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Applied and Environmental Microbiology, June 2006, p. 4078-4087, Vol. 72, No. 6
0099-2240/06/$08.00+0     doi:10.1128/AEM.02969-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Microbial Dioxygenase Gene Population Shifts during Polycyclic Aromatic Hydrocarbon Biodegradation

Sinéad M. Ní Chadhain,1,{dagger} R. Sean Norman,1,{dagger} Karen V. Pesce,1 Jerome J. Kukor,1,2,3 and Gerben J. Zylstra1,4*

Biotechnology Center for Agriculture and the Environment, Rutgers University, 59 Dudley Road, New Brunswick, New Jersey 08901-8520,1 Department of Environmental Sciences, Rutgers University, 14 College Farm Road, New Brunswick, New Jersey 08901-8551,2 New Jersey Agricultural Experiment Station, 88 Lipman Drive, New Brunswick, New Jersey 08901,3 Department of Biochemistry and Microbiology, Rutgers University, 76 Lipman Drive, New Brunswick, New Jersey 08901-85254

Received 15 December 2005/ Accepted 3 April 2006

The degradation of polycyclic aromatic hydrocarbons (PAHs) by bacteria has been widely studied. While many pure cultures have been isolated and characterized for their ability to grow on PAHs, limited information is available on the diversity of microbes involved in PAH degradation in the environment. We have designed generic PCR primers targeting the gene fragment encoding the Rieske iron sulfur center common to all PAH dioxygenase enzymes. These Rieske primers were employed to track dioxygenase gene population shifts in soil enrichment cultures following exposure to naphthalene, phenanthrene, or pyrene. PAH degradation was monitored by gas chromatograph with flame ionization detection. DNA was extracted from the enrichment cultures following PAH degradation. 16S rRNA and Rieske gene fragments were PCR amplified from DNA extracted from each enrichment culture and an unamended treatment. The PCR products were cloned and sequenced. Molecular monitoring of the enrichment cultures before and after PAH degradation using denaturing gradient gel electrophoresis and 16S rRNA gene libraries suggests that specific phylotypes of bacteria were associated with the degradation of each PAH. Sequencing of the cloned Rieske gene fragments showed that different suites of genes were present in soil microbe populations under each enrichment culture condition. Many of the Rieske gene fragment sequences fell into clades which are distinct from the reference dioxygenase gene sequences used to design the PCR primers. The ability to profile not only the bacterial community but also the dioxygenases which they encode provides a powerful tool for both assessing bioremediation potential in the environment and for the discovery of novel dioxygenase genes.

* Corresponding author. Mailing address: Biotechnology Center for Agriculture and the Environment, Rutgers University, 59 Dudley Rd., New Brunswick, NJ 08901-8520. Phone: (732) 932-8165. Fax: (732) 932-0312. E-mail: zylstra{at}aesop.rutgers.edu.

{dagger} These authors contributed equally to this paper.

Applied and Environmental Microbiology, June 2006, p. 4078-4087, Vol. 72, No. 6
0099-2240/06/$08.00+0     doi:10.1128/AEM.02969-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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