title: Isolation of oxygenase genes for indigo-forming activity from an artificially polluted soil metagenome by functional screening using Pseudomonas putida strains as hosts.
abstract: Metagenomes contain the DNA from many microorganisms, both culturable and non-culturable, and are a potential resource of novel genes. In this study, a 5.2-Gb metagenomic DNA library was constructed from a soil sample (artificially polluted with four aromatic compounds, i.e., biphenyl, phenanthrene, carbazole, and 3-chlorobenzoate) in Escherichia coli by using a broad-host-range cosmid vector. The resultant library was introduced into naphthalene-degrading Pseudomonas putida-derived strains having deficiencies in their naphthalene dioxygenase components, and indigo-forming clones on the indole-containing agar plates were screened. Cosmids isolated from 29 positive clones were classified by their various properties (original screening hosts, hosts showing indigo-forming activity, and digestion patterns with restriction enzymes), and six representative cosmids were chosen. Sequencing and in vitro transposon mutagenesis of the six cosmids resulted in the identification of genes encoding putative class B and D flavoprotein monooxygenases, a multicomponent hydroxylase, and a reductase that were responsible for the indigo-forming activity in the host cells. Among them, the genes encoding the multicomponent hydroxylase were demonstrated to be involved in phenol degradation. Furthermore, two genes encoding ring-cleavage dioxygenases were also found adjacent to the genes responsible for the indigo formation, and their functions were experimentally confirmed.
authors.affiliation: Department of Environmental Life Sciences, Graduate School of Life Sciences, Tohoku University, 2-1-1 Katahira, Sendai, 980-8577, Japan.
authors.forename: Hirofumi
authors.initials: H
authors.lastname: Nagayama
authors.forename: Tomonori
authors.initials: T
authors.lastname: Sugawara
authors.forename: Ryo
authors.initials: R
authors.lastname: Endo
authors.forename: Akira
authors.initials: A
authors.lastname: Ono
authors.forename: Hiromi
authors.initials: H
authors.lastname: Kato
authors.forename: Yoshiyuki
authors.initials: Y
authors.lastname: Ohtsubo
authors.forename: Yuji
authors.initials: Y
authors.lastname: Nagata
authors.forename: Masataka
authors.initials: M
authors.lastname: Tsuda
mesh_terms: D001419:Bacteria / Q000145:classification / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001673:Biodegradation, Environmental; D007203:Indigo Carmine / Q000378:metabolism*; D054892:Metagenome; D008969:Molecular Sequence Data; D010105:Oxygenases / Q000235:genetics* / Q000378:metabolism; D016958:Pseudomonas putida / Q000235:genetics* / Q000378:metabolism; D012988:Soil Microbiology; D012989:Soil Pollutants / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D012989:Soil Pollutants; D007203:Indigo Carmine; D010105:Oxygenases