Proteomic Analysis of Ethene-Enriched Groundwater Microcosms

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Environ. Sci. Technol. 2010, 44, 1594–1601

Proteomic Analysis of Ethene-Enriched Groundwater Microcosms from a Vinyl Chloride-Contaminated Site ADINA S. CHUANG,† YANG OH JIN,† LAURA S. SCHMIDT,† YALAN LI,‡ SAMUEL FOGEL,§ DONNA SMOLER,§ AND T I M O T H Y E . M A T T E S * ,† Department of Civil and Environmental Engineering, University of Iowa, Iowa City, IA, University of Iowa Proteomics Facility, Iowa City, IA, and Bioremediation Consulting, Inc., Watertown, MA

Received October 5, 2009. Revised manuscript received January 4, 2010. Accepted January 8, 2010.

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 shotgunmassspectrometry-basedproteomicmethodstoetheneenriched 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.

Introduction Vinyl chloride (VC), a known human carcinogen (1) and common groundwater contaminant (2), is frequently generated in groundwater by incomplete reductive dechlorination of the widely used chlorinated solvents tetrachloroethene (PCE) and trichloroethene (TCE), also common groundwater contaminants (2). At some sites, VC produced under anaerobic conditions migrates into aerobic groundwater and is subsequently degraded (3-5). At least three groups of * Corresponding author e-mail: [email protected]. † Department of Civil and Environmental Engineering, University of Iowa. ‡ University of Iowa Proteomics Facility. § Bioremediation Consulting, Inc. 1594

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microorganisms could participate in the observed aerobic attenuation of VC. Both methanotrophs (6) and “etheneotrophs” (ethene-assimilating bacteria) (7) are known to fortuitously oxidize VC during aerobic growth on methane and ethene, respectively. Methane and ethene can be produced under certain anaerobic conditions and comigrate with VC into aerobic zones. In the absence of methane and ethene, bacteria that use VC as a carbon and energy source (VC-assimilating bacteria 8-11) could also participate in VC biodegradation. Recent studies of etheneotrophic and VC-assimilating bacteria have focused on identifying VC and ethene biodegradation pathway intermediates, pathway enzymes, the genes encoding them, and their regulation (10, 12-18). All ethenenotrophic and VC-assimilating bacteria characterized thus far employ the enzymes alkene monooxygenase (AkMO; EtnABCD) and epoxyalkane:coenzyme M transferase (EaCoMT; EtnE) during the initial steps of the aerobic microbial VC and ethene biodegradation pathways (11, 14-17). AkMO, a member of the soluble di-iron monooxygenase (SDIMO) family of enzymes, converts ethene to epoxyethane and VC to chlorooxirane. These epoxide intermediates are subsequently metabolized by EaCoMT in a CoM-dependent manner (14-16). Several etheneotrophic strains have been observed to adapt to VC as a growth substrate after extended incubation with VC in the laboratory (19, 20), suggesting that etheneotrophs and VC-assimilators are closely related. However, genetic differentiation of etheneotrophs and VCassimilating bacteria is currently not possible. The ecology of methanotrophs has been well studied and a variety of cultivation-independent molecular tools are available (21). In contrast, the ecology of etheneotrophic and VC-assimilating bacteria is poorly understood and only a limited selection of molecular tools is available. Coleman et al. developed and applied a nested PCR assay for SDIMO genes to direct environmental samples from unpolluted and VC-contaminated sites, and to ethene-enriched microcosms (22). The AkMO alpha subunit gene (etnC) was not detectable in unpolluted and unenriched environmental samples, but was readily detected in ethene-enriched microcosms (22). In addition, etnC-like genes were the only SDIMO genes found in samples from two VC-contaminated sites (22). This suggests that etnC could be useful as a biomarker for etheneotrophic and VC-assimilating bacteria at contaminated sites. In another study, EtnE genes were amplified from streambed sediments and VC-fed sediment microcosms derived from a PCE-contaminated site undergoing reductive dechlorination to VC and ethene (23). These results suggest that observing etnE at a VC-contaminated site could also indicate the presence of etheneotrophic and VC-assimilating bacteria. We are interested in developing new technologies aimed at detecting the presence and functionality of etheneotrophic and VC-assimilating microorganisms in environmental samples. Such technologies hold potential value for use in site characterization and monitoring of aerobic VC bioremediation in the field. Approaches involving detection of mRNA transcripts and proteins are especially attractive as they would reveal metabolic functionality, rather than metabolic potential, and thus provide novel insights into the structure and function of active microbial communities. Proteins are particularly well-suited as bioindicators of functional microbes in the environment because they are directly involved in enzyme-catalyzed reactions, cell maintenance, and cellular response to environmental perturbations. Proteomic approaches are also not limited by the short 10.1021/es903033r

