Cloning and characterization of a Pseudomonas mendocina KR1 gene cluster encoding toluene-4-monooxygenase

Abstract

Pseudomonas mendocina KR1 metabolizes toluene as a carbon source by a previously unknown pathway. The initial step of the pathway is hydroxylation of toluene to form p-cresol by a multicomponent toluene-4-monooxygenase (T4MO) system. The T4MO enzyme system has broad substrate specificity and provides a new opportunity for biodegradation of toxic compounds and bioconversions. Its known activities include conversion of a variety of phenyl compounds into the phenolic derivatives and the complete degradation of trichloroethylene. We have cloned and characterized a gene cluster from KR1 that determines the T4MO activity. To clone the T4MO genes, KR1 DNA libraries were constructed in Escherichia coli HB101 by using a broad-host-range vector and transferred to a KR1 mutant able to grow on p-cresol but not on toluene. An insert consisting of two SaI fragments of identical size (10.2 kb) was shown to complement the mutant for growth on toluene. One of the Sac fragments, when cloned into the E. coli vector pUC19, was found to direct the synthesis of indigo dye. The indigo-forming property was correlated with the presence of T4MO activity. The T4MO genes were mapped to a 3.6-kb region, and the direction of transcription was determined. DNA sequencing and N-terminal amino acid determination identified a five-gene cluster, tmoABCDE, within this region. Expression of this cluster carrying a single mutation in each gene demonstrated that each of the five genes is essential for T4MO activity. Other evidence presented indicated that none of the tmo genes was involved in the regulation of the tmo gene cluster, in the control of substrate transport for the T4MO system, or in major processing of the products of the tmo genes. It was tentatively concluded that the tmoABCDE genes encode structural polypeptides of the T4MO enzyme system. One of the tmo genes was tentatively identified as a ferredoxin gene.Pseudomonas mendocina KR1 utilizes toluene as a sole carbon and energy source. Toluene is oxidized in this bacterial strain to protocatechuate, which is further oxidized through ortho ring cleavage to form substrates of tricarboxylic acid cycle (37). The initial step of this pathway is distinctively different from those of other known toluene pathways. Earlier studies have shown that in Pseudomonas putida Fl, toluene is initially oxidized to form (+)-cis-1(S),2(R)-dihydroxy-3-methylcyclohexa-3,5-diene (cis-toluene dihydrodiol) via a dioxygenation reaction (Fig. 1) (14, 19, 45). Subsequently, it was found that in P. putida mt-2, toluene is initially attacked at the methyl group by a monooxygenase enzyme system to form benzyl alcohol ( Fig. 1) (39). More recent studies have demonstrated that the initial monooxygenation reaction can also occur on the aromatic ring of toluene. In the toluene-degrading bacterium G4, this reaction hydroxylates toluene to form o-cresol ( Fig. 1) (33). However, in P. mendocina KR1, toluene is initially hydroxylated by a toluene-4-monooxygenase (T4MO) system to form p-cresol ( Fig. 1) (36). The T4MO enzyme ...

Indigo production by naphthalene-degrading bacteria

“…putida S12 and CA-3 (O' Connor et al 1997) and toluene-degrading Ps. mendocina KR1 (Yen et al 1991). The enzyme system responsible for indigo formation generally consists of one or more enzymes, typically monooxygenases, dioxygenases or hydroxylases (O'Connor et al 1997;Doukyu et al 1998).…”

“…The genes encoding these enzymes have been cloned from Pseudomonas and Rhodococcus spp. into Escherichia coli strains (Yen et al 1991;Hart et al 1992;Eaton and Chapman 1995) so as to produce indigo directly from either nutrient medium (Ensley et al 1983) or from glucose (Murdock et al 1993).…”

“…The genes encoding these enzymes have been cloned from Pseudomonas and Rhodococcus spp. into Escherichia coli strains (Yen et al 1991;Hart et al 1992;Eaton and Chapman 1995) so as to produce indigo directly from either nutrient medium (Ensley et al 1983) or from glucose (Murdock et al 1993).Recently, a method has been patented by O'riel and Kim (1998) for producing indigo and indirubin dyes using a recombinant Escherichia coli containing a gene encoding a phenol hydroxylase from Bacillus stearothermophilus. In the present study, we report on the formation and identi®ca-tion of indigo and indirubin pigments by one wild-type and four recombinant bacterial strains.…”

Induction of aromatic catabolic activity in Sphingomonas aromaticivorans strain F199

“…The linkage of these clusters is unusual, since we expect xylM to be necessary only for xylene and toluene degradation and genes on cluster M only to be necessary for p-cresol or vanillic acid degradation. Metabolites of toluene 4-sulfonate monooxygenase [4] and toluene-4-monooxygenase [55] feed into the pcresol catabolic pathway. Perhaps pNL1 encodes a novel oxygenase with a Xy1M component that produces aromatic metabolites that also feed into the p-cresol pathway.…”

“…The conversion of indole to indigo has been attributed to a variety of oxygenases that catalyze the oxidation of naphthalene [12], toluene [29], styrene [40,42], isopropylbenzene [11], p-cumate [9,10], m-xylene [8], toluene [55], or dimethyl sulfoxide [25]. A single oxygenase encoded on pNL1, comprised of the products of bphA1f, bphA2f, bphA3, and bphA4, is predicted to catalyze this reaction (see Discussion).…”