Identification of the monooxygenase gene clusters responsible for the regioselective oxidation of phenol to hydroquinone in mycobacteria

Abstract

Mycobacterium goodii strain 12523 is an actinomycete that is able to oxidize phenol regioselectively at the para position to produce hydroquinone. In this study, we investigated the genes responsible for this unique regioselective oxidation. On the basis of the fact that the oxidation activity of M. goodii strain 12523 toward phenol is induced in the presence of acetone, we first identified acetone-induced proteins in this microorganism by two-dimensional electrophoretic analysis. The N-terminal amino acid sequence of one of these acetone-induced proteins shares 100% identity with that of the protein encoded by the open reading frame Msmeg_1971 in Mycobacterium smegmatis strain mc(2)155, whose genome sequence has been determined. Since Msmeg_1971, Msmeg_1972, Msmeg_1973, and Msmeg_1974 constitute a putative binuclear iron monooxygenase gene cluster, we cloned this gene cluster of M. smegmatis strain mc(2)155 and its homologous gene cluster found in M. goodii strain 12523. Sequence analysis of these binuclear iron monooxygenase gene clusters revealed the presence of four genes designated mimABCD, which encode an oxygenase large subunit, a reductase, an oxygenase small subunit, and a coupling protein, respectively. When the mimA gene (Msmeg_1971) of M. smegmatis strain mc(2)155, which was also found to be able to oxidize phenol to hydroquinone, was deleted, this mutant lost the oxidation ability. This ability was restored by introduction of the mimA gene of M. smegmatis strain mc(2)155 or of M. goodii strain 12523 into this mutant. Interestingly, we found that these gene clusters also play essential roles in propane and acetone metabolism in these mycobacteria.

FIG. 1.

Identification of acetone-induced proteins. Two-dimensional electrophoretic protein profiles of extracts from M. goodii strain 12523 cells in the absence and presence of acetone (left and right panels, respectively) are shown. The two acetone-induced proteins whose N-terminal sequences were determined (A and B) are indicated by arrows.

FIG. 2.

Monooxygenase activities of wild-type and mutant strains toward phenol. Growing cells of wild-type 12523 and mc²155, the deletion mutant mc²155 ΔmimA, and the complemented strains mc²155 ΔmimA (mimA_sm) and mc²155 ΔmimA (mimA_go), which were induced with acetone, were reacted with phenol, and the monooxygenation product hydroquinone was quantified using HPLC. Bars represent the averages from three independent experiments, and error bars represent the standard deviations from the means.

FIG. 3.

Growth of wild-type and mutant strains on propane and related compounds. Cells of wild-type 12523 and mc²155, the deletion mutant mc²155 ΔmimA, and the complemented strains mc²155 ΔmimA (mimA_sm) and mc²155 ΔmimA (mimA_go) were cultivated on individual compounds as a source of carbon and energy for 8 days. Cell growth is expressed as cell dry weight per liter of medium. Bars represent the averages from two independent experiments, and error bars represent the standard deviations from the means.

FIG. 4.

Growth profiles of wild-type and mutant strains on acetone. Cells of wild-type 12523 (white circles) and mc²155 (white triangles), the deletion mutant mc²155 ΔmimA (squares), and the complemented strains mc²155 ΔmimA (mimA_sm) (black triangles) and mc²155 ΔmimA (mimA_go) (black circles) were cultivated on acetone as a source of carbon and energy. The growth of the deletion mutant mc²155 ΔmimA in the absence of acetone is indicated by crosses. Cell growth is expressed as cell dry weight per liter of medium. Plots represent the averages from two independent experiments, and error bars represent the standard deviations from the means.