caa a22 4500 605502056 CHVBK 20210128100603.0 cr unu---uuuuu 210128e20150501xx s 000 0 eng 10.1007/s00253-014-6322-2 doi (NATIONALLICENCE)springer-10.1007/s00253-014-6322-2 Isolation of oxygenase genes for indigo-forming activity from an artificially polluted soil metagenome by functional screening using Pseudomonas putida strains as hosts [Elektronische Daten] [Hirofumi Nagayama, Tomonori Sugawara, Ryo Endo, Akira Ono, Hiromi Kato, Yoshiyuki Ohtsubo, Yuji Nagata, Masataka Tsuda] 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. Springer-Verlag Berlin Heidelberg, 2015 Metagenome nationallicence Biodegradation nationallicence Oxygenase nationallicence Aromatic compound nationallicence Indigo nationallicence Nagayama Hirofumi Department of Environmental Life Sciences, Graduate School of Life Sciences, Tohoku University, 2-1-1 Katahira, 980-8577, Sendai, Japan aut Sugawara Tomonori Department of Environmental Life Sciences, Graduate School of Life Sciences, Tohoku University, 2-1-1 Katahira, 980-8577, Sendai, Japan aut Endo Ryo Department of Environmental Life Sciences, Graduate School of Life Sciences, Tohoku University, 2-1-1 Katahira, 980-8577, Sendai, Japan aut Ono Akira Department of Environmental Life Sciences, Graduate School of Life Sciences, Tohoku University, 2-1-1 Katahira, 980-8577, Sendai, Japan aut Kato Hiromi Department of Environmental Life Sciences, Graduate School of Life Sciences, Tohoku University, 2-1-1 Katahira, 980-8577, Sendai, Japan aut Ohtsubo Yoshiyuki Department of Environmental Life Sciences, Graduate School of Life Sciences, Tohoku University, 2-1-1 Katahira, 980-8577, Sendai, Japan aut Nagata Yuji Department of Environmental Life Sciences, Graduate School of Life Sciences, Tohoku University, 2-1-1 Katahira, 980-8577, Sendai, Japan aut Tsuda Masataka Department of Environmental Life Sciences, Graduate School of Life Sciences, Tohoku University, 2-1-1 Katahira, 980-8577, Sendai, Japan aut Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/10(2015-05-01), 4453-4470 0175-7598 99:10<4453 2015 99 253 https://doi.org/10.1007/s00253-014-6322-2 text/html Onlinezugriff via DOI BK010053 XK010053 XK010000 Metadata rights reserved Springer special CC-BY-NC licence nationallicence 1 research-article jats NATIONALLICENCE NATIONALLICENCE NL-springer NATIONALLICENCE 856 40 https://doi.org/10.1007/s00253-014-6322-2 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Nagayama Hirofumi Department of Environmental Life Sciences, Graduate School of Life Sciences, Tohoku University, 2-1-1 Katahira, 980-8577, Sendai, Japan aut NATIONALLICENCE 700 1- Sugawara Tomonori Department of Environmental Life Sciences, Graduate School of Life Sciences, Tohoku University, 2-1-1 Katahira, 980-8577, Sendai, Japan aut NATIONALLICENCE 700 1- Endo Ryo Department of Environmental Life Sciences, Graduate School of Life Sciences, Tohoku University, 2-1-1 Katahira, 980-8577, Sendai, Japan aut NATIONALLICENCE 700 1- Ono Akira Department of Environmental Life Sciences, Graduate School of Life Sciences, Tohoku University, 2-1-1 Katahira, 980-8577, Sendai, Japan aut NATIONALLICENCE 700 1- Kato Hiromi Department of Environmental Life Sciences, Graduate School of Life Sciences, Tohoku University, 2-1-1 Katahira, 980-8577, Sendai, Japan aut NATIONALLICENCE 700 1- Ohtsubo Yoshiyuki Department of Environmental Life Sciences, Graduate School of Life Sciences, Tohoku University, 2-1-1 Katahira, 980-8577, Sendai, Japan aut NATIONALLICENCE 700 1- Nagata Yuji Department of Environmental Life Sciences, Graduate School of Life Sciences, Tohoku University, 2-1-1 Katahira, 980-8577, Sendai, Japan aut NATIONALLICENCE 700 1- Tsuda Masataka Department of Environmental Life Sciences, Graduate School of Life Sciences, Tohoku University, 2-1-1 Katahira, 980-8577, Sendai, Japan aut NATIONALLICENCE 773 0- Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/10(2015-05-01), 4453-4470 0175-7598 99:10<4453 2015 99 253