10.1128/AEM.70.9.5290-5297.2004.

2004, 70(9):5290. DOI:Appl. Environ. Microbiol. Löffler and Lorenz AdrianFriedrich von Wintzingerode, Helmut Görisch, Frank E. Tina Hölscher, Rosa Krajmalnik-Brown, Kirsti M. Ritalahti, 

DehalococcoidesGenes Are Common in Reductive-Dehalogenase-Homologous Multiple Nonidentical

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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 2004, p. 5290–5297 Vol. 70, No. 90099-2240/04/$08.00�0 DOI: 10.1128/AEM.70.9.5290–5297.2004Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Multiple Nonidentical Reductive-Dehalogenase-Homologous GenesAre Common in Dehalococcoides

Tina Holscher,1 Rosa Krajmalnik-Brown,2 Kirsti M. Ritalahti,2 Friedrich von Wintzingerode,3Helmut Gorisch,1 Frank E. Loffler,2,4* and Lorenz Adrian1

Fachgebiet Technische Biochemie, Institut fur Biotechnologie, Technische Universitat Berlin, Berlin, Germany1;School of Civil and Environmental Engineering2 and School of Biology,4 Georgia Institute of Technology,

Atlanta, Georgia; and Institut fur Mikrobiologie und Hygiene,Universitatsklinikum Charite, Berlin, Germany3

Received 15 December 2003/Accepted 11 May 2004

Degenerate primers were used to amplify large fragments of reductive-dehalogenase-homologous (RDH)genes from genomic DNA of two Dehalococcoides populations, the chlorobenzene- and dioxin-dechlorinatingstrain CBDB1 and the trichloroethene-dechlorinating strain FL2. The amplicons (1,350 to 1,495 bp) corre-sponded to nearly complete open reading frames of known reductive dehalogenase genes and short fragments(approximately 90 bp) of genes encoding putative membrane-anchoring proteins. Cloning and restrictionanalysis revealed the presence of at least 14 different RDH genes in each strain. All amplified RDH genesshowed sequence similarity with known reductive dehalogenase genes over the whole length of the sequence andshared all characteristics described for reductive dehalogenases. Deduced amino acid sequences of seven RDHgenes from strain CBDB1 were 98.5 to 100% identical to seven different RDH genes from strain FL2, suggestingthat both strains have an overlapping substrate range. All RDH genes identified in strains CBDB1 and FL2were related to the RDH genes present in the genomes of Dehalococcoides ethenogenes strain 195 and Dehalo-coccoides sp. strain BAV1; however, sequence identity did not exceed 94.4 and 93.1%, respectively. The presenceof RDH genes in strains CBDB1, FL2, and BAV1 that have no orthologs in strain 195 suggests that thesestrains possess dechlorination activities not present in strain 195. Comparative sequence analysis identifiedconsensus sequences for cobalamin binding in deduced amino acid sequences of seven RDH genes. In conclu-sion, this study demonstrates that the presence of multiple nonidentical RDH genes is characteristic ofDehalococcoides strains.

Bacterial reductive dechlorination reactions play importantroles in the detoxification of chlorinated aromatic and aliphaticcompounds in contaminated environments (14, 23). Severalanaerobic bacteria use chlorinated compounds in their energymetabolism, coupling reductive dehalogenation to electrontransport phosphorylation (16, 25). The Dehalococcoides groupforms a bacterial cluster that is phylogenetically distant fromother dechlorinating bacteria (2, 31) and uses a spectrum ofchlorinated compounds as growth-supporting electron accep-tors, including chlorinated ethenes (13, 24, 31), chlorinatedbenzenes (2, 18), and polychlorinated dibenzodioxins (PC-DDs) (9). Four members of the Dehalococcoides group havebeen isolated. Three strains, Dehalococcoides ethenogenesstrain 195, Dehalococcoides sp. strain FL2, and Dehalococ-coides sp. strain BAV1, have been cultivated with chlorinatedethenes as growth-supporting substrates (12, 24, 25, 31),whereas Dehalococcoides sp. strain CBDB1 was isolated withchlorinated benzenes as terminal electron acceptors (2). Sev-eral strain-specific physiological differences exist. For instance,tetrachloroethene (PCE) is used as a metabolic electron ac-ceptor by strain 195 but not by strains FL2, BAV1, and CBDB1(2, 13, 25). Both strain 195 and strain FL2 use trichloroethene(TCE) for growth (24, 25, 31), while this compound is only

