Dehalococcoides mccartyi Strain JNA Dechlorinates Multiple

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Dehalococcoides mccartyi Strain JNA Dechlorinates Multiple Chlorinated Phenols Including Pentachlorophenol and Harbors at Least 19 Reductive Dehalogenase Homologous Genes Ashwana D. Fricker,†,§ Sarah L. LaRoe,‡,∥ Michael E. Shea,† and Donna L. Bedard*,† †

Department of Biological Sciences and ‡Department of Civil and Environmental Engineering, Rensselaer Polytechnic Institute, 110 8th Street, Troy, New York 12180, United States S Supporting Information *

ABSTRACT: Pentachlorophenol and other chlorinated phenols are highly toxic ubiquitous environmental pollutants. Using gas chromatographic analysis we determined that Dehalococcoides mccartyi strain JNA in pure culture dechlorinated pentachlorophenol to 3,5-dichlorophenol (DCP) via removal of the ortho and para chlorines in all of the three possible pathways. In addition, JNA dechlorinated 2,3,4,6tetrachlorophenol via 2,4,6-trichlorophenol (TCP) and 2,4,5-TCP to 2,4-DCP and 3,4-DCP, respectively, and dechlorinated 2,3,6-TCP to 3-chlorophenol (CP) via 2,5DCP. JNA converted 2,3,4-TCP to 3,4-DCP and 2,4-DCP by ortho and meta dechlorination, respectively. 2,3-DCP was dechlorinated to 3-CP, and, because cultures using it could be transferred with a low inoculum (0.5 to 1.5% vol/vol), it may act as an electron acceptor to support growth. Using PCR amplification with targeted and degenerate primers followed by cloning and sequencing, we determined that JNA harbors at least 19 reductive dehalogenase homologous (rdh) genes including orthologs of pcbA4 and pcbA5, pceA, and mbrA, but not tceA or vcrA. Many of these genes are shared with D. mccartyi strains CBDB1, DCMB5, GT, and CG5. Strain JNA has previously been shown to extensively dechlorinate the commercial polychlorinated biphenyl (PCB) mixture Aroclor 1260. Collectively the data suggest that strain JNA may be well adapted to survive in sites contaminated with chlorinated aromatics and may be useful for in situ bioremediation.

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INTRODUCTION Pentachlorophenol (PCP) and other chlorinated phenols are ubiquitous contaminants due to their extensive use as wood preservatives, biocides, and intermediates in the manufacture of dyes and pharmaceuticals. Currently the greatest sources of contamination are wood treatment facilities, landfills, and hazardous waste sites. More than 700 wood preserving sites have been identified in the United States, and many of these are contaminated with PCP.1,2 Furthermore, the CERCLA Public Access Database lists 152 sites other than wood preserving sites that are contaminated with PCP.3 PCP leaches from these sites contaminating the groundwater. Often contaminated groundwater plumes extend far beyond the boundaries of the site, impacting drinking water, private wells, streams, wetlands, and cropland and posing a particularly difficult challenge for cleanup.4 PCP is in the top 20% of pollutants, ranked by priority based on a combination of frequency, toxicity, and potential for human exposure, on the 2013 Priority List of Hazardous Substances,5 and the EPA has specified a maximum contamination level of 1 ppb for drinking water with a target goal of 0 ppb.6 Chlorinated phenols also harm benthic ecosystems because they partition into organic sediments where they persist,7 in part, due to their high toxicity to microorganisms. Chlorinated phenols can be absorbed through the skin, inhaled, or ingested. Their greatest toxicity results from their ability to uncouple mitochondrial oxidative © 2014 American Chemical Society

phosphorylation, and this activity increases with the level of chlorination.8 In mammals, chlorophenols can also cause damage to the liver and immune system.8 Pentachlorophenol is the most toxic congener9 and has been implicated in damaging mammalian developmental and reproductive systems.10 Consequently, there is great interest in the safe and effective removal of chlorinated phenols from groundwater and aquatic sediments. In 1986, Mikesell and Boyd showed that three anaerobic microbial enrichments could reductively dechlorinate PCP to phenol,11 demonstrating the potential of reductive dehalogenation for bioremediation of chlorinated phenols. Unfortunately, the organisms responsible were not isolated or identified. Since then, many organisms that reductively dechlorinate various chlorinated phenols have been described, but until 200712 only a few strains of Desulfitobacterium haf niense and Desulfitobacterium dehalogenans were known to dechlorinate PCP (reviewed in reference13). Several recent discoveries suggest that members of the species Dehalococcoides mccartyi are excellent candidates for reductive dehalogenation of PCP and other highly chlorinated Received: Revised: Accepted: Published: 14300

