Identity and Substrate Specificity of Reductive Dehalogenases ...

Identity and Substrate Specificity of Reductive Dehalogenases Expressed in Dehalococcoides-Containing Enrichment Cultures Maintained on Different Chlorinated Ethenes Xiaoming Liang, Olivia Molenda, Shuiquan Tang, Elizabeth A. Edwards Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario, Canada

Many reductive dehalogenases (RDases) have been identified in organohalide-respiring microorganisms, and yet their substrates, specific activities, and conditions for expression are not well understood. We tested whether RDase expression varied depending on the substrate-exposure history of reductive dechlorinating communities. For this purpose, we used the enrichment culture KB-1 maintained on trichloroethene (TCE), as well as subcultures maintained on the intermediates cis-dichloroethene (cDCE) and vinyl chloride (VC). KB-1 contains a TCE-to-cDCE dechlorinating Geobacter and several Dehalococcoides strains that together harbor many of the known chloroethene reductases. Expressed RDases were identified using blue native polyacrylamide gel electrophoresis, enzyme assays in gel slices, and peptide sequencing. As anticipated but never previously quantified, the RDase from Geobacter was only detected transiently at the beginning of TCE dechlorination. The Dehalococcoides RDase VcrA and smaller amounts of TceA were expressed in the parent KB-1 culture during complete dechlorination of TCE to ethene regardless of time point or amended substrate. The Dehalococcoides RDase BvcA was only detected in enrichments maintained on cDCE as growth substrates, in roughly equal abundance to VcrA. Only VcrA was detected in subcultures enriched on VC. Enzyme assays revealed that 1,1-DCE, a substrate not used for culture enrichment, afforded the highest specific activity. trans-DCE was substantially dechlorinated only by extracts from cDCE enrichments expressing BvcA. RDase gene distribution indicated enrichment of different strains of Dehalococcoides as a function of electron acceptor TCE, cDCE, or VC. Each chloroethene reductase has distinct substrate preferences leading to strain selection in mixed communities.

M

icrobial reductive dechlorination and organohalide respiration play a significant role in the natural and engineered attenuation of chlorinated ethenes, such as tetrachloroethene (PCE) and trichloroethene (TCE). However, microbial dechlorination via hydrogenolysis sometimes stalls at dichloroethene (DCE) and vinyl chloride (VC) which is problematic as these intermediates are more water-soluble and more toxic than PCE or TCE, particularly VC (1). Organisms that are known to be capable of respiring chlorinated ethenes to nontoxic ethene are restricted to some Dehalococcoides mccartyi strains (2–4). The genomes of two VC-respiring Dehalococcoides isolates were described previously (5), as well as the metagenome of the VC-respiring mixed culture KB-1 (6, 7). In addition to chlorinated ethenes, some D. mccartyi strains can also dechlorinate 1,2-dichloroethane (1,2DCA) (8), 1,2-dichloropropane (9), and a variety of chlorinated and brominated aromatic contaminants (10–12). Membrane-bound reductive dehalogenases (RDases) are key enzymes that mediate dechlorination reactions in organohalide respiring microorganisms. Many RDases and putative RDase sequences have been identified in D. mccartyi strains (13–21). To date, only a few RDases from D. mccartyi have been purified and partially characterized for their activities and substrate range, including TceA (22) and PceA (23) from D. mccartyi strain 195, and VcrA from D. mccartyi strain VS (14). In 2007, Adrian et al. (24) purified and characterized the first chlorobenzene RDase, CbrA, using clear native polyacrylamide gel electrophoresis (CN-PAGE) coupled with an in-gel enzymatic assay and a peptide sequencing by liquid chromatography-tandem mass spectrometry (LC-MS/ MS) system. This approach directly applied crude protein extracts onto polyacrylamide gels for dechlorination activity assays with no need for prior protein purification, which significantly reduced

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the amount of biomass and effort required for partial protein purification and subsequent functional characterization. A somewhat similar approach was also used to identify MbrA (25), an enzyme that dechlorinates TCE to trans-DCE. In 2013, Tang et al. (6) described a blue native polyacrylamide gel electrophoresis (BN-PAGE) method that significantly improved the recovery of dechlorinating activity in gel slices. Since then, several other reductive dehalogenases have been partially characterized with this approach, including chloroform and 1,1,1-trichlorethane and 1,1dichloroethane RDases from Dehalobacter (26) and a chloroform and 1,1,1-trichloroethane RDase from Desulfitobacterium (27), 1,2-dichloropropane dehalogenases from Dehalococcoides and Dehalogenimonas (28), and a trans-DCE RDase from Dehalogenimonas (O. Molenda and E. A. Edwards, unpublished data). Very recently, the PCE RDase from Sulfurospirillum was successfully heterologously expressed in Shimwellia (29), and the VC RDase

