands and Mediterranean Institute for Advanced Studies (IMEDEA, UIB-CSIC), Palma de Mallorca, Spain

    Balbina Nogales

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© 2015 Springer-Verlag Berlin Heidelberg

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Raberg, M., Heinrich, D., Steinbüchel, A. (2015). Analysis of PHB Metabolism Applying Tn5 Mutagenesis in Ralstonia eutropha . In: McGenity, T., Timmis, K., Nogales, B. (eds) Hydrocarbon and Lipid Microbiology Protocols. Springer Protocols Handbooks. Springer, Berlin, Heidelberg. https://doi.org/10.1007/8623_2015_110

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  • DOI: https://doi.org/10.1007/8623_2015_110

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  • Publisher Name: Springer, Berlin, Heidelberg

  • Print ISBN: 978-3-662-49138-6

  • Online ISBN: 978-3-662-49140-9

  • eBook Packages: Springer Protocols

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