APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 2004, p. 5290–5297 0099-2240/04/$08.00⫹0 DOI: 10.1128/AEM.70.9.5290–5297.2004 Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Vol. 70, No. 9
Multiple Nonidentical Reductive-Dehalogenase-Homologous Genes Are Common in Dehalococcoides Tina Ho ¨lscher,1 Rosa Krajmalnik-Brown,2 Kirsti M. Ritalahti,2 Friedrich von Wintzingerode,3 Helmut Go ¨risch,1 Frank E. Lo ¨ffler,2,4* and Lorenz Adrian1 Fachgebiet Technische Biochemie, Institut fu ¨r Biotechnologie, Technische Universita ¨t Berlin, Berlin, Germany1; School of Civil and Environmental Engineering2 and School of Biology,4 Georgia Institute of Technology, Atlanta, Georgia; and Institut fu ¨r Mikrobiologie und Hygiene, Universita ¨tsklinikum 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-dechlorinating strain CBDB1 and the trichloroethene-dechlorinating strain FL2. The amplicons (1,350 to 1,495 bp) corresponded 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 restriction analysis revealed the presence of at least 14 different RDH genes in each strain. All amplified RDH genes showed sequence similarity with known reductive dehalogenase genes over the whole length of the sequence and shared all characteristics described for reductive dehalogenases. Deduced amino acid sequences of seven RDH genes from strain CBDB1 were 98.5 to 100% identical to seven different RDH genes from strain FL2, suggesting that both strains have an overlapping substrate range. All RDH genes identified in strains CBDB1 and FL2 were related to the RDH genes present in the genomes of Dehalococcoides ethenogenes strain 195 and Dehalococcoides sp. strain BAV1; however, sequence identity did not exceed 94.4 and 93.1%, respectively. The presence of RDH genes in strains CBDB1, FL2, and BAV1 that have no orthologs in strain 195 suggests that these strains possess dechlorination activities not present in strain 195. Comparative sequence analysis identified consensus sequences for cobalamin binding in deduced amino acid sequences of seven RDH genes. In conclusion, this study demonstrates that the presence of multiple nonidentical RDH genes is characteristic of Dehalococcoides strains. cometabolically dechlorinated by strain BAV1 (13). Strain BAV1, however, respires vinyl chloride (VC) to ethene (13), whereas strains FL2 and 195 are unable to use VC as a metabolic electron acceptor (12, 32). Reductive dechlorination of chlorinated benzenes and PCDDs which had previously been shown for strain CBDB1 (2, 9, 18) was also recently demonstrated for strain 195 (11). However, different patterns of chlorobenzene dechlorination (3) and differences in the utilization of chlorobenzene and PCDD congeners have been observed for strain CBDB1 (2, 9, 17, 18) and strain 195 (11). For example, hexachlorobenzene (HCB) dechlorination by strain 195 mainly follows the pattern HCB3pentachlorobenzene (PeCB)31,2,3,5-tetrachlorobenzene (TeCB)31,3,5-trichlorobenzene (TCB) (11) that was also described for the Dehalococcoides-like bacterium DF-1 identified in a mixed culture (50). In contrast, HCB dechlorination in strain CBDB1 proceeds via the two isomers 1,2,4,5-TeCB and 1,2,3,5-TeCB and leads to the formation of dichlorobenzenes and 1,3,5-TCB (17, 18). In contrast to strain 195 and bacterium DF-1, strain CBDB1 is able to grow with 1,2,3-TCB and 1,2,4-TCB as the sole electron acceptors (2). Reductive dehalogenases catalyzing the reductive dechlorination of chlorinated ethenes, phenols, or benzoates have been isolated from several species that use chlorinated compounds as growth-supporting electron acceptors (10, 20, 28, 33, 34, 37, 42, 47). However, due to poor biomass yields, the isolation of catalytically active dehalogenases from Dehalococcoides is problematic (17, 28). Only the TCE dehalogenase and the PCE