Received 15 February 2015 Accepted 23 April 2015 Accepted manuscript posted online 1 May 2015 Citation Liang X, Molenda O, Tang S, Edwards EA. 2015. Identity and substrate specificity of reductive dehalogenases expressed in Dehalococcoides-containing enrichment cultures maintained on different chlorinated ethenes. Appl Environ Microbiol 81:4626 – 4633. doi:10.1128/AEM.00536-15. Editor: A. M. Spormann Address correspondence to Elizabeth A. Edwards, [email protected]. Supplemental material for this article may be found at http://dx.doi.org/10.1128 /AEM.00536-15. Copyright © 2015, American Society for Microbiology. All Rights Reserved. doi:10.1128/AEM.00536-15

Applied and Environmental Microbiology

July 2015 Volume 81 Number 14

RDase Activity and Expression

VcrA from Dehalococcoides was successfully expressed in E. coli (30), opening exciting new avenues for characterization of novel RDases. KB-1 is a Dehalococcoides-dominated microbial consortium that typically dechlorinates 0.5 mM PCE or TCE via cDCE and VC to ethene in days (31). It was the first such culture to be commercialized for use in bioaugmentation (Siremlabs), which has become a successful remediation strategy at many sites (32). In the present study, we applied the BN-PAGE approach to examine RDases in crude extract samples from KB-1 during TCE dechlorination to ethene, as well as from long-established subcultures maintained on cDCE and VC instead of TCE. The first objective was to determine which RDases were expressed as dechlorination progressed from TCE via cDCE and VC to ethene and to compare expression profiles to cultures enriched specifically on these intermediate substrates. We wanted to know if different enzymes were used at different steps of dechlorination in mixed cultures containing multiple competing enzymes and dechlorinating strains. The second objective was to compare and contrast activities of specific enzyme on a broader suite of possible chlorinated substrates, which included all of the chlorinated ethenes as well as 1,2-dichloroethane, to begin to explain strain selection in environmentally relevant mixed cultures. MATERIALS AND METHODS Cultures and growth conditions. The KB-1 parent enrichment culture was originally derived over 15 years ago from sediment from a TCEcontaminated site in southern Ontario, Canada, and has been maintained with TCE as terminal electron acceptor and methanol as electron donor in a defined prereduced mineral medium supplemented with vitamins (33). This consortium contains multiple D. mccartyi strains that comprise more than 50% of the culture that are able to dechlorinate PCE and TCE to ethene (34) and a Geobacter lovleyi strain that makes up 10 to 20% of the culture that dechlorinates PCE and TCE to cis-DCE (35, 36). This parent culture is referred to here as TCE/M_1998, with the naming convention indicating that TCE and methanol (“M”) were added as the electron acceptor and donor, respectively, and that the culture was first established in 1998. The KB-1 subculture VC/H2_2003_1 is a highly enriched subculture of TCE/M_1998 maintained with VC as the electron acceptor and H2 as the electron donor since 2003. PCR followed by denaturing gradient gel electrophoresis, cloning, and phylogenetic analyses revealed that this subculture is dominated by a D. mccartyi strain that grows on VC, and the yields on an electron-equivalent basis measured for this subculture indicated that this strain accounts for a major fraction of the culture biomass (34). The dominant nondechlorinating bacterium in this culture was identified as Sporomusa (37). Although maintained on VC and H2, this subculture is still capable of dechlorinating TCE and cis-DCE but not PCE (34). The KB-1 subcultures cDCE/M_2001 and cDCE/M_2003 have been maintained on cis-DCE as electron acceptor and methanol as electron donor over 10 years. They are also dominated by D. mccartyi strains (⬎80%), with nondechlorinating species, including Sporomusa, Acetobacterium, Methanomethylovorans, Methanosarcina, and others (37). The microbial composition of these enrichment cultures as determined by 16S rRNA sequencing has been relatively stable over many years, although shifts in the nondechlorinating populations have been observed from time to time (7). Culture and sample preparation for BN-PAGE. Prior to beginning specific experiments, active enrichment culture bottles containing ⬃150 ml of liquid were purged with N2/CO2 (80/20 [vol/vol]) to remove residual chlorinated ethenes, and left to starve for 5 days to promote the degradation of previously expressed RDases. Experimental cultures were then amended with chlorinated electron acceptors (30 mg/liter, aqueous concentration) and excess electron donor (methanol or H2). After 2 to 4 h


FIG 1 Sequential dechlorination of TCE in the KB-1 TCE/M_1998 culture. Protein extractions were carried three times over the course of degradation, as indicated by arrows at time points 1, 2, and 3.

