Microbes Environ. Vol. 23, No. 1, 1–12, 2008 http://wwwsoc.nii.ac.jp/jsme2/ doi:10.1264/jsme2.23.1 Minireview

Biodiversity of Dehalorespiring with Special Emphasis on /Dioxin Dechlorinators

AKIRA HIRAISHI1* 1Department of Ecological Engineering, Toyohashi University of Technology, Toyohashi 441–8580, Japan (Received December 30, 2007—Accepted January 18, 2008)

A wide variety of haloorganic compounds undergo reductive dehalogenation by certain anaerobic microorganisms. Metabolic reductive dehalogenation is coupled with energy-conserving respiratory electron transport in which a halo- genated compound is used as the terminal electron acceptor, the biological process called dehalorespiration or halores- piration. Dehalorespiring bacteria may play important roles in the geochemical cycle with organohalogens in nature and have great promise in their application to the of haloorganic contaminants derived from anthropo- genic sources. During the past decade, a number of dehalorespiring microorganisms, including a unique group of strictly dehalorespiring bacteria, “Dehalococcoides”, have been isolated and characterized at phylogenetic, physio- logic, and genetic levels. Also, new perspectives of dehalorespiring bacteria have emerged based on information about genomics and molecular microbial ecology. This review article focuses on up-to-date knowledge of the biodiversity of dehalorespiring bacteria and reductively dehalogenating microbial consortia with special emphasis on those capable of transforming polychlorinated biphenyls and dioxins. Key words: dehalorespiring bacteria, reductive dehalogenation, polychlorinated aromatics, Dehalococcoides, bioremediation

10) Introduction these unique microorganisms . As it has been estimated that there are more than 3,800 of organohalogens pro- Large amounts of structurally diverse haloorganic com- duced by living organisms and via natural abiogenic pounds have been and still are released into the environment processes53), much more attention should be paid toward the as a consequence of human and industrial activities. These geochemical cycle with halogenated compounds in which halogenated compounds are recognized as persistent pollut- anaerobic dehalogenating microorganisms may play impor- ants in general but possibly undergo microbial breakdown in tant ecological roles. nature. One of the most important biological processes Another important feature of reductive dehalogena- for this is reductive dehalogenation, which provides new tion is relevant to its application to environmental bio- insights into our understanding of the significance of remediation128). Most congeners of polychlorinated biphe- haloorganic compounds in microbial physiology and eco- nyls (PCBs), polychlorinated dibenzo-p-dioxins/furans logy21,67,93,113). The most important feature of microbial (PCDD/Fs), and other haloaromatics, as well as aliphatic reductive dehalogenation is the utilization of halogenated haloorganic compounds, are highly toxic and recalcitrant compounds as terminal electron acceptors for energy-con- contaminants whose potential risk to human health and wild serving anaerobic respiratory electron transport, i.e., the met- life should be taken into account31,55,104). Thus, the problem of abolic process called dehalorespiration, , or how to remedy organohalogen pollution is central to environ- chlororespiration. It has long been believed that most haloor- mental science and technology. Harnessing microbial reduc- ganic compounds are xenobiotics, and the occurrence of tive dehalogenation may offer scientifically sound and cost- these compounds in nature is due to contamination from effective bioremediation procedures, because this anaerobic anthropogenic sources. However, accumulated scientific process may work more efficiently than aerobic biodegrada- knowledge of reductive dehalogenation and dehalorespiring tion in removing halogen atoms from (poly)halogenated bacteria implies the importance of organohalides potentially compounds54,61,80,96,109). produced in natural environments, as well as anthropogenic During the past decade, a number of dehalorespiring ones, as physiological substrates for growth and survival of microorganisms, including a unique group of dehalorespir- ers, “Dehalococcoides”, have been isolated and character- + * Corresponding author. E-mail: [email protected]; Tel: 81– ized from phylogenetic, physiologic, and genetic points of + 532–44–6913; Fax: 81–532–44–6929. view81,113). Also, new perspectives of dehalorespiring bacteria Abbreviations: DCDD, dichlorodibenzo-p-dioxin; DCE, dichloroet- hene; DLG, Dehalococcoides-like group; MCDD, monochlorod- have emerged based on information about genomics and ibenzo-p-dioxin; PBDs, polychlorinated biphenyls/dioxins; PCB molecular microbial ecology. However, while microbial (s), polychlorinated biphenyl(s); PCE, tetrachloroethene, PCDD/ reductive dehalogenation of haloaliphatic compounds has F(s), polychlorinated dibenzo-p-dioxin(s)/furan(s); PeCDDs, pen- been studied extensively, less information is available on tachlorodibenzo-p-dioxins; RDase, reductive dehalogenase; TCE, haloaromatic-dehalogenating microorganisms. There have trichloroethene, TCDD, tetrachlorodibenzo-p-dioxin; TrCDD, in recent years been excellent review