Detection of a Bacterial Group Within the Phylum Chloroflexi And
Microbes Environ. Vol. 21, No. 3, 154–162, 2006 http://wwwsoc.nii.ac.jp/jsme2/
Detection of a Bacterial Group within the Phylum Chloroflexi and Reductive-Dehalogenase-Homologous Genes in Pentachlorobenzene- Dechlorinating Estuarine Sediment from the Arakawa River, Japan
KYOSUKE SANTOH1, ATSUSHI KOUZUMA1, RYOKO ISHIZEKI2, KENICHI IWATA1, MINORU SHIMURA3, TOSHIO HAYAKAWA3, TOSHIHIRO HOAKI4, HIDEAKI NOJIRI1, TOSHIO OMORI2, HISAKAZU YAMANE1 and HIROSHI HABE1*†
1 Biotechnology Research Center, The University of Tokyo, 1–1–1 Yayoi, Bunkyo-ku, Tokyo 113–8657, Japan 2 Department of Industrial Chemistry, Faculty of Engineering, Shibaura Institute of Technology, Minato-ku, Tokyo 108–8548, Japan 3 Environmental Biotechnology Laboratory, Railway Technical Research Institute, 2–8–38 Hikari-cho, Kokubunji-shi, Tokyo 185–8540, Japan 4 Technology Research Center, Taisei Corporation, 344–1 Nase, Totsuka-ku, Yokohama 245–0051, Japan
(Received April 21, 2006—Accepted June 12, 2006)
We enriched a pentachlorobenzene (pentaCB)-dechlorinating microbial consortium from an estuarine-sedi- ment sample obtained from the mouth of the Arakawa River. The sediment was incubated together with a mix- ture of four electron donors and pentaCB, and after five months of incubation, the microbial community structure was analyzed. Both DGGE and clone library analyses showed that the most expansive phylogenetic group within the consortium was affiliated with the phylum Chloroflexi, which includes Dehalococcoides-like bacteria. PCR using a degenerate primer set targeting conserved regions in reductive-dehalogenase-homologous (rdh) genes from Dehalococcoides species revealed that DNA fragments (approximately 1.5–1.7 kb) of rdh genes were am- plified from genomic DNA of the consortium. The deduced amino acid sequences of the rdh genes shared sever- al characteristics of reductive dehalogenases. The mixed culture could be maintained by transferring small inocu- la into fresh synthetic mineral medium containing either acetate or pyruvate, which supported the dechlorination of pentaCB by acting as an electron donor.
Key words: Persistent organic pollutants, Pentachlorobenzene, Chloroflexi, Reductive dehalogenation, Dehalococcoides
Persistent organic pollutants (POPs) is the common name ingly high levels25). To raise awareness of the need for glo- of a group of pollutants that have toxic properties, bioaccu- bal action on POPs, the Stockholm Convention on Persis- mulate, and resist degradation. POPs are semi-volatile and tent Organic Pollutants was held in May 200125). The global transported across international boundaries through air, wa- treaty included several measures, such as the prohibition ter, and migratory species25). As a result, POPs are deposited and elimination of the production, use, import, and export of far from their place of release, accumulating in terrestrial POPs, and a call for reduction of their release. Currently, 12 and aquatic ecosystems. A key environmental concern is types of POPs, i.e., aldrin, chlordane, dieldrin, endrin, hep- that some POPs have been found in polar regions at surpris- tachlor, hexachlorobenzene (hexaCB), mirex, toxaphene, polychlorinated biphenyls (PCB), polychlorinated dibenzo- + * Corresponding author; E-mail: [email protected], Tel: 81– p-dioxins (PCDD), polychlorinated dibenzofurans (PCDF), 29–861–4666, Fax: +81–29–861–4674 † Present address: National Institute of Advanced Industrial Science and DDT (1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane), 25) and Technology (AIST), Central 5–2, 1–1–1 Higashi, Tsukuba, are specifically listed in the treaty . Ibaraki 305–8565, Japan HexaCB, one of the POPs listed, was used as a pesticide, Pentachlorobenzene Dechlorination by Estuarine Sediment 155 fungicide, and seed protectant, and as a consequence has been detected all over the world in air, water, sediments, Materials and Methods and biota. The compound persists in the environment for many decades under natural conditions and shows hepato- Sampling and culture conditions carcinogenic activity10,20). According to an environmental In 2004, 820–1500 pg of hexaCB (g dry weight−1) was monitoring report published by the Japanese Ministry of the detected in Arakawa River sediment (http://www.env.go.jp/ Environment (2004) (
