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. o-, m-and p-xylenes, which are included in petroleum, are used as solvents in the production of chemicals and drugs, as thinners for paints and enamels, and as intermediates for the synthesis of numerous chemical compounds. They are therefore widespread environmental contaminants in groundwater and soil (4,11,27). In m-and p-xylenes, numerous bacteria that are able to utilize them as the sole carbon and energy source have been isolated, and details of their catabolic pathways have been well documented. The most elucidated examples are the catabolism of toluene, m-xylene, and p-xylene with the enzymes encoded on a transmissible TOL plasmid (pWW0) from Pseudomonas putida mt-2 (38, 39). Xylene monooxygenase is the first enzyme on this degradation pathway (14, 34). This enzyme consists of two polypeptide subunits encoded by the xylM and xylA genes. XylA, the NADH acceptor reductase component (34), has been characterized as an electron transport protein transferring reducing equivalents from NADH to XylM, which is the hydroxylase component located in the membrane (18). Toluene and m-and p-xylenes are, respectively, metabolized with XylMA to benzyl alcohol and m-and p-methylbenzyl alcohol. These respective alcohols are further metabolized to benzoate and m-and p-toluates via their corresponding aldehydes b...

Thermophilic bacteria are potential sources of novel Rieske non-heme iron oxygenases

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Degradation of a Mixture of Hydrocarbons, Gasoline, and Diesel Oil Additives by Rhodococcus aetherivorans and Rhodococcus wratislaviensis

“…The ability to degrade o-xylene is important since it is considered a difficult-to-degrade hydrocarbon and only a few Rhodococcus strains with this ability have been isolated (14,23,24,29,45). A nidA gene in R. wratislaviensis IFP 2016 was identified with 99% identity to a gene from Rhodococcus opacus TKN14 (29) that encodes the large subunit of a dioxygenase iron-sulfur protein. This gene is part of a group of genes (nidABEF) induced in R. opacus TKN14 by o-xylene.…”

Identification and characterization of o-xylene-degrading Rhodococcus spp. which were dominant species in the remediation of o-xylene-contaminated soils

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