title: Re-evaluation of dioxygenase gene phylogeny for the development and validation of a quantitative assay for environmental aromatic hydrocarbon degraders.
abstract: Rieske non-heme iron oxygenases enzymes have been widely studied, as they catalyse essential reactions initiating the bacterial degradation of organic compounds, for instance aromatic hydrocarbons. The genes encoding these enzymes offer a potential target for studying aromatic hydrocarbon-degrading organisms in the environment. However, previously reported primer sets that target dioxygenase gene sequences or the common conserved Rieske centre of aromatics dioxygenases have limited specificity and/or target non-dioxygenase genes. In this work, an extensive database of dioxygenase α-subunit gene sequences was constructed, and primer sets targeting the conserved Rieske centre were developed. The high specificity of the primers was confirmed by polymerase chain reaction analysis, agarose gel electrophoresis and sequencing. Quantitative polymerase chain reaction (qPCR) assays were also developed and optimized, following MIQE guidelines (Minimum Information for Publication of Quantitative Real-Time PCR Experiments). Comparison of the qPCR quantification of dioxygenases in spiked sediment samples and in pure cultures demonstrated an underestimation of the Ct value, and the requirement for a correction factor at gene abundances below 10(8) gene copies per g of sediment. Externally validated qPCR provides a valuable tool to monitor aromatic hydrocarbon degrader population abundances at contaminated sites.
authors.affiliation: School of Civil Engineering and Geosciences, Newcastle University, NE1 7RU, England, UK paola.meynet@ncl.ac.uk.
authors.forename: Paola
authors.initials: P
authors.lastname: Meynet
authors.affiliation: School of Civil Engineering and Geosciences, Newcastle University, NE1 7RU, England, UK.
authors.forename: Ian M
authors.initials: IM
authors.lastname: Head
authors.affiliation: School of Civil Engineering and Geosciences, Newcastle University, NE1 7RU, England, UK.
authors.forename: David
authors.initials: D
authors.lastname: Werner
authors.affiliation: School of Civil Engineering and Geosciences, Newcastle University, NE1 7RU, England, UK.
authors.forename: Russell J
authors.initials: RJ
authors.lastname: Davenport
mesh_terms: D001483:Base Sequence; D001673:Biodegradation, Environmental; D019117:Burkholderia / Q000235:genetics / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics*; D049308:Dioxygenases / Q000235:genetics*; D004777:Environment; D006841:Hydrocarbons, Aromatic / Q000378:metabolism*; D009161:Mycobacterium / Q000235:genetics / Q000378:metabolism; D010802:Phylogeny; D011549:Pseudomonas / Q000235:genetics / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods; D012240:Rhodococcus / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA
chemical_list: D017931:DNA Primers; D006841:Hydrocarbons, Aromatic; D049308:Dioxygenases
keywords: Rieske centre; dioxygenases; phylogeny; quantitative PCR