 2010 American Chemical Society

Published on Web 02/01/2010

half-life of RNA and bypass the challenges related to low correlations between mRNA and protein expressions that are typically associated with transcriptomics (24, 25). Proteomics has been used to characterize microbial communities in several different environments (reviewed in refs 25-27). Application of proteomics to environmental problems is still a burgeoning research area, but has yielded insights into phosphorus removal in wastewater treatment plants (28, 29), protein expression in mixed chloroethenedechlorinating cultures (30), uranium bioremediation (31), and biofilms associated with acid mine drainage (32-35). The objective of this study was to search for proteins (e.g., EtnC and EtnE) that would signal the presence and functionality of etheneotrophs in ethene-fed groundwater microcosms from a VC-contaminated site.

Materials and Methods EnvironmentalSampleCollection.VC-contaminatedgroundwater, collected from several monitoring wells at a site in MA on 10/03/2007 and 07/01/2008 following USEPA/540/S-95/ 504 low-flow purge procedures, was used to prepare ethenefed microcosms at Bioremediation Consulting, Inc. Previous work indicated that etheneotrophic bacteria, capable of cometabolic VC oxidation, are present within the VC plume at this site (36). Groundwater (40 mL) was added to sterile 160 mL serum bottles containing 60 mL of mineral salts media (pH 6.9, containing 288 mg/L KNO3, 120 mg/L KH2PO4, 200 mg/L Na2HPO4, 100 mg/L MgSO4, 20 mg/L CaCl2 · 2H2O, 40 mg/L KCl, 246 mg/L NaCl, 3 mg/L FeSO4 · 7H2O and 1 mL of a trace metals mixture per liter). The trace metals mixture consisted of ZnSO4 · 7 H2O (7 mg/mL), MnCl2 · 4 H2O (2 mg/ mL), H3BO3 (1.5 mg/mL), CoCl2 · 6 H2O (5 mg/mL), CuCl2 · 2 H2O (1 mg/mL), NiCl2 · 6 H2O (1 mg/mL) and Na2MoO4 · 2H2O (3 mg/mL). Ethene was added to an initial aqueous concentration of 2.85 mM. The bottles were incubated for 63-64 days at 22 °C ((1 °C) with 20 s of shaking by hand three days per week, and headspace samples (100 µL) were analyzed by gas chromatography in accordance with EPA method 5021A. Both flame ionization detection (for ethene measurements) and thermal conductivity detection (for % oxygen and % CO2 measurements) were used. Microcosms were declared positive for etheneotrophs if ethene was degraded, oxygen was consumed, and CO2 evolved after a 60 day incubation period. Following the completion of these analyses, the microcosms were shipped to our laboratory for molecular biology and proteomic analyses. Extraction of DNA and Proteins from Microcosms. Solids were harvested from microcosms by adding Tween80 (0.01% final concentration) to each 100 mL sample, and then centrifuging (10 000g, 5 min, 4 °C). Sample pellets used for DNA extraction were resuspended in 600 µL STE buffer and subjected to a beadbeating extraction procedure carried out as described previously (20). For protein extraction, sample pellets were washed once with 50 mM Tris/10 mM CaCl2 [pH 7.6], centrifuged (10 000g, 5 min, 4 °C), resuspended in 600 µL 50 mM Tris/10 mM CaCl2 [pH 7.6], transferred to a microfuge tube, and stored at -80 °C until further treatment, as described below and in the Supporting Information (SI). If large particulates were visible in resuspended pellets, proteins were extracted via sonication; if no large particulates were visible, proteins were extracted by either French press or guanidine lysis (Table 2). Extracted proteins were quantified with the Coomassie Plus - The Better Bradford Assay (Pierce Protein Research Products, Rockford, IL) against external BSA standards resuspended in the appropriate buffer per the manufacturer’s instructions without modification. PCR, Cloning, and DNA Sequencing of EtnC and EtnE Genes from Microcosms. EtnE genes were amplified from DNA extracts with CoM-F1L and CoM-R2E primers (expected size 891 bp) (17) as described in ref 15. EtnC genes were

