Biochemical and genetic bases of dehalorespiration



Some anaerobic bacteria can efficiently eliminate one or more halide atoms from halogenated compounds such as chlorophenols and chloroethenes through reductive dehalogenation. During this process, the bacteria utilize halogenated compounds as the terminal electron acceptors in their anaerobic respiration, called dehalorespiration, to yield energy for growth. Currently the genera of Desulfitobacterium and Dehalococcoides occupy the major part of the dehalorespiring isolates. The former can acquire energy not only by dehalorespiration but also by other respirations utilizing organic compounds and metals. In sharp contrast, the latter is specialized in dehalorespiration and plays a crucial role in the detoxification of chlorinated compounds in nature. From these bacteria, various reductive dehalogenases, which catalyze the dehalogenation reaction, were purified and their corresponding genes were identified. Most reductive dehalogenases exhibit similar features such as the presences of a Tat (twin arginine translocation) signal sequence, two Fe-S clusters, and a corrinoid cofactor. Some of dehalogenase-encoding genes are found to be flanked by insertion sequences. Thus, dehalogenase genes act as a catabolic transposon, and genetic rearrangements mediated by transposable elements occur well in dehalorespirers. Moreover, the genome sequences of some dehalorespiring bacteria provide many insights into the mechanism of dehalorespiration and the evolution of a dehalogenase gene. © 2008 The Japan Chemical Journal Forum and Wiley Periodicals, Inc. Chem Rec 8: 1–12; 2008: Published online in Wiley InterScience ( DOI 10.1002/tcr.20134


Human activity over the past century had caused enormous environmental pollution by halogenated aliphatic compounds, as well as halogenated aromatic compounds. Because of their toxicity, the persistence and bioaccumulation of these halides in the biosphere have caused enhanced public concern over the possible effects on human health. Therefore, various remediation approaches including physicochemical and biochemical processes have been applied to the environmental pollution. Among them, usage of dechlorinating bacteria is one of the promising approaches to restore contaminated subsurface environments. For example, rapid and complete in situ bioremediation approaches have been successfully applied to tetrachloroethene (also referred to as perchloroethene, PCE) and trichloroethene (TCE). PCE and TCE have been widely used as solvents in the dry-cleaning industry and as metal degreasing agents and are recognized among the most common groundwater contaminants due to spillage and leakage. Under anaerobic conditions, PCE is successively converted to TCE, dichloroethenes [DCEs, mainly cis-1,2-dichloroethene (cis-DCE)], vinyl chloride (VC), and the nontoxic end product ethene by microbial reductive dehalogenation (Fig. 1). In all reported examples of biologically catalyzed reductive dehalogenation, the halogen atoms are released as halide anions. In this process, chloroethenes are used as the terminal electron acceptors in their anaerobic respiration called dehalorespiration, halorespiration, or halidogenesis.1–3 This growth-coupled process would be more effective for in situ bioremediation rather than the co-metabolic or abiotic dehalogenation. The co-metabolic reductive dehalogenation is defined as a fortuitous reductive dehalogenation uncoupled to the growth.

Figure 1.

Reductive dechlorination pathways for chloroethenes by the dehalorespiring bacteria. The dashed arrow shows the co-metabolic dechlorination. The co-metabolic dechlorination activity of Dehalococcoides strains VS and KB1/VC has not been determined. GenBank accession numbers of 16S rDNA sequences are as follows. Desulfitobacterium sp. strain PCE1: X81032; Desulfitobacterium sp. strain KBC1: AB194704; Desulfitobacterium hafniense strain PCE-S: AJ512772; Desulfitobacterium hafniense strain TCE1: X95742; Desulfitobacterium hafniense strain Y51: AB049340; Dehalobacter restrictus strain PER-K23: U84497; Dehalobacter restrictus strain TEA: Y10164;98Sulfurospirillum multivorans: X82931; Sulfurospirillum halorespirans strain PCE-M2: AF218076;99Desulfuromonas michiganensis strain BB1: AF357915;100Desulfuromonas michiganensis strain BRS1: AF357914;100Geobacter lovleyi strain SZ: AY914177; Dehalococcoides ethenogenes strain 195: AF004928; Dehalococcoides sp. strain FL2: AF357918; Dehalococcoides sp. strain VS: AY323233; Dehalococcoides sp. strain BAV1: AY165308; Dehalococcoides sp. strain GT: AY914178; Dehalococcoides sp. strain KB1/VC: AY146779. PCE = perchloroethene; TCE = trichloroethene; DCE = dichloroethene; VC = vinyl chloride.