(when ca. 10 to 25% of the chlorinated substrates had degraded), cell pellets were collected from 15 to 45 ml of culture by centrifugation at 8,000 ⫻ g for 20 min at 4°C. For the TCE/M_1998 culture, cell pellets were collected three times during the dechlorination profile, by sacrificing 15 ml of culture at each time point, as shown in Fig. 1. In an anaerobic chamber (Coy), crude protein extracts were prepared from fresh cell pellets as described previously (6). Briefly, cells were lysed by vigorous bead beating in sample buffer containing 1% (wt/vol) digitonin, 50 mM BisTris, 50 mM NaCl, 10% (wt/vol) glycerol, and 50 mg of glass beads. The lysates were centrifuged at 10,000 ⫻ g for 10 min, and the supernatant containing the solubilized protein extracts was collected. Prior to loading extracts onto precast 4 to 16% acrylamide gradient gels (Invitrogen), extracts were amended with Coomassie blue G-250 at a final concentration of 0.25% (wt/vol). Duplicates were prepared for each experiment. BN-PAGE and staining. Electrophoresis was conducted in the NativePAGE Novex Bis-Tris gel system (Invitrogen) according to the method of Tang et al. (6). Briefly, 5 ␮l of NativeMark unstained protein standard (Invitrogen) was loaded to the first lane of precast gradient Bis-Tris gel (4 to 16%, 1.0 mm thick; Invitrogen), and 20 ␮l of crude protein extract was loaded onto each of the other lanes of the gel. Electrophoresis was run in an ice bath at 150 V for 60 min and 200 V for 45 min, followed by the staining of the first two lanes of the gel according to the Fast Coomassie G-250 staining protocol from Invitrogen. Protein quantification. Protein concentrations in crude extracts were determined using the Bradford assay (38) with bovine serum albumin (BSA) as a standard. We also estimated the amount of protein in gel slices using the method of Tang et al. (6). Briefly, gel images were acquired with G:BOX Chemi HR16 (Syngene) and band intensity digitized using ImageJ software (http://rsb.info.nih.gov/ij/). The protein concentration of each molecular weight (MW) marker in the ladder was established by comparing to band intensities of a series of BSA samples of known concentration, as illustrated in Fig. S1 in the supplemental material. The protein content in gel slices was estimated from band intensities obtained from gel images using ImageJ with the known concentrations for each band in the MW ladder used to calibrate protein content of unknown bands from samples on the same gel. All bands within a slice used in activity assays were summed to get an estimate of the total protein content in a specific gel slice. Dechlorination activity assays in gel slices. Unstained gel lanes were aligned with the corresponding lane stained with Coomassie blue G-250 to cut protein bands into distinct slices. Typically, a total of six individual gel slices were prepared per lane (see Fig. S2 to S8 in the supplemental material), and dechlorination activity assays in these slices were performed as previously described (6, 39) testing the following chlorinated substrates: TCE, cis-DCE, trans-DCE, 1,1-DCE, VC, and 1,2-DCA. In ad-


