title: Abundance of dioxygenase genes similar to Ralstonia sp. strain U2 nagAc is correlated with naphthalene concentrations in coal tar-contaminated freshwater sediments.
abstract: 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-microl 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 10(3) to (2.9 +/- 0.3) x 10(5) copies of nagAc-like dioxygenase genes per microg of DNA extracted from sediment samples. These values corresponded to (1.2 +/- 0.6) x 10(5) to (5.4 +/- 0.4) x 10(7) 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.
authors.affiliation: Center for Environmental Biotechnology, 676 Dabney Hall, The University of Tennessee, Knoxville, TN 37996, USA.
authors.forename: Hebe M
authors.initials: HM
authors.lastname: Dionisi
authors.forename: Christopher S
authors.initials: CS
authors.lastname: Chewning
authors.forename: Katherine H
authors.initials: KH
authors.lastname: Morgan
authors.forename: Fu-Min
authors.initials: FM
authors.lastname: Menn
authors.forename: James P
authors.initials: JP
authors.lastname: Easter
authors.forename: Gary S
authors.initials: GS
authors.lastname: Sayler
mesh_terms: D001673:Biodegradation, Environmental; D003033:Coal Tar; D004269:DNA, Bacterial / Q000032:analysis; D049308:Dioxygenases; D005618:Fresh Water / Q000032:analysis*; D005798:Genes, Bacterial; D019015:Geologic Sediments / Q000032:analysis*; D009097:Multienzyme Complexes / Q000235:genetics*; D009281:Naphthalenes / Q000032:analysis / Q000378:metabolism*; D010105:Oxygenases / Q000235:genetics*; D016133:Polymerase Chain Reaction; D043367:Ralstonia / Q000235:genetics*
chemical_list: D004269:DNA, Bacterial; D009097:Multienzyme Complexes; D009281:Naphthalenes; C031721:naphthalene; D003033:Coal Tar; D010105:Oxygenases; D049308:Dioxygenases; C038703:naphthalene dioxygenase