cometabolically dechlorinated by strain BAV1 (13). StrainBAV1, however, respires vinyl chloride (VC) to ethene (13),whereas strains FL2 and 195 are unable to use VC as a met-abolic electron acceptor (12, 32). Reductive dechlorination ofchlorinated benzenes and PCDDs which had previously beenshown for strain CBDB1 (2, 9, 18) was also recently demon-strated for strain 195 (11). However, different patterns of chlo-robenzene dechlorination (3) and differences in the utilizationof chlorobenzene and PCDD congeners have been observedfor strain CBDB1 (2, 9, 17, 18) and strain 195 (11). For exam-ple, hexachlorobenzene (HCB) dechlorination by strain 195mainly follows the pattern HCB3pentachlorobenzene(PeCB)31,2,3,5-tetrachlorobenzene (TeCB)31,3,5-trichloro-benzene (TCB) (11) that was also described for the Dehalo-coccoides-like bacterium DF-1 identified in a mixed culture(50). In contrast, HCB dechlorination in strain CBDB1 pro-ceeds via the two isomers 1,2,4,5-TeCB and 1,2,3,5-TeCB andleads to the formation of dichlorobenzenes and 1,3,5-TCB (17,18). In contrast to strain 195 and bacterium DF-1, strainCBDB1 is able to grow with 1,2,3-TCB and 1,2,4-TCB as thesole electron acceptors (2).

Reductive dehalogenases catalyzing the reductive dechlori-nation of chlorinated ethenes, phenols, or benzoates have beenisolated from several species that use chlorinated compoundsas growth-supporting electron acceptors (10, 20, 28, 33, 34, 37,42, 47). However, due to poor biomass yields, the isolation ofcatalytically active dehalogenases from Dehalococcoides isproblematic (17, 28). Only the TCE dehalogenase and the PCE

* Corresponding author. Mailing address: School of Civil and Envi-ronmental Engineering, 311 Ferst Dr., 3228 ES&T Building, GeorgiaInstitute of Technology, Atlanta, GA 30332-0512. Phone: (404) 894-0279. Fax: (404) 894-8266. E-mail: frank.loeffler@ce.gatech.edu.

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dehalogenase of D. ethenogenes strain 195 have been enrichedfrom mixed cultures containing strain 195 in a semipreparativemanner that allowed the initial characterization of these inter-esting enzyme systems (28). Nevertheless, purification of de-halogenases from Dehalococcoides will remain a major obsta-cle, and an integrated genetic and physiological approachseems most promising to shed light on the biochemistry andgenetics of reductive dechlorination. Dehalogenase-encodinggenes from different species have been identified following(partial) purification of the dechlorinating enzyme systems andpeptide sequencing. Examples include the PCE dehalogenasefrom Sulfurospirillum multivorans (36) (formerly Dehalospiril-lum multivorans [27]), the PCE dehalogenase from Desulfito-bacterium sp. strain Y51 (46), the ortho-chlorophenol dehalo-genase from Desulfitobacterium dehalogenans (47), and theTCE dehalogenase from strain 195 (29). Sequence comparisonof identified reductive dehalogenase genes revealed the pres-ence of several conserved motifs to which specific functionshave been attributed (reviewed in reference 43). The openreading frame (orf) encoding the catalytic subunit of the de-halogenase, designated orfA, is linked to a second open read-ing frame, orfB. orfB encodes a small hydrophobic B protein,possibly acting as a membrane anchor for the dehalogenase(36). The N termini of characterized reductive dehalogenasescontain a twin-arginine signal sequence, comprising the con-sensus motif RRXFXK followed by a stretch of hydrophobicresidues. Such signal sequences are involved in transportingcofactor-containing proteins across the cytoplasmic membraneand are proteolytically removed during protein maturation (6).Two iron-sulfur cluster binding (ISB) motifs characteristic ofbacterial ferredoxins (8) are located near the C-terminal end ofthe dehalogenases. Furthermore, dehalogenases contain highlyconserved tryptophane and histidine residues that might beinvolved in catalysis (43), and other conserved sequence blockswith as-yet-unknown function (38, 49) have been recognized.With the exception of the 3-chlorobenzoate dehalogenase ofDesulfomonile tiedjei (37), all biochemically characterized re-ductive dehalogenases apparently contain a corrinoid cofactor(10, 20, 30, 33, 35, 42). However, no consensus sequences forcorrinoid binding had been described in the encoding genespreviously (30, 36, 46, 47).

Access to whole-genome sequence data for D. ethenogenesstrain 195 (http://www.tigr.org/tdb/mdb/mdbinprogress.html)allowed for a systematic screening for dehalogenase sequences(48). Besides the TCE dehalogenase-encoding tceA gene, 17different reductive-dehalogenase-homologous (RDH) geneswere found in strain 195, and all share the above-describedfeatures characteristic of dehalogenases (44, 48). Hence, it washypothesized that the genomes of Dehalococcoides populationscontain multiple RDH genes, corresponding to the individualrange of chlorinated electron acceptors used by individualstrains. The goal of our study was to PCR amplify and identifygenes in the genomes of strain CBDB1 and strain FL2 thatshow high sequence similarity to known reductive dehaloge-nase genes. RDH genes found in the genome of strain BAV1were included in the analysis. Our results demonstrate thatmultiple nonidentical copies of RDH genes are common inDehalococcoides and that strain-specific differences exist. Fur-thermore, phylogenetic relationships of RDH genes and puta-tive cofactor binding sites were investigated.