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dition, a negative control (assay buffer alone) and a positive control containing assay buffer solution and 20 ␮l of the crude protein extract were also tested. Concentrations of dechlorination products were measured by gas chromatography with headspace analysis calibrated with external standards (40). Normalized dechlorinating activity in active gel slices between 146 and 242 kDa (typically slices 3, 4, and 5) was calculated by summing all measured dechlorination products in the active gel slice and dividing by the corresponding protein content estimated for the same gel region and the incubation time for the assays (24 h). LC-MS/MS analysis. Gel slices of interest were pretreated according to the protocol from the Advanced Protein Technology Center at SickKids Hospital, Toronto, Ontario, Canada (http://www.sickkids.ca/pdfs /Research/APTC/8129-in-geltryptic.pdf). Briefly, the stained gel bands were first destained with 50 mM ammonium bicarbonate and then treated with 50% acetonitrile–25 mM ammonium bicarbonate, reduced with 10 mM dithiothreitol, alkylated with 100 mM iodoacetamide, digested with 13 ng of trypsin/␮l prepared in 50 mM ammonium bicarbonate, extracted with 25 mM ammonium bicarbonate and 5% formic acid, and reconstituted in 0.1% formic acid solution after drying. The resulting peptides were loaded onto a 150 ␮m (inner diameter) precolumn (Magic C18; Michrom Biosciences) at 4 ␮l/min and separated over a 75 ␮m (inner diameter) analytical column packed into an emitter tip containing the same packing material. The peptides were eluted over 60 min at 300 nl/ min with a 0 to 40% acetonitrile gradient in 0.1% formic acid using an EASY n-LC nano-chromatography pump (Proxeon Biosystems, Odense, Denmark). The peptides were eluted into an LTQ-Orbitrap hybrid mass spectrometer (Thermo-Fisher, Bremen, Germany) operated in a datadependent mode. Mass spectra were acquired at a resolution of 60,000 (full-width/half-maximum [FWHM]) by Fourier transform mass spectrometry (FTMS), and tandem mass spectrometry (MS/MS) was carried out in the linear ion trap. Six MS/MS scans were obtained per MS cycle. Sequence database and searching. The KB-1 metagenome (Joint Genome Institute IMG Taxon Object ID 2013843002), including all known curated KB-1 dehalogenase sequences from multiple studies (6, 21, 41) (final database of 40,772 entries) was used as a sequence database to compare to mass spectra. All MS/MS samples were analyzed using Mascot (version 2.3.02; Matrix Science, London, United Kingdom). Mascot was searched with a fragment ion mass tolerance of 0.50 Da and a parent ion tolerance of 3.0 Da. Iodoacetamide derivative of cysteine was specified in Mascot as a fixed modification. Pyroglutamic acid from elimination at the N terminus, S-carbamoylmethylcysteine cyclization of the N terminus, deamidation of asparagine and glutamine, oxidation of methionine, and acetylation of the N terminus were specified in Mascot as variable modifications. Criteria for protein identification. Scaffold (version Scaffold_3.6.5; Proteome Software, Inc., Portland, OR) was used to validate MS/MSbased peptide and protein identifications. Peptide identifications were accepted if they could be established at ⬎80.0% probability, as specified by the Peptide Prophet algorithm (42). Protein identifications were accepted if they could be established at ⬎95.0% probability and contained at least nine identified peptides. Protein probabilities were assigned by the Protein Prophet algorithm (43). Proteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony. rdhA gene survey. Quantitative PCR (qPCR) was used to determine the relative concentrations of the rdhA genes in various KB-1 enrichment cultures using specific primers (see Table S1 in the supplemental material). Reaction mixtures (final volume, 20 ␮l) contained 10 ␮l of 2⫻ SYBR green supermix (Bio-Rad, Hercules, CA), forward and reverse primers (1 ␮M each), and 2 ␮l of template DNA. The amplification program included an initial denaturation step at 98°C for 2 min, followed by 40 cycles of 5 s at 98°C and 10 s at the corresponding annealing temperature (see Table S1 in the supplemental material). A final melting curve from 64 to 95°C at increments of 0.5°C/s was not performed but recorded. Quantifications were achieved by using 10-fold serial dilutions of plasmid DNA

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containing one copy of the 16S rRNA gene or the rdhA gene as calibrators. In addition to the cultures used for BN-PAGE, the following cultures were also included in the qPCR survey: TCE/M_1999, TCE/M_2001, TCE/ H2_2001, TCE/M_2010, VC/H2_2003_2, VC/M_2001, and VC/M_2004. These cultures were named using the same convention as the KB-1 parent enrichment culture, i.e., “electron acceptor/electron donor_year established” (and an additional number if more than one culture was established in a given year).

RESULTS AND DISCUSSION

Dechlorination assays in crude cell extracts. Each crude cell extract sample to be used for BN-PAGE analysis was also assayed for dechlorination activity as a positive control for the corresponding assays using samples from gel slices. Using the data from these crude cell assays, we estimated a minimum specific dechlorination rate in crude extracts from the total millimoles of the dechlorination products measured after 24 h divided by the protein content of the extract. These data revealed that, regardless of the culture or condition, extracts dechlorinated cis-DCE and 1,1-DCE the fastest and at comparable rates ranging from 3 to 23 nmol min⫺1 mg protein⫺1 over 24 h (Table 1). To make comparisons easier, we also reported the rate data as a percentage of the maximum rate measured for that preparation (Table 1). The substrate activity profile in the extracts from the parent culture TCE/M_1998 were similar regardless of sample time point and substrate, with perhaps the exception of slightly more activity on PCE at time point 1, consistent with the known presence and activity of Geobacter with PCE and TCE in KB-1. All extracts dechlorinated TCE at comparably high rates except extracts from cultures enriched on cDCE. These extracts were also the only ones to significantly dechlorinate trans-DCE. All extracts dechlorinated 1,2-DCA except the one from the VC enrichment. There was little to no activity on PCE in the extracts from the subcultures maintained on cDCE or VC (Table 1). Enzyme assays using BN-PAGE gel slices. After extracts were subjected to BN-PAGE to separate the proteins, the dechlorination activity was also measured in individual gel slices. As in previous studies (6), the region of highest activity was consistently found in slices situated between 146 and 242 kDa based on the MW ladder, regardless of chlorinated substrate or culture extract tested (see Fig. S2 to S8 in the supplemental material). The protein content in gel slices was quantified, and the resulting estimates of minimum specific dechlorinating activity in (nmol min⫺1 mg of protein⫺1) over a 24-h period in a given gel slice are also reported in Table 1. As with crude extracts, the highest activities in gel slices were found for the substrates 1,1-DCE and cDCE. Trends were similar to those in crude extracts, although the specific activities were generally 2- to 30-fold higher (Table 1), clearly indicating partial purification or enrichment of RDases as a result of BNPAGE separation. Identification of expressed RDases. Proteins from active gel slices were analyzed by LC-MS/MS, and peptides were identified by comparison to the KB-1 metagenome sequence database. These analyses detected a total of only five distinct RDases in any of the samples: KB1_VcrA, KB1_TceA, KB1_BvcA, KB1_GeoRD, and KB1_RdhA5, as well as many other Dehalococcoides proteins. (A complete list of all detected proteins for all samples is compiled in Table S2 in the supplemental material.) Focusing on the RDases expressed during sequential dechlorination of TCE via cDCE and VC in the TCE-fed KB-1 parent enrichment culture (TCE/ M_1998), we found that KB1_VcrA was consistently detected at