articles on the pro- trichlorodibenzo-p-dioxin; VC, vinyl chloride. cesses of microbial reductive dehalogenation of halo- 2 HIRAISHI aromatics4,44,45,49,132). The present review article focuses nated- or dichlorinated congeners are the end products in on up-to-date knowledge of the biodiversity of dehalorespir- general, and no complete dechlorination has so far been ing bacteria and reductively dehalogenating microbial con- reported. sortia with particular emphasis given to those capable of The redox potential for haloorganic compounds including transforming polychlorinated biphenyls/dioxins (PBDs). PBDs and chloroethenes (tetrachloroethene [PCE], trichloro- ethene [TCE], dichloroethene [DCE], and vinyl chloride 37) Biological significance of reductive dehalogenation [VC]) is relatively high, ranging from 260 to 570 mV . For example, if the dehalogenation of 1,2,3,4-tetachlorodibenzo- Reductive dehalogenation is defined as the removal of a p-dioxin (TCDD) to 2-monochlorodibenzo-p-dioxin halogen substituent from a molecule with the concomitant (MCDD) takes place with H2 as the electron donor via a addition of two electrons (Fig. 1)37,93). There are two modes pathway forming 1,2,3-trichlorodibenzo-p-dioxin (TrCDD) of reductive dehalogenation, called hydrogenolysis and and 2,3-dichlorodibenzo-p-dioxin (DCDD) as the intermedi- dichloroelimination, but the biological process takes place ates (see Fig. 1), the total reaction and ∆G°’ for this is given mostly as the hydrogenolytic reaction. Co-metabolic reduc- by: tive dehalogenation, which is of no benefit to the catalyzing + − 1,2,3,4-TCDD (C12H4Cl4O2)+6H +6e → 2-MCDD microorganisms, happens with enzymes that normally cata- (C12H7ClO2)+3HCl lyze other reactions. On the other hand, in metabolic reduc- tive dehalogenation, i.e., dehalorespiration, energy is con- ∆G°’=−469 kJ mol−1 served via an anaerobic respiratory process with a halogenated compound as the terminal electron acceptor and As described above and previously37,71), it is clear that the reductive dehalogenase (RDase) as the key enzyme involved. hydrogenotrophic redox process with PBDs as terminal elec- Dehalogenated end products from polyhalogenated com- tron acceptors can provide energy sufficient for the growth pounds during dehalorespiration differ among species and and survival of dehalogenating microorganisms. strains, as can be seen in the of tet- Although microorganisms capable of metabolic or co-met- rachloroethene (PCE). In the case of dehalorepiration with abolic reductive dehalogenation have been described as both PBDs and other polychlorinated aromatics, their monochlori- obligate and facultative anaerobes, all of the dehalorespiring

Fig. 1. Examples of reductive dehalogenation: hydrogenolytic dechlorination of chloroethenes (A) and 1,2,3,4-TCDD (B) found in “Dehalococ- coides” species. Biodiversity of Dehalorespiring Bacteria 3 bacteria so far described are strictly anaerobic, with the proximity to aerobic environments in wetland systems73). exception of Anaeromyxobacter (see below). As mentioned above, however, the redox potentials for organohalides are 2− Phylogeny of dehalogenating microorganisms much higher than that for the SO4 /H2S redox couple (E’0= − − −217 mV) and are comparable to the value for the NO3 /NO2 Anaerobic dehalogenating bacteria from different organo- couple (E’0=433 mV). Therefore, the reductive dehalogena- halogen-contaminated environments have been isolated as tion process itself may not always require strictly anoxic, pure cultures or highly enriched consortia and affiliated to low-potential conditions37). In fact, populations of “Dehalo- diverse phylogenetic groups (Fig. 2). Among these dehaloge- coccoides” and its phylogenetic relatives within the phylum nating bacteria, dehalorespiring species belong to three were detected together with ubiquinone-contain- major phyla, Chloroflexi, Firmicutes, and . ing aerobic bacteria at the surface of lake sediment at a broad There seems no correlation between the specificity for the 63) Eh gradient of 5 to −75 mV . In semi-aerobic fed-batch organohalides used by and the phylogenetic affiliations of composting reactors loaded with PCDD/F-contaminated soil, dehalorespiring isolates. Although reductive dehalogenation relatively high population densities of “Dehalococcoides” of PBDs by axenic cultures has so far been reported only in a (107 cells g−1 of dry soil) were found (ref. 95, unpublished phylogenetic group consisting of “Dehalococcoides” and its observations). Moreover, it has been suggested that reductive relatives, other dehalorespiring bacteria, such as those of Fir- dehalogenating activity of “Dehalococcoides” can be trig- micutes, may also have the potential to reductively dehaloge- gered in anaerobic environments located in close spatial nate PBDs in light of their ability to utilize some haloaromat-