July 22, 2014 October 22, 2014 November 6, 2014 November 7, 2014 dx.doi.org/10.1021/es503553f | Environ. Sci. Technol. 2014, 48, 14300−14308

Environmental Science & Technology

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distinct advantage and would be potentially useful in benthic ecosystems for in situ bioremediation. Therefore, we sought to determine whether strain JNA could also dechlorinate PCP and other chlorinated phenols. Knowledge of the number and diversity of rdhA genes in strain JNA would give an indication of this strain’s versatility for dehalogenation. It is also important to identify the genes that code for the RDases responsible for the dechlorination of chlorinated phenols because probes can then be designed and used to demonstrate the presence of bacteria capable of dechlorinating chlorophenols. The first step is to identify and sequence the rdhA genes. All reductive dehalogenases and RdhA proteins identified to date have similar primary structures. In D. mccartyi, these proteins are approximately 450 to 600 amino acids and have a twin arginine leader sequence at the N-terminus and two iron sulfur binding sites near the C-terminus. The rdhA genes encode the catalytic subunit, while the nearly always present adjacent rdhB genes are thought to encode a membrane anchor.15 The RdhA proteins are quite diverse. Hug et al. have classified 264 RDase and RdhA protein sequences from known organohalide respiring bacteria into 46 reductive dehalogenase ortholog groups (RD_OGs).15 The members of each RD_OG are nearly full length RdhA proteins that have at least 90% pairwise amino acid sequence identity and that fall into a highly supported clade on protein tree analyses.15 Of the 205 RdhA protein sequences curated for eight strains of D. mccartyi, 175 fall into 32 RD_OGs. Additionally, there are 30 RdhA proteins with no known orthologs, and each newly characterized D. mccartyi strain reveals more novel RdhA proteins; thus the diversity of RdhA proteins has yet to be exhausted.15 Here we characterize the dechlorination of seven chlorophenols by D. mccartyi strain JNA. We also report the sequences of 19 rdhA genes from this isolate and classify the RdhA proteins that they encode according to the classification system recently described by Hug et al.15

phenols. First, these strict anaerobes are obligate organohalide respiring bacteria (OHRB), meaning that their sole method of obtaining energy for growth is through the reductive removal of halogens from halogenated organic compounds.14 Second, to support this restrictive lifestyle, each of these organisms carries a unique set of between 10 and 36 reductive dehalogenase (RDase) genes, or, if the encoded protein has not been biochemically characterized, reductive dehalogenase homologous (rdhA) genes.14,15 Third, the rdhA genes of D. mccartyi appear to be ancient,14,15 i.e. they have had millions of years to evolve and diversify in order to use a wide variety of naturally occurring halogenated organic compounds as respiratory electron acceptors. Fourth, a recent review catalogued the sequences of 264 RDase and rdhA genes; 205 of these were found in D. mccartyi and only 59 in all other species, including Desulfitobacterium.15 Fifth, several strains of D. mccartyi have been shown to be capable of using highly chlorinated aromatic compounds such as hexachlorobenzene,16 polychlorinated dibenzodioxins,17 and polychlorinated biphenyls18−20 as respiratory electron acceptors. Sixth and finally, these organisms appear to be widely dispersed in both contaminated and pristine anoxic environments including sediments and aquifers.14 The substrates for most RdhA proteins are not yet known, but the most likely original substrates are naturally occurring compounds.21 Highly chlorinated phenols, including tetrachlorophenols and PCP, are not just industrial products but are also formed in nature. Specifically, they are produced by the combustion of fresh wood and have been found in remote pristine areas in sediment layers predating industrial use; thus they are presumed to have been produced by forest fires and subsequently widely distributed in ash.22−25 Accordingly, it is highly likely that at least some members of the species D. mccartyi have evolved RDases that can dechlorinate chlorinated phenols in order to exploit this potential energy source. To date, the only D. mccartyi strain tested for the ability to dechlorinate a wide variety of chlorinated phenols is CBDB1, which is capable of dechlorinating PCP and all tri- and tetrachlorinated phenols to less chlorinated phenols.12 This strain respires 2,3-dichlorophenol (2,3-DCP) and 2,3,4trichlorophenol (2,3,4-TCP) which are dechlorinated to 3chlorophenol (3-CP) and to 2,4-DCP plus 3,4-DCP, respectively.12 Two other strains of D. mccartyi have been reported to dehalogenate chlorinated phenols, but the reported substrate ranges were limited. Strain 195 removes ortho chlorines from 2,3-DCP, 2,3,4-TCP, and 2,3,6-TCP and uses the first two as respiratory electron acceptors but does not dechlorinate PCP.12 Dehalococcoides ribotype BTF08 in mixed culture slowly removed a meta chlorine from PCP to produce 2,3,4,6-tetrachlorophenol (TeCP).26 The only other chlorophenol tested, 2,3-DCP, was not dehalogenated. Clearly more studies of chlorinated phenol dehalogenation by D. mccartyi strains are warranted, especially by strains noted for their ability to use chlorinated aromatics for respiration. D. mccartyi strain JNA has the unusual ability to grow using selected polychlorinated biphenyl (PCB) congeners, including a hexachlorobiphenyl, as sole electron acceptors.18,19 In addition, JNA carries out extensive dechlorination of the highly chlorinated commercial PCB mixture Aroclor 1260, a process which comprises at least 80 distinct PCB dechlorination reactions.19 Since various halogenated aromatics are often found at the same polluted site, D. mccartyi strains that can dehalogenate a variety of halogenated compounds would have a

