Dehalogenation Activities and Distribution of Reductive Dehalogenase Homologous Genes in Marine Subsurface Sediments

Scientific ocean drilling explorations have revealed that marine subsurface sediments harbor remarkable numbers of microbial cells that account for approximately 1/10 to 1/3 of all living biota on Earth (20, 25, 33). Thermodynamic calculations of pore-water chemistry suggest that subseafloor microbial activities are generally supported by nutrient and energy supplies from the seawater and/or underlying basaltic aquifers (6, 7). Although sulfate, nitrate, Fe(III), Mn(IV), and bicarbonate are known to be potential electron acceptors for anaerobic microbial respiration in marine subsurface sediments (5), the incidence of both the dissimilatory dehalorespiration pathway and microbial activity in halogenated organic substrates remains largely unknown.

Previous molecular ecological studies using 16S rRNA gene sequences demonstrated that Chloroflexi is one of the most frequently detected phyla in subseafloor sediments of the Pacific Ocean margins (12-14). Some of the sequences within the Chloroflexi are closely related to sequences in the genus Dehalococcoides, which contains obligatory dehalorespiring bacteria that employ halogenated organic compounds as terminal electron acceptors (21, 29). The frequent detection of Dehalococcoides-related 16S rRNA genes from these environments implies the occurrence of dissimilatory dehalorespiration in marine subsurface sediments.

In this study, we detected and phylogenetically analyzed the reductive dehalogenase homologous (rdhA) genes, key functional genes for dehalorespiration pathways, from frozen sediment core samples obtained by Ocean Drilling Program (ODP) Leg 201 (Peru margin and eastern equatorial Pacific) (7, 14); Integrated Ocean Drilling Program (IODP) Expedition 301 (Juan de Fuca Ridge flank) (8, 24); Chikyu Shakedown Expedition CK06-06 (Northwest Pacific off Japan) (20, 23); and IODP Expedition 315 (Nankai Trough Forearc Basin off Japan) (Table 1). DNA was extracted using an ISOIL bead-beating kit (Nippon Gene, Japan) and purified using a MagExtractor DNA fragment purification kit (Toyobo, Japan) according to the manufacturer's instructions. To increase concentration, DNA was amplified by multiple displacement amplification using the phi29 polymerase supplied with a GenomiPhi kit (GE Healthcare, United Kingdom) (20). Putative rdhA genes were amplified by PCR using Ex Taq polymerase (TaKaRa, Japan) with degenerate primers RRF2 and B1R (17), dehaloF3, dehaloF4, dehaloF5, dehaloR2, dehaloR3, and dehaloR4 (32), and ceRD2S, ceRD2L, and RD7 (26) and the PCR conditions described in those studies. Amplicons of the approximate target size were gel purified and cloned into the pCR2.1 vector (Invitrogen, Japan). Sequence similarity was analyzed using FastGroupII web-based software (34), and sequences with a 95% identity were tentatively assigned to the same phylotype. Amino acid sequences were aligned by ClustalW (31), including known and putative reductive dehalogenase sequences in the genome of Dehalococcoides ethenogenes strain 195 (28), as well as several functionally characterized reductive dehalogenases from other species.

Putative rdhA genes were successfully detected by primer set RRF2-B1R in samples from the eastern equatorial Pacific (ODP site 1226, 3.2 and 46.7 m below the seafloor [mbsf]), the Peru margin (ODP site 1227, 0.3, 16.6, and 75.1 mbsf, and ODP site 1230, 0.3 mbsf), the Juan de Fuca Ridge flank (IODP site 1301, 2.5 mbsf), offshore from the Shimokita Peninsula of Japan (CK06-06 site C9001, 1.0, 13.5, 78.5, 191.5, 216.8, and 358.6 mbsf), and the Nankai Trough Forearc Basin off the Kii Peninsula of Japan (IODP site C0002, 1.9, 9.2, 20.2, and 66.6 mbsf) (Table 1). No amplification was observed in samples from several deep horizons at sites 1227, 1230, 1301, C9001, and C0002 (Table 1). A total of 92 clones of subseafloor putative rdhA genes were sequenced and classified into 32 phylotypes (Fig. 1). Phylogenetic analysis revealed that all of the detected putative rdhA sequences were related to those of Dehalococcoides.

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FIG. 1.

Phylogenetic tree based on the deduced amino acid sequences of rdhA genes, including sequences from marine subsurface sediments. Putative rdhA sequences from marine subsurface sediments (rdhA clones 1 to 32) are marked in red, while those of the Dehalococcoides genome are marked in blue. Clonal frequencies and sequence accession numbers are indicated in parentheses. Bootstrap values from 50% to 84% and 85% to 100% are indicated by open and solid circles at the branches, respectively. Asterisks indicate the following functionally characterized rdhA genes: pceA and prdA, tetrachloroethene reductive dehalogenase; tceA, trichloroethene reductive dehalogenase; vcrA and bvcA, vinyl chloride reductive dehalogenase; dcaA, 1,2-dichloroethane reductive dehalogenase; cprA, chlorophenol reductive dehalogenase; and cbrA, chlorobenzene reductive dehalogenase. The tree was constructed by a neighbor-joining (NJ) method based on an alignment of almost-complete rdhA amino acid sequences with pairwise gap deletion on MEGA version 4.0 software (30). The resulting tree was displayed using Interactive Tree Of Life (19). The scale bar represents 0.1 substitutions per amino acid position.