Fig. 2. Neighbor-joining phylogenetic tree of dehalogenating bacteria based on 16S rRNA gene sequences. The database accession numbers for 16S rRNA gene sequences are given in parentheses behind the organism names. Aquifex pyrophilus is used as the outgroup to root the tree. Bar=2% nucleotide substitution rate (Knuc). Abbreviations and symbols: O, obligate dehalorespirers; F, facultative dehalorespirers; +, bacteria showing co- metabolic reductive dehalogenation. 4 HIRAISHI ics and their presence in PCB-transforming microbial anaerobic, sulfur-reducing, acetate-oxidizing bacterium102). consortia11,139). Microorganisms capable of metabolic reduc- Much later, the PCE- and TCE-utilizing bacterium Desulfu- tive dehalogenation are classified into two physiological romonas chloroethenica (type strain: TT4BT) was added to types; facultatively dehalorespiring bacteria and obligately the genus76). Also, “Desulfuromonas michiganensis” strains dehalorespiring bacteria. BB1 and BRS1 were reported to grow by coupling the oxida- Apart from dehalorespiration, there have been a number of tion of acetate to the reduction of PCE and TCE, as well as reports on microbial co-metabolic reductive dehalogenation with fumarate and ferric iron, as electron accepters119). or dehalogenation of unknown metabolic significance64). Co- Geobacter. The genus Geobacter was established with a metabolic reductive dehalogenation is found in diverse phy- single species, Geobacter metallireducens, as being capable logenetic groups of bacteria (see Fig. 2) and methanogenic of coupling the complete oxidation of organic compounds to archaea18,38,39,91). Representatives of these bacteria are also the reduction of iron and other metals83). “Geobacter lovleyi” presented in Fig. 2. strain SZ was reported as a novel species of the genus that is able to reduce metals and dechlorinate PCE117). Trichloro- T= T= Facultatively dehalorespiring bacteria bacter thiogenes (type strain: K1 ATCC BAA-34 JCM 14045T), which is a trichloroacetic acid dechlorinator36), has The genera of facultatively dehalorespiring bacteria so far been transferred to the genus Geobacter as Geobacter described are those belonging to the class Deltaproteobacte- thiogenes97). ria (i.e., Anaeromyxobacter, , Desulfuromo- Sulfurospirillum. The genus Sulfurospirillum was first nas, Desulfovibrio, and Geobacter), the class Epsilonproteo- described with S. deleyianum as the type species without any bacteria, (i.e., Sulfurospirillum), and the phylum Firmicutes information on its dehalogenating activity108). Later, an addi- (i.e., Desulfitobacterium). Among the genera noted above, tional species, Sulfurospirillum halorespirans (type strain: Anaeromyxobacter, Desulfitobacterium, and Desulfomonile PCE-M2T=ATCC BAA-583T=DSM 13726T) was reported to consist of dehalorespiring species exclusively, whereas the be able to reduce PCE to cis-DCE84). A related dehalogenat- remaining genera encompass non-dehalogenating species in ing organism, Dehalospirillum multivorans (type strain: addition. These organisms are metabolically quite versatile DSM 12446), has been transferred to the genus Sulfurospiril- and greatly differ from one another in the spectrum of usable lum as Sulfurospirillum multivorans84). electron donors and acceptors. Although several species are Desulfitobacterium. One of the most characterized able to grow with haloaromatics as electron acceptors, there groups of facultatively dehalorespiring bacteria is the genus has been no available information on their utilization of Desulfitobacterium, which was created in 1994 with the PBDs, with the exception that Desulfitobacterium dehaloge- monotypic species Desulfitobacterium dehalogenans125). nans was reported to have the ability to dechlorinate hydrox- Desulfitobacterium dehalogenans strain JW/IU-DC1T ylated PCBs133). Representative genera of facultatively deha- (=ATCC 51507T=DSM 9161T) is able to reductively dechlo- lorespiring bacteria are described below. rinate ortho-dechlorinate 2,4-dichlorophenol, hydroxylated Anaeromyxobacter. The genus Anaeromyxobacter, which PCBs, and, to a lesser extent, chloroalkenes125,133). Later, sev- was described with the monotypic species Anaeromyxo- eral new species were added to the genus Desulfitobacte- bacter dehalogenans (type strain: 2CP-1T=ATCC BAA- rium. In most species, sulfite, thiosulfate, sulfur, nitrate, and 258T) by Sanford et al.106), is proposed for a group of faculta- fumarate are utilized as physiological electron acceptors, tively anaerobic aryl-halorespiring myxobacteria. This whereas sulfate is not. Haloorganic compounds used as ter- organism is able to grow with chlorophenols as well as 2- minal electron acceptors include chloroethenes, haloalkanes, bromophenol, nitrate, fumarate, and oxygen as terminal elec- and haloaromatics. However, the spectrum of usable organo- tron acceptors. halides differs among species, and no strain of Desulfitobac- Desulfomonile. The genus Desulfomonile with the mono- terium described to date is able to completely dechlorinate typic species (type strain: DCB- PCE and TCE to ethene. Several Desulfitobacterium strains 1T=ATCC 49306T=DSM 6799T) was described in 199035). are also able to use humic substances, metals, and metalloids This was the first report to describe metabolic reductive deh- as terminal electron acceptors27,46,98). alogenation by a pure culture of microorganisms. Des- Desulfitobacterium chlororespirans strain Co23T (=ATCC ulfomonile tiedjei strain DCB-1T is capable of dehalorespir- 700175T=DSM 11544T) was described as an anaerobic ing growth with 3-chlorobenzoate as an electron acceptor. grower by coupling the oxidation of lactate to the reductive