In fact, the dehalorespiring bacteria have been shown to play an important role in transformation and detoxification of chlorinated compounds such as PCE and TCE. However, it should be noted that the source of halogenated molecules is not only anthropogenic but is also of biological or geogenic origins.4–7 More than 3800 organohalogen compounds, mainly containing chlorine or bromine but a few with iodine and fluorine, are produced by living organisms or are formed during natural abiogenic processes.7 The production of highly reactive and toxic VC was also demonstrated in significant amounts in terrestrial environments.8,9 The existence of reductively dehalogenating microbes was detected in the bromophenol-producing marine sponge Aplysina aerophoba.10 Furthermore, it has been demonstrated that the mixed anaerobic microbial cultures or the pure cultures of several dehalorespiring Desulfitobacterium spp. were capable of the complete demethylation and dechlorination of fungal chlorinated hydroquinone metabolites.11 These studies revealed the critical involvement of microorganisms in the global halogen cycle.

Various anaerobic dehalorespiring bacteria have been increasingly isolated and identified. Among them, Desulfitobacterium strains are relatively easy to grow under laboratory conditions, allowing a number of isolations and making them promising model systems. On the other hand, Dehalococcoides strains usually grow slowly with a doubling time of 1 to 2 days and have complicated nutritional requirements. Dehalococcoides strains, however, have attracted much attention because laboratory and field studies demonstrated a significant role in bioremediation. To elucidate the mechanism of dehalorespiration, various studies have been extensively conducted by many workers.1,2,12–20 In our laboratory, the Desulfitobacterium hafniense strain Y51 was isolated from PCE-contaminated soil in Japan, based on its ability to dechlorinate PCE to cis-DCE efficiently.21 This review paper focuses on recent advances in biochemical and genetic studies on dehalorespiration.

Dehalorespiring Bacteria

All of the known dehalorespiring microorganisms are bacteria, and their dehalogenation capacities are highly strain-dependent. Anaerobic bacteria that can grow with chloroethenes as final electron acceptors include Dehalobacter, Dehalococcoides, Desulfitobacterium, Desulfuromonas, Geobacter, and Sulfurospirillum (Fig. 1). Members of the Desulfitobacterium spp. belonging to the phylum of Firmicutes are known as versatile microorganisms that can utilize electron acceptors other than chlorinated compounds. In the case of Desulfito. hafniense Y51, fumarate, thiosulfate, sulfite, nitrate, nitrite, dimethylsulfoxide (DMSO), trimethylamine N-oxide, As(V), and the soluble form of Fe(III) are confirmed to serve as growth-supporting electron acceptors.21,22 Most Desulfitobacterium isolates have the capability to reductively dechlorinate chloroethenes, chlorophenols, or both. Three Desulfitobacterium hafniense strains Y51, TCE1, and PCE-S, dechlorinate PCE to cis-DCE via TCE but do not dechlorinate chlorophenols.23,24 In contrast, Desulfitobacterium hafniense DCB-2 and Desulfitobacterium dehalogenans IW/IU-DC1 utilize chlorophenols in the dehalorespiration process but not chloroethenes.23,25–27Desulfitobacterium sp. strains PCE1 and KBC1 dechlorinate PCE to yield TCE and also dechlorinate chlorophenols.28,29

The well-studied organisms, Sulfurospirillum multivorans and Dehalobacter restrictus PER-K23 dechlorinate PCE to cis-DCE.30–33S. multivorans is a Gram-negative anaerobic spirillum, which belongs to the e-subdivision of proteobacteria. The Dehalobacter genus belongs to Firmicutes and is allied with the genus Desulfitobacterium; however, the dehalorespiration is sole system of energy production in the genus Dehalobacter.