MATERIALS AND METHODS

Cultivation. Dehalococcoides sp. strains CBDB1 and FL2 were cultivated un-der anaerobic conditions with TCBs (strain CBDB1) or TCE (strain FL2) asdescribed previously (1, 12, 17). The chloroorganic compounds were supplied viaa hexadecane phase (15). For substrate tests, the strains were incubated withdifferent chlorobenzenes or chloroethenes. Tests for enzyme activity towardschlorobenzenes or chloroethenes were performed with reduced methyl viologenas an artificial electron donor as previously described (17).

Preparation of genomic DNA. Genomic DNA of strain CBDB1 was extractedfrom 30 ml of culture fluid with a QIAGEN (Hilden, Germany) Mini kit accord-ing to the manufacturer’s instructions. Genomic DNA of strain FL2 was ex-tracted from 50 ml of culture fluid as described previously (12).

Amplification of RDH genes. The degenerate primers (forward primer RRF2and reverse primer B1R) were derived from alignments of RDH genes of D.ethenogenes strain 195 (19). PCR mixtures (30 �l) contained 0.05 to 3 ng oftemplate DNA (i.e., genomic DNA of strain CBDB1 or strain FL2), 0.5 �M eachprimer, 2.5 mM MgCl2, 0.25 mM each deoxynucleotide, 0.13 mg of bovine serumalbumin/ml, and 0.4 U of Taq DNA polymerase (Applied Biosystems, FosterCity, Calif.) in 1�-concentrated GeneAmp PCR buffer (Applied Biosystems).PCR was carried out with a GeneAmp PCR System 9700 (Applied Biosystems)with the following parameters: 130 s at 94°C; 30 cycles of 30 s at 94°C, 45 s at48°C, and 130 s at 72°C; and a final extension of 6 min at 72°C. The ampliconsfrom five reactions were combined and purified with a QIAGEN PCR purifica-tion kit according to the manufacturer’s recommendations. Purified PCR prod-ucts were cloned in TOP10 Escherichia coli cells by using a TA cloning kit(Invitrogen, Carlsbad, Calif.) according to the manufacturer’s instructions.Clones were screened for an insert of the expected size by colony PCR as follows.A small amount of cell material was transferred with a sterile toothpick to aplastic tube containing 50 �l of TE buffer (10 mM Tris-HCl [pH 8.0]–1 mMEDTA) and incubated at 95°C for 10 min. A volume of 3 �l of the suspension wassubsequently used as a template for PCR with primers targeting the polylinker ofthe cloning vector (51). PCR mixtures were prepared as described above, andamplification was carried out by using the following parameters: 130 s at 92°C; 30cycles of 30 s at 94°C, 1 min at 68°C, and 2 min at 72°C; and a final extension of6 min at 72°C. A total of 99 clones containing inserts of 1.7 kb were selected forfurther experiments. The 1.7-kb PCR products were digested with the restrictionenzyme MspI or HhaI at 35°C for 4 h. Digestion mixtures (20 �l) contained 10�l of the PCR product, 0.25 U of the restriction endonuclease (Promega Bio-sciences, Inc., San Luis Obispo, Calif.), and 0.1 mg of acetyl-bovine serumalbumin (Promega)/ml in restriction buffer (Promega). The resulting fragmentswere separated by electrophoresis for 2 h on 3% low-melting agarose gels.Plasmids containing inserts with different restriction patterns were extractedfrom the respective E. coli clones with a Qiaprep Spin Miniprep kit (QIAGEN),according to the manufacturer’s recommendations.

Sequencing of RDH genes. Sequence analysis was performed with an ABI 3100genetic analyzer (Applied Biosystems) using an ABI PRISM BigDye Terminatorversion 3.1 cycle sequencing kit. Single-stranded sequencing of the ends of the1.7-kb fragment was performed with the M13 reverse primer 5�-CAGGAAACAGCTATGAC-3� and the vector-targeted 3�-end primer (51). The resultingsequence information was used to design two additional internal primers for eachfragment to obtain the complete sequence of the insert.

Additional sequencing data. Three fragments of about 500 bp in length werePCR amplified from strain CBDB1 with primers targeting conserved motifsupstream of the ISB region of orfA (forward primer fdehal [5�-CARGGXACXCCXGARGA-3�] and reverse primer rdehal [5�-RSXCCRAARTCXATXGG-3�]); X indicates inosine). Another set of degenerate primers (forward primermern2 [5�-NNNTTYCAYGAYYTNGAYGAM-3�] and reverse primer mern5[5�-NCCNGCRTCDATNGGNNNNNN-3�]) was used for PCR amplificationwith genomic DNA of strain CBDB1 as a template. PCR products were clonedinto INVaF� E. coli cells with a TA cloning kit (Invitrogen, Karlsruhe, Germany),and inserts were sequenced (M. Meixner Sequencing Service, Berlin, Germany).One clone was selected for further experiments. To extend sequence informationof the 3� end, a new primer (mintF [5�-GCCGGGTGTATTTCAGGG-3�]) spe-cific to an internal region of the plasmid insert was used together with reverseprimer B1R for a new PCR with genomic DNA. The PCR product was directlysequenced. In a separate experiment, the primers 797F and 2490R targeting thetceA gene of D. ethenogenes strain 195 (29) were used for PCR with genomicDNA from strain CBDB1 or strain FL2 as a template.