Additional electron acceptors utilized are fumarate, sulfate, dechlorination of 3-chloro-4-hydroxybenzoate105). At the sulfite, thiosulfate, and nitrate. Another species of the genus, same time, Desulfitobacterium hafniense was described with Desulfomonile limimaris (type strain: DCB-MT=ATCC the type strain DCB-2T (=DSM 10664T)30), which is able to 700979T), is also able to grow with 3-chlorobenzoate115). dechlorinate pentachlorophenol and 3-chloro-4-hydroxy- Desulfovibrio. This genus is a group of the sulfate-reduc- phenylacetate but not PCE. Some other strains of Desulfito- ing bacteria and includes a dehalogenating species, Des- bacterium hafniense were reported to dechlorinate ulfovibrio dechloracetivorans (type strain: SF3T=ATCC pentachlorophenol22,79,122), 2,4,6-trichlorophenol24), 3-chloro- 700912T). This bacterium is able to grow by coupling the 4-hydroxy-phenylacetate51,52), and PCE51,120). Strain PCP-1 oxidation of acetate to the reductive dechlorination of 2- was isolated from a methanogenic consortium and classified chlorophenol114). originally as a new species of the genus Desulfitobacterium Desulfuromonas. This genus was created in 1976 with with the name Desulfitobacterium frappieri22). However, this the monotypic species Desulfuromonas acetoxidans as an organism has been reclassified as a strain of Desulfitobacte- Biodiversity of Dehalorespiring Bacteria 5 rium hafniense98). Desulfitobacterium metallireducens strain the other hand, strain CBDB1 grows depending upon chlo- 853-15AT (=ATCC BAA-636T=DSM 15288T), isolated from robenzenes, chlorophenols, and PCDD/Fs5,7,25). “Dehalococ- uranium-contaminated aquifer sediment, utilizes PCE, TCE, coides ethenogenes” strain 195 is able to reductively dehalo- 3-chloro-4-hydroxy-phenylacetate, Fe(III) citrate, Mn(IV) genate chloroethenes, chlorophenols, and PBDs5,43). oxide, humic substances, and elemental sulfur as terminal Therefore, “Dehalococcoides” strains are attracting interest electron acceptors for in the presence as model organisms for the study of the evolution and diver- of lactate as the carbon and energy source46). Some other sification of dehalorespiration. strains of Desulfitobacterium without species designation The group of “Dehalococcoides” strains represents a have been reported to reductively dechlorinate PCE124) and deeply branching lineage within the phylum Chloroflexi, but chlorophenols52,123). may not be always clustered with established species of the Chloroflexi as a coherent phylogenetic assembly depending Obligately dehalorespiring bacteria upon species included in the phylogenetic analysis. The taxo- nomic description of the genus is still incomplete, and no To date, strictly dehalorespiring microorganisms are lim- “Dehalococcoides” strains are available from any culture ited to only two bacterial genera, and “Dehalo- collection. Therefore, the name “Dehalococcoides” has yet coccoides”, which are members of the phyla Firmicutes and to be validated in the nomenclature. One of the major reasons Chloroflexi, respectively. They have a highly restricted life- for this situation is that it is not easy to cultivate and maintain style, deriving energy from the reductive dehalogenation of “Dehalococcoides” strains as axenic cultures. Due to poor haloaliphates or haloaromatics with hydrogen as the sole biomass yields, standard taxonomic and biochemical proce- electron donor. All of the isolates described to date are meso- dures are not applicable. Methods for the enrichment and philic and neutrophilic bacteria that live in freshwater/terres- cultivation of “Dehalococcoides” as well as other reductively trial environments. dechlorinating bacteria have been described in detail by Löf- Dehalobacter. The genus Dehalobacter was established fler et al.82). as being monotypic with Dehalobacter restrictus in 199866). One of the most striking observations on the properties of The type strain PER-K23T (=DSM 9455T) was isolated from “Dehalococcoides” is that the lipid fraction of two “Dehalo- a PCE-dechlorinating, packed bed-column filled with river coccoides” isolates, strains BAV1 and FL2, and a PCE-to- sediment and granular sludge65). This bacterium belongs to ethene-dechlorinating “Dehalococcoides”-containing con- the so-called low G+C Gram-positive group (the phylum Fir- sortium contained unusual phospholipid modifications, micutes), but its rod-type cells are stained Gram-negative, including furan fatty acids, and large amounts of ubiquinone- like most Desulfitobacterium species. Acetate serves as a 8131). Smaller amounts of menaquinone-5 were also found. A carbon source in a defined medium containing hydrogen as possible function for ubiquinone in “Dehalococcoides” is to the electron donor and PCE or TCE as an electron acceptor. protect against free radicals that are generated in the process The respiratory chain contained menaquinones and b-type of reductive dehalogenation. Since previous papers have cytochromes. Whereas Dehalobacter restrictus grows hydro- reported a limited distribution of ubiquinones in Alpha-, genotrophically solely with PCE or TCE as the electron Beta-, and Gammaproteobacteria among prokaryotes60), it is acceptor, Dehalobacter sp. strain TCA1 exhibites the reduc- clearly necessary to study quinone profiles of