title: Heterologous expression of polycyclic aromatic hydrocarbon ring-hydroxylating dioxygenase genes from a novel pyrene-degrading betaproteobacterium.
abstract: A betaproteobacterium within the family Rhodocyclaceae previously identified as a pyrene degrader via stable-isotope probing (SIP) of contaminated soil (designated pyrene group 1 or PG1) was cultivated as the dominant member of a mixed bacterial culture. A metagenomic library was constructed, and the largest contigs were analyzed for genes associated with polycyclic aromatic hydrocarbon (PAH) metabolism. Eight pairs of genes with similarity to the α- and β-subunits of ring-hydroxylating dioxygenases (RHDs) associated with aerobic bacterial PAH degradation were identified and linked to PG1 through PCR analyses of a simplified enrichment culture. In tandem with a ferredoxin and reductase found in close proximity to one pair of RHD genes, six of the RHDs were cloned and expressed in Escherichia coli. Each cloned RHD was tested for activity against nine PAHs ranging in size from two to five rings. Despite differences in their predicted protein sequences, each of the six RHDs was capable of transforming phenanthrene and pyrene. Three RHDs could additionally transform naphthalene and fluorene, and these genotypes were also associated with the ability of the E. coli constructs to convert indole to indigo. Only one of the six cloned RHDs was capable of transforming anthracene and benz[a]anthracene. None of the tested RHDs were capable of significantly transforming fluoranthene, chrysene, or benzo[a]pyrene.
authors.affiliation: Department of Environmental Sciences and Engineering, Gillings School of Global Public Health, University of North Carolina, Chapel Hill, North Carolina, USA. drsingle@ad.unc.edu
authors.forename: David R
authors.initials: DR
authors.lastname: Singleton
authors.forename: Jing
authors.initials: J
authors.lastname: Hu
authors.forename: Michael D
authors.initials: MD
authors.lastname: Aitken
mesh_terms: D003001:Cloning, Molecular; D016000:Cluster Analysis; D049308:Dioxygenases / Q000235:genetics* / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D007203:Indigo Carmine; D007211:Indoles / Q000378:metabolism; D054892:Metagenome; D008969:Molecular Sequence Data; D010616:Phenanthrenes / Q000378:metabolism; D010802:Phylogeny; D011084:Polycyclic Aromatic Hydrocarbons / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011721:Pyrenes / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D043362:Rhodocyclaceae / Q000201:enzymology* / Q000302:isolation & purification / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D017385:Sequence Homology; D012988:Soil Microbiology
chemical_list: D007211:Indoles; D010616:Phenanthrenes; D011084:Polycyclic Aromatic Hydrocarbons; D011721:Pyrenes; D011994:Recombinant Proteins; C031181:phenanthrene; C030374:indole; C030984:pyrene; D007203:Indigo Carmine; D049308:Dioxygenases