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Use of reporter transposons for tagging and detection of Mycobacterium sp. strain 1B in PAH-contaminated soil

Abstract

An environmental Mycobacterium able to degrade phenanthrene, pyrene and fluoranthene was transformed with an IS1096-based transposon marker system. Electroporation and subsequent delivery of the transposon enabled formation of constitutive lacZ transformants, with similar growth rates on pyrene and R2A media to the parental strain. A semi-selective medium was developed to recover and detect colonies of the transformed strain after inoculation into polycyclic aromatic hydrocarbon-contaminated soil. Microcosm experiments involving inoculation of the tagged Mycobacterium strain into a historically PAH-contaminated soil indicated survival when an appropriate carbon source was available. The results reported show that transposon systems developed for clinical mycobacterial isolates are also applicable for use in environmental isolates. The results also show that inoculated Mycobacterium strains could survive for at least 100 days at 106–107 cfu g−1 in the PAH-contaminated soil tested here.

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Abbreviations

PAH:

Polycyclic aromatic hydrocarbon

GC:

Gas chromatography

FID:

Flame ionization detection

Temp:

temperature

DCM:

Dichloromethane

PBS:

Phosphate buffered saline

MPN:

Most probable number

DMF:

Dimethylformamide

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Acknowledgements

C.D. was supported by Flinders University Research Scholarship. The vectors pEM32 and pEM42 were kind gifts from Edith Machowski, Molecular Mycobacteriology Research Unit, University of the Witwatersrand, South Africa.

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Correspondence to R. H. Bentham.

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Dandie, C.E., Bentham, R.H. & Thomas, S.M. Use of reporter transposons for tagging and detection of Mycobacterium sp. strain 1B in PAH-contaminated soil. Appl Microbiol Biotechnol 71, 59–66 (2006). https://doi.org/10.1007/s00253-005-0121-8

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Keywords

  • Polycyclic Aromatic Hydrocarbon
  • Pyrene
  • Mycobacterium
  • Electrocompetent Cell
  • Polycyclic Aromatic Hydrocarbon Degradation