Bacterial reductive dechlorination reactions play important roles in the detoxification of chlorinated aromatic and aliphatic compounds in contaminated environments (14, 23). Several anaerobic bacteria use chlorinated compounds in their energy metabolism, coupling reductive dehalogenation to electron transport phosphorylation (16, 25). The Dehalococcoides group forms a bacterial cluster that is phylogenetically distant from other dechlorinating bacteria (2, 31) and uses a spectrum of chlorinated compounds as growth-supporting electron acceptors, including chlorinated ethenes (13, 24, 31), chlorinated benzenes (2, 18), and polychlorinated dibenzodioxins (PCDDs) (9). Four members of the Dehalococcoides group have been isolated. Three strains, Dehalococcoides ethenogenes strain 195, Dehalococcoides sp. strain FL2, and Dehalococcoides sp. strain BAV1, have been cultivated with chlorinated ethenes as growth-supporting substrates (12, 24, 25, 31), whereas Dehalococcoides sp. strain CBDB1 was isolated with chlorinated benzenes as terminal electron acceptors (2). Several strain-specific physiological differences exist. For instance, tetrachloroethene (PCE) is used as a metabolic electron acceptor 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 * Corresponding author. Mailing address: School of Civil and Environmental Engineering, 311 Ferst Dr., 3228 ES&T Building, Georgia Institute of Technology, Atlanta, GA 30332-0512. Phone: (404) 8940279. Fax: (404) 894-8266. E-mail:
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dehalogenase of D. ethenogenes strain 195 have been enriched from mixed cultures containing strain 195 in a semipreparative manner that allowed the initial characterization of these interesting enzyme systems (28). Nevertheless, purification of dehalogenases from Dehalococcoides will remain a major obstacle, and an integrated genetic and physiological approach seems most promising to shed light on the biochemistry and genetics of reductive dechlorination. Dehalogenase-encoding genes from different species have been identified following (partial) purification of the dechlorinating enzyme systems and peptide sequencing. Examples include the PCE dehalogenase from Sulfurospirillum multivorans (36) (formerly Dehalospirillum multivorans [27]), the PCE dehalogenase from Desulfitobacterium sp. strain Y51 (46), the ortho-chlorophenol dehalogenase from Desulfitobacterium dehalogenans (47), and the TCE dehalogenase from strain 195 (29). Sequence comparison of identified reductive dehalogenase genes revealed the presence of several conserved motifs to which specific functions have been attributed (reviewed in reference 43). The open reading frame (orf) encoding the catalytic subunit of the dehalogenase, designated orfA, is linked to a second open reading 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 dehalogenases contain a twin-arginine signal sequence, comprising the consensus motif RRXFXK followed by a stretch of hydrophobic residues. Such signal sequences are involved in transporting cofactor-containing proteins across the cytoplasmic membrane and are proteolytically removed during protein maturation (6). Two iron-sulfur cluster binding (ISB) motifs characteristic of bacterial ferredoxins (8) are located near the C-terminal end of the dehalogenases. Furthermore, dehalogenases contain highly conserved tryptophane and histidine residues that might be involved in catalysis (43), and other conserved sequence blocks with as-yet-unknown function (38, 49) have been recognized. With the exception of the 3-chlorobenzoate dehalogenase of Desulfomonile tiedjei (37), all biochemically characterized reductive dehalogenases apparently contain a corrinoid cofactor (10, 20, 30, 33, 35, 42). However, no consensus sequences for corrinoid binding had been described in the encoding genes previously (30, 36, 46, 47). Access to whole-genome sequence data for D. ethenogenes strain 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, 17 different reductive-dehalogenase-homologous (RDH) genes were found in strain 195, and all share the above-described features characteristic of dehalogenases (44, 48). Hence, it was hypothesized that the genomes of Dehalococcoides populations contain multiple RDH genes, corresponding to the individual range of chlorinated electron acceptors used by individual strains. The goal of our study was to PCR amplify and identify genes in the genomes of strain CBDB1 and strain FL2 that show high sequence similarity to known reductive dehalogenase genes. RDH genes found in the genome of strain BAV1 were included in the analysis. Our results demonstrate that multiple nonidentical copies of RDH genes are common in Dehalococcoides and that strain-specific differences exist. Furthermore, phylogenetic relationships of RDH genes and putative cofactor binding sites were investigated.
