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Genomic Characterization of Dehalococcoides mccartyi Strain JNA That Reductively Dechlorinates Tetrachloroethene and Polychlorinated Biphenyls Shanquan Wang,†,∥ Kern Rei Chng,†,‡ Chen Chen,† Donna L. Bedard,*,§ and Jianzhong He*,† †

Department of Civil and Environmental Engineering, National University of Singapore, Singapore 117576 Computational and Systems Biology, Genome Institute of Singapore, Singapore 138672 § Department of Biological Sciences, Rensselaer Polytechnic Institute, 110 Eighth St., Troy, New York 12180, United States ‡

S Supporting Information *

ABSTRACT: Dehalococcoides mccartyi strain JNA detoxifies highly chlorinated polychlorinated biphenyl (PCB) mixtures via 85 distinct dechlorination reactions, suggesting that it has great potential for PCB bioremediation. However, its genomic and functional gene information remain unknown due to extremely slow growth of strain JNA with PCBs. In this study, we used tetracholorethene (PCE) as an alternative electron acceptor to grow sufficient biomass of strain JNA for subsequent genome sequencing and functional gene identification. Analysis of the assembled draft genome (1 462 509 bp) revealed the presence of 29 putative reductive dehalogenase (RDase) genes. Among them, JNA_RD8 and JNA_RD11 genes were highly transcribed in both PCE- and PCB-fed cultures. Furthermore, in vitro assays with crude cell lysate from PCE grown cells revealed dechlorination activity against both PCE and 2,2′,3,4,4′,5,5′-heptachlorobiphenyl. These data suggest that both JNA_RD8 and JNA_RD11 may be bifunctional PCE/PCB RDases. This study deepens the knowledge of organohalide respiration of PCBs and facilitates in situ PCBbioremediation with strain JNA.

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INTRODUCTION

Thus far, several bacterial genera have been implicated in reductive dechlorination of PCBs, including Dehalococcoides, Dehalobium, Dehalogenimonas, and Dehalobacter.5−13 Among them, Dehalococcoides exhibit the most extensive dechlorination activities on highly chlorinated PCB mixtures.9,11,12 Recently, a unique strain, Dehalococcoides mccartyi strain JNA, was isolated from the sediment-free JN culture by growth with 2,2′,3,3′,6,6′hexachlorobiphenyl (236-236-CB) as the sole electron acceptor.14 Strain JNA is capable of extensively dechlorinating PCBs via 85 distinct dechlorination reactions comprising 56 pathways and has shown great potential for PCB bioremediation.9,14 This strain can also dechlorinate chlorinated phenols including pentachlorophenol, suggesting that it may be particularly well adapted to thrive in sites contaminated with a variety of halogenated aromatics.15 PCR amplification with degenerate primers identified 19 rdhA genes in strain JNA.15 However, information on the genome and the PCB-RDase gene(s) of strain JNA is still limited. Many Dehalococcoides strains are able to respire chloroethenes,16−18 which have much higher bioavailability than PCBs and, therefore, might act as better substrates to support the growth of PCB-dechlorinating Dehalococcoides. Recently, Wang

The toxic polychlorinated biphenyls (PCBs) were massively produced as commercial mixtures (e.g., Aroclor 1260) for industrial use, and their poor handling, transportation and disposal resulted in contamination of the sediments of rivers, lakes and harbors worldwide.1 As persistent organic pollutants (POPs) posing significant health risk to humans and ecosystems, PCBs are ranked fifth on the 2013 National Priority List of Hazardous Substances prepared by the U.S. Environmental Protection Agency and the Agency for Toxic Substances and Disease Registry.2 Bioremediation through the use of organohalide-respiring bacteria is an economic and environmentally attractive strategy for cleanup of chlorinated organic compounds from contaminated sites. This technology has been successfully applied to removal of chloroethenes in situ.3 Reductive dehalogenases (RDases if biochemically characterized, otherwise RdhAs) catalyze the dehalogenation of chlorinated ethenes, and a variety of other chlorinated or brominated aliphatic, aromatic, and polycyclic compounds.4 These RDase enzymes are composed of two subunits: RdhA, the catalytic subunit, and RdhB, thought to be a membraneanchoring protein.4 The genome of each organohalide respiring bacterium encodes a suite of up to 36 RdhA proteins thought to be capable of dehalogenating a wide range of halogenated compounds.4 © XXXX American Chemical Society