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EXPERIMENTAL SECTION Chemicals. Analytical standards of all 19 chlorinated phenols were purchased from AccuStandard (North Haven, CT). A larger quantity of 2,3-DCP (98% purity) needed for respiking experiments was purchased from Sigma-Aldrich. Chlorophenol Studies. Minimal medium reduced with Ti(III) citrate was prepared as previously described19 and then spiked to a final concentration of 20 μM with a concentrated chlorophenol stock in acetone. Cultures were inoculated with 10% (v/v) of an active JNA culture grown with 2,2′,3,3′,6,6′hexachlorobiphenyl (236-236-CB) as the sole electron acceptor.19 Samples were incubated in the dark at 30 °C. Chlorinated phenol dechlorination was monitored weekly for 5 weeks. Each substrate was tested in triplicate. Abiotic controls were prepared and incubated identically to live samples but had no inoculum. No dechlorination was observed in these controls. For 2,3-DCP and 2,3,4-TCP (100% purity) which were added to the same cultures multiple times, we prepared filtersterilized 250 mM chlorophenol stock solutions in HPLC grade acetone (Fisher Scientific). We then used these stocks to prepare 1 mM feeding solutions of these chlorophenols in minimal medium in order to avoid introducing an excess of acetone to the cultures. Chlorinated Phenol Derivatization, Extraction, and Analysis by Gas Chromatography (GC). Prior to GC analysis, chlorophenols were acetylated with acetic anhydride 14301

dx.doi.org/10.1021/es503553f | Environ. Sci. Technol. 2014, 48, 14300−14308


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primer sets and further details are given in Supporting Information (SI), Table S1. Primers targeting RDase genes pceA, tceA, cbrA, and several other rdhA genes were designed using Primer3 Plus software33,34 and were synthesized by Integrated DNA Technologies (SI, Table S1). These primers were tested for specificity using Primer-BLAST on the National Center for Biotechnology Information web site. PCR conditions were optimized using genomic DNA from strains containing the appropriate gene. A single hot start PCR program was used for all primers with only the annealing temperature changed for various primers: PCR mixtures (19 μL) containing all reagents except Taq polymerase were incubated for 2 min at 94 °C, and then 1 μL containing 0.5 Units of Taq polymerase was added. Twenty-nine cycles of 45 s at 94 °C, 1 min at the annealing temperature, and 1 min at 72 °C were carried out, followed by final extension at 72 °C for 10 min and a final hold at 4 °C. Unless otherwise indicated, primers were used at a concentration of 0.25 μM. All negative results with a given primer were repeated at least once, usually with a higher DNA concentration. The PCR amplified rdhA genes were cloned and screened by restriction fragment length polymorphism (RFLP) analysis as described previously.18 Two to five clones representative of each unique RFLP group were grown, and their plasmids were isolated with a QIAGEN Spin Miniprep Kit used according to the manufacturer’s instructions. A single exception was the clone which yielded the RD_7 sequence which was the sole member of its RFLP group. The cloned rdhA/B genes were sequenced from both ends using the M13F and M13R primers of the vector, and, where necessary, internal primers were designed and used to complete the sequences. The sequences were then analyzed by the Basic Local Alignment Search Tool (BLAST) and translated using the online ExPASy translation tool (web.expasy.org/translate/).35 Chimeras and misprimed sequences were discarded. RdhA protein amino acid sequences that met the specified criteria for a particular RdhA protein ortholog group15 were assigned to that RD_OG or used to form a new ortholog group as appropriate. RdhA/B gene sequences were deposited in GenBank (accession numbers KJ580599−KJ580617), and the corresponding RdhA protein sequences were added to the curated reductive dehalogenase database.15