PCR was carried out using the degenerate primer set RRF2 DNA extraction (5'-SHMGBMGWGATTTYATGAARR-3') and B1R (5'- DNA was extracted from sediment samples according to CHADHAGCCAYTCRTACCA-3')14) as follows: 130 s at the method of Zhou et al. (1996)31), with some modifica- 94°C; 30 cycles of 1 min at 94°C, 1 min at 46°C, and 130 s tions. Briefly, 2 ml of the sediment slurry was centrifuged at 72°C; and 72°C for 6 min. These PCR products were re- and the pellet was suspended in 900 µl of DNA extraction covered after agarose gel electrophoresis using the QIAEX buffer (100 mM Tris-HCl, 100 mM EDTA, 100 mM sodi- II gel extraction kit (Qiagen) according to the manufactur- um phosphate buffer, 1.5 M NaCl, and 1% CTAB, pH 8.0). er’s instruction. Purified DNA fragments were cloned into Next, 10 µl of 10 µg proteinase K/µl was added to the sus- pT7 Blue T-Vector (Novagen), which was used to transform pension, which was then incubated for 30 min at 37°C. One- Escherichia coli DH5α according to published hundred microliters of 20% SDS was added and the mixture procedures22). Plasmid DNA was isolated and purified with was further incubated for 2 h at 65°C. After three cycles of the Quantum Prep plasmid miniprep kit (Bio-Rad) accord- freezing (−80°C for 30 min) and thawing (65°C for 10 min), ing to the manufacturer’s instructions. the suspension was extracted twice with phenol/chloroform (1:1) and once with chloroform/isoamyl alcohol (24:1). Iso- Sequencing and phylogenetic analysis propyl alcohol (0.7 volumes) was added to the recovered Cloned 16S rRNA genes and rdh genes were sequenced aqueous solution, the mixture was gently mixed, and DNA with an ABI PRISM 310 Genetic Analyzer (Applied Bio- was precipitated by centrifugation. The DNA-containing systems, Foster City, CA, USA) or a 3730xl DNA Analyzer precipitate was washed with 70% (vol/vol) ethanol and then (Applied Biosystems). The nucleotide sequences obtained treated with Tris-EDTA buffer containing RNase. were analyzed with DNASIS-Mac software (version 3.7; Hitachi Software Engineering, Tokyo, Japan). Homology PCR-DGGE searches were carried out with the BLASTN or BLASTX3) The V3 region of the bacterial 16S rRNA gene was PCR- program, and multiple sequence alignment with amplified using primer set 341F-GC (5'-CCTACGGGAG- CLUSTALW24). Phylogenetic trees were constructed by the GCAGCAG-3', with a GC clamp attached to the 5'-end) and neighbor-joining method21), and the topology of the trees 534R (5'-ATTACCGCGGCTGCTGG-3')19). The PCR con- was evaluated by bootstrapping with 1000 resamplings8). ditions reported by Muyzer et al. (1993)19) were followed. The nucleotide sequences were deposited at the DDBJ, EM- First, the samples were incubated for 10 min at 94°C, fol- BL, and GenBank databases under accession numbers lowed by two cycles of 1 min at 94°C, one cycle of 1 min at AB253368 to AB253381 and AB251615 to AB251618. 