Received: April 28, 2015 Revised: October 21, 2015 Accepted: November 9, 2015

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DOI: 10.1021/acs.est.5b01979 Environ. Sci. Technol. XXXX, XXX, XXX−XXX

Article

Environmental Science & Technology et al. described three new strains of D. mccartyi that dechlorinate Aroclor 1260, strains CG1, CG4, and CG5, each of which employs a different bifunctional RDase to catalyze chlorine removal from both PCBs and PCE.19 This finding has considerable ramifications for studying PCB dechlorination because it means that organisms having such a bifunctional RDase can be easily enriched with PCE without risk of losing their PCB dechlorination capability. We hypothesized that strain JNA might also have such a bifunctional PCE/PCBRDase. Accordingly, in this study we aimed to (1) determine if strain JNA can respire PCE, (2) sequence the JNA genome, (3) identify which RDases are transcribed during growth on PCE and PCBs, and (4) test if RDases expressed during growth on PCE can dechlorinate PCBs.

ing libraries were then prepared and multiplexed utilizing TruSeq DNA Sample Prep kit (V2) prior to 2 × 76 paired end sequencing by using Illumina Hiseq 2000 (Illumina, San Diego, CA). Bases with quality scores lower than 3 were trimmed off the 3′ ends of reads, and read pairs with a read shorter than 60 bp were discarded. Contig assembly was performed with SOAPdenovo (v1.05)24 and scaffolding with Opera (v1.4).25 Reads were down-sampled to around 100× coverage as this was seen to improve assembly statistics (N50, the shortest scaffold size such that the sum of scaffolds of equal size or longer, is at least 50% of the total size of all scaffolds). We tried different kmer sizes and the assembly with the least number of scaffolds and highest N50 was kept. GapCloser (v1.12)23 was utilized for in silico closing of gaps between contigs. Open reading frames (ORFs) were predicted using Prodigal (v2.60).26 Functional annotations were assigned by screening predicted ORFs with entries in the KEGG database27 using RapSearch (v2.12).28 The assignment of gene distribution for different categories was obtained from the RAST server.29 For the identification of RDase genes in the draft genome, 306 RDase genes were downloaded from the NCBI protein database, aligned with Clustal-Omega (v1.2.0)30 and converted into a hidden Markov model (HMM) that was used to search against the predicted ORFs with HMMER3 (v3.1b1, http://hmmer. org). Finally, the predicted RDase genes were blasted against the NCBI NR database for further confirmation. Whole genome phylogenetic analysis was performed by aligning genomes against the reference genome (D. mccartyi strain 195) using NUCmer (v3.23)31 to call single nucleotide polymorphisms (SNPs) (alignments shorter than 10 kbp were ignored). The corresponding reference based multiple alignment of genomes (restricted to variant positions) was analyzed using PHYML (v20100720)32 to construct maximum likelihood trees with 100 bootstrap replicates. Nucleotide Sequence Accession Numbers. This Whole Genome Shotgun project has been deposited at DDBJ/EMBL/ GenBank under accession number JSWM00000000. The version described in this paper is version JSWM01000000. Enzymatic Analyses of the PCB-RDase. After complete PCE dechlorination, cells were harvested from 1 L cultures by centrifugation (15 min, 10 000g, 4 °C) and resuspended in a buffer of 10 mM Tris-HCl and 1 mM dithiothreitol (pH 7.2). Crude cell lysates were obtained by disruption for 3 min at 40% amplitude with 5s/10s working/cooling pulse using a VCX 130 ultrasonicator (Sonics & Materials Inc., Newtown, CT). The amount of total proteins in crude cell lysates was quantified with Nanodrop 1000 (NanoDrop Technologies, Wilmington, DE). Cell collection and lysis were carried out aerobically in less than 30 min to minimize air exposure. In vitro enzyme dechlorination activity tests were performed under anaerobic conditions in 20 mL serum bottles. Each bottle contained 4 mL of assay solution (buffered with 100 mM Tris-HCl, pH 7.0) consisting of 20 mM of methyl viologen that was reduced by 15 mM Ti(III)-citrate, 0.2 mL of cell extract, and the chlorinated compound (18 μM 2345-245-CB, or 30 μM PCE) as described.21 Assay bottles were then incubated at 30 °C in the dark before PCB/PCE analyses.

