title: Characterization of novel polycyclic aromatic hydrocarbon dioxygenases from the bacterial metagenomic DNA of a contaminated soil.
abstract: Ring-hydroxylating dioxygenases (RHDs) play a crucial role in the biodegradation of a range of aromatic hydrocarbons found on polluted sites, including polycyclic aromatic hydrocarbons (PAHs). Current knowledge on RHDs comes essentially from studies on culturable bacterial strains, while compelling evidence indicates that pollutant removal is mostly achieved by uncultured species. In this study, a combination of DNA-SIP labeling and metagenomic sequence analysis was implemented to investigate the metabolic potential of main PAH degraders on a polluted site. Following in situ labeling using [(13)C]phenanthrene, the labeled metagenomic DNA was isolated from soil and subjected to shotgun sequencing. Most annotated sequences were predicted to belong to Betaproteobacteria, especially Rhodocyclaceae and Burkholderiales, which is consistent with previous findings showing that main PAH degraders on this site were affiliated to these taxa. Based on metagenomic data, four RHD gene sets were amplified and cloned from soil DNA. For each set, PCR yielded multiple amplicons with sequences differing by up to 321 nucleotides (17%), reflecting the great genetic diversity prevailing in soil. RHDs were successfully overexpressed in Escherichia coli, but full activity required the coexpression of two electron carrier genes, also cloned from soil DNA. Remarkably, two RHDs exhibited much higher activity when associated with electron carriers from a sphingomonad. The four RHDs showed markedly different preferences for two- and three-ring PAHs but were poorly active on four-ring PAHs. Three RHDs preferentially hydroxylated phenanthrene on the C-1 and C-2 positions rather than on the C-3 and C-4 positions, suggesting that degradation occurred through an alternate pathway.
authors.affiliation: CEA, DSV, Laboratoire de Chimie et Biologie des Métaux, Grenoble, France CNRS UMR 5249, Grenoble, France Université Grenoble Alpes, Grenoble, France.
authors.forename: Angelina
authors.initials: A
authors.lastname: Chemerys
authors.affiliation: CEA, DSV, Institut de Génomique, Genoscope, Evry, France CNRS-UMR 8030, Evry, France UEVE, Université d'Evry, Evry, France.
authors.forename: Eric
authors.initials: E
authors.lastname: Pelletier
authors.affiliation: CEA, DSV, Institut de Génomique, Genoscope, Evry, France CNRS-UMR 8030, Evry, France UEVE, Université d'Evry, Evry, France.
authors.forename: Corinne
authors.initials: C
authors.lastname: Cruaud
authors.affiliation: CEA, DSV, Laboratoire de Chimie et Biologie des Métaux, Grenoble, France CNRS UMR 5249, Grenoble, France Université Grenoble Alpes, Grenoble, France.
authors.forename: Florence
authors.initials: F
authors.lastname: Martin
authors.affiliation: CEA, DSV, Laboratoire de Chimie et Biologie des Métaux, Grenoble, France CNRS UMR 5249, Grenoble, France Université Grenoble Alpes, Grenoble, France.
authors.forename: Fabien
authors.initials: F
authors.lastname: Violet
authors.affiliation: CEA, DSV, Laboratoire de Chimie et Biologie des Métaux, Grenoble, France CNRS UMR 5249, Grenoble, France Université Grenoble Alpes, Grenoble, France yves.jouanneau@cea.fr.
authors.forename: Yves
authors.initials: Y
authors.lastname: Jouanneau
mesh_terms: D020562:Betaproteobacteria / Q000145:classification / Q000201:enzymology / Q000235:genetics*; D001711:Biotransformation; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D049308:Dioxygenases / Q000235:genetics* / Q000378:metabolism*; D004787:Environmental Pollution; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D014644:Genetic Variation; D054892:Metagenome; D008969:Molecular Sequence Data; D011084:Polycyclic Aromatic Hydrocarbons / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D012988:Soil Microbiology
chemical_list: D004269:DNA, Bacterial; D011084:Polycyclic Aromatic Hydrocarbons; D049308:Dioxygenases