Although the above-mentioned strains can utilize PCE or TCE as the electron acceptor, they cannot completely dechlorinate cis-DCE or VC to ethene. The genus Dehalococcoides, which is closely related to a member of the Chloroflexi phylum (green nonsulfur bacteria), is only known to reductively dechlorinate cis-DCE or VC to ethene (Fig. 1). Dehalococcoides ethenogenes 195 and Dehalococcoides sp. FL2 respectively dechlorinate PCE and TCE to ethene.34–36 However, these two strains are unable to use VC as the electron acceptor. Thus, the slow dechlorination of VC to ethene is considered to proceed in a co-metabolic fashion uncoupled to energy production.37 On the other hand, four other Dehalococcoides strains, BAV1, VS, GT, and KB1/VC, can use VC as the electron acceptor in their dehalorespiration and can dechlorinate VC to ethene efficiently.38–42 Among these four Dehalococcoides, strain GT and highly enriched strain KB-1/VC can dechlorinate TCE. In contrast, Dehalococcoides sp. CBDB1 has a different dechlorination spectrum. For instance, strain CBDB1 dechlorinates chlorobenzenes and dioxins such as 1,2,3-trichlorobenzene, 1,2,4-trichlorobenzene, 2,3-dichloro-p-dibenzodioxin, and 2,3,7,8-tetrachloro-p-dibenzodioxin.43,44 In the genus Dehalococcoides, the dehalorespiration is solely an energy preservation system. These isolates exhibit a metabolic specialization, using only H2 as an electron donor and chlorinated compounds as electron acceptors to support growth.

Ecological interactions among dehalorespiring bacteria and other members of the microbial community play an important role in anaerobic dehalogenation in nature. Most dehalorespiring bacteria are able to use H2 as an electron donor. Therefore, hydrogenotrophic populations, e.g., methanogens or sulfate-reducing bacteria, compete with dehalorespiring populations for H2.45–50 On the other hand, several studies demonstrated that the syntrophic relationship is present among dehalorespiring bacteria and other nondehalorespiring bacteria via interspecies hydrogen transfer, and this stimulates the reductive dechlorination. For instance, PCE dehalorespiring Desulfito. hafniense TCE1 was cocultured with sulfate-reducing Desulfovibrio fructosivorans in the presence of fructose, PCE, and sulfate.51,52 Under this condition, Desulfo. fructosivorans fermented fructose to yield H2, and the produced H2 was used by strain TCE1 as the electron donor. A positive effect of methanogens on the VC dechlorination by Dehalococcoides spp. was also reported.53 Although Methanosarcina spp. are generally regarded as aceticlastic methanoarchaea, they also oxidize acetate to CO2 and H2. The produced H2 drove the hydrogenotrophic dehalorespiration of VC to ethene by Dehalococcoides in the VC-dechlorinating consortia. Recently, improvement in the limited growth and dechlorination activity of Dehalo. ethenogenes 195 and Dehalo. sp. BAV1 was observed under conditions with high vitamin B12.54 Although vitamin B12 is produced by a wide variety of bacteria during their metabolic process, Dehalococcoides are known to be unable to biosynthesize it by a de novo pathway. Strain 195 cells grew with lactate as the electron donor 1.5 times greater in co- and tricultures with Desulfovibrio desulfuricans and/or Acetobacterium woodii than they grew alone.54Desulfo. desulfuricans is an organism that converts lactate to generate H2 and acetate, whereas A. woodii is an organism that generates vitamin B12de novo during its metabolic process.

Biochemistry of Reductive Dehalogenases

The reductive dechlorination reactions are catalyzed by the reductive dehalogenase. From PCE-dechlorinating Desulfito. hafniense strains Y51, TCE1 and PCE-S, and Dehalobacter restrictus PER-K23, PCE dehalogenase (PceA) enzymes, which dechlorinate PCE to cis-DCE via TCE, are purified and characterized. These PceA proteins exhibit nearly 100% sequence identity.55,56 In contrast, PceA of S. multivorans also catalyzes the same reaction; however, it shows only 27% sequence identity to the above-mentioned PceA.57 In the case of Dehalococcoides, the detailed studies on the biochemistry of reductive dehalogenases are hampered by difficulties in obtaining sufficient biomass. Two dominant reductive-dehalogenases were purified from strain 195, and their substrate specificities were determined. PCE dehalogenase catalyzes the dechlorination reaction from PCE to TCE, and TCE dehalogenase (TceA) catalyzes the dechlorination from TCE to VC.58 VcrA dehalogenase of strain VS catalyzes the dechlorination reaction of cis-DCE to ethene via VC.40