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MATERIALS AND METHODS Culture Transfer and Growth Conditions. Completely defined anaerobic mineral medium for bacterial cultivation was prepared as described.12,20 D. mccartyi strain JNA was continuously transferred in 160 mL serum bottles containing 100 mL medium supplemented with sodium acetate (10 mM) as the carbon source, hydrogen as the electron donor (5 × 104 Pa) and PCE (∼70 μmol/bottle) or 2345-245-CB (1.2 μmol/ bottle) as the electron acceptor. The PCB-fed culture for transcriptional analysis of RDase genes was inoculated from PCE-fed JNA (10% inoculum) in which PCE had been completely dechlorinated to TCE and DCEs. Experiments were set up in triplicate unless otherwise stated. Controls without PCE injection were set up in duplicate. Cultures were incubated at 30 °C in the dark without shaking. Analytical Techniques. Headspace samples of chlorinated ethenes were analyzed by gas chromatography (GC) (Agilent 6890N, Santa Clara, CA) using a flame ionization detector and a GS-GasPro column (30 m × 0.32 mm × 0.25 μm film thickness; J&W Scientific, Folsom, CA).13 PCBs were extracted with isooctane and quantified by the same model GC equipped with an electron capture detector (Agilent) and a DB-5 capillary column (30 m × 0.32 mm × 0.25 μm film thickness; J&W Scientific, Folsom, CA) as described.12 DNA/RNA Extraction, PCR Amplification, Quantitative Real-Time PCR (qPCR), and Reverse Transcription PCR (RT-qPCR). Cells were harvested by centrifugation (15 min, 10 000g, 4 °C). DNA and RNA for qPCR was extracted from 1 mL aliquots of 100 mL cultures using QIAGEN DNeasy Blood and Tissue Kit and RNeasy mini kit (QIAGEN, Hilden, Germany). RNA for RT-qPCR analyses was reverse transcribed into complementary DNA (cDNA) using a two-step reverse transcription-PCR Sensiscript kit as described.21 Luciferase mRNA (Promega, Madison, WI) was added as an internal reference transcript for mRNA losses during RNA isolation, reverse transcription and quantification.22 PCR amplifications with RDase gene-specific primers (Supporting Information Table S1) were conducted on a Mastercycler thermal cycler (Eppendorf, Hamburg, Germany) as described previously.21 The primers were designed with Primique.23 The qPCR (ABI 7500 Fast real-time PCR system; ABI, Foster, CAA) enumeration of Dehalococcoides cells and RDase genes was performed with the QuantiTect SYBR Green PCR kit.21 Genome Sequencing, Assembly and Annotation. Genomic DNA was extracted from 1 L PCE-fed cultures using QIAGEN Genomic-tip 500/G (QIAGEN, Hilden, Germany), and fragmented using adaptive focused acoustics ultrasound technology (Covaris, Woburn, MA). DNA sequenc-

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RESULTS Reductive Dechlorination of PCE to DCEs by Strain JNA. Strain JNA was inoculated (5% inoculum) into medium amended with PCE instead of PCBs as sole electron acceptor in order to determine if PCE could serve as an alternative B


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Environmental Science & Technology