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MATERIALS AND METHODS Cultivation. Dehalococcoides sp. strains CBDB1 and FL2 were cultivated under anaerobic conditions with TCBs (strain CBDB1) or TCE (strain FL2) as described previously (1, 12, 17). The chloroorganic compounds were supplied via a hexadecane phase (15). For substrate tests, the strains were incubated with different chlorobenzenes or chloroethenes. Tests for enzyme activity towards chlorobenzenes or chloroethenes were performed with reduced methyl viologen as an artificial electron donor as previously described (17). Preparation of genomic DNA. Genomic DNA of strain CBDB1 was extracted from 30 ml of culture fluid with a QIAGEN (Hilden, Germany) Mini kit according to the manufacturer’s instructions. Genomic DNA of strain FL2 was extracted from 50 ml of culture fluid as described previously (12). Amplification of RDH genes. The degenerate primers (forward primer RRF2 and 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 of template DNA (i.e., genomic DNA of strain CBDB1 or strain FL2), 0.5 M each primer, 2.5 mM MgCl2, 0.25 mM each deoxynucleotide, 0.13 mg of bovine serum albumin/ml, and 0.4 U of Taq DNA polymerase (Applied Biosystems, Foster City, 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 at 48°C, and 130 s at 72°C; and a final extension of 6 min at 72°C. The amplicons from five reactions were combined and purified with a QIAGEN PCR purification kit according to the manufacturer’s recommendations. Purified PCR products 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 a plastic tube containing 50 l of TE buffer (10 mM Tris-HCl [pH 8.0]–1 mM EDTA) and incubated at 95°C for 10 min. A volume of 3 l of the suspension was subsequently used as a template for PCR with primers targeting the polylinker of the cloning vector (51). PCR mixtures were prepared as described above, and amplification was carried out by using the following parameters: 130 s at 92°C; 30 cycles of 30 s at 94°C, 1 min at 68°C, and 2 min at 72°C; and a final extension of 6 min at 72°C. A total of 99 clones containing inserts of 1.7 kb were selected for further experiments. The 1.7-kb PCR products were digested with the restriction enzyme 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 Biosciences, Inc., San Luis Obispo, Calif.), and 0.1 mg of acetyl-bovine serum albumin (Promega)/ml in restriction buffer (Promega). The resulting fragments were separated by electrophoresis for 2 h on 3% low-melting agarose gels. Plasmids containing inserts with different restriction patterns were extracted from 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 3100 genetic analyzer (Applied Biosystems) using an ABI PRISM BigDye Terminator version 3.1 cycle sequencing kit. Single-stranded sequencing of the ends of the 1.7-kb fragment was performed with the M13 reverse primer 5⬘-CAGGAAAC AGCTATGAC-3⬘ and the vector-targeted 3⬘-end primer (51). The resulting sequence information was used to design two additional internal primers for each fragment to obtain the complete sequence of the insert. Additional sequencing data. Three fragments of about 500 bp in length were PCR amplified from strain CBDB1 with primers targeting conserved motifs upstream of the ISB region of orfA (forward primer fdehal [5⬘-CARGGXACX CCXGARGA-3⬘] and reverse primer rdehal [5⬘-RSXCCRAARTCXATXGG3⬘]); X indicates inosine). Another set of degenerate primers (forward primer mern2 [5⬘-NNNTTYCAYGAYYTNGAYGAM-3⬘] and reverse primer mern5 [5⬘-NCCNGCRTCDATNGGNNNNNN-3⬘]) was used for PCR amplification with genomic DNA of strain CBDB1 as a template. PCR products were cloned into 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 information of the 3⬘ end, a new primer (mintF [5⬘-GCCGGGTGTATTTCAGGG-3⬘]) specific to an internal region of the plasmid insert was used together with reverse primer B1R for a new PCR with genomic DNA. The PCR product was directly sequenced. In a separate experiment, the primers 797F and 2490R targeting the tceA gene of D. ethenogenes strain 195 (29) were used for PCR with genomic DNA from strain CBDB1 or strain FL2 as a template. Sequence analysis. Numbering of RDH genes from D. ethenogenes strain 195 was adopted from Villemur et al. (48). Reductive dehalogenase gene sequences from 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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FIG. 1. Schematic diagram of gene fragments amplified from Dehalococcoides sp. strain CBDB1 and strain FL2. (A) Sequences identified with the primer pair RRF2-B1R. (B) Sequence rdh9CBDB1 obtained with primers mern2 and mern5 and extended by additional PCR amplification with the primer pair mintF-B1R (shaded area). (C) Sequences amplified with the primer pair fdehal-rdehal. orfA represents the open reading frame encoding 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 codon of orfA). malnik-Brown et al. (19) (GenBank accession numbers AY553222 to AY553228) were included in the analysis. Sequences of PCR-amplified fragments were compared to other published sequences with the National Center for Biotechnology Information BLASTX search tool. Deduced amino acid sequences were obtained with the TRANSLATE program (http://us.expasy.org/tools/dna.html). Amino acid sequences were aligned with the ClustalW program located at the European Bioinformatics Institute website (http://www.ebi.ac.uk/clustalw/) or with QAlign (41). Phylogenetic trees (neighbor joining, maximum parsimony, default settings) were generated from nearly complete orfA genes (sequences extending from immediately downstream of the twin-arginine signal sequence to the 3⬘ end) by using MEGA version 2.1 (22). Nucleotide sequence accession numbers. The coding sequences of RDH genes and putative B gene fragments were deposited in GenBank under the accession numbers AY374229 to AY374244 (strain CBDB1), AY374245 to AY374255 (strain FL2), and AY165309 (strain FL2).