(Supelco, Bellefonte, PA) by the Fries reaction using a modification of the methods described by Boyd27 and Renberg and Lindström.28 Light exposure was minimized or eliminated for all steps. Because high pH promotes ionization of the chlorophenols, which in turn promotes their reaction with acetic anhydride, samples (1 mL) were treated with an equal volume of 0.2 M NaHCO3 to bring the final concentration of NaHCO3 to 0.1 M. Acetic anhydride (10 μL) was added, and the samples were immediately shaken by hand for 2−2.5 min. The acetylated chlorophenols were extracted with hexane (2 mL) by shaking in a horizontal position for 2 h. The hexane layers were transferred to separate vials, and traces of water and underivatized chlorinated phenols were removed by shaking with a few mg of anhydrous Na2SO4. The clear supernatants were then transferred to amber GC vials and analyzed using a 5890 series II gas chromatograph (Hewlett-Packard) fitted with a splitless injector, an autosampler (model 7673), a Ni63 electron capture detector (ECD), electronic pressure control, and a DB-1 capillary column (30 m, 0.25-μm phase thickness, 0.25 mm inner diameter; J&W Scientific; Agilent Technologies). The injection temperature was 270 °C, and the detector temperature was 300 °C. The initial pressure was 0.67 bar; this was raised at 6.83 bar/min to 3.76 bar, held for 5.5 min, then decreased to 1.6 bar, and held. The initial column flow was 0.873 mL of helium gas per min. The makeup gas was nitrogen. Data were collected with a PerkinElmer Network Chromatography Interface (model 901) and TotalChrom version 6.3.1 software (PerkinElmer). A retention time standard was prepared from all 19 chlorinated phenols. The chlorinated phenols in the standard were acetylated and used to optimize conditions for GC/ECD analysis. Optimal resolution was achieved using a 30 min temperature program: initial temperature, 60 °C, ramp at 20 °C per minute to 95 °C, ramp at 3 °C per minute to 160 °C, then ramp at 20 °C per minute to 270 °C. All chlorinated phenols except 2,4- and 2,5-DCP could be resolved with this program. The elution position of each chlorinated phenol was determined with separate standards of individual congeners. Detection and Quantitation. Qualitative analyses were carried out for PCP, all three tetrachlorophenols, and 2,3,6TCP. All products were identified by matching retention times to the standard. Quantitative analyses were carried out for 2,3DCP, 2,3,4-TCP, and their products. Calibration standards were prepared for 3-CP (5−500 μM), 2,3-DCP (5−200 μM), 2,4-DCP (5−250 μM), 3,4-DCP (5−250 μM), and 2,3,4-TCP (5−40 μM) and used to prepare quadratic calibration curves (4−10 point) to quantify the dechlorination of 2,3-DCP and 2,3,4-TCP. We could not reliably detect monochlorinated phenols below 2.5 μM (equivalent to 5 μM in samples prior to dilution during extraction) because of low detector response. Amplification, Cloning, Screening, and Sequencing of rdhA/B Genes. To determine the number and sequence diversity of rdhA genes present in strain JNA, we used degenerate primers to amplify rdhA/B genes from strain JNA and newly designed targeted primers to search for additional specific rdhA genes. Genomic DNA was extracted as described previously.29 A comparison of the sequences of the rdhA genes in the genomes of CBDB130 and strain 19531 with the primers RRF2 and B1R32 shows that many of these rdhA genes cannot be amplified by those primers. Therefore, we designed four new sets of degenerate primers intended to amplify rdhA genes that were not amplified by the original RRF2/B1R primers. These

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RESULTS AND DISCUSSION Dechlorination of Chlorophenols. We tested the ability of strain JNA to dechlorinate seven chlorinated phenols and determined their dechlorination pathways. All of the pentachlorophenol (PCP) (5 μM) was dechlorinated to 3,5-CP in 6 days. After this, PCP was respiked to 10 μM whenever depleted and was monitored daily to determine the dechlorination pathway. In this way we were able to visualize the main intermediates and products, most of which are shown in SI Figure S1. These are 2,3,4,5-tetrachlorophenol (2,3,4,5-TeCP), 2,3,5,6-TeCP, 2,3,5-TCP, 2,4,5-TCP, 3,4,5-TCP, and 3,5-DCP. Much smaller amounts of 2,4-DCP and 3,4-DCP were also produced. At several time points we also observed 2,3,4,6TeCP but not 2,4,6-TCP. We also monitored dechlorination at 8 h intervals for 3 days after respiking with PCP, but no further intermediates or monochlorophenols were detected. The results of experiments with each of the three tetrachlorophenols (see below) were also used to elucidate the dechlorination of PCP. Collectively, these data suggest three main routes of PCP 14302