It contains 1450 protein-coding genes, a number similar to those of the genomes of D. mccartyi strains 195,33 CBDB1,34 VS,35 CG1, CG4 and CG5 (Figure 2A).19 Whole genome phylogenetic analysis showed that strain JNA clusters with two other strains of PCB-dechlorinating bacteria, strains CBDB1 and CG5, into the Pinellas subgroup of D. mccartyi (Figure 2B). The gene distribution of strain JNA in different functional categories is similar to that of Pinellas strains CBDB1 and CG5 (Figure S1). However, strain JNA has significantly more genes in the category of “Cell wall and Capsule”, as do Cornell subgroup strains 195 and CG4. Furthermore, only strain JNA of the three Pinellas strains harbors two genes in subcategory of “Phages and Prophages” involving the phage packaging machinery (Figure S1; Table S2). Characterization of the putative RDase genes in the draft genome revealed 27 full-length and one partial rdhA genes present in strain JNA (Table S1). The partial rdhA gene (396 bp) shared 100% sequence identity with the 3′ ends of both the JNA_RD5 (KJ580603) and JNA_RD6 (KJ580604) genes (Figure S2) previously identified from strain JNA using degenerate primers.15 Hence JNA actually has 29 RDase genes. Each of these rdhA genes has an rdhB gene located adjacent to it (Table S1). Phylogenetic analysis of the translated proteins of all 212 rdhA genes of D. mccartyi strains JNA, 195,33 CBDB1,34 VS,35 CG1, CG4, CG5,19 and DCMB536 shows that strain JNA shares orthologs of many RdhA proteins with D. mccartyi strains CBDB1, DCMB5, and CG5, including orthologs of three functionally identified RDases: MbrA21 (JNA_RD5), PceA37 (JNA_RD8) and PcbA519 (JNA_RD11) (Figure S3 and Table S3). Transcription of Reductive Dehalogenase Genes in PCE-fed and in PCB-fed Strain JNA. To identify the genes responsible for PCE dechlorination, specific primers were designed for each rdhA gene (Table S1) and RT-PCR was used to determine which RDase genes were transcribed. Transcription of seven rdhA genes (i.e., JNA_RD1, JNA_RD5/6, JNA_RD8, JNA_RD10, JNA_RD11, and JNA_RD22) was observed in PCE-fed strain JNA (Figure S4). These transcribed rdhA genes were further monitored using RT-qPCR to quantify their transcription levels during reductive dechlorination of PCE to DCEs, which showed that two of the 29 rdhA genes (i.e., JNA_RD8 and JNA_RD11) were highly transcribed, achieving around 15 transcripts per cell (Figure 3A). JNA_RD8 and JNA_RD11 share 94 and 97% amino acid sequence identity, respectively, with the PCE-dechlorination-catalyzing PceA (from D. mccartyi 195, accession ID YP_181066) and PcbA5 (from D. mccartyi CG5, accession ID AII60305). These two RdhAs together with other PCE- and PCB-RDases form phylogenetic clades that are separate from those of other RDases identified in D. mccartyi: MbrA, CbrA, VcrA, BvcA and TceA (Figure 3B). The RdhA encoded by the third most highly transcribed rdhA gene, JNA_RD1 (achieving 4.2 transcripts per cell), shares 100% amino acid sequence identity with RdhA proteins cbdb1_A1455 (accession ID, YP_308398) of D. mccartyi CBDB1 and dcmb_1341 (YP_007484273) of D. mccartyi strain DCMB5; its function is unknown. Both the active transcription of the JNA_RD8 and JNA_RD11 RDase genes and the high amino acid sequence similarity of their translation products with known PCE-RDases suggest that these genes may function in catalyzing PCE dechlorination in strain JNA. To elucidate the transcription profiles of JNA_RD8 and JNA_RD11 genes grown with PCBs, RT-qPCR was employed

substrate to grow sufficient amounts of biomass for subsequent genome sequencing. After 14 days of incubation, more than half of the PCE (55.8%) was dechlorinated to TCE and cis-DCE; subsequently the PCE and TCE were dechlorinated to both cisand trans-DCEs in a ratio of 2.5 (±0.4):1 in 45 days (Figure 1A). No further dechlorination to lower chlorinated compounds (i.e., vinyl chloride or ethene) was observed after another three months of incubation.