RESULTS Amplification of RDH genes. Amplicons of the expected size of 1.7 kb were obtained with the primer pair RRF2-B1R by using genomic DNA from strain CBDB1 or strain FL2 as a template. A total of 99 clones, 51 for CBDB1 (designated rdhCBDB1) and 48 for FL2 (designated rdhFL2), were obtained. Restriction fragment length analysis distinguished 13 different patterns among the rdhCBDB1 clones and 14 distinct patterns among the rdhFL2 clones. Ten identical patterns were shared between the rdhCBDB1 and rdhFL2 clones, and inserts of representative rdhFL2 and rdhCBDB1 clones were sequenced. All sequences contained a nearly complete orfA (length of 1,350 to 1,495 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 an additional 250 to 260 nucleotides of the 5⬘ end, probably due to mispriming of the forward primer RRF2 used for PCR. One additional RDH sequence from strain CBDB1 (designated rdh9CBDB1) was obtained with the highly degenerate primers mern2 and mern5 (Fig. 1). The amplicon contained the complete 5⬘ end of orfA including the twin-arginine signal sequence motif, suggesting that the forward primer had bound about 180 nucleotides upstream of the actual target sequence. orfA of rdh9CBDB1 (rdhA9CBDB1) has a length of 1,488 bp and represents the first complete orfA obtained from strain CBDB1 (Fig. 1). Two additional 500-bp sequences, designated rdhA15CBDB1 and rdhA16CBDB1, were obtained from strain
CBDB1 with the primer pair fdehal-rdehal (Fig. 1). tceA-targeted primers yielded an amplicon with genomic DNA from strain FL2 that was very similar to the tceA gene of D. ethenogenes strain 195 (i.e., 99.3% sequence identity at the amino acid level). In contrast, the tceA gene was not found in strain CBDB1 when the same primers were used. Sequence analysis of RDH genes. rdhACBDB1 and rdhAFL2 genes showed highest similarity to a group of genes comprising the tceA gene of D. ethenogenes strain 195, the tceA gene of strain FL2, and tceA genes amplified previously from three chloroethene-dechlorinating enrichment cultures (GenBank accession numbers AAN85590, AAN85592, and AAN85594). A BLASTX search yielded best hits (E value range, 10⫺31 to 10⫺61) for full-length alignments of rdhA1CBDB1 to rdhA14CBDB1 (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 sequences, rdhA1–14CBDB1 and rdhA1–11FL2 were 27 to 33% identical to the tceA gene of strain 195. The 500-bp fragments rdhA15CBDB1 and rdhA16CBDB1 showed 30 and 21% identity to tceA, respectively. Some rdhACBDB1 and rdhAFL2 genes also shared high similarity (BLASTX E values of ⬍10⫺35) with sequence rdh63A (GenBank accession number AAO15649), an RDH gene derived from a 2-bromophenol-degrading microbial consortium (39). Hits with small E values (E value range, 10⫺10 to 10⫺30) were also obtained for alignments of rdhACBDB1 and rdhAFL2 genes with other known reductive dehalogenase genes, i.e., the PCE dehalogenase-encoding genes (pceA genes) from S. multivorans, Dehalobacter restrictus, and Desulfitobacterium spp. and the chlorophenol dehalogenase-encoding genes (cprA genes) from Desulfitobacterium spp. For other functionally assigned genes, e.g., a phosphoglycerate mutase from Clostridium tetanii as well as a periplasmic [Fe] hydrogenase and a molybdenum formylmethanofuran dehydrogenase subunit from Methanosarcina mazei, a BLASTX search resulted in only partial alignments with high E values (⬎10⫺4). Deduced amino acid sequences of nearly complete orfA genes from rdhCBDB1 and rdhFL2 sequences (404 to 489 residues) were used to generate a tree in which the 17 RDH genes (rdhA1–17DE) found in D. ethenogenes strain 195 (48), the
