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Journal of Bacteriology, September 2003, p. 5536-5545, Vol. 185, No. 18
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.18.5536-5545.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Epoxyalkane:Coenzyme M Transferase in the Ethene and Vinyl Chloride Biodegradation Pathways of Mycobacterium Strain JS60

Nicholas V. Coleman{dagger} and Jim C. Spain*

Air Force Research Laboratory-MLQL, Tyndall AFB, Florida 32403

Received 7 April 2003/ Accepted 3 July 2003

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 mc2155 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.

* Corresponding author. Mailing address: Air Force Research Laboratory-MLQL, Bldg. 1117, 139 Barnes Dr., Tyndall AFB, FL 32403. Phone: (850) 283-6058. Fax: (850) 283-6090. E-mail: Jim.Spain{at}tyndall.af.mil.

{dagger} Present address: School of Molecular and Microbial Biosciences, University of Sydney, NSW, 2006, Australia.

Journal of Bacteriology, September 2003, p. 5536-5545, Vol. 185, No. 18
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.18.5536-5545.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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