Figure 1. PCE dechlorination and PCE-dependent growth of strain JNA in pure culture. (A) PCE dechlorination to TCE and DCEs by strain JNA. (B) Coupled growth of strain JNA with PCE dechlorination. Controls are cultures without PCE-amendment. Error bars represent standard deviations of triplicate cultures.

Analysis of the cell density by qPCR measuring the copies of the 16S rRNA gene (assuming 1 copy per cell) revealed that dechlorination of PCE was coupled to the growth of strain JNA (Figure 1). The cell density increased from 5.6 × 106 cells/ml to 1.8 × 108 cells/ml with 1.1 μmol/mL chlorine removed after 25 days of incubation (Figure 1B). The average cell growth of strain JNA with PCE was 1.6 × 1014 cells per mole of chlorine removed. This is comparable to that of other PCEdechlorinating Dehalococcoides.17,19 No cell growth was observed in controls without PCE amendment. Genomic Characterization. DNA for sequencing the genome of strain JNA was extracted from cell biomass grown with PCE. Sequence assembly generated a total of six scaffolds (scaffold_1, 1 142 884 bp; scaffold_2, 160 842 bp; scaffold_3, 128 254 bp; scaffold_4, 28 271 bp; scaffold_5, 541 bp; scaffold_6, 541 bp) and one contig (1221 bp), and the N50 scaffold size is 1 142 884 bp. The assembled draft genome of strain JNA is 1 462 509 bp long, with a G+C content of 47.0%. C


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Figure 2. Comparative genomics characterization of strain JNA. (A) Genomic overview of strain JNA and previously sequenced Dehalococcoides mccartyi isolates (strains195, CBDB1, VS, BAV1, GT, CG1, CG4, and CG5) belonging to the three D. mccartyi phylogenetic subgroups. (B) Maximum-likelihood tree based on whole genome SNP analysis. The numbers marked at the branches are confidence values based on 100 bootstrap replications and only values less than 100 are noted. The scale bar indicates the number of substitutions per site.

Figure 3. (A) Transcription profiles of potential PCE-RDase genes in strain JNA and (B) phylogeny of JNA_RD8 and JNA_RD11 proteins and functionally identified RDase enzymes. Transcription changes (obtained via RT-qPCR by using gene-specific primers) at various time-points are plotted as number of copies per cell. The two putative RDases encoded by the rdhA genes most highly transcribed in PCE-fed strain JNA, that is, RD8 and RD11, are marked with solid circles. Numbers in parentheses are NCBI accession numbers. A phylogenetic tree of deduced amino acid sequences of JNA_RD8 and JNA_RD11 and all full-length identified RDases from Dehalococcoides was constructed using the maximum-likelihood method. The scale bar indicates the number of substitutions per site.

to quantify their transcription levels upon chlorine-removal from 2345-245-CB in strain JNA. Again both JNA_RD8 and JNA_RD11 genes coupled the gene transcription with chlorineremoval from 2345 to 245-CB, although nearly twice as much JNA_RD11 was transcribed (Figure S5). In Vitro Assay of PCE and PCB Dechlorination. In our previous study, each of three D. mccartyi strains employed a different bifunctional RDase to catalyze chlorine removal from both PCBs and PCE.19 To determine whether PCE-RDases of strain JNA catalyze PCB dechlorination, in vitro assays were conducted with crude cell lysates from PCE-fed JNA. The crude cell lysates in all bottles spiked with either 2345-245-CB

or PCE showed dechlorination activities with the same dechlorination profiles as those observed in the active cultures (Figure 4A,B), suggesting that PCB-RDases were induced by PCE in strain JNA. Similar to the PCB dechlorination specificities observed in live cultures,14 the 2345-245-CB was dechlorinated predominantly to 245-245-CB and 235-245-CB, and to a lesser extent to 245-24-CB and 245-25-CB (Figure 4B). The calculated PCB dechlorination rate (1.2 × 10−8 kat/g) (Figure 4C) of strain JNA’s crude cell lysate is comparable to those of PCB-dechlorinating strains CG1, CG4, and CG5 (Figure 4C).19 However, strain JNA’s PCE dechlorination rate is two to 3-fold lower than that of strains CG1, CG4, and CG5 D