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FIG. 2. Phylogenetic analysis of RDH genes from Dehalococcoides sp. strain CBDB1 (rdhA1–14CBDB1), Dehalococcoides sp. strain FL2 (rdhA1–11FL2), Dehalococcoides sp. strain BAV1 (rdhA1–7BAV1), and D. ethenogenes strain 195 (rdhA1–17DE) (48). The neighbor-joining tree shown was generated from amino acid sequences of nearly complete orfA genes (see Fig. 1 and text for details). Branching points supported by 85 to 100% of 1,000 bootstrap sampling events are indicated by solid circles. Open circles indicate 50 to 84% support by bootstrap sampling. Branching points supported by the maximum parsimony treeing method are marked with (P). Additional clones from strain FL2 (rdhA*FL2, sequences not determined) are indicated together with the rdhACBDB1 genes that have identical restriction patterns. Dotted double, triple, and quadruple lines indicate subclusters of highly similar genes from two, three, or four different Dehalococcoides strains, respectively (see the text for details). tceA, trichloroethene reductive dehalogenase, pceA, tetrachloroethene reductive dehalogenase, cprA,
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seven RDH genes (rdhA1–7BAV1) found in strain BAV1 (19), and reductive dehalogenase genes from other dechlorinating bacteria were included (Fig. 2). All Dehalococcoides RDH genes 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). A second cluster (cluster II) was composed of six rdhACBDB1 genes (rdhA1–6CBDB1), six rdhAFL2 genes (rdhA1–6FL2), two rdhABAV1 genes (rdhA1BAV1 and rdhA7BAV1) and five rdhADE genes (rdhA10–14DE). rdhA7CBDB1 could not be unambiguously assigned to either of the two clusters. rdhA16DE and rdhA17DE shared only limited similarity with rdhACBDB1 and rdhAFL2 genes but grouped with separate clusters comprising cprA genes and pceA genes from Desulfitobacterium spp. and Dehalobacter restrictus, respectively. Several rdhACBDB1 genes shared high sequence similarity with rdhAFL2 genes (Fig. 3). Six rdhACBDB1 genes with 98.5 to 99.8% identity to rdhAFL2 genes and one rdhACBDB1 gene with 100% identity to an rdhAFL2 gene based on the deduced amino acid sequences were identified. In addition, the restriction patterns of three FL2 clone library inserts matched three different restriction patterns of CBDB1 clone library inserts, indicating further pairs of highly similar sequences. Four rdhADE genes were identified that shared 86.5 to 94.4% identity with rdhACBDB1 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 one subcluster. A total of 10 subclusters, each comprising 2 to 4 RDH genes from different strains, were identified (Fig. 2). ISB motifs. Like other dehalogenases, all rdhACBDB1- and rdhAFL2-encoded amino acid sequences contained two ISB motifs in the C-terminal region (Fig. 3). The first ISB motif of all sequences corresponded to the conserved consensus sequence CXXCXXCXXXCP, found in bacterial ferredoxins (8). Variations of this pattern, however, were observed in the second ISB motif. In 14 out of 21 RDH genes in cluster I (Fig. 2), the second ISB motif shared the consensus CXXCXXC XXCP. In the other seven RDH genes of cluster I, the first two cysteine residues of the second ISB motif were separated by three, four, or six residues instead of two (Fig. 3). In all five RDH genes of cluster I with four or six residues between the first two cysteines of the second ISB (i.e., rdhA12–14CBDB1, rdhA10FL2, and rdhA2DE), a fifth cysteine residue was present upstream of the second ISB motif. This motif, CX4CXnCX2-3 CXXXCP, was also found in rdhA3DE and rdhA4DE. In all Dehalococcoides RDH genes outside of cluster I, the second ISB motif was characterized by a long stretch of residues (8 to 21 amino acids) separating the first two cysteine residues (i.e., rdhA1–7CBDB1, rdhA1–6FL2, rdhA7–A17DE, rdhA1–3BAV1, and rdhA7BAV1) (Fig. 3). All RDH genes that contained a second
chlorophenol reductive dehalogenase. The tceA cluster comprises the tceA genes of D. ethenogenes strain 195 (GenBank accession number AAF73916), strain FL2 (accession number AY165309), and three chloroethene-dechlorinating enrichment cultures (accession numbers AAN85590, AAN85592, and AAN85594). The scale bar represents 20% sequence divergence.