amplified with NVC105 and NVC106 primers (expected size 360 bp) as described in ref 22. PCR products were purified, cloned, and transformed into recombinant vectors for sequencing at the University of Iowa DNA facility. A total of 15 clones (seven etnC and eight etnE) were sequenced. Sequences were analyzed by BLAST (www.ncbi.nlm.nih.gov/ BLAST), compared with the PHYLIP DNA distance algorithm (37), and deposited under Genbank accession numbers GQ847806-GQ847821. Further details concerning these methods are available in the SI. Proteomics Analyses. We applied a targeted shotgun proteomics approach to identify protein biomarkers from etheneotrophic bacteria in ethene-fed microcosms. Protein extracts were separated by SDS-PAGE or strong cation exchange (SCX) chromatography. Following SDS-PAGE, four 2 × 10 × 1 mm3 gel slices were excised near the expected location of EtnC (∼58 kDa) and one 4 × 10 × 1 mm3 gel slice was excised near the expected location of EtnE (∼40 kDa) of each gel and subjected to in-gel tryptic digestion. These gel regions were identified in a previous proteomics study of VC-assimilating Nocardioides sp. strain JS614 (18) and were confirmed in preliminary proteomics experiments with ethene-grown JS614 protein extracts. Tools used for gel excisions were decontaminated with ethanol, Windex, and deionized water between samples. A blank section of each gel was analyzed as a negative control. Bovine glutamate dehydrogenase I, the only protein identified in negative controls, was also found in all samples. The presence of this contaminant was expected because it was used as an internal standard for the mass spectrometer. For SCX chromatography, proteins in solution were digested with trypsin. The resulting peptides were separated by SCX chromatography into eight different fractions. Peptides in aliquots from digested gel slices or in SCX fractions were further concentrated with centrifugal evaporation and separately analyzed by liquid chromatography coupled to electrospray-tandem mass spectrometry (LC-ES-MS/MS). Further details describing these methods are available in the SI. Analysis of Tandem Mass Spectra and Protein Identification. A protein sequence database named the “biomarker database”, containing primarily EtnC and EtnE sequences from cultivated strains and uncultured clones, was developed for this study. Details of the biomarker database contents and development can be found in the SI. We also used a protein sequence database containing 4905 entries of all predicted proteins encoded in the VC-assimilating strain JS614 genome (Genbank Acc.CP000508-CP000509) in our bioinformatic analyses. Protein identifications were made by comparing MS/MS spectra to theoretical spectra generated from either the JS614 genome or the biomarker database. MS/MS spectra Thermo RAW files were searched against the biomarker database and the JS614 genome with the Thermo Bioworks v3.1.1 software package (Thermo Scientific), which employs the SEQUEST algorithm (38). The following parameters were used: enzyme: trypsin, up to four missed cleavages allowed, parent mass tolerance 3.0 AMU, and fragment mass tolerance 0.5 AMU. For gel-resolved samples, peptide masses in SEQUEST searches included a fixed modification of carbamidomethylation. Protein identifications were considered significant if the following criteria were simultaneously met for at least two tryptic peptides: (1) SEQUEST XCorr values of 1.5 (+1), 2.5 (+2), 3.5 (+3), and (2) SEQUEST delta Cn values equal or greater to 0.08.