Sequence analysis. Numbering of RDH genes from D. ethenogenes strain 195was adopted from Villemur et al. (48). Reductive dehalogenase gene sequencesfrom other organisms were obtained from GenBank (http://www.ncbi.nlm.nih.gov). RDH sequences of Dehalococcoides sp. strain BAV1 obtained by Kraj-

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malnik-Brown et al. (19) (GenBank accession numbers AY553222 to AY553228)were included in the analysis. Sequences of PCR-amplified fragments werecompared to other published sequences with the National Center for Biotech-nology Information BLASTX search tool. Deduced amino acid sequences wereobtained with the TRANSLATE program (http://us.expasy.org/tools/dna.html).Amino acid sequences were aligned with the ClustalW program located at theEuropean Bioinformatics Institute website (http://www.ebi.ac.uk/clustalw/) orwith QAlign (41). Phylogenetic trees (neighbor joining, maximum parsimony,default settings) were generated from nearly complete orfA genes (sequencesextending from immediately downstream of the twin-arginine signal sequence tothe 3� end) by using MEGA version 2.1 (22).

Nucleotide sequence accession numbers. The coding sequences of RDH genesand putative B gene fragments were deposited in GenBank under the accessionnumbers AY374229 to AY374244 (strain CBDB1), AY374245 to AY374255(strain FL2), and AY165309 (strain FL2).

RESULTS

Amplification of RDH genes. Amplicons of the expected sizeof 1.7 kb were obtained with the primer pair RRF2-B1R byusing genomic DNA from strain CBDB1 or strain FL2 as atemplate. A total of 99 clones, 51 for CBDB1 (designatedrdhCBDB1) and 48 for FL2 (designated rdhFL2), were obtained.Restriction fragment length analysis distinguished 13 differentpatterns among the rdhCBDB1 clones and 14 distinct patternsamong the rdhFL2 clones. Ten identical patterns were sharedbetween the rdhCBDB1 and rdhFL2 clones, and inserts of rep-resentative rdhFL2 and rdhCBDB1 clones were sequenced. Allsequences contained a nearly complete orfA (length of 1,350 to1,495 bp), missing only the first 45 to 60 nucleotides at the 5�end of the RDH genes, and about 90 nucleotides of orfB (Fig.1). The one exception, orfA of rdh10FL2 (rdhA10FL2) lacked anadditional 250 to 260 nucleotides of the 5� end, probably due tomispriming of the forward primer RRF2 used for PCR.

One additional RDH sequence from strain CBDB1 (desig-nated rdh9CBDB1) was obtained with the highly degenerateprimers mern2 and mern5 (Fig. 1). The amplicon containedthe complete 5� end of orfA including the twin-arginine signalsequence motif, suggesting that the forward primer had boundabout 180 nucleotides upstream of the actual target sequence.orfA of rdh9CBDB1 (rdhA9CBDB1) has a length of 1,488 bp andrepresents the first complete orfA obtained from strainCBDB1 (Fig. 1). Two additional 500-bp sequences, designatedrdhA15CBDB1 and rdhA16CBDB1, were obtained from strain

CBDB1 with the primer pair fdehal-rdehal (Fig. 1). tceA-tar-geted primers yielded an amplicon with genomic DNA fromstrain FL2 that was very similar to the tceA gene of D. etheno-genes strain 195 (i.e., 99.3% sequence identity at the aminoacid level). In contrast, the tceA gene was not found in strainCBDB1 when the same primers were used.

Sequence analysis of RDH genes. rdhACBDB1 and rdhAFL2

genes showed highest similarity to a group of genes comprisingthe tceA gene of D. ethenogenes strain 195, the tceA gene ofstrain FL2, and tceA genes amplified previously from threechloroethene-dechlorinating enrichment cultures (GenBankaccession numbers AAN85590, AAN85592, and AAN85594).A BLASTX search yielded best hits (E value range, 10�31 to10�61) for full-length alignments of rdhA1CBDB1 tordhA14CBDB1 (rdhA1–14CBDB1) and rdhA1FL2 to rdhA11FL2