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ISB motif with closely linked cysteine residues (cluster I) formed a coherent group of related genes, whereas the other Dehalococcoides RDH genes that contained a second ISB motif with a distant cysteine did not form a phylogenetically consistent group (Fig. 2). Cobalamin-binding consensus sequences. A consensus sequence 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 between the two ISB motifs, whereas the SXL and twin-glycine motifs were located downstream of the second ISB motif. Amino acid stretches between the three conserved motifs of the consensus were longer in rdhA12DE (DXHXXG-X50-SXL-X42-GG) than those described for a group of known cobalamin-dependent enzymes from other prokaryotes (e.g., DXHXXG-X41-SXLX26–28-GG) (26). In addition, two rdhACBDB1 genes, three rdhAFL2 genes, and one rdhABAV1 gene (rdhA3CBDB1 and rdhA4CBDB1, rdhA1–3FL2, and rdhA1BAV1; cluster II) contained the consensus sequence DXXHXXG (Fig. 3). The SXL motif was found in rdhA3CBDB1, rdhA4CBDB1, and rdhA1–3FL2; rdhA1BAV1 contained a serine and a leucine residue separated by five residues in the corresponding region. Four of these genes contained the twin-glycine motif, whereas two genes had a single glycine residue in the corresponding position. N-terminal region of RDH genes. Deduced amino acid sequences of all gene fragments amplified with the primer pair RRF2-B1R contained stretches of hydrophobic residues at their N-terminal ends that were similar to those in known reductive dehalogenases (data not shown). The forward primer RRF2 targets the consensus motif RRXFXK of twin-arginine signal sequences, indicating that all amplified genes contained this sequence, though the exact nucleotide composition is not known due to the degenerate nature of this primer. rdhA9CBDB1, a sequence amplified with the primers mern2 and mern5, contained the complete N-terminal end of orfA, including the motif RRDFMK that is also present in tceA and 13 of the 17 RDH genes in strain 195. orfB sequences. The obtained N-terminal orfB fragments (rdhBCBDB1 and rdhBFL2) encoded mainly hydrophobic amino acid residues, similar to the respective regions of orfB genes linked to known dehalogenase genes, e.g., tceB of D. ethenogenes strain 195 or pceB of S. multivorans. The obtained rdhBCBDB1 and rdhBFL2 fragments were small (⬃90 bp) and therefore did not allow calculation of a phylogenetic tree with high bootstrap values. However, the calculations with deduced amino acid sequences of these small fragments supported the established subclusters of highly similar orfA genes from the different strains. Six rdhBCBDB1 gene fragments were 100% identical to six rdhBFL2 fragments, analogous to the corresponding highly similar pairs of rdhACBDB1 and rdhAFL2 genes. Furthermore, a stable grouping of the other orfB fragments analogous to the
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corresponding orfA subclusters was obtained; branch points were supported by 75 to 93% bootstrap sampling events in all cases except one (bootstrap support for grouping of rdhB11DE with rdhB5CBDB1/rdhB4FL2 was 29%). Physiological tests. In media supplied with 1,2,3-TCB/1,2,4TCB or PeCB as an electron acceptor and inoculated with a TCE-grown culture of strain FL2, no dechlorination products were detected within 6 months of incubation. Also, no dechlorination 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 with methyl viologen as an electron donor. Strain CBDB1 was specifically tested for TceA activity, i.e., dechlorination of TCE to VC (28, 29). However, no VC formation was observed. DISCUSSION Thirty novel RDH genes were amplified from Dehalococcoides sp. strain CBDB1 and Dehalococcoides sp. strain FL2 that all differ from the 17 RDH genes identified in the genome of D. ethenogenes strain 195 and the seven RDH genes amplified from Dehalococcoides sp. strain BAV1. Although the substrate specificities of the amplified genes cannot be inferred, there is strong evidence that all RDH genes identified in the four Dehalococcoides strains represent true homologs (genes with a common ancestry) of the biochemically characterized TCE dehalogenase gene of strain 195. The evidence is as follows. (i) All amplified RDH genes share 27 to 33% sequence identity with the tceA gene at the amino acid level over the whole length of the sequence (449 to 489 residues for all rdhACBDB1 and rdhAFL2 genes except rdhA10FL2, which is 404 residues in length), which allows the inference of a homologous relationship as previously described by Brenner et al. (7) and Rost (40). (ii) All RDH genes share two functional domains that determine the structure and/or localization of the protein, i.e., the N-terminal twin-arginine signal sequence and the ISB region near the C terminus comprising two separate ISB motifs. (iii) All RDH genes are organized in operons with a downstream orfB gene