Sample type

340 ⫾ 9 0.70 495 ⫾ 0.8 0.38 550 ⫾ 9 0.48 280 ⫾ 20 0.37 185 ⫾ 6 0.26 210 ⫾ 20 0.43 170 ⫾ 0.3 0.05

Protein concn (␮g/ml or ␮g/slice)a

0.33 ⫾ 0.21 (8) NM 0.25 ⫾ 0.01 (3) NM 0.29 ⫾ 0.01 (3) NM 0.8 ⫾ 0.1 (3) NM 0 (0) NM 1.1 (3) 6.8 0 (0) NM

PCE

1.5 ⫾ 0.1 (38) 5.6 3.7 ⫾ 0.1 (45) 4.5 4.9 ⫾ 0.3 (46) 3.6 12 ⫾ 0.4 (54) 5.8 0.57 ⫾ 0.05 (6) 0.79 1.9 (5) 3.2 2.3 ⫾ 0.4 (74) 3

TCE

4.0 ⫾ 0.1 (100) 6.9 8.3 ⫾ 0.04 (100) 130.5 11 ⫾ 0.3 (100) 74.8 23 ⫾ 1 (100) 181 4.3 ⫾ 0.7 (48) 4.7 SL 101.3 3.1 ⫾ 0.2 (100) 38

cis-DCE

0.16 ⫾ 0.01 (4) 2.9 0.29 ⫾ 0.01 (4) 4.6 0.47 ⫾ 0.01 (4) 2.3 0.52 ⫾ 0.03 (2) 4.1 5.1 ⫾ 0.1 (57) 6.1 SL 57.0 0.03 ⫾ 0.00 (1) 0.83

trans-DCE

3.9 ⫾ 0.1 (98) 92 7.7 ⫾ 0.1 (9) 177.0 10 ⫾ 0.3 (96) 81.1 20 ⫾ 0.03 (87) 134 9.0 ⫾ 0.03 (100) 18 40 (100) 31.7 2.9 ⫾ 0.1 (94) 82

1,1-DCE

0.78 ⫾ 0.01 (20) 14 2.0 ⫾ 0.02 (25) 12.8 2.8 ⫾ 0.1 (26) 10.8 4.8 ⫾ 0.2 (21) 16 0.53 ⫾ 0.03 (6) 1.2 27 (68) 17.8 0.78 ⫾ 0.01 (25) 67

VC

0.91 ⫾ 0.02 (20) 4.8 4.0 ⫾ 0.1 (48) 16.6 5.4 ⫾ 0.01 (51) 6.9 10 ⫾ 0.4 (43) 8.3 2.5 ⫾ 0.07 (28) 2.8 25 (63) 15.2 0.06 ⫾ 0.00 (2) 2.7

1,2-DCA

Dechlorination activity (nmol min⫺1 mg of protein⫺1) by chlorinated substrateb

CFE Gel slice CFE Gel slice CFE Gel slice CFE Gel slice CFE Gel slice CFE Gel slice CFE Gel slice

TABLE 1 Protein-normalized dechlorination activity determined in cell-free extracts (CFE) and active gel slices over a 24-h period

Culture

TCE-fed TCE/M_1998; time point 1 TCE-fed TCE/M_1998; time point 2 TCE-fed TCE/M_1998; time point 3 cis-DCE-fed TCE/M_1998 cis-DCE-fed cDCE/M_2003 cis-DCE-fed cDCE/M_2001 VC-fed VC/H2_2003_1

a The protein concentrations in cell-free extracts (CFE) were measured in triplicate and are reported as averages ⫾ the standard deviations; the protein concentrations in single gel slices were estimated from the band intensity (see the text). b Values for CFE (% of maximum) are reported as averages ⫾ the ranges of duplicate analyses; for gel slices, the values are from single measurements. Percentages are indicated in parentheses. NM, not measured; SL, sample lost. Shading indicates a dechlorinating activity of ⬍6% of the maximum observed for that sample.