(rdhA1–11FL) with the characterized tceA gene of strain 195.As shown by pairwise alignments of deduced amino acid se-quences, rdhA1–14CBDB1 and rdhA1–11FL2 were 27 to 33%identical to the tceA gene of strain 195. The 500-bp fragmentsrdhA15CBDB1 and rdhA16CBDB1 showed 30 and 21% identity totceA, respectively. Some rdhACBDB1 and rdhAFL2 genes alsoshared high similarity (BLASTX E values of �10�35) withsequence rdh63A (GenBank accession number AAO15649), anRDH gene derived from a 2-bromophenol-degrading micro-bial consortium (39). Hits with small E values (E value range,10�10 to 10�30) were also obtained for alignments ofrdhACBDB1 and rdhAFL2 genes with other known reductivedehalogenase genes, i.e., the PCE dehalogenase-encodinggenes (pceA genes) from S. multivorans, Dehalobacter restrictus,and Desulfitobacterium spp. and the chlorophenol dehaloge-nase-encoding genes (cprA genes) from Desulfitobacterium spp.For other functionally assigned genes, e.g., a phosphoglyceratemutase from Clostridium tetanii as well as a periplasmic [Fe]hydrogenase and a molybdenum formylmethanofuran dehy-drogenase subunit from Methanosarcina mazei, a BLASTXsearch resulted in only partial alignments with high E values(�10�4).

Deduced amino acid sequences of nearly complete orfAgenes from rdhCBDB1 and rdhFL2 sequences (404 to 489 resi-dues) were used to generate a tree in which the 17 RDH genes(rdhA1–17DE) found in D. ethenogenes strain 195 (48), the

FIG. 1. Schematic diagram of gene fragments amplified from Dehalococcoides sp. strain CBDB1 and strain FL2. (A) Sequences identified withthe primer pair RRF2-B1R. (B) Sequence rdh9CBDB1 obtained with primers mern2 and mern5 and extended by additional PCR amplification withthe primer pair mintF-B1R (shaded area). (C) Sequences amplified with the primer pair fdehal-rdehal. orfA represents the open reading frameencoding the catalytically active reductive dehalogenase, and orfB is the open reading frame encoding a small hydrophobic B protein. Tat signal,twin-arginine translocation signal sequence including the RRXFXK consensus motif. The scale bar indicates nucleotide position (1 � start codonof orfA).

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seven RDH genes (rdhA1–7BAV1) found in strain BAV1 (19),and reductive dehalogenase genes from other dechlorinatingbacteria were included (Fig. 2). All Dehalococcoides RDHgenes except rdhA16DE and rdhA17DE grouped together (Fig.2). Furthermore, seven rdhACBDB1 genes (rdhA8–14CBDB1),five rdhAFL2 genes (rdhA7–11FL2), and three rdhABAV1 genes(rdhA4–6BAV1) formed a cluster with six rdhADE genes(rdhA1–6DE) and the tceA gene subcluster (cluster I; Fig. 2). Asecond cluster (cluster II) was composed of six rdhACBDB1

genes (rdhA1–6CBDB1), six rdhAFL2 genes (rdhA1–6FL2), twordhABAV1 genes (rdhA1BAV1 and rdhA7BAV1) and five rdhADE

genes (rdhA10–14DE). rdhA7CBDB1 could not be unambigu-ously assigned to either of the two clusters. rdhA16DE andrdhA17DE shared only limited similarity with rdhACBDB1 andrdhAFL2 genes but grouped with separate clusters comprisingcprA genes and pceA genes from Desulfitobacterium spp. andDehalobacter restrictus, respectively.

Several rdhACBDB1 genes shared high sequence similaritywith rdhAFL2 genes (Fig. 3). Six rdhACBDB1 genes with 98.5 to99.8% identity to rdhAFL2 genes and one rdhACBDB1 gene with100% identity to an rdhAFL2 gene based on the deduced aminoacid sequences were identified. In addition, the restriction pat-terns of three FL2 clone library inserts matched three differentrestriction patterns of CBDB1 clone library inserts, indicatingfurther pairs of highly similar sequences. Four rdhADE geneswere identified that shared 86.5 to 94.4% identity withrdhACBDB1 and rdhAFL2 genes, thus forming four subclusters(Fig. 2). Also, one rdhABAV1 gene (rdhA5BAV1; cluster I)shared 90.8 to 93.1% sequence identity with the genes of onesubcluster. A total of 10 subclusters, each comprising 2 to 4RDH genes from different strains, were identified (Fig. 2).

ISB motifs. Like other dehalogenases, all rdhACBDB1- andrdhAFL2-encoded amino acid sequences contained two ISBmotifs in the C-terminal region (Fig. 3). The first ISB motif ofall sequences corresponded to the conserved consensus se-quence CXXCXXCXXXCP, found in bacterial ferredoxins(8). Variations of this pattern, however, were observed in thesecond ISB motif. In 14 out of 21 RDH genes in cluster I (Fig.2), the second ISB motif shared the consensus CXXCXXCXXCP. In the other seven RDH genes of cluster I, the first twocysteine residues of the second ISB motif were separated bythree, four, or six residues instead of two (Fig. 3). In all fiveRDH genes of cluster I with four or six residues between thefirst two cysteines of the second ISB (i.e., rdhA12–14CBDB1,rdhA10FL2, and rdhA2DE), a fifth cysteine residue was presentupstream of the second ISB motif. This motif, CX4CXnCX2-3

