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Department of Life Sciences, National Chung Hsing University, Taichung, Taiwan, ROC
Correspondence
Mei-Chin Lai
mclai{at}dragon.nchu.edu.tw
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Published online ahead of print on 18 July 2003 as DOI 10.1099/ijs.0.02761-0.
The GenBank/EMBL/DDBJ accession numbers for the 16S rDNA sequences of strains K1F9705bT, K1F9705c and O1F9704a determined in this study are AF347025, AF321115 and AY234332, respectively.
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first proposed the genus Methanocalculus, with Methanocalculus halotolerans, a hydrogenotrophic, halotolerant methanogen that was isolated from an oil-producing well, as the type species. Recently, Methanocalculus pumilus, a heavy-metal-tolerant methanogen that was isolated from a waste-disposal site (Mori et al., 2000), and Methanocalculus taiwanensis strains P2F9704aT and P2F9705, isolates from an estuary in Eriln Shi, near Wang-gong, Taiwan (Lai et al., 2002), were reported. All these new Methanocalculus isolates are hydrogenotrophic methanogens with an irregularly coccoid shape. They all require acetate for growth and grow optimally at mesophilic temperatures and neutral pH. The following Methanocalculus species grow in a relatively restricted NaCl range: M. taiwanensis and M. pumilus grow optimally at 1 % NaCl and can only tolerate up to 4 and 7 % NaCl, respectively (Mori et al., 2000; Lai et al., 2002). In contrast, M. halotolerans grows optimally at 5 % NaCl and can tolerate salt concentrations of 0–12 %. Such a wide salt-tolerance range is the widest reported to date for any hydrogenotrophic methanogen (Ollivier et al., 1998; Garcia et al., 2000). In this study, we report the characterization of three novel methanogenic strains, K1F9705bT, K1F9705c and O1F9702c, that were isolated from an estuary and a marine fishpond in Taiwan. Phylogenetic evidence, DNA–DNA hybridization and phenotypic characteristics showed that these three strains constitute a novel species of the genus Methanocalculus, for which we propose the name Methanocalculus chunghsingensis sp. nov.
Sampling sites were the estuary environment at Eriln Shi, Wong-gong, Taiwan, and a nearby marine water aquaculture fishpond. Water temperature in this subtropical environment was 30–34 °C during summer and salinity at this estuary was around 1 % (w/v). The water source of the aquaculture fishpond was mixed sea water and groundwater, which was pumped into a man-made pond for the mixed cultivation of Chanos chanos (white mullet) and Meretrix lusoria (Lai & Chen, 2001). Strains K1F9705bT and K1F9705c were isolated from sediment samples of the aquaculture fishpond, whereas strain O1F9704a was isolated from a sediment sample from the estuary. Samples were collected in a stainless-steel sampling basket. They were then transferred immediately to a sterile Oak-Ridge bottle that had been equilibrated overnight inside a Coy anaerobic chamber with an N2 : CO2 ratio of 4 : 1. The bottles were filled to the top, sealed and returned to the laboratory for processing within 3 h.
The modified anaerobic technique of Hungate (Balch et al., 1979; Sowers & Noll, 1995) was utilized in this study. Sterilized media were prepared under an oxygen-free N2/CO2 (4 : 1) atmosphere. MB medium contained the following [(l deionized water)-1]: MgCl2.6H2O, 1·0 g; KCl, 0·5 g; NaCl, 5 g; CaCl2.2H2O, 0·1 g; K2HPO4, 0·4 g; NH4Cl, 1·0 g; cysteine.HCl, 0·25 g; NaHCO3, 4·0 g; yeast extract, 2 g; tryptone, 2 g; and resazurin, 1 mg. Vitamin solution (Wolin et al., 1963) and trace-element solution without tungstate (Ferguson & Mah, 1983) were each added to a final concentration of 1 % (v/v). The pH of MB medium was 7·0. MB/W medium, made from MB medium, was prepared with 1 % (v/v) trace-element solution that contained tungstate (Na2WO4, 0·3 mg l-1). Minimal medium (MM) was MB medium without yeast extract or tryptone. All constituents except for NaHCO3, cysteine.HCl (which was added after the solution was cooled) and sulfide were dissolved in water, boiled and cooled under an oxygen-free atmosphere of N2/CO2 (4 : 1). The medium was distributed into serum bottles (Wheaton Scientific) or Hungate tubes (Bellco Glass) under the same atmosphere. The anaerobic tubes were then sealed and autoclaved at 121 °C for 20 min. Sodium sulfide, from a sterilized, anoxic stock solution, was added to a final concentration of 1 mM before inoculation. For solid roll-tube medium, agar was added at 20 g l-1. To measure the effect of pH on growth, the N2 : CO2 ratio in the gas phase and the concentration of NaHCO3 in the medium were modified to obtain pH values between 6·0 and 8·1.