the available tive dechlorination of 1,1,1-trichloroethane to chloroethane “Dehalococcoides” cultures more extensively. via the oxidation of hydrogen or formate as the electron Uncultured Chloroflexi. It is noteworthy that a relatively donor66,116). A co-culture of Dehalobacter sp. and Sedimento- diverse group of uncultured dehalogenating bacteria, desig- bacter sp. enriched from soil was reported to be able to nated herein as the “Dehalococcoides”-like group (DLG), reductively dechlorinate β-hexachlorocyclohexane126). exists as the phylogenetic sister cluster of “Dehalococ- “Dehalococcoides”. The genus “Dehalococcoides” was coides” within the phylum Chloroflexi. The existence of this first described in 1997 with “Dehalococcoides ethenogenes” novel dehalorespiring group, of which the PCB-dechlorinat- strain 195 as a strictly anaerobic bacterium capable of reduc- ing bacteria designated as o-1734) and DF-1135,136) are tively dechlorinating PCE to VC and ethene88,89). Later, addi- representatives, has been demonstrated using enrichment tional axenic cultures designated as “Dehalococcoides” spp. cultures from PBD- and chlorobenzene-contaminated were isolated, including strains CBDB17), VS32), BAV156,57), sites41,42,62,63,68,107,129,138–140). FL258), and GT118). This group of bacteria is quite unique in Although no taxonomic proposals with any axenic cultures having small coccoid cells, being resistant to penicillin-type of the DLG bacteria have been made to date, the cultures antibiotics, and exhibiting a strict tendency to use the respira- containing o-17 and DF-1 are highly enriched dehalogenat- tory mode with a set of H2 and organohalides as physiologi- ing cultures giving a nearly single band of the PCR-amplified cal substrates. It is also of special interest that, although 16S 16S rRNA gene upon denaturing gradient gel electrophoresis rRNA genes and housekeeping genes sequenced from “Deh- (DGGE). The bacterium o-17 requires acetate for reductive alococcoides” strains are >98% identical in nucleotide ortho-dechlorination of a PCB congener, 2,3,5,6-CB, and 34) sequence and >85% identical at the amino acid level, respec- growth . A mixture of H2:CO2 (80:20 at 101 kPa) may not tively, different strains utilize different ranges of haloorganic support dechlorination or growth of the bacterium o-17. On compounds. That is, the usable halogenated compounds are the other hand, the bacterium DF-1 reductively dechlorinates 1,1-DCE, cis-DCE, trans-DCE, and VC for strain BAV1; PCB congeners with doubly flanked chlorines when supplied 136) TCE and cis-DCE for strain FL2; TCE, 1,1-DCE, cis-DCE, with formate or H2-CO2 (80:20) . It has been shown that and VC for strain GT; and cis-DCE and VC for strain VS. On both of the bacteria are able to reductively dechlorinate chlo- 6 HIRAISHI

Table 1. Anaerobic enrichment cultures and microcosms capable of reductive dechlorination of PCBs and PCDD/Fs Enrichment culture Main dechlorinators detecteda Reductive dechlorination Reference Congener(s) used Main end product(s) PCB-dechlorinating culture from: Estuarine sediment DLG 2,3,5,6-CB 3,5-CB 68 Estuarine sediment DLG (DF-1) 2,3,4,5-CB 2,3,5-CB 136 Estuarine sediment DLG 2,2',3,4,4',5'-CB 2,2',4,4',5-CB, 2,2',4,4'-CB 129 Estuarine sediment DLG, “Dehalococcoides” Aroclor 1260 41 Marine/estuarine sediment DLG 2,3,4.5-CB 2,3,5-CB, 2,4,5-CB 138 Marine/estuarine sediment DLG, Dehalobacter 2,3,4.5-CB 2,4-CB, 2,5-CB 139 River sediment DLG (o-17) 2,3,5,6-CB 34 River sediment “Dehalococcoides” 2,2',4,5,5'-CB 2,2'4,5'-CB 42 River sediment “Dehalococcoides” 2,2',3,3'4,6'-CB 2,2',3',4,6'-CB 42 River sediment DLG 2,2',3,'4,6'-CB 2,2',4,6'-CB 42 River sediment “Dehalococcoides” Aroclor 1260 2,2',4,4'-CB, 2,2',4,6'-CB 15 River sediment “Dehalococcoides” Aroclor 1260 16 Soil Desulfitobacterium, Dehalobacter Kanechor-300/400 11 PCDD/F-dechlorinating culture from: Estuarine sediment DLG 1,2,3,4-TCDD 1,2,4-TrCDD, 1,3-DCDD, 2-MCDD 9 Esturarine/marine sediment DLG 1,2,3,4-TCDD 8 River sediment “Dehalococcoides” PCDD/F mixture 25 River sediment “Dehalococcoides” 1,2,4-TrCDD, 1,2,3-TrCDD 1,3-DCDD, 2,3-DCDD, 2-MCDD 12 River sediment “Dehalococcoides” 1,2,4-TrCDD, 1,2,3-TrCDD 1,3-DCDD, 2,3-DCDD, 2-MCDD 40 River sediment DLG PCDD/F mixture 140 Soil/lake sediment DLG PCDD/F mixture 62 a DLG, “Dehalococcoides”-like group, including the o-17/DF-1 bacteria. roethenes in addition to haloaromatics87,92). It might be sug- the second open reading frame orfB, which encodes a small gested that the DLG bacteria play more important roles than hydrophobic B protein as the membrane anchor subunit of “Dehalococcoides” in the transformation of PBDs in natural RDases. The N termini of RDases contain a twin-arginine ecosystems, because of the more frequent detection of the signal sequence with the consensus motif RRXFXX, which corresponding 16S rRNA gene clones in PBD-contaminated is involved in transporting cofactor-containing proteins environments (see Table 1). across the cytoplasmic membrane. Actually, TCE-RDase of “Dehalococcoides ethenogenes”85), the chlorobenzene- 6,69) Genomic diversity of dehalorespiration RDase of “Dehalococcoides ethenogenes” strain CBDB1 , and PceA-RDase of Desulfitobacterium hafniense strain The entire genome structure of “Dehalococcoides