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Figure 2. Dechlorination pathways for 2,3,4,6-TeCP, 2,3,4,5-TeCP, and 2,3,6-TCP by strain JNA. Cells grown with 236-236-CB as sole electron acceptor were used to inoculate triplicate fresh cultures spiked to a concentration of 20 μM with 2,3,4,6-TeCP (panel A), 2,3,4,5TeCP (panel B), or 2,3,6-TCP (panel C). Colors indicate the different pathways inferred from the experimental data for each substrate. Figure 1. Proposed dechlorination pathways of PCP by D. mccartyi strain JNA. The primary product was 3,5-DCP. Other products and intermediates were visualized by repeatedly feeding with PCP so that intermediates would accumulate. Intermediates that accumulated to the greatest extent are in pink and blue boldface. Open pink and blue arrows indicate major pathways, yellow arrows indicate secondary pathways, and dotted green arrows indicate minor pathways. A very small amount of 2,4-DCP formed, possibly from a very minor route of 2,3,4,6-TeCP → 2,4,6-TCP → 2,4-DCP (not shown because 2,4,6TCP was not detected). See text for further information.

2,3,6-TCP was ortho dechlorinated to 2,5-DCP by day 13 and to 3-CP by day 21 (Figure 2). There was no detectable 2,3,6-TCP after 21 to 28 days. In contrast, 2,3,4-TCP was converted by ortho and meta dechlorination to 3,4-DCP and 2,4-DCP, respectively. Initially these products appeared in approximately the same quantities, but after respiking the production of 2,4-CP leveled off while the 3,4-CP continued to increase until it was more than five times the concentration of the 2,4-DCP (Figure 3). This indicates that the ortho dechlorination reaction was favored over that of the doubly flanked meta dechlorination. No other products were detected. The only dichlorophenol tested, 2,3-DCP, was dechlorinated to 3-CP and could be transferred with a low inoculum (0.5 to 1.5% vol/vol) and spiked repeatedly (Figure 4). Upon repeated additions the 2,3-DCP was dechlorinated at higher rates, indicative of growth. When the 3-CP had accumulated to 140 μM, the cultures were transferred to fresh medium for a second

dechlorination through 2,3,4,5-TeCP and 2,3,5,6-TeCP to 3,5DCP as shown by the blue and pink pathways in Figure 1. We cannot rule out the possibility that some 2,5-DCP, which comigrates with 2,4-DCP, was produced from the dechlorination of 2,4,5-TCP or 2,3,5-TCP. However, no such product was produced from either of these trichlorophenols when they were formed as intermediates in separate experiments with tetrachlorophenols (Figure 2 and text below). All three tetrachlorophenols were substrates, but 2,3,4,6TeCP was dechlorinated the most rapidly. At 6 days both 2,4,6TCP and 2,4,5-TCP were present. A week later all of the 2,4,6TCP had been dechlorinated while a corresponding amount of 2,4-DCP/2,5-DCP and a small amount of 3,4-DCP had appeared. Because of the stoichiometry, we interpreted this as ortho dechlorination of all of the 2,4,6-TCP to 2,4-DCP and ortho dechlorination of a small amount of the 2,4,5-TCP to 3,4DCP. The latter three congeners persisted without further dechlorination for the next 3 weeks until the experiment was terminated. The dechlorination pathways are shown in Figure 2. The second TeCP, 2,3,5,6-TeCP, was ortho dechlorinated to 2,3,5-TCP by 2 to 3 weeks and subsequently to 3,5-DCP which was not further dechlorinated. The 2,3,4,5-TeCP was partially meta dechlorinated to 2,4,5-TCP within 6 days, and this was the sole product seen until 5 weeks at which time the para dechlorination product 2,3,5-TCP also appeared (Figure 2). No other products were detected.