encoding a putative membrane anchor. (iv) The high number of RDH genes (17 RDH genes in strain 195, at least 14 RDH genes in both strains CBDB1 and FL2, and 7 RDH genes in strain BAV1) makes convergent evolution unlikely. Sequence comparison of RDH gene fragments from all four Dehalococcoides isolates demonstrated that highly similar genes are shared among strains and that unique RDH genes that distinguish different Dehalococcoides strains exist. The subclusters of highly similar genes can be interpreted as orthologs, i.e., homologs derived by a speciation event (45). The 16S rRNA genes of strain CBDB1 (GenBank accession number 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 to the tceA gene of D. ethenogenes strain 195; see the legend of Fig. 2 for other designations). Also included is sequence rdh63A, an RDH gene obtained from a bacterial consortium (39). ClustalW alignments were manually corrected to align putative cofactor binding sites. Conserved residues of the two ISB motifs and cobalamin-binding consensus sequences are highlighted in gray. Boxes mark cobalamin-binding motifs in rdhA3CBDB1 and rdhA4CBDB1, rdhA1–3FL2, rdhA12DE, and rdhA1BAV1. In rdhA12DE and rdhA1BAV1, the SXL motif could not be unambiguously located (see the text for details).
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are 100% identical and share 98.5% sequence identity with the 16S rRNA gene of strain 195 (accession number AF004928). The topology of each orthologous group of RDH genes from strains CBDB1, FL2, and 195 is consistent with 16S rRNA gene-based phylogeny, supporting the idea that strains FL2 and CBDB1 are more closely related to each other than to strain 195. The 16S rRNA gene of strain BAV1 (accession number AY165308) exhibits only one base difference (99.9% identity) from the 16S rRNA genes from strains CBDB1 and FL2. However, no stronger relationship was identified between rdhABAV1 genes and RDH genes from strains CBDB1 and FL2 than between rdhABAV1 genes and rdhADE genes. Analysis of orfB gene fragments from all four Dehalococcoides isolates showed that sequence relationships between orfB genes correspond to those of orfA genes, suggesting that both genes have coevolved and are functionally linked. In conclusion, our data provide evidence that RDH gene duplication and divergence occurred in a common Dehalococcoides ancestor and not after the speciation into the currently known strains. This is important because it indicates that the presence of multiple RDH genes in Dehalococcoides spp. is due not to rapid adaptation to the presence of anthropogenic halogenated compounds released within the last century but to much older evolutionary events. The high sequence identity of the tceA genes from strains 195 and FL2, compared to the other orthologous RDH genes, might indicate a lateral gene transfer event. The presence of multiple nonidentical RDH genes in Dehalococcoides strains is consistent with the observation that the different strains use different chlorinated electron acceptors. Several RDH genes are present in individual strains that do not have an ortholog among the known genes of the other strains. Many of the RDH genes found in strains CBDB1, FL2, and BAV1 do not have orthologs in the completely sequenced genome of strain 195, suggesting that these strains possess dechlorination activities not present in strain 195. The presence or absence of tceA genes is consistent with the observed activity of the different strains. TCE supports growth of strain FL2 and strain 195; accordingly, the tceA gene was found in these strains. Strain BAV1 (13) and strain CBDB1 do not grow with TCE. Consistently, the tceA gene was not found in strain BAV1 (12) or strain CBDB1. Strains CBDB1 and 195, but not strain FL2, dechlorinate chlorinated benzenes, suggesting that chlorobenzene reductive dehalogenases are present only in strains CBDB1 and 195. However, specific culture conditions might be necessary to induce chlorobenzene dechlorination in strain FL2. Substrate-dependent differential induction of dehalogenases has been demonstrated in Desulfitobacterium spp. (reviewed in reference 44). In strain CBDB1, different 1,2,3TCB, PeCB, and HCB dechlorination rates were obtained with cells pregrown on different chlorobenzene congeners as electron acceptors, suggesting that dehalogenases are induced by their respective substrates (i.e., electron acceptors) (18). In contrast, PCE dechlorination was constitutive in cultures of strain 195 grown on TCE, 1,1-DCE, or dichloroethane (32). All RDH genes contain the conserved functional domains characterized in reductive dehalogenases (16, 43). The presence of a twin-arginine signal sequence in RDH genes is consistent with the hypothesis that these genes are involved in respiratory reductive dehalogenation, because such motifs are predominantly found in membrane-associated proteins in-
APPL. ENVIRON. MICROBIOL.