etheno- Y51121) are located on the exterior of the cytoplasmic mem- genes” strain 195110), “Dehalococcoides” sp. strain brane. Most RDases contain a corrinoid cofactor and 2 iron- CBDB177), and Desulfitobacterium hafniense strain Y5199) sulfur clusters. In fact, the RDase activity of most dehalore- has been published in the literature. In addition, genomic spiring species is light-reversibly inhibited by alkyl iodides, information about several other dehalorespiring organisms indicating the involvement of corrinoids as a cofactor69,86). including Anaeromyxobacter dehalogenans strain 2CP-1T, The rdh genes are mostly adjacent to the genes for transcrip- “Dehalococcoides” sp. strain BAV1, Desulfitobacterium tional regulators such as two-component regulatory systems hafniense strain DCB2T, and “Geobacter lovleyi” strain SZ or transcriptional regulators of the MarR-type, indicating that has become available from databases. The chromosome of the expression of the rdhAB genes is tightly regulated. “Dehalococcoides ethenogenes” strain 195 is 1.46 Mb in size It is noteworthy that the chromosomes of “Dehalococ- and harbors 1,591 predicted coding sequences (CDSs). coides ethenogenes” strain 195110) and “Dehalococcoides” “Dehalococcoides” sp. strain CBDB1 has a 1.39 Mb chro- sp. strain CBDB177) contain at least 18 and 32 RDase-homol- mosome on which 1,458 predicted CDSs are identified. The ogous genes, respectively, although the functions of these chromosome of strain CBDB1 is the smallest among those of genes have not yet been fully elucidated. Such multi-enzyme the free-living prokaryotes so far reported. On the other systems for reductive dehalogenation in the “Dehalococ- hand, Desulfitobacterium hafniense strain Y51 has a much coides” strains indicate their strict tendency to use dehalores- larger chromosome (5.7-Mbp) harboring 5,060 predicted piration with a number of organohalides as terminal electron CDSs. acceptors. This physiological trait contrasts with those of Sequence comparisons of RDase-homologous genes (rdh), Desulfitobacterium hafniense strain Y51, which contains together with information about purified RDase proteins, only 2 rdh genes but is more physiologically versatile with have revealed common features of RDases with the presence utilization of a larger set of specialized electron donors and of some conserved motifs to which specific functions are acceptors for anaerobic respiration99). Another strain of Des- attributed6,70,85,86,112,113,121). The open reading frame (orf) ulfitobacterium hafniense, DCB2T, studied for whole encoding the catalytic subunit of RDases, orfA, is linked to genome sequences, contains at least 9 RDase-homologous Biodiversity of Dehalorespiring Bacteria 7 genes. Most of the respiration-related genes found in the community. As already mentioned above, the involvement of Desulfitobacterium hafniense strains are absent in the “Deh- DLG bacteria in the dechlorination of PCBs was clearly alococcoides” strains. demonstrated by studying highly enriched cultures contain- Twelve of the 18 rdhAB pairs present in “Dehalococcoides ing the representative DLG bacteria o-1734) and DF-1136). ethenogenes” strain 195 are orthologous in “Dehalococ- Watts et al.129) developed specific PCR primers optimized for coides” sp. strain CBDB177,110). The amino acid sequence the detection of o-17 and DF-1 and other closely related bac- identities of the orthologs are 86–95% between the two, teria. Using these PCR primer sets, they detected the o-17/ although the position and orientation are conserved only in 6 DF-1 bacteria as the main dechlorinating organisms in sedi- of the orthologs. Two functionally identified rdhA genes ment microcosms exhibiting active dechlorination of PCBs. found in strain 195, pceA and tceA, encoding PceA-RDase The addition of different concentrations of bicarbonate had and TceA-RDase, respectively, are absent in strain CBDB1. profound effects on the dechlorination of 2,3,4,5-CB in sedi- The PCE-RDase gene is also involved in the production of ment cultures containing the DLG organisms as putative 2,3-dichlorophenol-RDase in strain 195, suggesting naturally dechlorinators139). occurring chlorophenols to be candidates for the original Bedard et al.15), by contrast, showed based on the analysis substrates for PceA48). One of the 32 RDase-homologous of a 16S rRNA gene clone library that “Dehalococcoides” genes present in the genome of strain CBDB1 is the organisms but not the DLG bacteria were present in river- cbdbA84 gene (cbrA), encoding chlorobenzene-RDase6). sediment-enrichment cultures dechlorinating a commercially Orthologs of cbdbA84 have been found neither in strains 195 produced PCB mixture, Aroclor 1260. Further work using and BAV1 nor among the other “Dehalococcoides” cultures PCR with group-specific primers ruled out any involvement studied so far. Besides RDase genes, the “Dehalococcoides” of known dechlorinators other than “Dehalococcoides” in the strains share a number of genes involved in respiratory elec- dechlorination16). Group-specific PCR analyses of anaerobic tron transport including those predicted to encode different enrichment cultures with a PCB mixture from uncontami- multi-subunit complexes of hydrogenase. nated soil showed the presence of Desulfitobacterium