Results Presence of Etheneotrophs in VC-Contaminated Groundwater Samples. Ethene-fed groundwater microcosms (63 day incubation period) are currently used to assess the presence or absence of etheneotrophic bacteria in VC-contaminated VOL. 44, NO. 5, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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TABLE 1. Analysis of Ethene-Fed Microcosms (prepared on 10/ 03/2007)a sample

EtnC gene

EtnE gene

etheneotrophs

RB-46 RB-47D RB-58D RB-58I RB-63D RB-63I RB-64I RB-73 RV-EE

+ + + + +

+ + + + + + + +

+ + + + + + +

a Presence “+” or absence “-” EtnC and EtnE genes was determined by PCR, while the presence “+” or absence “-” of ethenotrophs was determined from a culture-based technique, both of which are described in the Materials and Methods.

groundwater samples from a site in MA (36, 39). Ethene-fed microcosm tests at two different time points (10/03/2007 and 07/01/2008) suggest that etheneotrophs were present in many of the groundwater samples at both time points (Tables 1 and 2). Ethene-fed groundwater microcosms that tested negative for etheneotrophs were used as negative controls in this study. Detection of AkMO and EaCoMT Genes in Ethene-Fed Groundwater Microcosms. PCR analysis for etnC and etnE was conducted with DNA extracted from nine ethene-fed groundwater microcosms (prepared on 10/03/2007) to determine if the presence of these genes correlated with culture-based determination of etheneotroph presence. A band of the expected size of the etnE PCR product (891bp) was detected in eight microcosms, while a band of the expected size of the etnC PCR product (360 bp) was detected in five microcosms. Seven of these microcosms tested positive for etheneotrophs (Table 1). A microcosm that tested negative for etheneotrophs (RB-58I) was also negative for the presence of both etnC and etnE. The absence of etnC did not correlate with the absence of etheneotrophs in samples RB-58D and RB-63D. However, bands that were not of the expected size were often observed in the etnC PCRs (data not shown), suggesting that the etnC PCR assay could be further optimized. To confirm PCR band identity and expand the etnC and etnE sequence database for proteomic analysis, PCR bands of the expected size were cloned and sequenced from selected 10/03/2007 microcosms. This resulted in seven partial etnC and five partial etnE sequences from the RB-73 microcosm sample and three partial etnE sequences from the RB-63I microcosm. The seven partial (357 bp) etnC sequences from the RB-73 microcosm were 98.3-99.7% identical to each other, and 91.4-92.0% identical to the top BLAST hit (SDIMO alpha subunit from an uncultured bacterium; Genbank Acc. No. DQ264663). The sequences were also 91-92.3% identical to the etnC in ethene-assimilating Mycobacterium flavescens NBB1 (Genbank Acc. No. DQ264721). The five partial (865 bp) etnE sequences from the RB-73 microcosm were 99.0-99.8% identical to each other, and 94% identical to the partial etnE from VC-assimilating Mycobacterium strain JS61. The three partial (873 bp) etnE sequences from the RB-63I microcosm were 99.8-99.9% identical to each other and 97.2% identical to etnE (873 bp) from VC-assimilating Nocardioides sp. strain JS614. These sequences were appended to the “biomarker database” that was used to analyze peptide MS/MS spectra from protein extracts described below. Detection of AkMO and EaCoMT Polypeptides in EtheneFed Groundwater Microcosms. We hypothesized that if active microbes were expressing etnC and/or etnE in these 1596

