title: Isolation and characterization of o-xylene oxygenase genes from Rhodococcus opacus TKN14.
abstract: 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.
authors.affiliation: Marine Biotechnology Institute, 3-75-1 Heita, Kamaishi, Iwate 026-0001, Japan.
authors.forename: Takahiro
authors.initials: T
authors.lastname: Maruyama
authors.forename: Masaharu
authors.initials: M
authors.lastname: Ishikura
authors.forename: Hironori
authors.initials: H
authors.lastname: Taki
authors.forename: Kazutoshi
authors.initials: K
authors.lastname: Shindo
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Kasai
authors.forename: Miyuki
authors.initials: M
authors.lastname: Haga
authors.forename: Yukie
authors.initials: Y
authors.lastname: Inomata
authors.forename: Norihiko
authors.initials: N
authors.lastname: Misawa
mesh_terms: D001483:Base Sequence; D003360:Cosmids; D017931:DNA Primers; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D049308:Dioxygenases / Q000235:genetics; D006899:Mixed Function Oxygenases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D005810:Multigene Family; D009281:Naphthalenes / Q000378:metabolism; D016366:Open Reading Frames; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D015183:Restriction Mapping; D012240:Rhodococcus / Q000201:enzymology / Q000235:genetics*; D014158:Transcription, Genetic
chemical_list: D017931:DNA Primers; D004269:DNA, Bacterial; D009281:Naphthalenes; C031721:naphthalene; D006899:Mixed Function Oxygenases; D049308:Dioxygenases; C026267:4-xylene methylhydroxylase