Isolation and characterization of o-xylene oxygenase genes from Rhodococcus opacus TKN14.
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.
Maruyama
Takahiro
T
Marine Biotechnology Institute, 3-75-1 Heita, Kamaishi, Iwate 026-0001, Japan.
Ishikura
Masaharu
M
Taki
Hironori
H
Shindo
Kazutoshi
K
Kasai
Hiroaki
H
Haga
Miyuki
M
Inomata
Yukie
Y
Misawa
Norihiko
N
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
D017931:DNA Primers; D004269:DNA, Bacterial; D009281:Naphthalenes; C031721:naphthalene; D006899:Mixed Function Oxygenases; D049308:Dioxygenases; C026267:4-xylene methylhydroxylase