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microcosms, then the polypeptides EtnC and EtnE will be detectable using proteomic techniques. To test this hypothesis, proteins were extracted from a total of 14 ethene-fed groundwater microcosms (prepared on 07/01/2008) and subsequently analyzed by a proteomics workflow developed in our laboratory (SI Figure S1). Of these microcosms, 12 were declared positive for etheneotrophs by culture-based methods (Table 2). MS/MS spectra (e.g., SI Figures S2 and S3), when searched with SEQUEST against the biomarker database using our selected filtering criteria, resulted in the identification of EtnC and EtnE polypeptides in three and seven enrichment samples, respectively (Table 2), that were homologous to deduced EtnC and EtnE sequences from cultivated VC- and ethene-assimilating strains (SI Tables S1 and S2). EtnC and EtnE polypeptides were not identified in two control samples that tested negative for etheneotrophs (Table 2). Proteins from Shewanella sediminis (GroEL chaperonin), Geobacter bemidjiensis (GroEL chaperonin), and Clostridium leptum (iron-only hydrogenase maturation protein) were identified in the RB-73 microcosm. These genomes were added to the biomarker database to increase its size and assess the possibility of false protein identifications (see SI). However, identification of these proteins in the RB-73 microcosm is not unexpected as these strains were isolated from soil and related strains harboring similar proteins could be scavenging trace nutrients in these microcosms. Evaluation of EtnC and EtnE as Biomarkers of Etheneotrophic VC-Oxidizing Bacteria. We evaluated the uniqueness of, and the phylogenetic relationships between, the EtnC and EtnE peptides observed in the microcosms to determine the effectiveness of our protein biomarker identification approach and to provide insight into the diversity of the expressed polypeptides in the microcosms. Peptide uniqueness was evaluated by a BLASTp search of EtnC and EtnE peptides we identified against the NCBI nonredundant protein sequence database. A phylogenetic tree of EtnC sequences reveals two distinct clusters, one containing the JS614 EtnC and a few environmental clones, and the other containing the remainder of the EtnC sequences isolated to date (Figure 1). In the RB-46 microcosm, two of the three EtnC peptides identified were unique to VC-assimilating strain JS614 (NANLAEPR and VYGALDSNVR), with the third being a nonunique JS614 EtnC peptide (SI Table S1). None of the EtnC peptides identified in the RB-60 (six peptides) and RB-73 microcosms (eight peptides) were unique to any particular EtnC, but instead were shared between the Mycobacterium strains JS623 and JS60 EtnE sequences. We identified four JS614 EtnE peptides (one unique) from the RB-46 microcosm, two JS614 EtnE peptides from the RB-52I microcosm, nine JS614 EtnE peptides (one unique) from the RB-58I microcosm, thirteen JS614 EtnE peptides (two unique) from the RB-63I microcosm, and four JS614 EtnE peptides (one unique) from the RB-64I microcosm (SI Table S2). EtnE peptides identified in the RB-46, RB-58I, RB63I, and RB-73 microcosms (from 07/01/2008) also matched with expected peptides of the etnE sequences we retrieved from the 10/03/2007 RB-63I and RB-73 microcosms. In addition, SEQUEST returned the deduced EtnE sequence from uncultured clone RB63IE01 as a significant match in the RB-63I microcosm (data not shown). None of the peptides we observed were unique to any single cloned etnE sequence, however, the peptide PSDAPAPQAAVPIFPEVLGANIDALNYEVGR, identified from the 07/01/2008 RB-63I microcosm (SI Table S2), is shared by the expected product of three etnE clones sequenced from 10/03/2007 RB-63I microcosm. Three JS623 EtnE peptides (one unique) were identified in the RB-60 microcosm and seven JS623 EtnE peptides (one unique) were identified in the RB-73 microcosm (SI Table