65°C, and one cycle of 2 min at 72°C (i). Second, the an- nealing temperature was subsequently decreased by 1°C ev- Composition of the basal salt medium and culture ery second cycle until it reached 55°C, at which point 20 ad- conditions ditional cycles were performed (ii). Third, one cycle of 10 Several synthetic media were tested for their ability to min at 72°C was carried out (iii). The PCR products were stably maintain the pentaCB-dechlorinating activity of an applied to 8% (wt/vol) polyacrylamide gels containing a inoculum obtained from the Arakawa estuary sediment. A 30–70% denaturing gradient of formamide and urea and basal medium containing 10 g NaCl, 0.2 g KH2PO4, 0.27 g electrophoresed at 60°C for 6 h in 1×TAE buffer (40 mM NH4Cl, 4.5 g MgCl2·6H2O, 0.52 g KCl, 0.15 g CaCl2·2H2O, Tris-acetate, 1 mM EDTA, pH 8.0) at a constant voltage of 0.25 g cystein·HCl·H2O, 2.5 g NaHCO3, 0.001 g resazurin, 130 V with a DCode System (Bio-Rad, Hercules, CA, 0.5 ml vitamin mixture, and 1 ml mineral mixture (L−1) was USA). The gels were stained with ethidium bromide (0.5 µg selected. The vitamin mixture consisted of 4 mg p-ami- ml−1) and visualized with a UV transilluminator. Excised nobenzoate, 1 mg biotin, 10 mg nicotinic acid, 5 mg pan- DNA fragments were amplified using the PCR conditions tothenic acid, 15 mg pyridoxine, 10 mg thiamine, and 10 mg (ii) described above, purified using a QIAEX II gel extrac- cobalamine (100 ml−1), and the mineral mixture of 1.5 g tion kit (Qiagen, Hilden, Germany), and then cloned into FeCl2, 70 mg ZnCl2, 100 mg MnCl2·4H2O, 6 mg H3BO3, pT7 Blue T-Vector (Novagen, Madison, WI, USA). 190 mg CoCl2·6H2O, 2 mg CuCl2·2H2O, 24 mg NiCl2·6H2O, −1 36 mg NiMoO4·2H2O, and 10 ml HCl (7.7 M) (100 ml ). Construction of the clone library Acetate, pyruvate, or benzoate was added to the basal medi- PCR products of the 16S rRNA gene were amplified un- um as an electron donor at a final concentration of 5 mM. der the same conditions as described above. For rdh genes, The inoculum (1% vol/vol) and 30 ml of sterile synthetic Pentachlorobenzene Dechlorination by Estuarine Sediment 157 basal medium were dispensed into 100-ml glass bottles in could not be established because only trace amounts of the an anaerobic glove box, 0.8 mM Ti (III) citrate was added to latter two compounds were detected (data not shown). It each bottle as a reducing agent, and pentaCB was directly also remains to be determined whether pentaCB was added to the cultures as crystals at a final concentration of dechlorinated in the sediments to di- or monochloroben- 250 µM. The bottles were sealed with Teflon-coated butyl- zens. rubber septa and aluminum caps, the headspace was ex- changed with N2-CO2-H2 (3:1:1, vol/vol) as described PCR-DGGE and clone library analyses above, and the sample bottles were incubated at 30°C in the A PCR-DGGE analysis was carried out to investigate dark. changes in the microbial population in the sediment before and after incubation with pentaCB (Fig. 1). The former and Chemicals latter sediment samples were designated as AR0 (0 months All chlorobenzenes (98–99% purities) used in this study of incubation) and AR5 (5 months of incubation), respec- were purchased from Sigma-Aldrich (St. Louis, MO, USA) tively. Two bands, A and B, in the DGGE gel (Fig. 1) were or Tokyo Kasei Kogyo (Tokyo, Japan). All other chemicals reproducibly detected in repeated experiments only when used were of the highest purity (>98%) commercially avail- DNA extracted from AR5 was used as a template. Hence, able and were purchased from Merck (Whitehouse Station, these two bands were excised and sequenced. The nucle- NJ, USA), Sigma-Aldrich, Kanto Chemical (Tokyo, Japan), otide sequences of bands A and B exhibited 96.8 and 99.9% Wako Pure Chemical (Osaka, Japan), or Nacalai Tesque identity to that of an uncultured Chloroflexi bacterial iso- (Kyoto, Japan). late, ODP1176A6H_1_B (AY191351), and Clostridium litorale (X77845), respectively. Results and Discussion Furthermore, we investigated changes in the bacterial Detection of dechlorinated metabolites of pentaCB We tried to enrich the pentaCB-dechlorinating mixed cul- ture from Arakawa estuarine sediment by initially adding 500 µM of pentaCB to the sediment samples. To prevent