as the Genome analyses of halorespiring bacteria have revealed most frequent dechlorinator and Dehalobacter as the second the presence of RDase-encoding regions of potentially for- most common type; neither “Dehalococcoides” nor the DLG eign origin. In “Dehalococcoides ethenogenes” strain 195, was detected in any culture11). Microcosm studies of PCB the majority of reductive dehalogenase genes, including dechlorination by other investigators showed that different tceA, were probably acquired by several gene transfer members within the phylum Chloroflexi including “Dehalo- events103). Intrachromosomal or interchromosomal transfer of coccoides” and the DLG bacteria exhibited a limited range of tceAB among “Dehalococcoides” strains has also been sug- PCB congener specificities41,42), suggesting the importance of gested by studying the environmental distribution of the synergistic interactions of different species of microorgan- gene75). The VC-RDase genes, vcrA and bvcA, are highly isms for enhanced dechlorination. unusual in that the third position of codons in the genes is biased toward the nucleotide T, despite the absence of any PCDD/F-dehalogenating bacteria and consortia tRNAs matching codons that end in T90). This abnormality in the codon usage of VC-RDase genes suggests that the former Studies on the microbial reductive dehalogenation of genes differ from most other “Dehalococcoides” genes in PCDD/Fs as well as of PCBs started appearing in the 1990’s, evolutionary history, possibly being acquired by lateral using anaerobic sediment microcosms and mixed transfer. cultures2,3,13,14,19,20,26,47,72,127), although no phylogenetic infor- mation about PCDD/F-dechlorinating enrichment cultures PCB-dehalogenating bacteria and consortia had been available until the discovery of the ability of “Deh- alococcoides” to dechlorinate PCDD/Fs. In 2003, Bunge et Microbial reductive dehalogenation of halogenated biphe- al.25) reported that “Dehalococcoides” sp. strain CBDB1, nyls as well as of other haloorganic compounds has been which was originally described as a chlorobenzene-halore- extensively studied using anaerobic sediment cultures and spiring bacterium7), was able to reductively dechlorinate microcosms17,23,28,33,74,78,94,100,101,130,134,137). Most of the earlier 1,2,3,7,8-pentachlorodibenzo-p-dioxin (PeCDD) and 1,2, reports provided little or no definitive information about deh- 3,4-TCDD with 2,7(2,8)-DCDD and 2-MCDD as the end alogenating microorganisms in the cultures in terms of quan- products, respectively. This was the first report to demon- tity and quality. Since the discovery of “Dehalococcoides”, strate PCDD/F dechlorination by axenic cultures. They also culture-independent molecular approaches have been regu- showed the presence of a Dehalococcoides species in four larly made for the phylogenetic identification and character- dioxin-dechlorinating enrichment cultures from freshwater ization of microorganisms involved in the dechlorination. sediment highly contaminated with PCDD/Fs. The chloroet- Information about the so far described PCB-dechlorinating hene-halorespiring bacterium “Dehalococcoides etheno- anaerobic cultures and microcosms in which dechlorinators genes” strain 195 has also been found to dechlorinate PBDs, were identified is given in Table 1. The dechlorination of such as 1,2,3,4-TCDD, 2,3,4,5,6-PeCDD, and 2,3,4,5,6-CB, PCBs by axenic cultures has so far been reported only for and 1,2,3,4-tetrachloronaphthalene43), although whether “Dehalococcoides ethenogenes” strain 19543). these reactions are metabolic reductive dechlorination has A pioneering study by Holoman et al.68) showed that 16S remained unsolved. There have been no reports available on RNA gene clones corresponding to the DLG bacteria, desig- the reductive dechlorination of PCDD/Fs by any other axenic nated as RFLP-17, were present in a PCB-dechlorinating cultures of dehalorespiring bacteria. 8 HIRAISHI

Like the research into PCB-dehalogenating microorgan- alogenation of naturally produced bromophenols has been isms, studies on the the dechlorination of PCDD/Fs by sedi- demonstrated10). Thus, it is likely that dehalorespiring bacte- ment cultures have produced different results with respect to ria have modified the substrate specificity of their RDase the microbial populations involved in reductive dechlorina- systems with increases in the number and level of different tion (Table 1). A TrCDD-dechlorinating river-sediment cul- halogenated xenobiotics that have been released into the ture yielded “Dehalococcoides” sp. as a putative dehalore- environment as a consequence of human and industrial activ- spiring species12). A “Dehalococcoides”-containing mixed ities. culture reductively dechlorinated 1,2,4-TrCDD and 1,2,3- Although genomic and molecular approaches have pro- TrCDD, forming the main products 1,3-DCDD and 2- vided fundamental data on dehalorespiring bacteria, informa- MCDD from the former congener and 2,3-DCDD from the tion about in situ dehalorespiring populations including the latter, with carbon isotope fractionation taking place during DLG bacteria is fragmentary. Also, most of the multiple the sequential reductive dechlorination40). On the other hand, RDase-homologous genes found in the available axenic