Physiological and molecular genetic analyses of vinyl chloride and ethene biodegradation in Nocardioides sp. strain JS614
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Nocardioides sp. strain JS614 utilizes vinyl chloride and ethene as carbon and energy sources. JS614 could be influential in natural attenuation and biogeochemical ethene cycling, and useful for bioremediation, biocatalysis and metabolic engineering, but a fundamental understanding of the physiological and genetic basis of vinyl chloride and ethene assimilation in strain JS614 is required. Alkene monooxygenase (AkMO) activity was demonstrated in whole-cell assays and epoxyalkane:coenzyme M transferase (EaCoMT) activity was detected in JS614 cell-free extracts. Pulsed-field gel electrophoresis revealed a 290-kb plasmid (pNoc614) in JS614. Curing experiments and PCR indicated that pNoc614 encodes vinyl chloride/ethene-degradation genes. JS614 vinyl chloride/ethene catabolic genes and flanking DNA (34.8 kb) were retrieved from a fosmid clone. AkMO and EaCoMT genes were found in a putative operon that included CoA transferase, acyl-CoA synthetase, dehydrogenase, and reductase genes. Adjacent to this gene cluster was a divergently transcribed gene cluster that encoded possible coenzyme M biosynthesis enzymes. Reverse transcription-PCR demonstrated the vinyl chloride- and ethene-inducible nature of several genes. Genes encoding possible plasmid conjugation, integration, and partitioning functions were also discovered on the fosmid clone.
KeywordsLinear plasmid Alkene oxidation Bioremediation Vinyl chloride
We thank Anthony Hay, Ruth Richardson, and Steve Zinder for use of their laboratories and for technical advice. We thank Michelle Detwiler at RPCI for her DNA sequencing expertise and persistence in attempts to sequence the hairpin loop. We also thank Juli Rubin, Brian Weisenstein, and Linda Rankin for technical assistance. The US Strategic Environmental Research and Development Program funded this work.
- Bucher JR, Cooper G, Haseman JK, Jameson CW, Longnecker M, Kamel F, Maronpot R, Matthews HB, Melnick R, Newbold R, Tennant RW, Thompson C, Waalkes M (2001) Ninth report on carcinogens. In: US Department of Health and Human Services, National Toxicology Program. Available via http://ehis.niehs.nih.gov/roc/ninth/known/vinylchloride.pdf.
- Clark DD, Allen JR, Ensign SA (2000) Characterization of five catalytic activities associated with the NADPH:2-ketopropyl-coenzyme M [2-(2-ketopropylthio)ethanesulfonate] oxidoreductase/carboxylase of the Xanthobacter strain Py2 epoxide carboxylase system. Biochemistry 39:1294–1304PubMedGoogle Scholar
- Lane DJ (1991) 16S/23S rRNA sequencing. In: Stackbrandt E, Goodfellow M (eds) Nucleic acid techniques in bacterial systematics. Wiley, Chichester, pp 177–203Google Scholar
- Leahy JG, Batchelor PJ, Morcomb SM (2003) Evolution of the soluble diiron monooxygenases. FEMS Microbiol Rev 27:449–479Google Scholar
- Saeki H, Furuhashi K (1994) Cloning and characterization of a Nocardia corallina B-276 gene cluster encoding alkene monooxygenase. J Ferment Bioeng 78:399–406Google Scholar
- Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring HarborGoogle Scholar
- Squillace PJ, Moran MJ, Lapham WW, Price CV, Clawges RM, Zogorski JS (1999) Volatile organic compounds in untreated ambient groundwater of the United States, 1985–1995. Environ Sci Technol 33:4176–4187Google Scholar
- Zhou NY, Chion CK, Leak DJ (1996) Cloning and expression of the genes encoding the propene monooxygenase from Xanthobacter Py2. Appl Microbiol Biotechnol 44:582–588Google Scholar