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Phenotype-Based Identification of Key Enzymes for Polycyclic Aromatic Hydrocarbon (PAH) Metabolism from Mycobacteria Using Transposon Mutagenesis and a PAH Spray Plate

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Hydrocarbon and Lipid Microbiology Protocols

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Abstract

Despite considerable knowledge of bacterial aromatic hydrocarbon metabolism, the key enzymes and their pleiotropic and epistatic behaviors responsible for mycobacterial high-molecular-weight (HMW) polycyclic aromatic hydrocarbon (PAH) metabolism remain poorly understood. In this chapter, we describe a rapid, low-cost, high-throughput screening for function-dependent selection of mutants with metabolic discrepancies or defects in the metabolism of HMW-PAHs, which consists of (1) preparation of electrocompetent mycobacterial cells; (2) construction of a transposition mutant library, using a Tn5-based transposon; (3) phenotypic screening, using a PAH spray plate method; and (4) rescue cloning of transposed genomic DNA. The protocol may also be applicable to other PAH-degrading mycobacterial strains or actinobacteria.

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Acknowledgments

We thank John B. Sutherland and Dongryeoul Bae for critical review of the manuscript. The views presented in this article do not necessarily reflect those of the Food and Drug Administration.

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Authors and Affiliations

  1. Division of Microbiology, National Center for Toxicological Research/U.S. FDA, 3900 NCTR Road, Jefferson, AR, 72079, USA

    Seong-Jae Kim, Ohgew Kweon & Carl E. Cerniglia

Authors
  1. Seong-Jae Kim
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  2. Ohgew Kweon
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  3. Carl E. Cerniglia
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Corresponding author

Correspondence to Carl E. Cerniglia .

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

    Balbina Nogales

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Kim, SJ., Kweon, O., Cerniglia, C.E. (2015). Phenotype-Based Identification of Key Enzymes for Polycyclic Aromatic Hydrocarbon (PAH) Metabolism from Mycobacteria Using Transposon Mutagenesis and a PAH Spray Plate. 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_57

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  • DOI: https://doi.org/10.1007/8623_2015_57

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  • Publisher Name: Springer, Berlin, Heidelberg

  • Print ISBN: 978-3-662-49135-5

  • Online ISBN: 978-3-662-49137-9

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