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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

  • Seong-Jae Kim
  • Ohgew Kweon
  • Carl E. CernigliaEmail author
Protocol
Part of the Springer Protocols Handbooks book series (SPH)

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.

Keywords

Mycobacterium PAH metabolism PAH spray plate Phenotype Transposon mutagenesis 

Notes

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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Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Seong-Jae Kim
    • 1
  • Ohgew Kweon
    • 1
  • Carl E. Cerniglia
    • 1
    Email author
  1. 1.Division of MicrobiologyNational Center for Toxicological Research/U.S. FDAJeffersonUSA

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