Enrichment was begun immediately after the samples were brought to the laboratory; the methanogens were isolated and purified as described previously (Lai & Chen, 2001; Lai et al., 2002). Samples (5 ml) from sediments of the marine water aquaculture fishpond and the estuary of Eriln Shi, Taiwan, were inoculated into eight bottles of MB medium (45 ml) that contained methanol, trimethylamine, acetate or formate as methanogenic substrates. After 1 month incubation at room temperature, methanogenesis occurred in all enrichments. Four successive transfers further enriched the formate-grown cells; this culture was then inoculated into roll-tube MB agar medium for further isolation. Under fluorescent microscopy, three fluorescent colonies with different morphologies were picked and transferred to 5 ml MB medium with formate in a Coy anaerobic chamber. One was a small, white, pinpoint colony with a light-yellow, raised central area (strain K1F9705bT). The second colony was yellowish, circular and opaque (strain K1F9705c). The third was a circular, round colony (strain O1F9704a). Methane-producing cultures from these single colonies were purified further by combining an increased NaCl concentration (1–10 %) in MB medium with serial dilution until contamination by non-methanogens was not detectable. These isolates were deposited in the Oregon Collection of Methanogens, USA, and DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen), Germany, as strains K1F9705bT (=OCM 772T=DSM 14646T), K1F9705c (=OCM 666) and O1F9704a (=OCM 747).
Unless otherwise indicated, phenotypic and morphological characteristics were studied as described by Lai et al. (2002). Catabolic substrates tested under an N2/CO2 (4 : 1) atmosphere were sodium formate (100 mM), sodium acetate (50 mM), trimethylamine (40 mM), methanol (50 mM), ethanol (48 mM), propan-2-ol (48 mM), isobutanol (48 mM) and butan-2-ol (48 mM). Utilization of H2 was tested by pressurizing the culture tubes with H2 (100 %, 200 kPa). Utilization of substrates was determined in MB/W medium by monitoring methane production by GC with flame-ionization detection (Lai et al., 1999). Specific growth rates were calculated from methane production, which was analysed by linear regression of the logarithm of the total amount of methane that accumulated over time. Inocula were grown under conditions that were similar to the experimental conditions.
Sensitivity to ampicillin, penicillin, spectinomycin, kanamycin, tetracycline and chloramphenicol (each at 100 µg ml-1) was tested in MB/W medium with sodium formate (100 mM) plus acetate (20 mM) at 37 °C. Whole-cell proteins were extracted from cell pellets that were lysed by adding loading buffer that contained 4 % SDS at a ratio of 1 ml buffer per OD unit. An OD unit was the amount of cells formed in 1 ml culture with an OD540 of 1·0. Surface-layer proteins were isolated according to the protocol of König (1995). SDS-PAGE was performed as described by Laemmli (1970) and Coomassie blue R-250 was used to visualize protein. An Olympus BH-2 microscope was used for phase-contrast microscopy. Preparation for negative staining and ultrathin sectioning was performed as described previously (Lai & Shih, 2001). Electron micrographs were taken by using model JEM-1200EX II and 200cx transmission electron microscopes (JEOL).