volved in respiratory electron transport (6, 43). Furthermore, deduced amino acid sequences of all RDH genes contain two ISB motifs and could therefore form two spatially linked ironsulfur clusters. Iron-sulfur clusters have been detected in most reductive dehalogenases and probably mediate electron transfer to the active site containing the corrinoid (5, 34, 47). Indications for the involvement of a corrinoid cofactor in catalysis have been found for most reductive dehalogenases from bacteria that couple reductive dechlorination to growth (16, 43). Recently, a norpseudovitamin B12 was identified as the cofactor of the PCE dehalogenase of S. multivorans (21); however, a corrinoid binding motif was not identified in the amino acid sequence of the protein (36). From subsets of cobalamin-dependent methyltransferases and isomerases of different bacterial species including E. coli, Propionibacterium freudenreichii subsp. shermanii, and Clostridium sp., a cobalamin-binding consensus sequence (DXHXXG. . .SXL. . .GG) has been established (26). In these methyltransferases and isomerases, the dimethylbenzimidazole ligand of the cobalt center is replaced by the conserved histidine residue of the consensus sequence. In our study, such a cobalamin-binding consensus sequence could be identified in a group of seven Dehalococcoides RDH genes. Other RDH genes, including all genes encoding characterized reductive dehalogenases, lack the cobalamin-binding consensus sequence. Electron paramagnetic resonance spectroscopy data indicated that the chlorophenol dehalogenase of Desulfitobacterium dehalogenans and the PCE dehalogenase of S. multivorans contain the corrinoid in the base-off form (21, 34, 47). This finding raises the question of whether the RDH genes containing the cobalamin-binding consensus sequence bind the corrinoid cofactor differently and exhibit a different catalytic mechanism. However, cobalamin binding by histidine ligation does not determine a specific reaction mechanism, since different catalytic mechanisms have been identified in methyltransferases and isomerases (26). Moreover, other subfamilies of cobalamin-dependent methyltransferases and isomerases lack the cobalamin binding consensus and do not bind cobalamin via histidine coordination (4, 26). Though we did not identify the function of the Dehalococcoides RDH genes, this study provides essential sequence data that are useful for the identification of Dehalococcoides reductive dehalogenase genes from peptide fragments and for the design of primers for transcription analysis of specific RDH genes. ACKNOWLEDGMENTS This work was supported by the Deutsche Forschungsgemeinschaft project number AD 178/1 to L.A., by a National Science Foundation CAREER award (award number 0090496) to F.E.L., and, in part, by the U.S. Strategic Environmental Research and Development Program. D. S. Mern was involved in initial experiments with the cloning of gene rdh9CBDB1. We thank G. Wagner for technical assistance, B. Lynch for gene sequencing, Y. Sung for supplying cultures of Dehalococcoides sp. strain FL2, and U. Lechner for communicating fdehal and rdehal primer sequences. REFERENCES 1. Adrian, L., W. Manz, U. Szewzyk, and H. Go ¨risch. 1998. Physiological characterization of a bacterial consortium reductively dechlorinating 1,2,3- and 1,2,4-trichlorobenzene. Appl. Environ. Microbiol. 64:496–503. 2. Adrian, L., U. Szewzyk, J. Wecke, and H. Go ¨risch. 2000. Bacterial dehalorespiration with chlorinated benzenes. Nature 408:580–583. 3. Adrian, L., and H. Go ¨risch. 2002. Microbial transformation of chlorinated benzenes under anaerobic conditions. Res. Microbiol. 153:131–137.
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