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Figure 4. In vitro assays confirm PCB and PCE RDase activity in PCE-fed JNA. Crude cell lysates of PCE-fed strain JNA catalyzed chlorine removal from (A) PCE and (B) 2345-245-CB in the same dechlorination patterns as their active PCE- and PCB-dechlorinating cultures. Error bars represent standard deviations of triplicate assays. (C) Calculation of PCB- and PCE-dechlorination rates in the crude cell lysates.

transcription during growth on both PCE and PCBs, (2) its high amino acid sequence identity to PcbA5, and (3) the dechlorination of 2345-245-CB in the in vitro assay when grown with PCE. In contrast, the high transcription of JNA_RD8 during growth with 2345-245-CB (Figure S5B) was a surprise because it suggests that JNA_RD8 may also play a role in PCB dechlorination. There is no precedent for the catalysis of PCBs by PceA or its orthologs. In fact, when strain CG5 was grown with PCE, the gene for CG5_RD20, an ortholog of PceA, was the second most highly transcribed rdhA gene, but this gene was not transcribed when CG5 was grown with PCBs, suggesting that CG5_RD20 plays no role in PCB dechlorination.19 JNA_RD8 differs from CG5_RD20 by a single amino acid. In addition, based on transcriptional and proteomic data, Fung and colleagues proposed that PceA in D. mccartyi strain 195 acts as a bifunctional RDase that dechlorinates chlorinated phenols as well as PCE.40 Also, an ortholog of PceA in CBDB1, cbdb_A1588, was the most abundant RdhA protein expressed when CBDB1 was grown with 2,3-dichlorophenol.41 JNA_RD8 also differs from the CBDB1 PceA ortholog by a single amino acid, and strain JNA dechlorinates pentachlorophenol and other chlorinated phenols including 2,3-dichlorophenol with a specificity nearly identical to that of strain CBDB1.15,42 Consequently, Fricker and colleagues proposed that strain JNA uses the JNA_RD8 RDase to dehalogenate chlorinated phenols.15 This study has identified both JNA_RD8 and JNA_RD11 as potential candidates for PCE/PCB RDases. Further studies will be needed to conclusively determine whether both or only one of these RDases carries out PCB dechlorination in strain JNA as well as whether one or both of these enzymes is bifunctional. These studies should use native PAGE gel electrophoresis to resolve RDases expressed during growth on PCBs as well as growth on PCE followed by in gel assays for PCE and PCB dechlorination and subsequent protein sequencing to defini-

(Figure 4C); this may be because strains CG1, CG4, and CG5 have had years to acclimate to PCE while JNA has not.

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DISCUSSION

Analysis of RDase Genes in Strain JNA. The draft genome of strain JNA provides essential insight into the metabolic networks of PCB-dechlorinating bacteria, especially the key functional genes for organohalide respiration. Strain JNA shares 18 identical or nearly identical RdhA proteins with multiple-substrate-utilizing strains CBDB1 and DCMB538 (Table S3), suggesting that JNA may also have the ability to dehalogenate a broad spectrum of chlorinated aromatics. In particular, seven of the RdhA proteins that JNA shares with strain DCMB5 have no known orthologs in any other Dehalococcoides genome. However, it is notable that JNA lacks an ortholog of the chlorobenzene dechlorinating RDase CbrA that both CBDB1 and DCMB5 share.38,39 JNA also shares 17 RdhA orthologs with strain CG5 (Table S3), the only other isolate that dechlorinates Aroclor 1260 by Dechlorination Process N. The genome of strain JNA possesses orthologs of PceA, PcbA4, and PcbA5 RDases, but no orthologs of other chlorinated ethene RDases, namely TceA, BvcA, or VcrA. This is consistent with our finding that in strain JNA the dechlorination of PCE does not proceed beyond dichloroethenes. The genome sequencing and transcriptional analyses suggest that strain JNA may use two bifunctional RDases, JNA_RD8 and JNA_RD11, to catalyze chlorine removal from both PCE and PCBs. Both of these enzymes are orthologs of RDases previously shown to catalyze PCE dechlorination, namely PceA37 from strain 195 and the recently identified bifunctional PCE/PCB RDases PcbA4 and PcbA5 from the Aroclor 1260 dechlorinating strains CG4 and CG5, respectively. 19 JNA_RD11 has 97% amino acid sequence identity with PcbA5 and 95% with PcbA4. Three lines of evidence suggest that JNA_RD11 is a bifunctional PCE/PCB RDase: (1) its high E



