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 hereRhodococcus genes mediating oxygenation in the first step ofo -xylene degradation.Rhodococcus opacus TKN14, isolated from soil contaminated witho -xylene, was able to utilizeo -xylene as the sole carbon source and to metabolize it too -methylbenzoic acid. A cosmid library from the genome of this strain was constructed inEscherichia coli . A bioconversion analysis revealed that a cosmid clone incorporating a 15-kb NotI fragment had the ability to converto -xylene intoo -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 fromRhodococcus sp. strain I24. Complementation studies, usingE. coli expressing various combinations of individual open reading frames, revealed that a gene (namednidE ) for rubredoxin (Rd) and a novel gene (namednidF ) encoding an auxiliary protein, which had no overall homology with any other proteins, were indispensable for the methyl oxidation reaction ofo -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 correspondingcis -dihydrodiols. All thenidABEF genes were transcriptionally induced inR. opacus TKN14 by the addition ofo -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 byRhodococcus species as the first key enzyme.