TABLE 2. Analysis of Proteins Extracted from Ethene-Fed Microcosms (Prepared on 07/01/2008) using French Press (FP), Sonication (SN), or Guanidine (GU) Lysis Methodsa sample

lysis method

protein separation method

total protein loaded (µg)b

total JS614 Protein IDs

EtnC Biomarker

EtnE biomarker

etheneotrophsd

I-A RB-46 RB-47D RB-47I RB-52I RB-58D RB-58I RB-60 RB-61 RB-63D RB-63I RB-64I RB-73 RV-EE

GU GU SN SN SN SN SN SN SN FP SN SN FP SN

SCX SCX gel gel gel gel gel gel gel gel gel gel gel gel

8.6 29.5 0.5 1.7 1.0 1.3 1.5 11.8 0.9 1.2 2.0 2.4 5.5 1.3

0 18c 0 11c 1 0 3 17c 0 0 5 1 0 0

+ + + -

+ + + + + + + -

+ + + + + + + + + + + +

a When gel separations were used, proteins were excised from regions of the gel where, based on a previous study (18), the EtnE (∼40 kDa) and the EtnC (∼58 kDa) were observed to migrate. EtnC and EtnE identifications (using the biomarker database) and total JS614 protein IDs (using the JS614 genome) from LC-ES-MS/MS analysis of excised gel slices or from LC-ES-MS/MS of peptides extracted using SCX spin columns (SCX) are shown. Protein IDs were filtered with XCorr values of at least 1.5 (+1), 2.5 (+2), 3.5 (+3); deltaCn values of at least 0.08; and at least two unique peptide identifications. b Total protein loaded on SCX column or in polyacrylamide gel lanes. c Results combined from duplicate analysis on LC-ES-MS/ MS. Duplicates were performed because initial run on LC-ES-MS/MS was not optimized (i.e., HPLC leaks and ESI spray problems). d Presence “+” or absence “-” of etheneotrophs as determined from the culture-based technique described in materials and methods.

S2). A phylogenetic tree of EtnE sequences indicates four EtnE clusters with >95% bootstrap support (Figure 2). The deduced EtnE sequences from the RB-63I microcosm cluster with the EtnE from JS614. The tree also indicates that the deduced EtnE sequences from the RB-73 microcosm cluster with the EtnE sequences found in Mycobacterium strains JS61 and JS622 (Figure 2). Detection of Additional Polypeptides Associated with the Aerobic VC and Ethene Biodegradation Pathways in Ethene-Fed Microcosms. The Nocardioides sp. strain JS614 genome, currently the only complete genome of a VCassimilating microorganism, contains deduced protein sequences of all VC and ethene biodegradation pathway enzymes employed by JS614. We subjected MS/MS spectra to a SEQUEST search against the JS614 genome. Peptides in seven of the microcosms were matched to the products of the following JS614 genes: Noca_4807 (etnC), Noca_4810 (etnE), Noca_4813, Noca_4814, Noca_4822, Noca_4827, and Noca_4833 (SI Table S3). In JS614, these genes reside in a large cluster that also includes genes encoding EaCoMT (etnE) and AkMO (etnABCD). In addition to etnC and etnE, Noca_4814, Noca_4822, Noca_4827, and Noca_4833 were found to be expressed in response to VC, ethene, and epoxyethane in strain JS614 (18) and therefore further indicate the presence of a functional VC and/or ethene biodegradation pathway in several microcosms.

Discussion We have demonstrated that detecting biomarkers from etheneotrophic bacteria (i.e., EtnC and EtnE) in groundwater microcosms with proteomic methods is feasible. The EtnC and EtnE peptides we observed were closely related to those expressed by VC-assimilating bacteria. PCR amplification and sequencing of EtnC and EtnE genes from the microcosms further validate these results. However, the combination of PCR and proteomic methods were used primarily to validate the presence of EtnC and EtnE polypeptides and not to evaluate differences in etheneotroph presence/absence between the 10/3/07 and 7/1/08 microcosm experiments (e.g., the 10/3/07 RB-47D microcosm was positive for etheneotrophs, whereas the 7/1/08 RB-47D microcosm was