All reductive dehalogenases characterized so far are associated with the cytoplasmic membrane, reinforcing their role in membrane-associated electron transport-coupled phosphorylation. The N-terminal region of most reductive dehalogenases contains the Tat (twin arginine translocation) consensus sequence “RRXFXK,” which is involved in the translocation of the protein into or across the cytoplasmic membrane.59 In this translocation system, the newly synthesized secretory protein together with a cofactor, in some cases with the aid of a chaperone, are folded in the cytoplasm and translocated without energy consumption. The Tat sequences are proteolytically cleaved during protein maturation. In the case of Desulfito. hafniense strain Y51, the processed PceA that lost the N-terminal 39 aa is located in the periplasm, while unprocessed protein with the leader peptide is present in the cyloplasm.55 Thus, mature PceA is functional at the periplasm. The localization of the PceA of S. multivorans was also studied.60 When the cells were grown with pyruvate plus fumarate, a major part of the enzyme was either localized in the cytoplasm or was membrane-associated facing the cytoplasm. In cells grown on pyruvate or formate as electron donors and PCE or TCE as electron acceptor, most of the enzyme was detected at the periplasmic side of the cytoplasmic membrane. These results indicate that the localization of the enzyme is dependent on the electron acceptor utilized in this spirillum.

Most reductive dehalogenases contain a corrinoid, a derivative of vitamin B12, at the catalytic center as a cofactor. Co(I) corrinoid in its free form has previously been shown to reductively dechlorinate PCE and other chlorinated ethenes in homogeneous aqueous solutions.61,62 The involvement of a Co(I) corrinoid in the catalytic activity of reductive dehalogenases has been demonstrated by the reversible inactivation of corrinoid using propyl iodides. The importance of the corrinoid in PceA dehalogenase is also supported by the fact that the lack of the corrinoid cofactor results in loss of the PCE-dechlorination ability in S. multivorans.63 The corrinoid cofactor of PceA of S. multivorans was purified, and the structure was determined to be norpseudovitamin B12.64 In contrast, the corrinoid isolated from the PceA of Dehalobacter restrictus PER-K23 had properties the same as those of commercially available cobalamin.56 The PceA dehalogenase of Desulfito. hafniense Y51 and S. multivorans was heterologously expressed in Escherichia coli, but dechlorinating activity was not observed in both cases.55,57 It was suggested that the enzyme was not folded properly and/or that the proper cofactor was absent in E. coli. All functionally characterized reductive dehalogenase lack the cobalamin-binding consensus sequence “DXHXXG…SXL…GG,” which is identified in the subsets of cobalamin-dependent methyltransferases and isomerases.65 Recently, Hölscher et al. identified such a motif in a group of seven reductive dehalogenase homologues of Dehalococcoides.66

Consensus sequences similar to the binding motifs for two Fe-S clusters are in the C-terminal region of the reductive dehalogenases. In the case of Desulfito. hafniense Y51, the Fe-S cluster-binding motif is comprised of “CXXCXXCXXXCP” and “GXXCXXCXXXCS.”55 The existence of Fe-S clusters of PceA of Dehalobacter restrictus PER-K23 and ortho-chlorophenol reductase (CprA) of Desulfito. dehalogenans IW/IU-DC1 was investigated by electron paramagnetic resonance spectroscopy.56,67,68 It was demonstrated that the former contained two [4Fe-4S] clusters and that the latter contained one [4Fe-4S] and one [3Fe-4S] clusters.

As a feasible reaction mechanism for the reductive dechlorination of PCE, the schemes involving one corrinoid and two Fe-S clusters are proposed (Fig. 2).1,69–73 In Scheme 1, the one-electron transfer from the Co(I) corrinoid to PCE yields a trichlorovinyl radical and a chloride ion. The trichlorovinyl radical is reduced to the anion by electron transfer from one of the Fe-S clusters, and the anion combines with a proton to yield TCE. The Co(II) corrinoid is reduced back to the Co(I) state by the other Fe-S cluster. In Scheme 2, the initially generated trichlorovinyl radical reacts with the Co(II) corrinoid, forming the trichlorovinyl-Co(III) corrinoid. The trichlorovinyl-Co(III) corrinoid is then reduced by electron transfer from one of the Fe-S clusters, resulting in the Co(II) corrinoid and a trichlorovinyl anion that combines with a proton to yield TCE. McCaoulley et al. estimated that PceA from S. multivorans achieves a rate enhancement of 4800-fold over the reaction with a nonenzymatic cofactor-dependent reaction.62,71,74

Figure 2.