the enrichment of dechlorinating bacteria growing well on spe- cific synthetic media, Arakawa River water was used in- stead of the defined synthetic medium, e.g., for cultivation of Dehaloccoides strains. After 5 months of incubation, the amount of pentaCB in the sediment was approximately 10% of the initial value (data not shown). GC-MS analysis showed two less-chlorinated metabolites of pentaCB in the sample after 5 months of incubation, but not after 1 or 3 months of incubation. The mass spectrum of one metabolite exhibited fragment ions at m/z 216 (M+, 100), 179 (16), 143 (10), 108 (24), 84 (18), and 74 (36) at a retention time of 4.7 min (the relative intensities expressed as percentages are given in parentheses). This fragmentation pattern of the me- tabolite was identical to that of authentic 1,2,4,5-tetrachlo- robenzene (tetraCB). The mass spectrum of another metab- olite showed fragment ions at m/z 180 (M+, 100), 145 (27), 109 (40), 84 (19), and 74 (48) at a retention time of 4.0 min. Its fragmentation pattern was identical to that of authentic Fig. 1. Change in DGGE profiles for partial 16S rRNA gene frag- 1,2,4,-trichlorobenzene (triCB). These results suggested that ments in sediment samples before and after incubation with pen- the sediment sample may have dechlorinated pentaCB by a taCB. Lanes: 1, AR0 (0 months of incubation); 2, AR5 (after 5 pathway generating the metabolites 1,2,4,5-tetraCB and months of incubation). The nucleotide sequences of DGGE bands A and B exhibited 96.8 and 99.9% identity to that of an uncul- 1,2,4,-triCB. However, a quantitative relationship of the tured chloroflexi bacterium isolate, ODP1176A6H_1_B amounts of pentaCB, 1,2,4,5-tetraCB, and 1,2,4,-triCB (AY191351), and Clostridium litorale (X77845), respectively. 158 SANTOH et al. populations in AR0 and AR5, especially those of the phy- Furthermore, in other PCB- or PCDD/Fs-dechlorinating mi- lum Chloroflexi, by amplifying partial 16S rRNA gene frag- crobial communities, the predominant dechlorinating bacte- ments, which were used to construct two clone libraries. ria comprised diverse Dehalococcoides-related species, From these two libraries, 50 clones were randomly selected rather than the typical Dehalococcoides species27,30). Those and sequenced (Table 1). No fundamental differences in results suggest that such uncultured Dehalococcoides rela- phylogenetic composition between AR0 and AR5 were ob- tives may be the primary dechlorinating anaerobic bacteria served except for the phyla Chloroflexi, Firmicutes, and in a wide variety of chloroaromatics-polluted environments. Deltaproteobacteria. In the AR0 library, 46% of the clones Further studies will be necessary in order to test this hypoth- were affiliated with the phylum Firmicutes and 16% with esis. Deltaproteobacteria. However, the proportion of these bac- rdh teria decreased in the clone library from AR5; instead, bac- Detection of genes in the pentaCB-dechlorinating teria from the phylum Chloroflexi became increasingly sediment sample common (26%) (Table 1). Until now, there has been little information available on To obtain a more detailed picture of the affiliation of the reductive dehalogenases from Dehalococcoides-like deha- AR5 clones within the phylum Chloroflexi, we constructed lorespiring bacteria, because such bacteria have not been a phylogenetic tree of the Chloroflexi clones and their rela- isolated as pure cultures. Hence, using the DNA sample tives, including the Dehalococcoides group (Fig. 2). None from AR5, which contains Dehalococcoides-like bacteria of the clones fell into the cluster within the known dehalore- rather than Dehalococcoides species (Fig. 2), PCR was per- spiring Dehalococcoides strains, e.g., strains 195, CBDB1, formed with a degenerate primer set designated for amplify- and BAV1; however, five clones (CLA-4, 33, 36, 72, and ing putative reductive dehalogenase genes of Dehalococ- 76) formed a cluster with an uncultured bacterium, DF-1, coides species14) (Fig. 3). Amplicons with the expected sizes which has the ability to dechlorinate PCB and hexaCB28). (approximately 1.5–1.7 kb) were detected in AR5 but not in Also, we determined the position of clone CLA-46, which AR0 (Fig. 3). The AR5-derived amplicons were purified was related to DGGE band A (CLA-0) (Fig. 2). Hiraishi et and clone libraries were constructed, from which four al. (2005a, b)11,12) demonstrated that Dehalococcoides-like clones were randomly selected and sequenced. All four se- bacteria, which form a phylogenetic cluster different from quences contained a nearly complete rdhA, lacking only that of typical Dehalococcoides species, play important about 45–60 nucleotides at the 5'-end (Fig. 3a). The se- roles in the dechlorination of PCDD/Fs in the microcosm. quenced rdhA regions were designated as rdh1, rdh3, rdh4, and rdh6. The deduced amino acid sequences of these four rdhA genes showed 30–50% identity with rdh genes from Table 1. Phylogenetic classification of 16S rRNA gene clones in the strains 195, CBDB1, and BAV1 (data not shown), and con- AR0 and AR5 libraries tained two iron-sulfur cluster (ISB) motifs in their C-termi- Number of clones nal region14) (Fig. 4). Furthermore, the first ISB motif of all Phylum/class (percentages of clones) four RDHs possessed the conserved consensus sequence AR0 (0 months) AR5 (5 months) CXXCXXCXXXCP, which is found in 4Fe-4S clusters of 5) Bacteroidetes 5 (10) 7 (14) bacterial ferredoxins (Fig. 4a). In contrast, for the second Chloroflexi 3 (6) 13 (26) ISB motif, the consensus motif of the 4Fe-4S cluster, Deinococcus-Thermus 1 (2) 0 (0) CXXCXXCXXCP, was conserved in RDH1 (Fig. 4a), Firmicutes 23 (46) 14 (28) whereas in RDH4 and RDH6 the first cysteine residue of Fusobacteria 1 (2) 0 (0) the motif was lacking (Fig. 4b). A similar tendency was Proteobacteria observed in PceA from Sulfurospirillum multivorans Alphaproteobacteria 01 (2)(PceASm in Fig. 4b), PceA from Desulfitobacterium sp. Betaproteobacteria 01 (2)Y51 (PceADy), and CprA from Desulfitobacterium dehalo- Gammaproteobacteria 1 (2) 1 (2) genans (CprADd) (Fig. 4b). Electron paramagnetic reso- Deltaproteobacteria 8 (16) 2 (4) nance (EPR) analysis of CprADd strongly suggested the Epsilonproteobacteria 1 (2) 1 (2) presence of one 4Fe-4S cluster and one 3Fe-4S cluster, Spirochaetes 1 (2) 0 (0) consistent with two ISB motifs CXXCXXCXXCP and Unidentified 6 (12) 10 (20) CXXCXXCP26). Thus, the second ISB motif, CXXCXXCP, Total 50 50 in both RDH4 and RDH6, may correspond to the 3Fe-4S Pentachlorobenzene Dechlorination by Estuarine Sediment 159
Fig. 2. The 16S rRNA gene-based tree reflecting the phylogenetic positions of the uncultured clones from AR5 (after 5 months incubation with pentaCB) within the phylum Chloroflexi. Aquifex pyrophilus was used as the outgroup. The scale bar corresponds to 0.02 estimated nucleotide substitutions per position. Support for individual nodes is shown as percent values of 1000 replications of a bootstrap analysis. The sequence of DGGE band A shown in Fig. 1 was represented as CLA-0. The accession numbers of 16S rRNA gene sequence data are shown in parenthe- ses. Nucleotide sequences obtained from the AR5 clone library (CLA series in this figure) were deposited at the DDBJ, EMBL, and GenBank databases under the accession numbers AB253368 to AB253381. cluster. However, in RDH3, the first and third cysteine resi- and analyzed; nonetheless, the results suggest that Dehalo- dues of the motif were lacking (Fig. 4b). coccoides-like bacteria may also have rdh genes similar to In this experiment, only four rdh clones were selected those from Dehalococcoides species. Supposing that a Deh- 160 SANTOH et al.
