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 108 gene copies per g of sediment. Externally validated qPCR provides a valuable tool to monitor aromatic hydrocarbon degrader population abundances at contaminated sites.
Highlights
Many aromatic hydrocarbons are EPA priority pollutants (USEPA 2001, 2010), due to their high residence time in the environment (Cerniglia 1992), and their toxic effects on biota, from microbes to humans, increase with their molecular size (Miller and Miller 1981; Fetzer 2003; Benigni and Bossa 2011)
Four major lineages were identified in the phylogeny of 209 dioxygenase gene sequences from aromatic hydrocarbondegrading bacteria
Lineage 1, lineage 2 and lineage 3 were dominated by previously classified (Gibson and Parales 2000; Wackett 2002; Iwai et al 2011) PAH dioxygenases from Gram-negative bacteria (PAH-GN), toluene/biphenyl dioxygenases (T/B) and PAH dioxygenases from Gram-positive bacteria (PAH-GP) respectively (Fig. 1, and Table S1 in Supporting Information)
Summary
Many aromatic hydrocarbons are EPA priority pollutants (USEPA 2001, 2010), due to their high residence time in the environment (Cerniglia 1992), and their toxic effects on biota, from microbes to humans, increase with their molecular size (Miller and Miller 1981; Fetzer 2003; Benigni and Bossa 2011). The RHOs are multicomponent enzymes with an αnβn composition (Karlsson et al 2003; Parales 2003; Ferraro, Gakhar and Ramaswamy 2005), where the α-subunit contains the catalytically relevant components and determines substrate specificity The genes encoding these dioxygenases have been found in a wide range of aromatic-degrading bacteria (Habe and Omori 2003), either on plasmid or chromosomal DNA (Kweon et al 2008). The classification schemes for RHOs proposed so far are mainly based on their occurrence in Gram-positive or Gram-negative bacteria (Cebron et al 2008), substrate specificity (Wackett 2002; Iwai et al 2011) or on each component of the RHO and their combined modular structure (Batie, Ballou and Correll 1991; Kweon et al 2008) These classifications are generally constructed on the basis of homologies in the amino acid sequences of the components of each enzyme and their subunit structure, and included both mono- and dioxygenases. Recent attention has been focused on classification schemes based on the amino acid sequences of RHO α-subunits, due to their higher evolutionary plasticity compared to other RHO components and the fact that they contain the catalytically relevant metal centres (Wackett 2002; VilchezVargas et al 2013)
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