cul- Ahn et al.9) reported that 1,2,3,4-TCDD-dechlorinating sedi- tures have not yet been fully studied in terms of their actual ment microcosms contained DLG bacteria as the main functions and regulatory relationships. No RDase genes dechlorinators. In this case, the co-existence of halogenated involved in the reductive dehalogenation of PBDs have been aromatic compounds with structural similarity to PCDD/Fs identified. For a better understanding of the biology of deha- stimulated the dechlorination of 1,2,3,4-TCDD/F by the lorespiring bacteria, it is necessary to characterize many microcosms. Further work using 1,2,3,4-TCDD-dechlorinat- more enrichment and axenic cultures at the molecular and ing sediment cultures confirmed the involvement of the DLG genomic level. In particular, exploration by both metage- bacteria in the dechlorination8). Shiang Fu et al.111) investi- nomic and traditional culture-dependent approaches should gated the effect of respiratory conditions and priming com- elucidate the significance of the DLG bacteria and other deh- pounds on the dechlorination patterns of heptachlorod- alorespiring bacteria undescribed so far in natural dehaloge- ibenzo-p-dioxins using estuarine sediment-eluted cultures. nation processes. 16S rRNA gene-targeted PCR-DGGE analyses indicated sig- The structure of dehalogenating microbial consortia is of nificant shifts of microbial community structure in response great importance in connection with their utilization in engi- to terminal electron accepting processes as well as to the neered bioremediation, because PBDs and other haloaro- presence of the priming compounds. matic contaminants may undergo complete degradation by In semi-anaerobic microcosms seeded with PCDD/F-con- specific combinations of dehalogenating bacteria and other taminated sediment or soil, all of the congeners were totally physiologically different microorganisms having different removed without significant accumulation of less chlorinated metabolic pathways1). In natural systems, hydrogen as the congeners as intermediate or end products, where the DLG electron donor becomes available to obligately dehalorespir- bacteria rather than “Dehalococcoides” predominated as ing bacteria through anaerobic degradation or fermentation putative dechlorinators62,140). The apparent complete dechlo- of organic substrates. Therefore, the co-existence of syner- rination of PCDD/Fs might be due to a combination of gistic hydrogen producers and the concentration of hydrogen reductive dehalogenation of the polychlorinated congeners produced are the key factors for reductive dehalogenation29). and subsequent oxidative degradation of the dehalogenated For example, interspecies transfer of H2 produced via acetate products. Despite the presence of the DLG organisms as the oxidation by methanogens triggers hydrogenotrophic dechlo- major dechlorinators in the parent PCDD/F-dechlorinating rination of VC to ethene by “Dehalococcoides”59). A syner- microcosms, enrichment cultures with fthalide or 1,2,3- getic dehalogenating process with “Dehalococcoides” and a trichlorobenzene therefrom contained “Dehalococcoides” hydrogen-producing fermenting bacterium exists in the species as putative dechlorinators50,62). One of these “Dehalo- above-noted culture TUT2264 (unpublished data). Also, coccoides”-containing cultures, designated TUT2264, was less-halogenated aromatics as the products of reductive deha- able to dechlorinate PCE and TCE in addition to logenation might be good substrates for subsequent oxidative haloaromatics50). degradation and/or unknown metabolic processes, resulting in the complete detoxification of haloaromatics. An assess- Concluding remarks and perspectives ment of the bioremediation potential of dehalogenating con- sortia in situ will require a greater understanding of the syn- The biodiversity and universal occurrence of dehalorespir- ergistic and competitive interactions of key microorganisms. ing bacteria in nature indicate that they have evolved to pref- erably adapt to organohalides and have important ecological Acknowledgements niches in a geochemical cycle that was not taken into consid- This work was carried out as a part of “The Project for Develop- eration in the past. The fact that the phylogenetic assembly of ment of Technologies for Analyzing and Controlling the Mecha- “Dehalococcoides” and the DLG bacteria represent a distinct nism of Biodegrading and Processing” which was supported by the lineage within the deeply branching phylum Chloflexi sug- New Energy and Industrial Technology Development Organization gests that the invention of dehalorespiration itself was not an (NEDO), Japan. event under the stress of anthropogenic pollution but occurred in relatively ancient times. Of particular interest in References this context is that naturally occurring chlorophenols are sug- 1) Abraham, W.R., B. Nogales, P.N. Golyshin, D.H. Pieper, and K.N. gested to be candidates for the original substrates for the Timmis. 2002. Polychlorinated biphenyl-degrading microbial com- “Dehalococcoides” PCE-RDase48). Microbial reductive deh- munities in soils and sediments. Curr. Opin. Microbiol. 5:246–253. Biodiversity of Dehalorespiring Bacteria 9

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