all time points and was always the most abundant RDase with the highest number of peptide hits and the greatest percent coverage (Fig. 2), in agreement with previous results for KB-1 (6). KB1_VcrA was also listed as the third most abundant protein among those detected in active gel slices in all cultures (see Table S2 in the supplemental material). KB-1_TceA was also detected at all time points at lower abundance (⬃15%) compared to VcrA (Fig. 2). KB1_GeobRD was detected, although barely, only at time point 1, very early during TCE dechlorination (Fig. 2), a finding consistent with Geobacter’s inability to dechlorinate beyond cDCE. When this same enrichment culture was atypically fed cDCE instead of TCE, similar abundances of both VcrA and TceA were again detected, along with KB1_RdhA5 (Fig. 2). No BvcA was detected in these extracts. Two KB-1 subcultures established over a decade ago on cDCE were also assayed. Unlike any of the other cultures examined, samples from these cultures showed highest specific activity on trans-DCE (Table 1), both in crude extracts and in gel slices. LC-MS/MS analysis of these samples detected KB1_BvcA and KB1_VcrA with peptide hits for BvcA greater than those for VcrA (Fig. 2). One VC enrichment culture, VC/H2_2003_1, was assayed. In this culture, the dechlorinating activity was greatest with 1,1-DCE, VC, TCE, and cis-DCE, with little activity on 1,2-DCA and trans-DCE (Table 1). KB-1_VcrA was the only RDase detected in this culture by LC-MS (Fig. 2); thus, we can infer that KB-1_VcrA is responsible for this activity profile. The activity observed in the cDCE culture extracts on trans-DCE and 1,2-DCA must therefore be attributed to KB1_BvcA. KB1_VcrA (DQ177519) shares 97.1% amino acid identity (15 differences out of 519) with the characterized VcrA from D. mccartyi strain VS that dechlorinates VC, cDCE, and to a lesser extent TCE, and not PCE (14, 44). KB1_TceA (KP085026) shares 97.3% amino acid identity (15 differences/560) with the characterized TceA from D. mccartyi strain 195, which is known to dechlorinate TCE, all three DCE isomers, VC (slowly), and 1,2-DCA, but not PCE (22). KB1_RdhA5 (DQ177510) is homologous to DET1545 in D. mccartyi strain 195, where it was found expressed during starvation, although its substrate is not known (45). KB1_BvcA (DQ177511) shares 99.0% amino acid identity (5 differences/516) with BvcA from D. mccartyi strain BAV1, that was previously shown to dechlorinate all three DCE isomers, VC, 1,2-DCA, and TCE, but not PCE (6). The data collected here clearly demonstrated that the two well-known VC-dechlorinating dehalogenases, BvcA and VcrA, that are critical for site remediation have distinct substrate preferences, with BvcA showing significantly more activity on trans-DCE and 1,2-DCA, while VcrA shows more activity on TCE. It also is apparent that all steps in the dechlorination of TCE to ethene can be catalyzed by the same enzyme, VcrA, explaining why intermediates cDCE and VC do not accumulate significantly in cultures like KB-1. These protein expression profiles are consistent with previously reported transcriptional results for KB-1. Early transcriptional studies had detected genes encoding BvcA and VcrA in KB-1 cultures dechlorinating TCE, cis-DCE, VC, and 1,2-DCA and had also found that BvcA gene transcripts were more abundant with the substrate cDCE (17). Subsequent microarray data that found genes for VcrA most highly transcribed in KB-1 cultures in general, with vcrA being the only RDase gene transcribed in KB-1 cultures grown on VC (41). These microarray data also revealed transcription of the Geobacter RDase gene in a TCE-in-


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FIG 2 Identity, number of peptide hits (y axis), and coverage (the percentages are indicated in each bar) of the RDases identified in most active gel slices from KB-1 enrichment cultures. Three protein samples (TP#1, TP#2, and TP#3) were collected during TCE dechlorination via cDCE and VC, as shown in Fig. 1. The same TCE-enriched culture was also fed cDCE and proteins collected from the active gel slice for comparison. Subcultures enriched on cDCE and VC were also compared. For each protein sample, the gel slices that exhibited highest dechlorinating activity were analyzed by LC-MS/MS. VcrA was detected in all samples, BvcA only in cultures enriched on cDCE. The Geobacter RDase was only detected transiently as the KB1 parent culture dechlorinated TCE. KB1_RdhA5, homologous to DET 1545, was only detected in the KB1 parent culture when it was fed cDCE instead of TCE.

