Methanothermococcus Okinawensis Sp. Nov., a Thermophilic, Methane-Producing Archaeon Isolated from a Western Pacific Deep-Sea Hydrothermal Vent System
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International Journal of Systematic and Evolutionary Microbiology (2002), 52, 1089–1095 DOI: 10.1099/ijs.0.02106-0 Methanothermococcus okinawensis sp. nov., a thermophilic, methane-producing archaeon isolated from a Western Pacific deep-sea hydrothermal vent system Subground Animalcule Ken Takai, Akira Inoue and Koki Horikoshi Retrieval (SUGAR) Project, Frontier Research System for Extremophiles, Japan Author for correspondence: Marine Science & Ken Takai. Tel: j81 468 67 3894. Fax: j81 468 66 6364. Technology Center, 2-15 e-mail: kent!jamstec.go.jp Natsushima-cho, Yokosuka 237-0061, Japan A novel thermophilic, methane-producing archaeon was isolated from a deep- sea hydrothermal vent chimney at the Iheya Ridge, in the Okinawa Trough, Japan. The cells were highly motile, irregular cocci, with a polar bundle of flagella. Growth was observed between 40 and 75 SC (optimum 60–65 SC; 30 min doubling time) and between pH 45 and 85 (optimum pH 6–7). The isolate was a strictly anaerobic autotroph capable of using hydrogen and carbon dioxide as sole sources of energy and carbon. Formate can serve as an alternative energy source. The GMC content of the genomic DNA was 335 mol%. Phylogenetic analysis based on 16S rDNA sequences and DNA–DNA hybridization analysis indicated that the isolate was closely related to members of the genera Methanococcus and Methanothermococcus. This isolate, however, could be differentiated from the previously described species of these genera on the basis of its physiological and molecular properties. The name Methanothermococcus okinawensis sp. nov is proposed, with the type strain IH1T (UJCM 11175TUDSM 14208T). Keywords: deep-sea hydrothermal vent, thermophile, methanogen, Methanococcus, archaea INTRODUCTION anococcus infernus MET were isolated from deep-sea hydrothermal vent environments from the East Pacific Hyperthermophilic or thermophilic methanogens have Rise, Guaymas Basin and the Mid-Atlantic Ridge been isolated from a variety of marine hydrothermal (Jeanthon et al., 1998, 1999; Jones et al., 1983b, 1989; systems (Burggraff et al., 1990; Huber et al., 1982, Zhao et al., 1988). In addition, Methanococcus thermo- 1989; Jeanthon et al., 1998, 1999; Jones et al., 1983b, lithotrophicus ST22 and Methanococcus sp. strains 1989; Kurr et al., 1991; Stetter, 1996; Zhao et al., vp183 and vp21, closely related to Methanococcus 1988) and sub-sea-floor oil reservoirs (Nilsen & thermolithotrophicus SN-1T, were isolated from deep Torsvik, 1996; Orphan et al., 2000). Except for one sub-sea-floor oil reservoir waters in the North Sea isolate, Methanopyrus kandleri (Huber et al., 1989; (Nilsen & Torsvik, 1996) and off the shore of California Kurr et al., 1991), all of the methanogens isolated from (Orphan et al., 2000). Based on their widespread marine hydrothermal vent systems and sub-sea-floor occurrence, hyperthermophilic or thermophilic mem- oil reservoirs are members of the order Methanococ- bers of the Methanococcales are likely to be cosmo- cales. Methanococcus thermolithotrophicus SN-1T and politan micro-organisms inhabiting global marine Methanococcus igneus Kol 5T were isolated from hydrothermal vent systems and subsurface oil reservoir coastal hydrothermal systems of Italy and Iceland, environments like the hyperthermophilic members of respectively (Burggraff et al., 1990; Huber et al., 1982). the order Thermococcales (L’Haridon et al., 1995; Methanococcus jannaschii JAL-1T, Methanococcus vul- Slobodkin et al., 1999; Stetter et al., 1993; Takai et al., canius M7T, Methanococcus fervens AG86T and Meth- 2000; Zillig et al., 1983). However, no thermophilic ................................................................................................................................................. methanogen has yet been cultivated from the hy- The GenBank/EMBL/DDBJ accession number for the 16S rDNA sequence of drothermal vent systems located in the Western Pacific, strain IH1T is AB057722. although a number of microbiological surveys have 02106 # 2002 IUMS Printed in Great Britain 1089 K. Takai, A. Inoue and K. Horikoshi been performed in hydrothermal vent fields in the MMJ (described below) medium supplemented with 10 mM Western Pacific. sodium thiosulfate (Na#S#O$ ; 5H#O) and 10 mM magnetite (Fe$O%) under a gas phase of 80% H# and 20% CO# Historically, the systematics of methanococci has been (300 kPa). The cultures were incubated at 70 and 85 mCin hindered by the absence of information on the re- dry ovens on-board ship. The bottles of MMJ medium liability of phenotypic characters (Keswani et al., incubated at 70 mC became turbid after 2 days, but no 1996). Based on the comparison of nearly complete growth was observed at 85 mC. To obtain a pure culture, the 16S rRNA sequences from the members of Methano- dilution-to-extinction technique was employed (Baross, coccus, Boone et al. (1993) recommended that the 1995). genus Methanococcus should be further subdivided Sources of organisms. Methanococcus (Methanocaldococcus) into four genera to reduce the