For phylogenetic analysis, DNA was isolated by the general procedure of Jarrell et al. (1992). Approximately 30 ng DNA was used as a template for PCR amplification of an approximately 1300 bp portion of the 16S rRNA gene. PCR amplification primers used were 5'-CGACTAAGCCATGCGAGTC-3' and 5'-GTGACGGGCGGTGTGTGCAAG-3'. The sequences were checked by the Check-Probe program from the Ribosomal Database Project (Maidak et al., 1996); they corresponded respectively to positions 34–52 and 1340–1320 in the 16S rDNA nucleotide sequence of M. halotolerans. The following primers were used for sequencing: 5'-CGACTAAGCCATGCGAGTC-3', 5'-GCTCAGTAACACGTGGATAACC-3', 5'-GGATTACAGGGTTTCACTCCTACC-3' and 5'-GTGACGGGCGGTGTGTGCAAG-3' (M. halotolerans SEBR 4845T, GenBank accession no. AF033672: sequence positions 34–52, 78–99, 609–632 and 1340–1320, respectively). The resulting sequences of strains K1F9705bT, K1F9705c and O1F9704a were assembled to produce contiguous rDNA sequences of approximately 1254 bp (positions 58–1311), 1251 bp (58–1308) and 1262 bp (58–1319), respectively. Gene sequences of archaea were obtained from the Ribosomal Database Project and GenBank. The similarity matrix obtained was based on the analytical results of the Ribosomal Database Project (http://rdp.cme.msu.edu/html/). Multiple sequence alignments were analysed by the CLUSTAL W package at the Biology Workbench (http://workbench.sdsc.edu/). Distances were computed with the CLUSTAL tree package at the same website by using the neighbour-joining model and were fed to the program DRAWGRAM of the PHYLIP package version 3.5c (Felsenstein, 1993). Bootstrap confidence analysis was performed with the SEQBOOT program of the PHYLIP package by using 500 replicates.
DNA–DNA hybridization experiments were performed by using the dot-blot technique (Sambrook & Russell, 2000) with the VersiTag fluorescein labelling system (NEN Life Science). The type strain of M. pumilus, MHT-1T, was kindly provided by Dr Koji Mori of Gifu University, Japan, and the type strain of M. halotolerans, SEBR 4845T, was kindly provided by Dr David Boone from the Oregon Collection of Methanogens. M. taiwanensis P2F9704aT and P2F9705 were obtained from our own culture stock. Cells of Methanocalculus species were harvested at late-exponential phase and used for DNA isolation. DNA was isolated and purified by a modification of the methods of Johnson (1985) and Jarrell et al. (1992). Target DNA (500 ng), denatured by 0·8 M NaOH, was blotted on to a Hybond-N+ nylon membrane (Amersham Biosciences) and labelled DNA was reassociated in a solution that contained 50 % formamide, 5x Denhardt's solution (50x Denhardt's solution contains 1 % Ficoll, 1 % polyvinylpyrrolidone and 1 % BSA) with 0·5 % (w/v) SDS in 5x SSC buffer (1x SSC buffer is 0·15 M sodium chloride plus 0·015 M sodium citrate). After incubation overnight at 42 °C, the blots were analysed with a Renaissance nucleic acid chemiluminescence reagent with an anti-fluorescein horseradish peroxidase-conjugate detection system supplied by NEN Life Science. Hybridization signals were detected by autoradiography. Triplicate tests were performed for each assay and self-hybridization of the probe with homologous target DNA was set to 100 %.
To determine G+C content, DNA of Methanocalculus species was isolated and purified as described above. The melting temperature of purified DNA was measured by using the method of Marmur & Doty (1962) with the slight modification of Jan et al. (1999). DNA (50 µg) was dissolved in 1x SSC buffer (pH 7·0). This DNA solution was then placed in a cuvette and heated from 60 to 95 °C in increments of 2 °C. At each temperature, A260 was measured by using a Cary 100 UV-vis spectrophotometer (Varian). The G+C content was computed by using the formula Tm=69·3+0·41x(G+C). For comparison and accuracy, Tm values of DNA from M. pumilus and M. halotolerans were determined by the same method.