Proposed perchloroethene dechlorination mechanisms catalyzed by reductive dehalogenases with a corrinoid and two Fe-S clusters. The trichlorovinyl radical formed by one-electron transfer by the Co(I) corrinoid is reduced by one of the Fe-S centers (Scheme 1) or reacts with the Co(II) corrinoids (Scheme 2).

Gene Clusters of Reductive Dehalogenases

Although dehalorespiring bacteria are classified into the evolutionarily distant species, it is noteworthy that the reductive dehalogenase systems are closely related to each other. Most reductive dehalogenase genes are always organized in an operon comprised of at least two genes (Fig. 3). In the case of Desulfito. hafniense Y51, the PCE dehalogenase encoding the pceA gene is flanked by the pceB gene encoding a protein containing a three-transmembrane domain.55,75 Although the function of PceB has not been experimentally verified, this protein has been assumed to act as a membrane anchor protein of PceA on the periplasmic face of the inner membrane (Fig. 4). This is supported by the fact that PceA is located in the periplasm even without an obvious transmembrane domain as described.

Figure 3.

Organization of the reductive dehalogenase genes. An identical pattern indicates similar genes as described in the inner box. GenBank accession numbers of these genes are as follows. Desulfitobacterium hafniense strain Y51 pce genes: AY706985; Desulfitobacterium hafniense strain TCE1 pce genes: AJ439608; Desulfitobacterium sp. strain PCE-S pce genes: AY216592; Dehalobacter restrictus strain PER-K23 pce genes: AJ439607; Sulfurospirillum multivorans pce genes: AF022812; Dehalococcoides ethenogenes strain 195 tce genes: CP000027; Dehalococcoides sp. strain VS vcr genes: AY322364; Desulfitobacterium dehalogenans strain IW/IU-DC1 cpr genes: AF115542. Desulfitobacterium hafniense strain DCB 2 cpr genes can be obtained from the Joint Genomic Institute (

Figure 4.

Schematic presentation of maturation and transport processes of PCE reductive dehalogenase (PceA) in Desulfitobacterium hafniense strain Y51. PCE = perchloroethene; TCE = trichloroethene; DCE = dichloroethene; Tat = twin arginine translocation.

Downstream of pceB, the pceC, and pceT genes exist in strain Y51. PceC is a protein with unknown functions, but similar to those of the NirI/NosR family membrane-binding transcriptional regulators involved in nitrous oxide respiration (Fig. 3).76,77 PceC contains a five-transmembrane domain, a flavin mononucleotide-binding domain, and a C-terminal polyferredoxin-like domain. The cprC gene of Desulfito. dehalogenans IW/IU-DC1 and the vcrC gene of Dehalococcoides sp. strain VS also encode the NirI/NosR-type protein.40,78 Moreover, the pceABC genes of strain Y51 and the vcrABC genes of strain VS are co-transcribed, implying that these genes should have important roles in dehalorespiration.40,79 On the other hand, the PceT of strain Y51 is similar to a trigger factor involved in protein-folding. As described, PceA is translocated to the periplasm only if the PceA precursor is previously folded properly with the corrinoid cofactor. Therefore, PceT might contribute to the correct folding of the PceA precursor protein during a Tat secretion process (Fig. 4).

In the genome of Desulfito. dehalogenans IW/IU-DC1 and Desulfito. hafniense DCB2, the larger components of the ortho-chlorophenol reductive dehalogenase gene clusters were identified. The cpr regulon contains eight genes: cprT, cprK, cprZ, cprE, cprB, cprA, cprC, and cprD (Fig. 3).78 The transcription of the cpr genes is regulated by CprK, a member of the CRP-FNR (cAMP-binding protein/fumarate nitrate reduction regulatory protein) family regulators that are ubiquitous in bacteria.78,80 Recent in vivo and in vitro studies revealed that high-affinity interaction of a chlorinated aromatic compound with an effector domain of CprK triggers binding of CprK to an upstream target DNA sequence called the “dehalobox,” which leads to transcriptional activation of the cpr gene cluster.80,81 Moreover, the crystal structures of the oxidized Desulfito. hafniense DCB2 CprK with a ligand 3-chloro-4-hydroxyphenylacetate and that of the reduced Desulfito. dehalogenans IW/IU-DC1 CprK (both proteins are 89% identical) without the ligand were solved.82 Thus comparison of both structures permits identifying the allosteric changes induced by ligand binding.