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ACKNOWLEDGMENTS This study was supported by the Science and Engineering Research Council, Agency for Science, Technology and Research (A*STAR) (Project No.: 102 101 0025) as well as by the National Research Foundation, Prime Minister’s Office, Singapore under its Competitive Research Programme (Award No. NRF-CRP 5-2009-05).

tively identify which JNA_RdhAs are responsible for PCB and PCE dechlorination. Potential of Strain JNA for PCB Remediation. Strain JNA may be a promising candidate for the cleanup of PCBcontaminated sites as it is one of only two pure strains, JNA and CG5, capable of extensively dechlorinating complex PCB mixtures via the environmentally relevant Dechlorination Process N.14,19 In this process, strain JNA mainly removes chlorines from flanked meta-positions of 34-, 234-, 235-, 236-, 245-, 2345-, 2346-, and 2356-chlorophenyl rings. However, the extremely low water solubility of PCB mixtures results in their low bioavailability for growing PCB-dechlorinating bacteria, including strains JNA and CG5, posing challenges for applying them in PCB bioremediation.43 In this study, we determined that PCE, which is much more soluble in water (1.5 g/L for PCE vs 0.0027−0.42 ng/L for PCB mixtures),44 supports respiration in strain JNA (Figure 1) and that cells grown with PCE could dechlorinate PCBs (Figure 4). In addition, cells grown with PCE grew to comparatively higher cell density. Therefore, PCE was identified as an appropriate alternative electron acceptor to accelerate the growth and boost the biomass of strain JNA. Technologies for PCE-bioremediation with D. mccartyi have been well established.3,18 Therefore, the results from this study may promote efficient PCB bioremediation with strain JNA by providing sufficient amounts of active PCB-dechlorinating biomass for bioaugmentation in situ at PCB-contaminated sites.