CXXXCP, was also found in rdhA3DE and rdhA4DE. In allDehalococcoides RDH genes outside of cluster I, the secondISB motif was characterized by a long stretch of residues (8 to21 amino acids) separating the first two cysteine residues (i.e.,rdhA1–7CBDB1, rdhA1–6FL2, rdhA7–A17DE, rdhA1–3BAV1, andrdhA7BAV1) (Fig. 3). All RDH genes that contained a second

FIG. 2. Phylogenetic analysis of RDH genes from Dehalococcoidessp. strain CBDB1 (rdhA1–14CBDB1), Dehalococcoides sp. strain FL2(rdhA1–11FL2), Dehalococcoides sp. strain BAV1 (rdhA1–7BAV1), andD. ethenogenes strain 195 (rdhA1–17DE) (48). The neighbor-joiningtree shown was generated from amino acid sequences of nearly com-plete orfA genes (see Fig. 1 and text for details). Branching pointssupported by 85 to 100% of 1,000 bootstrap sampling events areindicated by solid circles. Open circles indicate 50 to 84% support bybootstrap sampling. Branching points supported by the maximum par-simony treeing method are marked with (P). Additional clones from strainFL2 (rdhA*FL2, sequences not determined) are indicated together withthe rdhACBDB1 genes that have identical restriction patterns. Dotted dou-ble, triple, and quadruple lines indicate subclusters of highly similar genesfrom two, three, or four different Dehalococcoides strains, respec-tively (see the text for details). tceA, trichloroethene reductive de-halogenase, pceA, tetrachloroethene reductive dehalogenase, cprA,

chlorophenol reductive dehalogenase. The tceA cluster comprises thetceA genes of D. ethenogenes strain 195 (GenBank accession numberAAF73916), strain FL2 (accession number AY165309), and threechloroethene-dechlorinating enrichment cultures (accession numbersAAN85590, AAN85592, and AAN85594). The scale bar represents20% sequence divergence.

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ISB motif with closely linked cysteine residues (cluster I)formed a coherent group of related genes, whereas the otherDehalococcoides RDH genes that contained a second ISB mo-tif with a distant cysteine did not form a phylogenetically con-sistent group (Fig. 2).

Cobalamin-binding consensus sequences. A consensus se-quence for cobalamin binding DXHXXG. . .SXL. . .GG (26)was found in the C-terminal region of rdhA12DE (cluster II)(Fig. 3). The consensus motif DXHXXG was located betweenthe two ISB motifs, whereas the SXL and twin-glycine motifswere located downstream of the second ISB motif. Amino acidstretches between the three conserved motifs of the consensuswere longer in rdhA12DE (DXHXXG-X50-SXL-X42-GG) thanthose described for a group of known cobalamin-dependentenzymes from other prokaryotes (e.g., DXHXXG-X41-SXL-X26–28-GG) (26). In addition, two rdhACBDB1 genes, threerdhAFL2 genes, and one rdhABAV1 gene (rdhA3CBDB1 andrdhA4CBDB1, rdhA1–3FL2, and rdhA1BAV1; cluster II) con-tained the consensus sequence DXXHXXG (Fig. 3). The SXLmotif was found in rdhA3CBDB1, rdhA4CBDB1, and rdhA1–3FL2;rdhA1BAV1 contained a serine and a leucine residue separatedby five residues in the corresponding region. Four of thesegenes contained the twin-glycine motif, whereas two genes hada single glycine residue in the corresponding position.

N-terminal region of RDH genes. Deduced amino acid se-quences of all gene fragments amplified with the primer pairRRF2-B1R contained stretches of hydrophobic residues attheir N-terminal ends that were similar to those in knownreductive dehalogenases (data not shown). The forward primerRRF2 targets the consensus motif RRXFXK of twin-argininesignal sequences, indicating that all amplified genes containedthis sequence, though the exact nucleotide composition is notknown due to the degenerate nature of this primer.rdhA9CBDB1, a sequence amplified with the primers mern2 andmern5, contained the complete N-terminal end of orfA, in-cluding the motif RRDFMK that is also present in tceA and 13of the 17 RDH genes in strain 195.

orfB sequences. The obtained N-terminal orfB fragments(rdhBCBDB1 and rdhBFL2) encoded mainly hydrophobicamino acid residues, similar to the respective regions of orfBgenes linked to known dehalogenase genes, e.g., tceB of D.ethenogenes strain 195 or pceB of S. multivorans. The ob-tained rdhBCBDB1 and rdhBFL2 fragments were small (90 bp)and therefore did not allow calculation of a phylogenetic tree withhigh bootstrap values. However, the calculations with deducedamino acid sequences of these small fragments supported theestablished subclusters of highly similar orfA genes from the dif-ferent strains. Six rdhBCBDB1 gene fragments were 100% identicalto six rdhBFL2 fragments, analogous to the corresponding highlysimilar pairs of rdhACBDB1 and rdhAFL2 genes. Furthermore, astable grouping of the other orfB fragments analogous to the

corresponding orfA subclusters was obtained; branch points weresupported by 75 to 93% bootstrap sampling events in all casesexcept one (bootstrap support for grouping of rdhB11DE withrdhB5CBDB1/rdhB4FL2 was 29%).