Under phase-contrast microscopy, refractive areas were observed in all three new methanogenic isolates (Fig. 1). This is similar to M. taiwanensis and M. pumilus. Cells of strain K1F9705bT were non-motile and irregularly coccoid, with a diameter of 1·1–1·6 µm (Fig. 2a). When observed by transmission electron microscopy (TEM) with negative staining, the transparent bag of surface (S)-layers tended to separate from the cells (Fig. 2a). This indicated that the proteinaceous cell-wall structure of strain K1F9705bT was very sensitive to physical and chemical treatment and lysed easily. Cells of strain K1F9705c were non-motile and irregularly coccoid, with a diameter of 1·0–1·5 µm (Fig. 2b). Several homogeneous, heavily stained granules were observed both in negative and ultrathin preparations (Fig. 2b). It was suggested that these heavily stained granules were polyphosphate (Jacob J. Goldberg, electron microscopist; personal communication). Cells of strain O1F9704a were weakly motile with several flagella and irregularly coccoid, with a diameter of 0·8–1·1 µm (Fig. 2c). Interestingly, cell morphology of strains K1F9705bT and K1F9705c showed wrinkle bands and heavily stained granules in negative-stain preparations under TEM. Of the two, strain K1F9705c tended to accumulate larger amounts of heavily stained granules and cells of this strain tended to clump together.
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). Negative-stain TEM of K1F9705bT and O1F9704a clearly showed a hexagonally arranged pattern of S-layer protein for both strains. The centre-to-centre spacing of the morphological units of the S-layer lattice of strain K1F9705bT was about 24 nm. Isolation and characterization of the S-layer protein from Methanocalculus species indicated that M. taiwanensis P2F9704aT, strains K1F9705bT, K1F9705c and O1F9704a were different from each other and were each composed of two different subunits, whereas the S-layers of M. halotolerans and M. pumilus were composed of a single subunit. The molecular masses of S-layer proteins of strains K1F9705bT, K1F9705c, O1F9704a (these were different from the known masses of S-layer proteins in M. halotolerans, M. pumilus and M. taiwanensis) varied from 88 to 102·2 kDa and are summarized in Table 1.
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Halotolerance was observed in strain K1F9705bT. Although cells grew optimally in medium that contained 1 % NaCl, these cells tolerated up to 12 % NaCl (Fig. 3). Under optimal growth conditions (temperature, pH and NaCl), the doubling time of strain K1F9705bT was approximately 7 h. Optimal temperature and pH for growth of strain K1F9705b and O1F9704a were similar to those of strain K1F9705bT. There were slight variations in salt tolerance among these three strains; both K1F9705bT and K1F9705c grew optimally in 1 % NaCl, but the optimal salt concentration for strain O1F9704a was 0·5 % (Fig. 3). They all tolerated high salt concentrations. Among them, strain K1F9705bT was more halotolerant than the others, as it tolerated up to 12 % NaCl, whereas strains K1F9705c and O1F9704a tolerated up to 10 and 8 %, respectively.
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). However, their protein profiles were distinct from those of M. halotolerans and M. pumilus, and were related more closely to that of the former than to that of the latter (Fig. 4).
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). The sequence of strain K1F9705c was identical to that of strain K1F9705bT, and the sequence of strain O1F9704a had 99·9 % similarity to those of the other two isolates. Phylogenetic analysis indicated that all known Methanocalculus species formed a cluster (>98 % similarity), but were distant from other methanogens (<90·7 % similarity).
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), but were higher than that of M. halotolerans (43 mol%) (Ollivier et al., 1998). In DNA–DNA hybridization experiments, DNA of strain K1F9705bT exhibited 37 % hybridization with that of M. halotolerans, 29 % hybridization with that of M. pumilus and <10 % hybridization with the DNA of M. taiwanensis. Among the novel strains, the DNA of strain K1F9705bT exhibited 85 % hybridization with that of strain K1F9705c and 80 % hybridization with that of strain O1F9704a.
All Methanocalculus species reported to date are mesophilic, neutrophilic and hydrogenotrophic, and can only utilize H2/CO2 and formate as catabolic substrates. The three new isolates reported here were similar to the other three known Methanocalculus species in catabolic substrates, optimum temperature and pH for growth (Table 1
). However, results of the cell morphology, halotolerance, protein profile, S-layer protein, phylogenetic and DNA–DNA hybridization analyses indicated that these three new isolates are distinct from M. halotolerans, M. pumilus and M. taiwanensis. Therefore, we conclude that these three isolates represent a novel species of the genus Methanocalculus, for which we propose the name Methanocalculus chunghsingensis sp. nov., to include strains K1F9705bT, K1F9705c and O1F9704a. Strain K1F9705bT (=OCM 772T=DSM 14646T) is designated as the type strain. DNA–DNA hybridization data revealed a high level of DNA relatedness among strains K1F9705bT, K1F9705c and O1F9704a (up to 80 %), which is indicative of strains of the same genospecies. Also, cell morphology, protein profile, molecular mass of S-layer protein, halotolerance and effects of tungsten (Table 1) revealed that they are three separate strains.