genetic diversity within jannaschii strain JAL-1T (l JCM 10045T), Methanococcus T one genus. More recently, Whitman et al. (2001) have (Methanothermococcus) thermolithotrophicus strain SN-1 (l JCM 10549T) and Methanococcus maripaludis strain JJT proposed the creation of two families and four genera T within the order Methanococcales. According to the (l JCM 10722 ) were obtained from the Japan Collection newly proposed taxonomy, the mesophilic methano- of Microorganisms (JCM, Wako, Japan). All strains were cultivated under optimal conditions as described previously cocci, including the as-yet undescribed ‘Methanococ- (Huber et al., 1982; Jones et al., 1983a, b). cus aeolicus’, belong to the genus Methanococcus and Methanococcus jannaschii, Methanococcus infernus, Culture medium and conditions. The novel isolate was Methanococcus vulcanius and Methanococcus fervens routinely cultivated in a standard medium, which was MMJ medium supplemented with 5 mM calcium chloride (CaCl#), belong to the genus Methanocaldococcus (Whitman et 10 mM sodium thiosulfate (Na#S#O$ ; 5H#O) and 10 mM al., 2001). Methanococcus igneus is assigned to the magnetite (Fe O ). MMJ medium consisted of the following $ "% genus Methanotorris and Methanococcus thermo- components (l− MJ synthetic seawater): 1 ml vitamin lithotrophicus to the genus Methanothermococcus solution (Balch et al., 1979), 50 mg sodium selenite, 30 mg (Whitman et al., 2001). This classification fits well the sodium tungstate, 1 mg resazurin, 20 g NaHCO$,0n5g phylogenetic relationships associated with thermoph- cysteine hydrochloride and 0n5gNa#S ; 9H#O (Sako et al., ily among the order Methanococcales recently pro- 1996; Takai et al., 1999). To prepare the standard medium, vided by Keswani et al. (1996) and Jeanthon et al. 50 mg sodium selenite, 30 mg sodium tungstate and 1 mg (1999). However, both of the genera Methanotorris resazurin were dissolved in 1 l MJ synthetic seawater and the Methanothermococcus pH of the medium was adjusted to around 7n0 with NaOH and are represented by single before autoclaving. After autoclaving, a concentrated sol- species and the phylogenetic affiliation of the deeply ution of vitamins (Balch et al., 1979), NaHCO$, thiosulfate, branched, mesophilic ‘Methanococcus aeolicus’ is still magnetite, cysteine hydrochloride (pH adjusted to 7n0) and uncertain. Hence, additional isolates will be helpful in Na#S (pH adjusted to 7n0) were added to the medium. These elucidating the phylogenetic organization and tax- solutions were sterilized separately by autoclaving except for onomy of the order Methanococcales. the vitamin solution, which was filter-sterilized. The pH of the medium was adjusted with H SO or NaOH in an In this study, we succeeded in isolating a thermophilic # % anaerobic chamber under 90% N# and 10% H# if needed. methanogen from a deep-sea hydrothermal vent chim- The medium was dispensed at 20% of the total bottle or tube ney at the Iheya Ridge, in the Okinawa Trough, Japan. volume and the tubes and bottles were sealed tightly with Phylogenetic analysis revealed that the novel isolate is butyl rubber stoppers under a gas phase of 80% H# and a member of the order Methanococcales. The novel 20% CO# at 300 kPa. In order to test the oxygen sensitivity isolate had physiological properties very similar to of the isolate, the gas phase was replaced by a gas mixture of those of Methanothermococcus thermolithotrophicus, 80% H#,15%CO# and 5% O# or 80% H#,19%CO# and but it is more closely related phylogenetically to the 1% O#. mesophile ‘Methanococcus aeolicus’. Based on its All experiments described below were conducted in dupli- physiological properties and the results of DNA–DNA cate. In an attempt to examine whether other potential hybridization analysis, the isolate can be described as a electron donors and acceptors supported or stimulated novel species of Methanothermococcus that we have growth in place of H# and CO#, organic compounds such as named Methanothermococcus okinawensis sp. nov. yeast extract, peptone, tryptone, Casamino acids, amino acids, formate, acetate, methanol, ethanol, dimethyl sulfide and trimethylamine were tested in the presence or absence of METHODS H# and CO#. The potential requirement for thiosulfate, magnetite, elemental sulfur, selenate, selenite, tungstate and Sample collection. A sample from a deep-sea hydrothermal nitrogen sources (10 mM NH%Cl, NaNO# or NaNO$) for vent chimney was obtained from the hydrothermal field at growth was also tested. To test the effect of pH on growth, Iheya Ridge in the Okinawa Trough, Japan (27m 47n220h N, the pH of MMJ medium was adjusted to various values with 126m 53n900h E) at a depth of 972 m by means of the manned 10 mM acetate\acetic acid buffer (pH 4–5), MES (pH 5–6), submersible Shinkai 2000 in dive F1194, performed in June PIPES (pH 6–7), HEPES (pH 7–7n5) or Tris (pH 8–9n5). 2000. This deep-sea hydrothermal vent site is the same site After autoclaving, the pH of the medium was checked and from which Thermosipho japonicus was isolated (Takai & readjusted with H#SO% or NaOH to the desired pH at room Horikoshi, 2000). The tip of the chimney was brought to the temperature. The pH was found to be stable during the sea surface in a sample box.