Physiological tests. In media supplied with 1,2,3-TCB/1,2,4-TCB or PeCB as an electron acceptor and inoculated with aTCE-grown culture of strain FL2, no dechlorination productswere detected within 6 months of incubation. Also, no dechlo-rination of chlorobenzene congeners (1,2,3-TCB; 1,2,4-TCB;1,2,3,4-TeCB; 1,2,3,5-TeCB; 1,2,4,5-TeCB; PeCB; and HCB)by whole cells of strain FL2 was observed in activity tests withmethyl viologen as an electron donor. Strain CBDB1 was spe-cifically tested for TceA activity, i.e., dechlorination of TCE toVC (28, 29). However, no VC formation was observed.

DISCUSSION

Thirty novel RDH genes were amplified from Dehalococ-coides sp. strain CBDB1 and Dehalococcoides sp. strain FL2that all differ from the 17 RDH genes identified in the genomeof D. ethenogenes strain 195 and the seven RDH genes ampli-fied from Dehalococcoides sp. strain BAV1. Although the sub-strate specificities of the amplified genes cannot be inferred,there is strong evidence that all RDH genes identified in thefour Dehalococcoides strains represent true homologs (geneswith a common ancestry) of the biochemically characterizedTCE dehalogenase gene of strain 195. The evidence is asfollows. (i) All amplified RDH genes share 27 to 33% se-quence identity with the tceA gene at the amino acid level overthe whole length of the sequence (449 to 489 residues for allrdhACBDB1 and rdhAFL2 genes except rdhA10FL2, which is 404residues in length), which allows the inference of a homolo-gous relationship as previously described by Brenner et al. (7)and Rost (40). (ii) All RDH genes share two functional do-mains that determine the structure and/or localization of theprotein, i.e., the N-terminal twin-arginine signal sequence andthe ISB region near the C terminus comprising two separateISB motifs. (iii) All RDH genes are organized in operons witha downstream orfB gene encoding a putative membrane an-chor. (iv) The high number of RDH genes (17 RDH genes instrain 195, at least 14 RDH genes in both strains CBDB1 andFL2, and 7 RDH genes in strain BAV1) makes convergentevolution unlikely.

Sequence comparison of RDH gene fragments from all fourDehalococcoides isolates demonstrated that highly similargenes are shared among strains and that unique RDH genesthat distinguish different Dehalococcoides strains exist. Thesubclusters of highly similar genes can be interpreted as or-thologs, i.e., homologs derived by a speciation event (45). The16S rRNA genes of strain CBDB1 (GenBank accession num-ber AF230641) and strain FL2 (accession number AF357918)

FIG. 3. Alignment of the C-terminal region of deduced amino acid sequences of RDH genes from Dehalococcoides strains (tceA DE refers tothe tceA gene of D. ethenogenes strain 195; see the legend of Fig. 2 for other designations). Also included is sequence rdh63A, an RDH geneobtained from a bacterial consortium (39). ClustalW alignments were manually corrected to align putative cofactor binding sites. Conservedresidues of the two ISB motifs and cobalamin-binding consensus sequences are highlighted in gray. Boxes mark cobalamin-binding motifs inrdhA3CBDB1 and rdhA4CBDB1, rdhA1–3FL2, rdhA12DE, and rdhA1BAV1. In rdhA12DE and rdhA1BAV1, the SXL motif could not be unambiguouslylocated (see the text for details).

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are 100% identical and share 98.5% sequence identity with the16S rRNA gene of strain 195 (accession number AF004928).The topology of each orthologous group of RDH genes fromstrains CBDB1, FL2, and 195 is consistent with 16S rRNAgene-based phylogeny, supporting the idea that strains FL2and CBDB1 are more closely related to each other than tostrain 195. The 16S rRNA gene of strain BAV1 (accessionnumber AY165308) exhibits only one base difference (99.9%identity) from the 16S rRNA genes from strains CBDB1 andFL2. However, no stronger relationship was identified betweenrdhABAV1 genes and RDH genes from strains CBDB1 and FL2than between rdhABAV1 genes and rdhADE genes. Analysis oforfB gene fragments from all four Dehalococcoides isolatesshowed that sequence relationships between orfB genes corre-spond to those of orfA genes, suggesting that both genes havecoevolved and are functionally linked. In conclusion, our dataprovide evidence that RDH gene duplication and divergenceoccurred in a common Dehalococcoides ancestor and not afterthe speciation into the currently known strains. This is impor-tant because it indicates that the presence of multiple RDHgenes in Dehalococcoides spp. is due not to rapid adaptation tothe presence of anthropogenic halogenated compounds re-leased within the last century but to much older evolutionaryevents. The high sequence identity of the tceA genes fromstrains 195 and FL2, compared to the other orthologous RDHgenes, might indicate a lateral gene transfer event.