Genomes of Dehalorespiring Bacteria

Currently, the genome sequences of Dehalo. ethenogenes 195 (CP000027), Dehalo. sp. CBDB1 (AJ965256), Dehalo. sp. BAV1 (CP000688), Desulfito. hafniense DCB-2 (AAAW00000000), Desulfito. hafniense Y51 (AP008230), Anaeromyxobacter dehalogenans 2CP-C (CP000251),83,84 and Geobacter lovleyi SZ (AAVG00000000)85 are available.

Detailed analyses of the genomes of these dehalorespirers provided much insight into their biological characteristics.86–89 The genome of Desulfito. hafniense Y51 is a 5,727,534 bp circular chromosome harboring 5060 predicted protein-coding sequences (CDSs) (Table 1). Among them, 25% are not related to any known function. The genome contains only two reductive dehalogenase genes, including the previously characterized pceA, of which the number is fewer than the number reported in most other dehalorespiring strains. The second reductive dehalogenase gene of strain Y51 is very similar to the reductive dehalogenase termed crdA of Desulfito. hafniense PCP-1, which exhibits dechlorinating activity toward several polychlorophenols, such as 2,4,6-TCP.90 Also, crdA showed no homology with any known dehalogenases, indicating a new type of reductive dehalogenase. However, it is uncertain whether strain Y51 dechlorinates polychlorophenols.

Table 1. Comparison of the genomes of three dehalorespiring strains.
StrainDesulfitobacterium hafniense Y51Dehalococcoides ethenogenes 195Dehalococcoides sp. CBDB1
  1. a

    GenBank accession numbers of genome sequences are as follows. Desulfitobacterium hafniense strain Y51: AP008230; Dehalococcoides ethenogenes strain 195: CP000027; Dehalococcoides sp. strain CBDB1: AJ965256. Modified from Nonaka et al.88 and Seth-Smith et al.89 with permission.

Taxonomic positionClostridiaChloroflexiChloroflexi
Size (bp)5,727,5341,469,7201,395,502
G + C content (%)47.447.048.9
No. of rRNA operons (16S-23S-5S)611
No. of predicted CDSs5,0601,5911,458
No. of dehalogenase genes21732

In the genome of strain Y51, several genes required for the reductive dehalogenase were found. First, the genome encodes the complete corrinoid biosynthetic pathway and the corrinoid scavenging pathway required by the reductive dehalogenases (Fig. 4). Second, four tatA-like genes and a tatC-like gene are present in the genome of Y51, as they are situated in Bacillus subtilis.91 Tat components are considered to be required by the translocation of PceA with a Tat signal to the periplasm as described above.

The large genome of strain Y51 indicates a more versatile microorganism that can utilize a larger set of electron acceptors.88 A large family of 59 DMSO reductase A CDSs is present in operons with CDSs for Fe-S accessory proteins and anchor proteins. Also present are CDSs for fumarate, sulfate, sulfite, nitrate, and nitrite reductases. These, together with at least six c-type cytochromes, are likely to be responsible for the wide range of electron acceptors available to strain Y51.

On the other hand, the Dehalo. ethenogenes 195 and Dehalo. sp. CBDB1 have a relatively small genome with a narrow metabolic repertoire.86,87 These genomes share a high degree of genomic similarity and synteny in nearly all housekeeping genes. The complete 1,469,720 bp genome of strain 195 revealed 17 potential genes that appeared homologous to genes encoding biochemically purified reductive dehalogenases (Table 1). On the other hand, 32 putative reductive dehalogenase genes were identified in the complete 1,395,502 bp genome of strain CBDB1 (Table 1). Neither strain 195 nor strain CBDB1 encodes the genes that specify de novo corrinoid biosynthesis; however, both genomes contain the corrinoid salvage operon. This lack agrees with the fact that they require vitamin B12 for growth. The Dehalococcoides genome showed a close relationship between most of the reductive dehalogenase genes and the genes encoding transcription regulators such as two-component regulatory systems or MarR-type transcriptional regulators, indicating that expression of these genes is highly regulated. Recent proteomic studies of strain 195 indicated that genes predicted to encode four reductive dehalogenases including TceA, a periplasmic [Ni/Fe] hydrogenase, and a putative formate dehydrogenase exhibited the highest overall expression levels.92,93 Interestingly, the formate does not donate electrons for reductive dechlorination and cells lack formate dehydrogenase activity.92