Figure 3. Dechlorination of 2,3,4-TCP to 2,4-DCP and 3,4-DCP by strain JNA. Left panel: Triplicate cultures of strain JNA were transferred from a culture dechlorinating 236-236-CB (1% vol/vol) and incubated with 2,3,4-TCP (navy squares). Products: 3,4-DCP, pink circles; 2,4-DCP, blue triangles. Additional 2,3,4-TCP was added at the times indicated by arrows. The figure shows one of three replicates. The other replicates showed similar dechlorination patterns. Right panel: The structures of the substrate and products. 14303



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→ 3-CP, whereas CBDB1 dechlorinated all dichlorophenols except 3,5-CP to monochlorophenols.12 Aside from CBDB1 and JNA, the only other bacterium reported to extensively dechlorinate PCP is Desulfitobacterium haf niense strain PCP-1 which dechlorinates PCP to 3-CP.36 Prominent intermediates are 2,3,5,6-TeCP and 3,4,5-TCP. Implications and Prospects for Bioremediation. Clearly chlorophenol dehalogenation merits more study in additional strains of D. mccartyi to determine how widespread this ability is and whether there is evidence of in situ dechlorination of chlorinated phenols by these organisms. D. mccartyi strain JNA is a promising candidate to include in a consortium for in situ remediation of highly chlorinated phenols in anaerobic environments, not only because of its demonstrated capability, but also because this species has been highly effective for in situ remediation of chlorinated ethenes.37,38 Additional strains would be needed to further dechlorinate the dichlorophenols that are the terminal products for most congeners in strain JNA. Ideally these would include strains that can utilize 3,5-DCP and other DCPs for respiration and a strain that can mineralize phenol. Thermodynamic tables indicate that the dehalogenation of di- and monochlorophenols are energetically favorable,39 so it is likely that such strains exist and can be found. Identification of rdhA Genes. We used two approaches to screen for and identify rdhA genes in strain JNA. First, we designed specific primers to target and amplify particular rdhA genes, especially those which have typically not been amplified by degenerate primers. These included the genes cbrA, pceA, and tceA, as well as CBDB_1503. The primer sequences and annealing temperatures are given in SI, Table S1. Using the targeted primers, we determined that JNA has an ortholog of the pceA gene but not the cbrA gene (SI, Figure S2). We also determined that orthologs of the tceA, vcrA, and CBDB_1503 rdhA genes are not present (SI, Figures S3, S4, and S5). In addition, we used four newly designed degenerate primer sets to amplify partial rdhA/B genes (SI, Table S1) and generated several clone libraries from these. We confirmed the presence of an orthologous pceA gene with degenerate primers. Ultimately we identified and sequenced 19 rdhA genes in strain JNA. We assigned 13 of the JNA RdhA proteins to RD_OGs (Figure 5 and SI, Table S2). Orthologs of three of the RdhA proteins, JNA_RD16−JNA_RD18, have been found in only one other strain, D. mccartyi strain DCMB5; therefore, each creates a new RD_OG. Orthologs of two other RdhA proteins, RD14 and RD15, have previously been found in only two to three other strains and had not previously been included in the RdhA database. The inclusion of these new ortholog groups expands the number of RD_OGs for D. mccartyi from 32 to 37. Another RdhA protein, JNA_RD19, has no ortholog in any identified strain as of present and is therefore novel. The clones belonging to one RFLP group yielded only a short sequence when amplified by the degenerate primers. This short sequence aligned with RdhA sequences in RD_OG 35. Two of the recently discovered PCB RDases that dechlorinate dozens of highly chlorinated PCBs, PcbA4 and PcbA5, from D. mccartyi strains CG4 and CG5, respectively,20 also belong to RD_OG35. To determine a more complete sequence for the JNA RdhA in RD_OG35, we manually designed targeted primers to amplify near full length genes coding for RD_OG 35 RdhA proteins (SI, Table S1) and amplified a 1563 bp sequence from JNA, including most of the rdhA gene and part of the rdhB gene. This sequence, JNA_RD11, shares 97%

Figure 4. Dechlorination of 2,3-DCP to 3-CP over time. Triplicate cultures of strain JNA were transferred from a 2,3-CP dechlorinating culture (0.5% vol/vol) and incubated with 2,3-DCP (navy triangles). They were repeatedly fed more 2,3-DCP whenever the substrate was depleted (arrows). The sole product was 3-CP (pink diamonds).