In the alignment of the subseafloor rdhA sequences, we observed two Fe-S cluster-binding motifs as a conserved structure of previously reported reductive dehalogenases (29). The sequences were amplified with primer RRF2 containing the N-terminal twin arginine translocation (Tat) signal sequence and primer B1R containing the rdhB genes encoding a putative dehalogenase membrane anchor protein (17). Thus, the dehalogenases of subseafloor bacteria have a structural framework similar to that of known dehalogenases from terrestrial Dehalococcoides species. However, BLASTP analysis showed that similarities among subseafloor rdhA sequences and previously reported dehalogenase sequences were generally low, ranging from 33.06% to 64.27%. Some sequences were affiliated, with relatively high bootstrap values, with subseafloor rdhA clusters I and II, which are clearly distinct from the rdhA sequences of Dehalococcoides and other known species (Fig. 1). In addition, we were unable to detect subseafloor rdhA genes using other primer sets targeting cprA- and pceA-like genes (26, 32). These results indicate that most subseafloor rdhA genes are distinct from those reported from terrestrial environments, a trend that corroborates the results of a metagenomic survey of subseafloor microbial communities at the Peruvian site (3). However, it is worth noting that the RRF2 and B1R primers used in this study are based on the rdhA sequences present in Dehalococcoides (17) and that sequence retrieval is probably biased by primer mismatch. It is thus likely that there are still unexplored functional genes related to the dehalorespiration pathways in marine subsurface sediments.

An interesting finding of the functional gene survey is that the subseafloor rdhA homologues are preferentially detected in shallow sediments. At site C9001 off Japan, the sedimentation ratio is considerably higher than at other sites (54 to 95 cm per 1,000 years) (unpublished data), and rdhA genes were successfully detected in horizons as deep as 358 mbsf (Table 1). The rdhA genes were also detected in sediments from the open ocean at site 1226, which contained very low concentrations (<0.2%) of organic matter (7). This may be because halogenated compounds are derived not only from terrestrial environments but also from the seawater overlying the sediments. In addition, a diverse range of marine organisms, such as phytoplankton, mollusks, algae, polychaetes, jellyfish, and sponges, are known to produce halogenated organic compounds (11). For example, the amount of brominated organic compounds in the ocean has been estimated at 1 to 2 million tons per year (10). Since these halogenated compounds are generally recalcitrant or not metabolizable by aerobic microorganisms in the seawater column (15), they are effectively buried in marine subsurface sediments. In fact, debromination of brominated phenols in marine, estuarine, or intertidal strait sediments has been reported (4, 9, 16, 22), and a brominated phenol-dehalogenating microbial community has been observed in the marine sponge Aplysina aerophoba, which produces bromophenolic metabolites (1).

We also observed reductive dehalogenation activity in subseafloor sediment slurry from site C0002 in the Nankai Trough (Fig. 2; also see the supplemental material). The slurry sample was prepared by mixing sediment samples from 1.9, 4.7, 9.2, 13.4, 20.2, 30.0, 66.6, and 155.4 mbsf. During the initial incubation with 2,4,6-tribromophenol (2,4,6-TBP) for 179 days, 2,4,6-TBP was completely converted to phenol. We then supplemented the same incubation slurry with 2,4,6-TBP and once again observed dehalogenation activity (Fig. 2A). During the incubation, 2,4-dibromophenol and 4-bromophenol were produced as intermediates (Fig. 2C), suggesting that ortho debromination occurred in preference to para debromination, as observed previously in marine sponge habitats (1). The maximum phenol production rate during the second incubation was calculated to be 0.094 μM per 1 cm³ of sediment per day (Fig. 2A).

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FIG. 2.

Dehalogenation activities of subseafloor microbes. (A) Debromination of 2,4,6-TBP in a subseafloor sediment slurry from site C0002 in the Nankai Trough Forearc Basin. Arrow indicates the timing of 2,4,6-TBP supplementation. (B) Dechlorination of TCE in the same slurry sample. Sterilized control sediment slurries did not exhibit phenol and/or cis-DCE production (data not shown). (C) Potential debromination pathway of 2,4,6-TBP (solid arrows) and (D) potential dechlorination pathway of TCE (solid arrows) observed. The pathways indicated by dashed arrows were not observed in this experiment.

Using the same sediment slurry sample, we also observed dehalogenation activity of trichloroethene (TCE), a substantial pollutant in the natural environment. During an incubation lasting more than 200 days, TCE was almost entirely converted to cis-dichloroethene (cis-DCE) (Fig. 2B). The subsequent dechlorination step of cis-DCE, which is presumably from cis-DCE to monochloroethene, was not observed during the incubation. The rate of cis-DCE production was calculated as 0.045 μM per 1 cm³ of sediment per day.

In conclusion, the observed molecular and activity data suggest that metabolically active dehalorespiring microbes are well represented in marine subsurface sediments and that these microbes may be widely distributed in Pacific Ocean margin sediments. Given the relatively high in vitro activity rates, we expect that subseafloor dehalorespiring microbes play important ecological roles in the biogeochemical cycles of chlorine, iodine, and bromine, as well as in halogenated carbon substrates. The distribution of in situ activity rates, chemical and geophysical constraints, metabolic characteristics of the individual dehalorespiring phylotypes, and genetic and enzymatic mechanisms of the microbes remain to be clarified. Nevertheless, the findings of this study provide new evidence of microbial functioning in the subseafloor ecosystem.

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