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.

Contamination of groundwater with vinyl chloride (VC), a known human carcinogen, is a common environmental problem at plastics manufacturing, dry cleaning, and military sites. At many sites, there is the potential to cleanup VC groundwater plumes with aerobic VC-oxidizing microorganisms (e.g., methanotrophs, etheneotrophs, and VC-assimilating bacteria). Environmental biotechnologies that reveal the presence and activity of VC-oxidizing bacteria in contaminated groundwater samples would provide valuable lines of evidence that bioremediation of VC is occurring at a site. We applied targeted shotgun mass spectrometry-based proteomic methods to ethene-enriched groundwater microcosms from a VC-contaminated site. Polypeptides from the enzymes alkene monooxygenase (EtnC) and epoxyalkane:CoM transferase (EtnE), both of which are expressed by aerobic etheneotrophs and VC-assimilating bacteria, were identified in 7 of the 14 samples analyzed. Bioinformatic analysis revealed that 2 EtnC and 5 EtnE peptides were unique to deduced EtnC and EtnE sequences from two different cultivated strains. In addition, several partial EtnE genes sequenced from microcosms matched with observed EtnE peptides. Our results have revealed broader etheneotroph functional gene diversity and demonstrate the feasibility, speed, and accuracy of applying a targeted metaproteomics approach to identifying protein biomarkers from etheneotrophs in complex environmental samples.

Enzymes expressed in response to vinyl chloride, ethene, and epoxyethane by Nocardioides sp. strain JS614 were identified by using a peptide mass fingerprinting (PMF) approach. PMF provided insight concerning vinyl chloride biodegradation in strain JS614 and extends the use of matrix-assisted laser desorptionionization time of flight mass spectrometry as a tool to enhance characterization of biodegradation pathways.Vinyl chloride (VC), a known human carcinogen (2) and groundwater contaminant (21), is often generated in groundwater by the incomplete reduction of chlorinated solvents. Diverse bacterial genera, including Mycobacterium (3,12), Nocardioides (3,18), Ochrobactrum (5), Pseudomonas (5, 22), and Ralstonia (9), use both VC and ethene as carbon and energy sources. Several strains appear to use the same enzymes to metabolize both VC and ethene (4,12,18). Alkene monooxygenase (AkMO) oxidizes VC to chlorooxirane (12,22) and ethene to epoxyethane (3,7,8,18). Epoxyalkane:coenzyme M transferase (EaCoMT) participates in further metabolism of both epoxyethane (4, 6, 18) and chlorooxirane (4). An unknown number of enzymatic steps catalyze the conversion of these epoxides to acetyl coenzyme A (acetyl-CoA) (7). Elucidating the remaining enzymes and intermediates of aerobic VC and ethene biodegradation will facilitate development of molecular tools for detecting and differentiating VC-and ethene-assimilating bacteria in the environment. The completion of the Nocardioides sp. strain JS614 genome sequence (http://genome.ornl.gov/microbial/noca/) provides opportunities to use new approaches to identify enzymes involved in VC and ethene biodegradation. In this study, proteomic techniques were used to rapidly and accurately identify enzymes expressed in response to VC, ethene, and epoxyethane in strain JS614.Chemicals, media, growth conditions, and protein extraction methods are described elsewhere (18). Sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) analysis was performed by the method of Laemmli (15) with extracts (10 to 50 g protein) from VC-, ethene-, epoxyethane-, and acetategrown cells. Polyacrylamide gels were stained with Bio-Safe Coomassie blue (Bio-Rad Laboratories, Inc.). Visual inspection (Fig. 1) revealed several polypeptides expressed in response to ethene, epoxyethane, and VC that were not expressed in response to acetate, suggesting that they were directly involved in VC, ethene, and epoxyethane metabolism. Polypeptide bands from all lanes in each numbered section of the gel were excised and digested with bovine trypsin (Promega Corp.). The resulting monoisotopic peptide fragment masses were analyzed with a Bruker BiflexIII matrix-assisted laser desorption-ionization time of flight (MALDI-TOF) mass spectrometer in positive-ion/reflector mode, using an ␣-cyano-4-hydroxycinnamic acid matrix (14,19). Peptide mass fingerprints (PMFs) of digested polypeptide bands (Fig. 2) were compared to the PMF of a control gel fragment that had no contact with cell extracts, and matching masses within a 0...

Vinyl chloride (VC) is a common groundwater pollutant and known human carcinogen that is commonly produced from the incomplete reductive dechlorination of tetrachloroethene and trichloroethene. A possible approach to clean-up VC-contaminated sites is with microorganisms that grow on VC and/or ethene as a carbon and energy source. However, little is known about the biochemical pathways involved in VCassimilation within these strains and their distribution and activity in situ in the environment. This work uses mass-spectrometry-based proteomics to contribute to the understanding of these microbial communities in both pure cultures and in the environment. The biochemical pathways of VC and ethene oxidation in Nocardioides sp. strain JS614 were studied using proteins identified with a peptide mass fingerprinting approach. New insights into a previously proposed pathway were made using mass spectrometry (MS)-based protein identifications, and potential protein biomarkers for the presence and activity of VC-assimilating bacteria in the environment were identified. Techniques to extract and identify proteins from various environmental samples such as activated sludge, sediments, soils, and water samples were developed based on preliminary experiments with protein extraction from strain JS614. These techniques were extended to 14 ethene-enrichment cultures derived from VC-contaminated groundwater to detect the presence and diversity of VC-and/or ethene-assimilating bacteria with previously identified protein biomarkers. VC-assimilating organisms can evolve under laboratory conditions from bacteria that grow on ethene but very little is known about the molecular changes involved. Tandem mass spectrometry and spectral counting were applied to three variants of ethene-grown Mycobacterium strain JS623 (a wild type and two VC-adapted variants) resulting in the identification of 174 differentially expressed proteins. The results of this v ABSTRACT Vinyl chloride (VC) is a common groundwater pollutant and known human carcinogen that is commonly produced from the incomplete reductive dechlorination of tetrachloroethene and trichloroethene. A possible approach to clean-up VC-contaminated sites is with microorganisms that grow on VC and/or ethene as a carbon and energy source. However, little is known about the biochemical pathways involved in VCassimilation within these strains and their distribution and activity in situ in the environment. This work uses mass-spectrometry-based proteomics to contribute to the understanding of these microbial communities in both pure cultures and in the environment. The biochemical pathways of VC and ethene oxidation in Nocardioides sp. strain JS614 were studied using proteins identified with a peptide mass fingerprinting approach. New insights into a previously proposed pathway were made using mass spectrometry (MS)-based protein identifications, and potential protein biomarkers for the presence and activity of VC-assimilating bacteria in the environment were identified. Techniques to extract a...