time (1.5% vol/vol) and dechlorinated all of the dichlorophenol to 3-CP in 10 to 17 days. These data suggest that this congener supports growth of JNA as it does for strains CBDB1 and 195.12 For the seven chlorinated phenols that we studied, the most frequent site of dechlorination was an ortho chlorine flanked by a meta chlorine. For example, when PCP was the initial substrate, 2,3,4,5-TeCP and later 3,4,5-TCP were major dechlorination products. In addition, 2,3,5,6-TeCP and 2,3,4,6-TeCP were both ortho dechlorinated. However, this was not the case for all congeners. Notably, there was no ortho dechlorination of 2,3,4,5-TeCP when it was the initial substrate, but instead there was removal of the doubly flanked meta or para chlorine (Figure 2). Furthermore, when 2,4,5-TCP was produced by doubly flanked meta dechlorination of 2,3,4,5TeCP, it was not further dechlorinated, but when it was produced by flanked ortho dechlorination of 2,3,4,6-TeCP it was ortho dechlorinated to 3,4-DCP (Figure 2). These data suggest that various chlorinated phenols may induce the expression of different RDases which, in turn, determine whether a product will be further dechlorinated, and, if so, which chlorine will be removed. In particular, the data suggest that PCP, 2,3,4,6-TeCP, 2,3,5,6-TeCP, 2,3,6-TCP, 2,3,4-TCP, and 2,3-DCP can all induce the expression of flanked ortho dechlorination, while 2,3,4,5-TeCP and 2,3,5-TCP cannot. The data also suggest that 2,5-DCP and 2,4,6-TCP (or perhaps 2,3,4,6-TeCP), but not 2,4,5-TCP, may induce the expression of unflanked ortho dechlorination leading to the production of 3-CP and 2,4-DCP, respectively. The broad chlorophenol substrate specificity exhibited by strain JNA was very similar to that reported for CBDB1,12 but there were a few differences. For strain JNA the major product of PCP dechlorination was 3,5-DCP (Figure 1). Only very small amounts of 2,4-DCP/2,5-DCP and 3,4-DCP were produced. This means that the predominant dechlorination of PCP in strain JNA resulted from ortho and doubly flanked para dechlorination. In contrast, strain CBDB1 dechlorinated PCP to a mixture of 2,4-DCP, 3,4-DCP, and 3,5-DCP as well as small amounts of 3-CP and 4-CP, with no dominant product reported.12 Also, after being spiked with 2,3,4-TCP several times, JNA strongly favored ortho dechlorination of this substrate, producing 3,4-DCP and 2,4-DCP in a 5:1 ratio, whereas no such preference was reported for CBDB1. Finally, strain JNA carried out dechlorination to monochlorophenols in only two cases: 2,3-DCP → 3-CP and 2,3,6-TCP → 2,5-DCP 14304



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Figure 5. Bootstrapped Maximum Likelihood tree of putative JNA RDase enzymes and their orthologs. The protein sequences were aligned with MUSCLE, and evolutionary analyses were conducted in MEGA6 with 100 bootstraps.40,41 The evolutionary history was inferred by using the Maximum Likelihood method based on the Le_Gascuel_2008 model.42 The tree with the highest log likelihood is shown. Initial tree(s) for the heuristic search were obtained by applying the Neighbor-Joining method to a matrix of pairwise distances estimated using a JTT model. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 110 amino acid sequences. All positions with less than 95% site coverage were eliminated. That is, fewer than 5% alignment gaps, missing data, and ambiguous bases were allowed at any position. There were a total of 373 positions in the final data set. Branches that appeared more than 70% of the time are labeled. RD_OGs are labeled outside the tree. RDases for which function has been identified are enclosed in boxes. New OGs are shown in red.

JNA harbors orthologs of PceA45 and MbrA46 which are both tetrachloroethene (PCE) dehalogenases, so it can likely dechlorinate PCE and perhaps trichloroethene (TCE). However, because it lacks TceA and VcrA (a vinyl chloride dehalogenase) it may not be able to completely dechlorinate PCE and TCE to ethene. We had previously used the degenerate primers RRF2 and B1R to amplify and clone rdhA/B genes from the JN enrichment culture, the mixed culture from which strain JNA is derived. Most of the rdhA genes found in the parent culture are still present, but several were not amplified from JNA despite repeated attempts. These included genes coding for members of RD_OGs 10, 19, and 3615 as well as rdhA JN7_d30, and rdhA JN7_d80 which are completely novel, and rdhA JN7_Pd3 which has an ortholog only in D. mccartyi strain GY5047 (accession numbers KM065435−KM065437). We designed primers for these latter genes (SI, Table S1) but were unable to amplify them from JNA genomic DNA, even after 40−50 cycles, confirming that they were lost during the purification of strain JNA.

