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The extended lag period associated with vinyl chloride (VC) starvation in VC- and ethene-assimilating Nocardioides sp. strain JS614 was examined. The extended lag periods were variable (3-7 days), only associated with growth on VC or ethene, and were observed in VC- or ethene-grown cultures following 24 h carbon starvation and mid-exponential phase cultures grown on non-alkene carbon sources (e.g. acetate). Alkene monooxygenase (AkMO) and epoxyalkane:coenzyme M transferase (EaCoMT) are the initial enzymes of VC and ethene biodegradation in strain JS614. Reverse-transcription PCR confirmed that the AkMO gene etnC was expressed in response to epoxyethane, a metabolic intermediate of ethene biodegradation. Epoxyethane (0.5 mM) eliminated the extended lag period in both starved and mid-exponential phase cultures, suggesting that epoxyethane accumulation activates AkMO expression in strain JS614. AkMO activity in ethene-grown cultures was not detected after 6.7 h of carbon starvation, while 40% of the initial EaCoMT activity remained after 24 h. Acetate eliminated the extended lag period in starved cultures but not in mid-exponential phase cultures suggesting that acetate reactivates extant AkMO in starved VC- or ethene-grown cultures. The imbalance between AkMO and EaCoMT activities during starvation likely contributes to the extended lag period by delaying epoxide accumulation and subsequent AkMO induction.

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.