negative). In is currently unclear whether these differences are biological or the result of variability in the methods employed. Because etheneotrophs can evolve into VC-assimilating bacteria in both VC-fed pure cultures and in VC-fed enrichments (19, 20), and appear to use the same enzymes (e.g., AkMO and EaCoMT) to metabolize both ethene and VC (15-17), it is possible that some of the EtnC and/or EtnE peptides we observed originated from VC-assimilating bacteria. Therefore, this work provides a foundation for future proteomic studies of etheneotrophs and VC-assimilators in unenriched environmental samples (e.g., groundwater). However, because these microcosms were fed ethene, we could not distinguish between VC-assimilating bacteria and etheneotrophs in this study. Additional research is required to determine if there are protein biomarkers that differentiate between these two closely related bacterial groups. Here, PCR analysis of the microcosms suggested that, with the exception of the 10/03/2007 RB-64I sample, EtnE gene presence correlated with culture-based determination of etheneotroph presence (Table 1). Therefore, using the EtnE gene and/or polypeptide as a sole biomarker of VCassimilating and/or etheneotrophic bacteria appears promising based on the results presented here, but should be done with caution as propene-assimilating bacteria are also known to employ this gene (40). Interestingly, 2 EtnC and 5 EtnE peptides were unique to inferred etnC and etnE gene products sequenced from two different VC-assimilating isolates (JS614 and JS623) (SI Table S2). This suggests that at least two distinct etheneotrophic strains were active in the microcosms. However, it is important to note that even though unique peptides were identified, the EtnC and EtnE polypeptides are not likely to be identical to those expressed in cultivated strains. Because the amount of proteomic data required to identify a protein only covers a portion of the polypeptide (18-47% amino acid coverage for the EtnC and EtnE polypeptides identified in this study) the amino acid sequence variability in other regions of the polypeptides remains unknown. The presence of unique JS614 EtnE peptides in four different microcosms is also significant with respect to EtnE diversity. The JS614 VOL. 44, NO. 5, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 1. Phylogenetic tree depicting the relationship between deduced EtnC sequences from both cultivated strains and environmental clones. An alignment of 95 amino acids (excluding gaps) was made with ClustalX and converted into a neighbor-joining tree, which was visualized with MEGA4 (45) using the AmoC from the propene-assimilating Gordonia rubripertincta strain B276 as the outgroup. The filled diamonds represent VC-assimilating strains. The filled circles at nodes indicate bootstrap values of 95%, whereas the open circles at nodes indicate bootstrap values of 75-95%. Bootstrap values lower than 75% are not shown. The bar represents 5% sequence difference. EtnE gene and the EtnE genes sequenced from the RB-63I microcosm cluster separately from all other VC-assimilating and etheneotrophic EtnE sequences (Figure 2). This indicates that there is greater EtnE diversity among etheneotrophs than is currently represented in pure culture. When conducting an environmental proteomics study, appropriate protein extraction, separation, and identification strategies must be carefully chosen and optimized. Given the numbers and types of cells, variations among different environments and sampling techniques, and broad range of proteins, effective protein extraction and separation can be very challenging. Here, we chose to analyze ethene-enriched groundwater microcosms from a VC-contaminated site in an effort to minimize the potential effects of low protein abundance that could be a factor in unenriched environmental samples. Several cell lysis and protein extraction 1598

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methods (i.e., guanidine, sonication, and French press lysis) were successfully applied to the microcosms. However, there were five instances where we did not observe peptide biomarkers in a microcosm that tested positive for etheneotrophs (I-A, RB-47I, RB-58D, RB-63D, and RV-EE). The targeted biomarkers were likely present in these microcosms but below our method detection limits. Inefficient protein extraction could have affected peptide detection in our proteomics workflow. However, a direct comparison of the efficiencies of the selected protein extraction techniques was not possible because the initial amount of protein available for extraction varied among the microcosms. Methods to concentrate microbial cells and/or proteins (e.g., by filtration) from direct environmental samples should be considered in future investigations to address potential problems with inefficient protein extraction.