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REFERENCES

(1) Falandysz, J.; Rose, M.; Fernandes, A. R. Mixed poly-brominated/ chlorinated biphenyls (PXBs): Widespread food and environmental contaminants. Environ. Int. 2012, 44, 118−127. (2) Agency for Toxic Substances and Disease Registry. ATSDR 2013 Substance Priority List, 2013. www.atsdr.cdc.gov/spl/. (3) Löffler, F. E.; Edwards, E. A. Harnessing microbial activities for environmental cleanup. Curr. Opin. Biotechnol. 2006, 17, 274−284. (4) Hug, L. A.; Maphosa, F.; Leys, D.; Löffler, F. E.; Smidt, H.; Edwards, E. A.; Adrian, L. Overview of organohalide-respiring bacteria and a proposal for a classification system for reductive dehalogenases. Philos. Trans. R. Soc., B 2013, 368 (1616).2012032210.1098/ rstb.2012.0322 (5) Fennell, D. E.; Nijenhuis, I.; Wilson, S. F.; Zinder, S. H.; Häggblom, M. M. Dehalococcoides ethenogenes strain 195 reductively dechlorinates diverse chlorinated aromatic pollutants. Environ. Sci. Technol. 2004, 38, 2075−2081. (6) Yan, T.; LaPara, T. M.; Novak, P. J. The effect of varying levels of sodium bicarbonate on polychlorinated biphenyl dechlorination in Hudson River sediment cultures. Environ. Microbiol. 2006, 8, 1288− 1298. (7) Yan, T.; LaPara, T. M.; Novak, P. J. The reductive dechlorination of 2,3,4,5-tetrachlorobiphenyl in three different sediment cultures: evidence for the involvement of phylogenetically similar Dehalococcoides-like bacterial populations. FEMS Microbiol. Ecol. 2006, 55, 248− 261. (8) Bedard, D. L.; Bailey, J. J.; Reiss, B. L.; Jerzak, G. V. Development and characterization of stable sediment-free anaerobic bacterial enrichment cultures that dechlorinate Aroclor 1260. Appl. Environ. Microbiol. 2006, 72, 2460−2470. (9) Bedard, D. L.; Ritalahti, K. M.; Löffler, F. E. The Dehalococcoides population in sediment- free mixed cultures metabolically dechlorinates the commercial polychlorinated biphenyl mixture Aroclor 1260. Appl. Environ. Microbiol. 2007, 73, 2513−2521. (10) May, H. D.; Miller, G. S.; Kjellerup, B. V.; Sowers, K. R. Dehalorespiration with polychlorinated biphenyls by an anaerobic ultramicrobacterium. Appl. Environ. Microbiol. 2008, 74, 2089−2094. (11) Adrian, L.; Dudková, V.; Demnerová, K.; Bedard, D. L. Dehalococcoides” sp. strain CBDB1 extensively dechlorinates the commercial polychlorinated biphenyl mixture Aroclor 1260. Appl. Environ. Microbiol. 2009, 75, 4516−4524. (12) Wang, S.; He, J. Phylogenetically distinct bacteria involve extensive dechlorination of Aroclor 1260 in sediment-free cultures. PLoS One 2013, 8, e59178. (13) Wang, S.; He, J. Dechlorination of commercial PCBs and other multiple halogenated compounds by a sediment-free culture containing Dehalococcoides and Dehalobacter. Environ. Sci. Technol. 2013, 47 (18), 10526−10534. (14) LaRoe, S. L.; Fricker, A. D.; Bedard, D. L. Dehalococcoides mccartyi strain JNA in pure culture extensively dechlorinates Aroclor 1260 according to polychlorinated biphenyl (PCB) Dechlorination Process N. Environ. Sci. Technol. 2014, 48, 9187−9196. (15) Fricker, A. D.; LaRoe, S. L.; Shea, M. E.; Bedard, D. L. Dehalococcoides mccartyi strain JNA dechlorinates multiple chlorinated phenols including pentachlorophenol and harbors at least 19 reductive dehalogenase homologous genes. Environ. Sci. Technol. 2014, 48, 14300−14308. (16) Maymó-Gatell, X.; Chien, Y. T.; Gossett, J. M.; Zinder, S. H. Isolation of a bacterium that reductively dechlorinates tetrachloroethene to ethene. Science 1997, 276, 1568−1571.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.est.5b01979. Five figures showing (1) RAST assignment of gene distribution in D. mccartyi strain JNA and six other D. mccartyi strains. (2) Relationship of partial rdhA gene sequence with those of two previously identified rdhA genes in strain JNA. (3) Phylogeny of RdhA proteins in D. mccartyi strain JNA and seven other D. mccartyi strains. (4) PCR screening of all rdhA genes in strain JNA to determine which are transcribed when grown on PCE. (5) Transcription of JNA_RD8 and JNA_RD11 genes is associated with PCB dechlorination in PCB-fed culture of strain JNA (PDF) Three tables showing (1) RdhA and rdhB gene sequences and quantitative real-time PCR (qPCR) primers specifically targeting each rdhA gene of D. mccartyi strain JNA. (2) List of annotated genes in the genome of D. mccartyi strain JNA. (3) RDase ortholog group assignments of RdhA proteins in D. mccartyi strain JNA and comparison of orthologous sequences in other D. mccartyi strains (XLS)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Present Address ∥

(S.W.) Department of Civil and Environmental Engineering, University of Illinois at Urbana−Champaign, 205 North Mathews Ave., Urbana Illinois L61802, United States. Notes

The authors declare no competing financial interest. F


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