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Applied and Environmental Microbiology, December 2005, p. 7705-7715, Vol. 71, No. 12
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.12.7705-7715.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Isolation and Characterization of o-Xylene Oxygenase Genes from Rhodococcus opacus TKN14

Takahiro Maruyama,^1, Masaharu Ishikura,^1, Hironori Taki,¹ Kazutoshi Shindo,² Hiroaki Kasai,¹ Miyuki Haga,¹ Yukie Inomata,¹ and Norihiko Misawa^1*

Marine Biotechnology Institute, 3-75-1 Heita, Kamaishi 026-0001,¹ Department of Food and Nutrition, Japan Women's University, Mejirodai, Bunkyo-ku, Tokyo 112-8681, Japan²

Received 20 April 2005/ Accepted 27 July 2005

o-Xylene is one of the most difficult-to-degrade environmental pollutants. We report here Rhodococcus genes mediating oxygenation in the first step of o-xylene degradation. Rhodococcus opacus TKN14, isolated from soil contaminated with o-xylene, was able to utilize o-xylene as the sole carbon source and to metabolize it to o-methylbenzoic acid. A cosmid library from the genome of this strain was constructed in Escherichia coli. A bioconversion analysis revealed that a cosmid clone incorporating a 15-kb NotI fragment had the ability to convert o-xylene into o-methylbenzyl alcohol. The sequence analysis of this 15-kb region indicated the presence of a gene cluster significantly homologous to the naphthalene-inducible dioxygenase gene clusters (nidABCD) that had been isolated from Rhodococcus sp. strain I24. Complementation studies, using E. coli expressing various combinations of individual open reading frames, revealed that a gene (named nidE) for rubredoxin (Rd) and a novel gene (named nidF) encoding an auxiliary protein, which had no overall homology with any other proteins, were indispensable for the methyl oxidation reaction of o-xylene, in addition to the dioxygenase iron-sulfur protein genes (nidAB). Regardless of the presence of NidF, the enzyme composed of NidABE was found to function as a typical naphthalene dioxygenase for converting naphthalene and various (di)methylnaphthalenes into their corresponding cis-dihydrodiols. All the nidABEF genes were transcriptionally induced in R. opacus TKN14 by the addition of o-xylene to a mineral salt medium. It is very likely that these genes are involved in the degradation pathways of a wide range of aromatic hydrocarbons by Rhodococcus species as the first key enzyme.

Present address: Tokyo Research Laboratory, Tosoh Corporation, 2743-1 Hayakawa, Ayase, Kanagawa 252-1123, Japan.

Present address: Research and Development Operations, Yamaha Motor Co., Ltd., 2500 Shingai, Iwata, Shizuoka 438-8501, Japan.

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