FIGURE 2. Phylogenetic tree depicting the relationship between deduced EtnE sequences from both cultivated strains and environmental clones. An alignment of 263 amino acids (excluding gaps) was made with ClustalX and converted into a neighbor-joining tree, which was visualized with MEGA4 (45) using the XecA from the propene-assimilating Xanthobacter autotrophicus Py2 as the outgroup. The filled diamonds represent VC-assimilating strains. The filled circles at nodes indicate bootstrap values of 95%, whereas the open circles at nodes indicate bootstrap values of 75-95%. Bootstrap values lower than 75% are not shown. The bar represents 5% sequence difference. We evaluated two different protein separation strategies prior to LC-ES-MS/MS. SDS-PAGE separation, followed by band excision in a desired mass range, and subsequent trypsin digestion was a suitable approach for the groundwater microcosms. In more complex environmental samples (e.g., soils and sediments), gel-based protein separation is often complicated by the coextraction of compounds such as phenolics or humic acids that interfere with staining (41, 42). However, our gel-based proteomic methods are relatively rapid and inexpensive in comparison to other approaches, and could therefore be useful for characterizing active microbial communities in samples that are relatively free of interfering compounds (e.g., groundwater microcosms and enrichment cultures). An alternative proteomics approach we tested, which involved trypsin digestion of proteins in solution and separation of peptides via SCX chromatography, was also successful. This technique is similar to the multidimensional protein identification technology (MudPIT) (43). MudPIT is a preferred approach in environmental proteomics studies, but the labor and expense involved could preclude widespread use in bioremediation studies. We found that our “offline” SCX peptide separation strategy was inexpensive in comparison to MudPIT, but still labor intensive because

several fractions were generated during the SCX chromatography step. Given the relatively small size of the biomarker database, we chose a conservative threshold for protein identifications by requiring that at least two peptides meet specific XCorr and deltaCn filter cutoffs in SEQUEST. If filtering parameters were less rigorous, more peptide identifications (and more protein identifications) would have been deemed “valid” in the microcosms. Future work should focus on determining an optimal threshold for maximizing the number of protein identifications in environmental samples and determining if there are peptides that are unique to VC-assimilating bacteria. Identification of unique EtnC and EtnE peptides in environmental samples will also help to resolve the presence and diversity of etheneotrophs and VC-assimilating bacteria at contaminated sites. Database size and composition is another important factor to consider in environmental proteomic investigations. Using an abridged database greatly decreased our analysis time and minimized the possibility of false protein identifications (data not shown). If larger databases are to be searched, the possibility of false protein identifications must be taken into account (44). A limitation of our targeted protein biomarker VOL. 44, NO. 5, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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approach is the availability of functional gene sequences in public databases (e.g., etnC and etnE). Protein identifications in microcosms were made using an abridged database composed of protein sequences from cultivated VC-, ethene-, and propene-assimilating strains available in UniProt databases and by sequencing PCR products amplified from the microcosms. Continued sequencing of functional genes from VC-contaminated environments and VC-assimilating enrichment cultures will provide additional database support for making effective protein identification from environmental samples.

Acknowledgments This work was supported by the National Science Foundation under Award No. 0738040. A.S.C. was also supported by a NSF Graduate Research Fellowship, the University of Iowa Carver College of Medicine, and a University of Iowa Presidential Fellowship. Proteomic data analysis was provided by the Michigan State University Proteomics Core.

Supporting Information Available Detailed PCR and proteomics methods; two figures with example mass spectra; two tables depicting all etnC and etnE peptides observed in ethene-fed microcosms; and a table of additional proteins identified that are homologous to VCassimilating Nocardioides sp. JS614. This material is available free of charge via the Internet at http://pubs.acs.org.

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