The presence of multiple nonidentical RDH genes in Deha-lococcoides strains is consistent with the observation that thedifferent strains use different chlorinated electron acceptors.Several RDH genes are present in individual strains that donot have an ortholog among the known genes of the otherstrains. Many of the RDH genes found in strains CBDB1, FL2,and BAV1 do not have orthologs in the completely sequencedgenome of strain 195, suggesting that these strains possessdechlorination activities not present in strain 195. The pres-ence or absence of tceA genes is consistent with the observedactivity of the different strains. TCE supports growth of strainFL2 and strain 195; accordingly, the tceA gene was found inthese strains. Strain BAV1 (13) and strain CBDB1 do not growwith TCE. Consistently, the tceA gene was not found in strainBAV1 (12) or strain CBDB1. Strains CBDB1 and 195, but notstrain FL2, dechlorinate chlorinated benzenes, suggesting thatchlorobenzene reductive dehalogenases are present only instrains CBDB1 and 195. However, specific culture conditionsmight be necessary to induce chlorobenzene dechlorination instrain FL2. Substrate-dependent differential induction of de-halogenases has been demonstrated in Desulfitobacterium spp.(reviewed in reference 44). In strain CBDB1, different 1,2,3-TCB, PeCB, and HCB dechlorination rates were obtained withcells pregrown on different chlorobenzene congeners as elec-tron acceptors, suggesting that dehalogenases are induced bytheir respective substrates (i.e., electron acceptors) (18). Incontrast, PCE dechlorination was constitutive in cultures ofstrain 195 grown on TCE, 1,1-DCE, or dichloroethane (32).

All RDH genes contain the conserved functional domainscharacterized in reductive dehalogenases (16, 43). The pres-ence of a twin-arginine signal sequence in RDH genes is con-sistent with the hypothesis that these genes are involved inrespiratory reductive dehalogenation, because such motifs arepredominantly found in membrane-associated proteins in-

volved in respiratory electron transport (6, 43). Furthermore,deduced amino acid sequences of all RDH genes contain twoISB motifs and could therefore form two spatially linked iron-sulfur clusters. Iron-sulfur clusters have been detected in mostreductive dehalogenases and probably mediate electron trans-fer to the active site containing the corrinoid (5, 34, 47). Indi-cations for the involvement of a corrinoid cofactor in catalysishave been found for most reductive dehalogenases from bac-teria that couple reductive dechlorination to growth (16, 43).Recently, a norpseudovitamin B12 was identified as the cofac-tor of the PCE dehalogenase of S. multivorans (21); however,a corrinoid binding motif was not identified in the amino acidsequence of the protein (36). From subsets of cobalamin-de-pendent methyltransferases and isomerases of different bacte-rial species including E. coli, Propionibacterium freudenreichiisubsp. shermanii, and Clostridium sp., a cobalamin-bindingconsensus sequence (DXHXXG. . .SXL. . .GG) has been es-tablished (26). In these methyltransferases and isomerases, thedimethylbenzimidazole ligand of the cobalt center is replacedby the conserved histidine residue of the consensus sequence.In our study, such a cobalamin-binding consensus sequencecould be identified in a group of seven Dehalococcoides RDHgenes. Other RDH genes, including all genes encoding char-acterized reductive dehalogenases, lack the cobalamin-bindingconsensus sequence. Electron paramagnetic resonance spec-troscopy data indicated that the chlorophenol dehalogenase ofDesulfitobacterium dehalogenans and the PCE dehalogenase ofS. multivorans contain the corrinoid in the base-off form (21,34, 47). This finding raises the question of whether the RDHgenes containing the cobalamin-binding consensus sequencebind the corrinoid cofactor differently and exhibit a different cat-alytic mechanism. However, cobalamin binding by histidine liga-tion does not determine a specific reaction mechanism, sincedifferent catalytic mechanisms have been identified in methyl-transferases and isomerases (26). Moreover, other subfamilies ofcobalamin-dependent methyltransferases and isomerases lack thecobalamin binding consensus and do not bind cobalamin via his-tidine coordination (4, 26).

Though we did not identify the function of the DehalococcoidesRDH genes, this study provides essential sequence data that areuseful for the identification of Dehalococcoides reductive dehalo-genase genes from peptide fragments and for the design of prim-ers for transcription analysis of specific RDH genes.

ACKNOWLEDGMENTS

This work was supported by the Deutsche Forschungsgemeinschaftproject number AD 178/1 to L.A., by a National Science FoundationCAREER award (award number 0090496) to F.E.L., and, in part, bythe U.S. Strategic Environmental Research and Development Pro-gram.

D. S. Mern was involved in initial experiments with the cloning ofgene rdh9CBDB1. We thank G. Wagner for technical assistance, B.Lynch for gene sequencing, Y. Sung for supplying cultures of Dehalo-coccoides sp. strain FL2, and U. Lechner for communicating fdehaland rdehal primer sequences.

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