duced KB-1 culture (41). KB1_GeobRD (JX081248) is the only RDase sequence present (though duplicated) in the genome of Geobacter lovleyi strain KB-1; thus, it must dechlorinate PCE and TCE to the product cis-DCE. Finally, transcription of the gene encoding KB1_RdhA5 was highest in starved KB-1 cultures (41). The detection of this protein in the cDCE-fed TCE enrichment suggests that at least a subpopulation of Dehalococcoides in this culture was responding to starvation. Other proteins detected in active gel slices. In addition to RDases, many other proteins were detected in the gel slices when searches were done against the KB-1 metagenome (see Table S2 in the supplemental material). In all of the cultures tested, chaperonin GroEL (Dehalococcoides, DCKB1_11070) was the protein with the highest protein hits and coverage, followed by chaperone protein DnaK (Dehalococcoides, DCKB1_174500). Other relatively high-abundance non-RDase proteins included Dehalococcoides glyceraldehyde-3-phosphate dehydrogenase, Dehalococcoides electron transport complex, C subunit, and Dehalococcoides 10-methylenetetrahydrofolate reductase. One interesting observation from the LC-MS/MS data is the importance of having a good database against which to match peptide hits. The KB-1 metagenome came from the parent culture grown on TCE. The expression data above and the rdhA qPCR survey results described in the next paragraph indicate that multiple Dehalococcoides strains exist in the various KB-1 enrichment cultures. The protein hits to the KB-1 metagenome in Table S2 in the supplemental material show good matches for non-RDase proteins for the TCE and VC enrichments but very poor matches to the cDCE enrichments, which is further evidence of diverging strains, particularly in the cDCE enrichments. To further investigate the distribution of Dehalococcoides strains in different subcultures, we measured the relative abundance of a selected dehalogenase genes in enrichment cul-

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tures maintained on different electron acceptors and donors, as described below. Abundance of rdhA genes in enrichment cultures. The abundance of the genes for five Dehalococcoides RDases and one Geobacter RDase in DNA samples from multiple cultures is summarized in Table 2. These six RDases were selected because they had been detected from BN-PAGE experiments either in the present study or previously (6). The qPCR data are presented as the ratio of the measured rdhA copies to the corresponding 16S rRNA gene copies to estimate copies per genome in the particular sample. The data were sorted by the electron acceptor amended to each enrichment culture. A few features stand out from this analysis. First, both rdhA1 and rdhA5 were detected in all cultures at a ratio of ⬃1 (from 0.56 to 1.53, or about 1 considering the error of qPCR analyses), indicating that these genes are present in all Dehalococcoides strains in these cultures (Table 2). The functions of the RDases encoded by these two genes are unknown; however, their presence in all strains provides a fortuitous internal control for the other rdhA sequences. Similarly, the Geobacter rdh gene (rdhGeo) was only detected in cultures enriched on TCE; consistent with expectations for where Geobacter will survive. In VC enrichment cultures, the only known chloroethene-dechlorinating RDase gene detected was vcrA, with a ratio ranging from 0.74 to 4.85 with an average 2.16; neither bvcA nor tceA were detected in any of these enrichments (Table 2). This agrees well with our proteomic data, in which KB1_VcrA was the only RDase detected in the gel slice derived from VC/H2_2003_1 culture (Fig. 2). Ratios of vcrA to 16S rRNA greater than one perhaps suggest more than one copy of this gene per genome, which is being further investigated in these cultures. The qPCR survey also found bvcA to be essentially present only in the cDCE enrichments, also in agreement with the proteomic data (Fig. 2). The average ratios of rdh to 16S rRNA




TABLE 2 Distribution of reductive dehalogenase genes in enrichment cultures derived from a common parent culture TCE/M_1998 but maintained on distinct electron acceptors since the year indicated in the culture designationa Electron acceptor

Electron donor

DNA extraction date (mo-day)

VC enrichment cultures VC/H2_2003-1 VC/H2_2003-2 VC/H2_2003-2 VC/M_2004 VC/M_2001 VC/H2_2003-1 Avg

VC VC VC VC VC VC

Hydrogen Hydrogen Hydrogen Hydrogen MeOH Hydrogen

cDCE enrichment cultures cDCE/M_2003 cDCE/M_2001 cDCE/M_2001 cDCE/M_2003 cDCE/M_2001 Avg

cDCE cDCE cDCE cDCE cDCE

TCE enrichment cultures TCE/M_1999 TCE/M_1998 TCE/M_2010 TCE/M_1998 TCE/H2_2001 TCE/ME_2001 TCE/ME_2001 Avg