All irregularly coccoid methanogens within the order Methanomicrobiales possess hexagonal S-layer lattices that consist of glycoprotein subunits with an Mr that ranges from 90 000 to 155 000. Among them, the family Methanocorpusculaceae has an S-layer glycoprotein with an Mr that ranges from 90 000 to 94 000, and those of the family Methanomicrobiaceae range from 101 000 to 155 000 (Zellner et al., 1999). There are no discussions of S-layer protein and whole-cell protein profile for M. halotolerans or M. pumilus (Ollivier et al., 1998; Mori et al., 2000). We have isolated the S-layer protein of all Methanocalculus species; the molecular masses of S-layer protein of these strains were different and were located within the range 86 000–112 000 (Table 1). Also, analysis of the whole-cell protein profiles of M. chunghsingensis, M. halotolerans, M. taiwanensis and M. pumilus showed different patterns (Fig. 4).
Most halotolerant or halophilic methanogenic species that have been described are methylotrophic and it was suggested by Ollivier et al. (1994) that the use of methylotrophic substrates by methane-producing bacteria in halophilic environments predominates over H2 and acetate utilization. Ollivier et al. (1998) reported the first hydrogen-oxidizing methanogen, M. halotolerans, from an oil-producing well. This organism grows optimally at 5 % NaCl (w/v) and tolerates up to 12 % NaCl, a range that is the widest reported to date for any hydrogenotrophic methanogen. Recently, Mori et al. (2000) reported that M. pumilus grew optimally at 1·0 % NaCl and did not grow at 10 % NaCl. The three new isolates that we report here have an optimal NaCl range of 0·5–1·0 %, but they tolerate up to 8–12 % NaCl (for strain K1F9705bT grown in 12 % NaCl, the specific growth rate is 0·021 h-1); hence, they are halotolerant, hydrogenotrophic methanogens. Most methylotrophic, halotolerant/halophilic methanogens are adapted for growth over a narrow NaCl concentration range, whereas hydrogenotrophic Methanocalculus species, such as M. halotolerans and M. chunghsingensis, are well-adapted for growth over a broad range of NaCl concentrations (0–2·0 M). This may indicate their better adaptation to an environment where the NaCl concentration fluctuates.
Description of Methanocalculus chunghsingensis sp. nov.
Methanocalculus chunghsingensis (chung.hsing.en'sis. N.L. masc. adj. chunghsingensis from Chung Hsing University, to honour the university where this research was performed).
Irregularly coccoid cells, non-motile or weakly motile with flagella. Obligately anaerobic cells; Gram-negative. Cell wall is composed of proteinaceous, SDS-sensitive, S-layer subunits with relative molecular masses that range from 88 100 to 102 200. Catabolic substrates used include H2/CO2 and formate, but not acetate, methanol, trimethylamine, dimethylamine, ethanol, propan-2-ol, isobutanol or butan-2-ol. Cells are mesophilic; growth occurs at 20–45 °C, with optimum at 37 °C. Cells grow over a wide pH range, from 6·0 to 8·0; optimal pH is 7·2. Cells grow well at 0–12 % NaCl; optimal NaCl concentration for growth is 1·0 %. Acetate is required for growth. Tungsten is highly stimulatory for some strains. Growth is inhibited completely by chloramphenicol and partially by tetracycline, but not by ampicillin, penicillin, kanamycin or spectinomycin. DNA G+C content of cells is about 50·0 mol%.
The type strain is K1F9705bT (=OCM 772T=DSM 14646T). Isolated from a marine water aquaculture fishpond near Wong-Kong, Taiwan. Reference strains are K1F9705c and O1F9704a.
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), K1F9705c (
) and O1F9704a (
). Cells were grown on MB/W medium with sodium formate as the catabolic substrate and acetate. Specific growth rates were calculated from methane production values and are the means of triplicate cultures.