amino acid sequence identity with PcbA5 and 96% with PcbA4. Considering that strain JNA dechlorinates highly chlorinated PCBs19 we speculate that JNA_RD11 may be a PCB RDase. If so, the RD_OG35 primers may be useful as probes for PCB dechlorinators. JNA shares orthologs of 13 RdhA proteins with strains CBDB1, GT, and the Aroclor 1260 respiring strain CG5 (Figure 5 and Table S2). JNA also shares 11 RdhA orthologs with DCMB543 which was enriched from a site contaminated with high levels of polychlorinated dibenzodioxins.17 Furthermore, many of the RdhA orthologs that JNA shares with these strains are either identical or differ by only one or two amino acids from the corresponding proteins in the other four strains (Table S2). Both CBDB1 and DCMB5 are noted for dechlorinating a variety of halogenated aromatics including chlorinated dibenzodioxins and dibenzofurans, suggesting that the same may be true for JNA and CG5. However, JNA apparently lacks CbrA, which dehalogenates chlorinated benzenes44 and is found in both CBDB1 and DCMB5. 14305



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Chlorophenol Reductive Dehalogenases. A PCP dehalogenase, CprA3, has been partially purified from Desulfitobacterium hafniense PCP-1 and shown to be restricted to ortho dechlorination; it dechlorinates PCP to 2,3,4,5-TeCP, 2,3,4,5-TeCP to 3,4,5-TCP, and 2,3,5,6-TeCP to 2,3,5-TCP, but it does not further dechlorinate these TCP products.48 A second reductive dehalogenase, CprA5, removes meta and para chlorines and dechlorinates 3,4,5-TCP to 3,5-DCP and 3,5DCP to 3-CP.49 Hence, the activity of both of these dehalogenases is required to dechlorinate PCP to 3-CP.48,50 Although Desulf itobacterium haf niense strain PCP-1 dechlorinates PCP by pathways similar to those in strain JNA, CprA3 and Cpr5 are not orthologous to any of the RdhA proteins of strain JNA that we identified or to those of any other D. mccartyi strain.15 Furthermore, none of the chlorophenol reductive dehalogenases that have been identified in Desulfitobacterium or any other genus are closely related to any of the D. mccartyi RdhA proteins;15 therefore, the chlorophenol dehalogenases must have evolved separately in D. mccartyi. Transcriptional51 and proteomic52 data for D. mccartyi strains CBDB1 and 195 suggested that PceA, whose initially identified substrate is tetrachloroethene (PCE),45 may also dechlorinate 2,3-DCP. Based on this, Fung and colleagues51 suggested that PceA is responsible for the dechlorination of at least some chlorinated phenols in D. mccartyi strains CBDB1 and 195. JNA encodes an ortholog of PceA, JNA_RD8, whose sequence differs at a single amino acid from that of CBDB1; therefore, this enzyme may also be responsible for some chlorinated phenol dehalogenation in strain JNA. However, as in Desulfitobacterium haf niense strain PCP-1, multiple RDases may be responsible for the chlorophenol dechlorination in strain JNA, thus additional rdhA genes in strain JNA may also encode chlorinated phenol RDases. The identification of the RDase(s) that carry out the dehalogenation of PCP and chlorinated phenols in D. mccartyi is an important area for future studies. In summary, we have demonstrated that D. mccartyi strain JNA can dehalogenate a broad spectrum of chlorinated phenols including PCP and all three tetrachlorophenols to less toxic congeners. Taken together with its broad substrate range for PCBs, this suggests that strain JNA may be especially well adapted to survive and even thrive at sites contaminated with multiple chlorinated aromatics and may be useful for in situ bioremediation.

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Anchor QEA, 4300 Route 50, Suite 202, Saratoga Springs, NY 12866. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Professors Stephen Zinder, Lorenz Adrian, and Frank Löffler for genomic DNA from D. mccartyi strains 195, CBDB1, and GT for PCR positive controls.

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ASSOCIATED CONTENT

S Supporting Information *

Chromatogram showing products of PCP dechlorination, agarose gels showing PCR products from targeted primers for various RDase genes, table of primers used in this study, table of RdhA proteins translated from rdhA genes in JNA, the ortholog groups to which they belong, and their closest orthologs. This material is available free of charge via the Internet at http://pubs.acs.org.

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Present Addresses §

Department of Microbiology, 175 Wing Hall, Wing Rd., Cornell University, Ithaca, NY, 14853. 14306



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Dehalococcoides mccartyi Strain JNA Dechlorinates Multiple

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