Comparative genomics of Desulfito. hafniense Y51 and Dehalo. ethenogenes 195 allow narrowing down the potential candidates implicated in the dechlorination process.86,88 In the case of reductive dehalogenase gene clusters, PceB and PceT of strain Y51 have no obvious orthologs in strain 195, providing some circumstantial evidence that the membrane-anchoring mechanism is not conserved or dispensable and that the PceT trigger factor-like folding chaperone might not be essential or may be complemented by a nonhomologous protein. Nonaka et al. identified the closest 18 reciprocal best hits homologous CDSs that showed more homology to each other than they showed to any other paralogous sequence from another strain.88 Surprisingly, PceA was not included in this group. Included in the group of 18 reciprocal best hits were the PceC-like putative transcription regulator, the large subunit and the maturation factor of a Hup-type [Ni-Fe] hydrogenase, and the multiple subunits of corrinoid transport systems, which clearly highlight the importance of scavenging corrinoid cofactors from the environment.

Rearrangements of pce Gene Cluster

Genomic rearrangements play an important role in the evolution of dehalogenating bacteria. Comparative sequence analysis revealed the vestiges of an interchromosomal rearrangement during evolution. The pceABCT gene cluster of Desulfito. hafniense Y51 is surrounded by two nearly identical copies of ISDesp, which includes the tnpA gene encoding the IS256 type transposase (Fig. 5). The direct repeat sequence is found just upstream of the first ISDesp and downstream of the second ISDesp. Thus, the pceABCT gene cluster seems to be inserted into the chromosome of strain Y51 as a composite transposon. During subculturing without chloroethenes, a circular molecule carrying an entire pceABCT gene cluster and two copies of terminal ISDesp was detected at low frequency in strain Y51, indicating that the catabolic transposon can still function and be excised from the chromosome.94 During subculturing, strain Y51 also gave rise spontaneously to two types of PCE-nondechlorinating variants termed SD (small deletion) and LD (large deletion) at relatively high frequency (Fig. 5). The SD variant was generated from the deletion of a single ISDesp1 where a part of ISDesp1 is a promoter region of the pceABC gene cluster; thereby, this variant abolished the pceABC transcription and accordingly the PCE-dechlorination capability. The LD variant came from the homologous recombination between ISDesp1 and ISDesp2, resulting in the excision of the pceABCT gene cluster and one copy of ISDesp to form a circular molecule. Thus, in the absence of chloroethenes, several modes of genetic rearrangement in strain Y51 occur around the pceABCT gene cluster. In the presence of 1 mM chloroform (CF), a significant growth inhibition of strain Y51 was observed.79 However, CF did not affect the growth of PCE-nondechlorinating SD and LD variants. Therefore, the PCE-nondechlorinating variants, mostly the LD variant, became predominant when wild-type Y51 was cultivated in the presence of 1 mM CF. Moreover, such a growth inhibitory effect was also observed in the presence of carbon tetrachloride (CT) at 1 mM, but not carbon dichloride even at 1 mM. The difference in the growth inhibition between wild-type Y51 and the SD/LD variants seems to be caused by the presence or absence of three proteins encoded by pceA, pceB, and pceC. Our hypothesis is that one of these three proteins may interact with CF or CT to inhibit the respiratory chain of strain Y51.

On the other hand, the 9.9 kb catabolic transposon, Tn-Dha1 containing the pceABCT, tnpA2, and tatA genes surrounded by two identical copies of ISDha1, was isolated from the Desulfito. hafniense TCE1 (Fig. 3).95 The pceABCT genes of strain TCE1 shared a 99.7% identity with those of strain Y51. Although the information about the strain TCE1 itself after genetic rearrangement is not available, circular intermediates containing the pceABCT gene cluster and either one or two copies of ISDha1 are detected.95 These results suggest that these strains acquired the PCE-dechlorination ability via transposition of the composite transposon at a different gene locus. Indeed, in strains Y51 and TCE1, and Dehalobacter restrictus PER-K23, the pceABCT genes including the intergenic regions are more than 99% identical in their DNA sequence, suggesting horizontal transfer between Dehalobacter restrictus and Desulfito. hafniense.

Figure 5.

Model of the transposition events of ISDesp1 and TnDesp2 leading to the emergence of PCE/TCE nondechlorinating variants of Desulfitobacterium hafniense strain Y51. PCE = perchloroethene; TCE = trichloroethene; DCE = dichloroethene; SD = small deletion variant; LD = large deletion variant; Wt = wild type.

