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Applied and Environmental Microbiology, July 2004, p. 3988-3995, Vol. 70, No. 7
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.7.3988-3995.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Abundance of Dioxygenase Genes Similar to Ralstonia sp. Strain U2 nagAc Is Correlated with Naphthalene Concentrations in Coal Tar-Contaminated Freshwater Sediments

Hebe M. Dionisi,1,{dagger} Christopher S. Chewning,1 Katherine H. Morgan,1 Fu-Min Menn,1 James P. Easter,1 and Gary S. Sayler1,2*

Center for Environmental Biotechnology,1 Department of Microbiology, The University of Tennessee, Knoxville, Tennessee 379962

Received 10 December 2003/ Accepted 26 March 2004

We designed a real-time PCR assay able to recognize dioxygenase large-subunit gene sequences with more than 90% similarity to the Ralstonia sp. strain U2 nagAc gene (nagAc-like gene sequences) in order to study the importance of organisms carrying these genes in the biodegradation of naphthalene. Sequencing of PCR products indicated that this real-time PCR assay was specific and able to detect a variety of nagAc-like gene sequences. One to 100 ng of contaminated-sediment total DNA in 25-µl reaction mixtures produced an amplification efficiency of 0.97 without evident PCR inhibition. The assay was applied to surficial freshwater sediment samples obtained in or in close proximity to a coal tar-contaminated Superfund site. Naphthalene concentrations in the analyzed samples varied between 0.18 and 106 mg/kg of dry weight sediment. The assay for nagAc-like sequences indicated the presence of (4.1 ± 0.7) x 103 to (2.9 ± 0.3) x 105 copies of nagAc-like dioxygenase genes per µg of DNA extracted from sediment samples. These values corresponded to (1.2 ± 0.6) x 105 to (5.4 ± 0.4) x 107 copies of this target per g of dry weight sediment when losses of DNA during extraction were taken into account. There was a positive correlation between naphthalene concentrations and nagAc-like gene copies per microgram of DNA (r = 0.89) and per gram of dry weight sediment (r = 0.77). These results provide evidence of the ecological significance of organisms carrying nagAc-like genes in the biodegradation of naphthalene.

* Corresponding author. Mailing address: Center for Environmental Biotechnology, 676 Dabney Hall, The University of Tennessee, Knoxville, TN 37996. Phone: (865) 974-8080. Fax: (865) 974-8086. E-mail: sayler{at}utk.edu.

{dagger} Present address: CENPAT-CONICET, Puerto Madryn, Chubut, Argentina.

Applied and Environmental Microbiology, July 2004, p. 3988-3995, Vol. 70, No. 7
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.7.3988-3995.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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