Epoxyalkane: coenzyme M transferase in the ethene and vinyl chloride biodegradation pathways of mycobacterium strain JS60

Abstract

Mycobacterium strains that grow on ethene and vinyl chloride (VC) are widely distributed in the environment and are potentially useful for biocatalysis and bioremediation. The catabolic pathway of alkene assimilation in mycobacteria is not well characterized. It is clear that the initial step is a monooxygenase-mediated epoxidation that produces epoxyethane from ethene and chlorooxirane from VC, but the enzymes involved in subsequent transformation of the epoxides have not been identified. We investigated epoxyethane metabolism in Mycobacterium strain JS60 and discovered a coenzyme M (CoM)-dependent enzyme activity in extracts from VC- and ethene-grown cells. PCR amplifications using primers targeted at epoxyalkane:CoM transferase (EaCoMT) genes yielded part of the JS60 EaCoMT gene, which was used to clone an 8.4-kb genomic DNA fragment. The complete EaCoMT gene (etnE) was recovered, along with genes (etnABCD) encoding a four-component monooxygenase and two genes possibly involved in acyl-CoA ester metabolism. Reverse transcription-PCR indicated that the etnE and etnA genes were cotranscribed and inducible by ethene and VC. Heterologous expression of the etnE gene in Mycobacterium smegmatis mc(2)155 using the pMV261 vector gave a recombinant strain capable of transforming epoxyethane, epoxypropane, and chlorooxirane. A metabolite identified by mass spectrometry as 2-hydroxyethyl-CoM was produced from epoxyethane. The results indicate that the EaCoMT and monooxygenase enzymes encoded by a single operon (etnEABCD) catalyze the initial reactions in both the VC and ethene assimilation pathways. CoM-mediated reactions appear to be more widespread in bacteria than was previously believed.

FIG. 1.

Growth of Mycobacterium strain JS60 on ethene (A) and VC (B) as sole carbon and energy sources. ○, biomass measured as OD₆₀₀; Δ, cumulative amount of substrate consumed; □, cumulative amount of chloride produced. Growth rates (0.080 h⁻¹ with ethene, 0.017 h⁻¹ with VC) were calculated by plotting an exponential curve through a subset of the OD₆₀₀ data (results not shown). The inoculum for both experiments was a frozen stock of washed, VC-grown cells. Data are averages of three replicates, and error bars are the standard deviations.

FIG. 2.

Effect of CoM on epoxyethane metabolism in JS60 cell extracts. ○, ethene-grown cell extract with CoM; □, VC-grown cell extract with CoM; ⋄, acetate-grown cell extract with CoM; ▿, ethene-grown cell extract with no cofactor; Δ, VC-grown cell extract with no cofactor. Data are the averages of three independent experiments, and error bars are the standard deviations.

FIG. 3.

Schematic diagram of genes on the 8,364-bp NheI restriction fragment cloned from Mycobacterium strain JS60. orf1 and orf2 are likely to be involved in acyl-CoA ester metabolism, etnE encodes an EaCoMT, and the etnABCD genes encode a putative four-component alkene monooxygenase.

FIG. 4.

Sequence alignment of EaCoMT proteins from Mycobacterium strain JS60, Rhodococcus strain B-276, and Xanthobacter strain Py2. Identical residues are shaded black, while similar residues are shaded gray. The conserved histidine (220) and cysteine (222, 343) residues likely to be involved in zinc binding are boxed.

FIG. 5.

RT-PCR analysis of etnEA expression in strain JS60. Lanes: A, acetate-grown cells; B, VC-grown cells; C, ethene-grown cells; D, acetate-grown cells (no RT); E, VC-grown cells (no RT); F, ethene-grown cells (no RT); G, DNA template; H, no template.

FIG. 6.

Chlorooxirane transformation in cell extract reactions. ○, buffer alone; □, buffer, CoM, and cell extract from strain mc²155(pMV261); Δ, buffer, CoM, and cell extract from strain mc²155(pMV-CoM). Data from two experiments are shown. Chlorooxirane was quantified by the absorbance (550 nm) of its 4-(p-nitrobenzyl)pyridine adduct. Initial concentrations of chlorooxirane in the assays ranged from 0.2 to 0.3 mM.

FIG. 7.

MS analysis of metabolites formed from epoxyethane during EaCoMT reaction. (A) Zero time sample; (B) 30-min sample; (C) 1-h sample.

FIG. 8.

Proposed pathways of VC and ethene assimilation in Mycobacterium strains. Intermediates that have not been identified are in brackets, and hypothetical reactions are indicated by dotted lines.