TCE TCE TCE TCE TCE TCE TCE

Enrichment culture

Ratio of rdh to 16S rRNA gene copies vcrA

bvcA

tceA

rdhA1

rdhA5

rdhGeo

Jan-11 Jan-11 Oct-11 Oct-11 Oct-11 May-13

1.87 0.74 2.08 4.85 1.42 1.98 2.16

⬍DL ⬍DL ⬍DL ⬍DL ⬍DL ⬍DL ⬍DL

⬍DL ⬍DL NA NA NA ⬍DL ⬍DL

0.91 1.21 NA NA NA NA 1.06

0.49 0.64 NA NA NA NA 0.56

⬍DL ⬍DL ⬍DL ⬍DL ⬍DL NA ⬍DL

MeOH MeOH MeOH MeOH MeOH

Jun-11 Jun-11 Oct-11 May-13 May-13

0.75 0.59 1.16 0.49 0.53 0.70

0.71 0.49 0.78 1.81 2.08 1.17

⬍DL ⬍DL NA ⬍DL ⬍DL ⬍DL

1.07 1.02 NA NA NA 1.05

2.80 0.27 NA NA NA 1.53

⬍DL ⬍DL ⬍DL NA NA ⬍DL

MeOH MeOH MeOH MeOH Hydrogen MeOH/EtOH MeOH/EtOH

Jan-11 Oct-11 Apr-11 May-13 Jan-11 Apr-7 Oct-9

0.51 1.05 0.37 0.68 0.40 2.27 0.39 0.81

0.01 0.00 0.00 0.00 0.31 0.02 0.00 0.05

0.08 0.16 0.00 0.02 0.02 NA 0.28 0.09

1.37 0.94 0.94 NA 1.41 NA 0.81 1.09

0.98 0.44 0.73 NA 0.69 NA 0.61 0.69

0.95 1.34 0.27 NA 3.50 0.98 0.91 1.33

a

Values are the ratio of rdh gene copies to 16S rRNA gene copies for Dehalococcoides (vcrA, bvcA, tceA, rdhA1, and rdhA5) or Geobacter (rdhGeo). MeOH, methanol; EtOH, ethanol. DL, detection limit; NA, not analyzed.

genes in the cDCE enrichments (bvcA, 1.17; vcrA, 0.70) are both close to 1, and this may indicate that one Dehalococcoides stain harbors both genes, although the average value for vcrA (0.7; see Table 2) versus the ratios for bvcA (1.17), rdhA1 (1.05), and rdhA5 (1.53) suggests that some of the Dehalococcoides in these cultures do not contain vcrA. In fact, looking at the data for cDCE enrichments over time (2011 versus 2013 extraction dates) suggests that strains containing bvcA (0.49 to 0.81 in 2011 and 1.81 to 2.08 in 2013) are increasing relative to vcrA (0.59 to 1.16 in 2011 and 0.49 to 0.53 in 2013). Considering TCE enrichment cultures, we see dominance of the vcrA gene and various fractions of bvcA and tceA genes ranging from undetected up to 30% of the Dehalococcoides population. Considering specifically culture TCE/M_1998, no bvcA was detected, and tceA was detected in 2 to 16% of the Dehalococcoides population, depending on the sample date (Table 2), a finding that was again consistent with our LC-MS/MS analysis indicating substantially more peptide hits to VcrA than to TceA and none to BvcA in extracts from this culture (Fig. 2). From the distribution of rdh genes in Table 2, it is clear that multiple strains of Dehalococcoides exist in each enrichment culture. Long-term enrichment on specific electron acceptors has led to consistent patterns in strain distributions. In the VC enrichments, strains containing bvcA and tceA are absent, whereas strains containing the vcrA gene prevail. The abundance ratios suggest that strains may even contain two or more copies of vcrA per genome. In the TCE enrichment cultures, the distribution of Dehalococcoides rdhA genes is similar to that in the VC enrichments, except that there these enrichments maintain subpopula-


tions at lower abundance that contain either the bvcA or the tceA gene or both. Finally, amending the KB-1 parent culture with cDCE has selected for Dehalococcoides populations containing bvcA. The presence of multiple strains of Dehalococcoides has been long suspected in these sets of cultures, from sporadic detection of tceA and bvcA to difficulties in genome assembly (7). These data indicate that selection of subpopulations is driven in large part by the nature of the most abundant available terminal electron acceptors. ACKNOWLEDGMENTS We thank Laura Hug for the curation of the RDase database and Alfredo Perez de Mora, Line Lomheim, Anna Lacourt for carrying out some of the qPCR experiments. S.T. and O.M. received awards from the Government of Ontario through the Ontario Graduate Scholarships in Science and Technology and the Natural Sciences and Engineering Research Council of Canada (NSERC PGS D). Financial support was provided by the Government of Canada through Genome Canada and the Ontario Genomics Institute (2009-OGI-ABC-1405) and the Government of Ontario through the ORF-GL2 program.

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