Genomic analysis of Dehalo. ethenogenes 195 revealed that the tceAB genes are located in a putative integrated element.86 Moreover, the gene DET0076, which exists downstream of the tceAB genes, encodes a protein that is highly similar to a resolvase (Fig. 3). The distribution of tceAB in ethene-producing enrichment cultures originating from diverse geographic locations in the USA was investigated.96 Sequence identity was interrupted downstream of tceB and upstream or downstream of the DET0076, suggesting that intra- or interchromosomal transfer of tceAB had occurred. Analysis of codon usage of the vcrA and bvcA genes encoding VC reductive dehalogenase showed that these genes are highly unusual, characterized by a low fraction of G + C at the third position.97 The comparatively high level of abnormality in the codon usage of vcrA and bvcA genes suggests an evolutionary history that is different from that of most other Dehalococcoides genes. These data suggest that mobile elements play an important role in the arrangement and thereby the evolution of the reductive dehalogenase genes of Dehalococcoides.

Concluding Remarks

Anaerobic dehalorespiring bacteria have received considerable attention because of their important roles in bioremediation. From a variety of dehalorespirers, the key enzymes, reductive dehalogenases, and their corresponding genes have been identified and characterized. The features of these terminal reductases are similar. Among the various genetic events including mutation, recombination, and gene transfer, the transposable elements seem to be highly involved in the evolution and distribution of genes responsible for dechlorination. Moreover, completion of the genome sequences of Desulfitobacterium spp. and Dehalococcoides spp. has greatly increased our knowledge of their physiology and metabolism and has enabled us to identify similarities and differences between these ecologically important dehalorespiring bacteria. Additionally, these genome sequences open up exciting new avenues of research and permit proteomic and microarray approaches to studying the metabolic capabilities of these bacteria. However, many aspects of the dehalorespirers still remain to be elucidated. Little or nothing is known about the biochemical function except for reductive dehalogenases. In the case of Desulfito. hafniense Y51, no biochemical information is available for the other components such as the PceB, PceC, and PceT. The gene transfer and expression system should be established to allow in-depth investigations of these proteins. In the future, engineering of novel enzymes which exhibit enhanced activities and/or expanded substrate specificities might help in the further promotion of in situ applications for bioremediation.

Biographical Information

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Taiki Futagami was born in Fukuoka, Japan, in 1980. He received his B.S. degree in 2003 and his M.S. degree in 2005 from the Graduate School of Bioresource and Bioenvironmental Sciences of Kyushu University. In 2005, he became a Ph.D. student of Kyushu University, and a JSPS Research Fellow. He is currently studying the molecular mechanism of dehalorespiration.

Biographical Information

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Masatoshi Goto was born in Fukuoka, Japan, in 1966. He received his B.S. degree in 1990, his M.S. degree in 1992, and his Ph.D. degree in 1995 from the Department of Agricultural Chemistry, Kyushu University. He became Assistant Professor of Kyushu University in 1995. In 2001, he joined the Chakrabarty laboratory of the University of Illinois at Chicago for a year as a visiting research scholar of MEXT. His current research interests include the molecular breeding of filamentous fungi and dehalorespiring bacteria.

Biographical Information

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Kensuke Furukawa is a professor of Department of Food and Bioscience, Beppu University since April 2007. He was born in Kumamoto in 1944. He received his Ph.D. in microbiology from Kyushu University in 1974 and worked for Fermentation Research Institute, Agency of Industrial Science and Technology, MITI (1966–1989) before he joined Kyushu University in 1989, where he managed the Laboratory of Applied Microbiology as a professor in the Graduate School of Bioresource and Environmental Science. His primary research interests are the exploration and molecular breeding of microorganisms capable of degrading a wide variety of the environmental pollutants. He has conducted biochemical and genetic works on microbial degradation of organic mercurials, polychlorinated biphenyls, trichloroethene, tetrachloroethene, and so on. His current interest also includes the evolutionary and molecular engineering of oxygenases and dehalogenases. He served (or serves) as an editor or as a member of the editorial boards of Journal of Bacteriology, Applied Microbiology and Biotechnology, Journal of General and Applied Microbiology, the Journal of Fermentation and Bioengineering, Journal of Environmental Biotechnology, and others.