(b)
(a)
Fig. 3. (a) Reductive-dehalogenase-homologous (rdh) gene fragments amplified from the known dehalorespiring Dehalococcoides strains with the primer pair RRF2-B1R. The conserved dehalogenase features are also shown: the Tat signal peptide RRDFMK, two 4Fe-4S clusters (ISB motifs) in the C-terminal portion, and a conserved motif WYEW within the Dehalococcoides rdhB gene. Conserved amino acid residues in dehalogenases are labeled with an asterisk. (b) PCR amplification of the rdh gene fragments with the primer pair RRF2-B1R. DNA extracted from AR0 (before incubation; lane 1) or AR5 (after 5 months incubation; lane 2) was used as a template.
(a)
(b)
Fig. 4. Alignments of the C-terminal region of the deduced amino acid sequences of rdhA genes. Alignment of RdhAs with two ISB motifs cor- responding to two 4Fe-4S clusters (a) and one 4Fe-4S and one 3Fe-4S cluster (b). Sequence abbreviations, species, and accession numbers are as follows: TceADe Dehalococcoides ethenogens 195, AF228507; VcrADv Dehalococcoides sp. VS, AY322364; PceASm Sulfurospirillum multivorans, AF022812; CprADd Desulfitobacterium dehalogenans, AF115542; PceADy, Desulfitobacterium sp. Y51, AB070709. Rdh1, Rdh3, Rdh4, and Rdh6 (AB251615 to AB251618) are reductive dehalogenase homologues from this study. Pentachlorobenzene Dechlorination by Estuarine Sediment 161
(a) functions of various Dehalococcoides rdh genes, including the reductive dehalogenase gene for pentaCB.
Effect of electron donors on pentaCB dechlorination The DGGE and clone library analyses indicate that fur- ther enrichment of the mixed culture is necessary for identi- fying and isolating pentaCB-dechlorinating bacteria. How- ever, repeated transfer of the sediment sample to sterile river water containing electron donors resulted in a loss of dechlorination activity. Therefore, several types of synthetic basal salt media were screened for their ability to maintain stable subcultivation. After 34 kinds of synthetic media, (b) with different constituents at varying concentrations (NaCl: −1 −1 1, 5, or 10 g (L ); MgCl2·6H2O: 0.42, 2.2, or 4.5 g (L ); re- ducing agents: 1 mM Na2S or 0.8 mM Ti (III) citrate; pen- taCB addition: as DMF solution or as crystal), had been tested (data not shown), the synthetic medium described in the Materials and Methods was chosen. Using this synthetic medium, we then set out to identify which of three electron donors (acetate, pyruvate, or benzoate) was most effective in mediating dechlorination of pentaCB in the microbial communities. Compared to the sterilized control sample, 5 mM acetate resulted in a significant decrease in the concen- tration of pentaCB after 2 months of incubation (Fig. 5a). (c) Supplementation with pyruvate resulted in more than 80% pentaCB depletion within 2 months of incubation (Fig. 5b), whereas benzoate was ineffective (Fig. 5c). These results indicated that either acetate or pyruvate can be used as an electron donor in the pentaCB-dechlorinating consortium. In conclusion, we obtained a microbial consortium that reductively dechlorinates about 90% of 250 µM pentaCB. The occurrence of bacteria within the phylum Chloroflexi and the detection of rdh genes in the mixed culture suggest that a bacterial group related to members of Dehalococ- coides species plays a primary role in anaerobic dechlorina- Fig. 5. Depletion of pentachlorobenzene by sediment-free mixed cultures in the presence of 5 mM acetate (a), pyruvate (b), and tion in the sediment. However, the use of such bacteria to benzoate (c) as a sole electron donor (diamond). The control decontaminate POP-polluted environments awaits the de- (square) shows the depletion rate with sterilized sediment-free velopment of techniques to isolate uncultured Dehalococ- mixed cultures. The rate of pentaCB depletion was calculated by coides-like bacteria from mixed cultures. setting the peak area for the molecular ion of pentaCB (m/z=250) at 0 weeks incubation at 100%. The results are averages of dupli- cate determinations. Acknowledgements We thank K. Watanabe and Y. 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