INTERNATIONALJOURNAL OF SYSTEMATICBACTERIOLOGY, July 1992, p. 463-468 Vol. 42, No. 3 0020-7713/92/030463-06$02.00/0 Copyright 0 1992, International Union of Microbiological Societies

Characterization of Three Thermophilic Strains of (‘ ‘”) thermophila sp. nov. and Rejection of Methanothrix (“Methanosaeta”) thermoacetophila YOICHI KAMAGATA,l* HIROKO KAWASAKI,* HIROSHI OYAIZU,3 KAZUNORI NAKAMURA,’ EIICHI MIKAMI,’ GINRO END0,4 YOSUKE KOGA,’ AND KAZUHIDE YAMASAT02 Fermentation Research Institute, Agency of Industrial Science and Technology, Tsukuba, Ibaraki 305’; Institute of Applied Microbiology2and Department of Agricultural Chernisty, The University of Tokyo, Tobo 113; Department of Civil Engineering, Tohoku Gakuin University, Tagajo-shi, Miyagi 9854; and Department of Chemistry, University of Occupational and Environmental Health, Japan, Yahatanishi-ku, Kitakyushu 807, Japan

Three thermophilic Methanothrix (“Methanosaeta”) strains, strains PTT (= DSM 6194,) (T = type strain), CALS-1 (= DSM 3870), and 2-517 (= DSM 4774), were characterized chemotaxonomicallyand compared with five mesophilic strains, (“Methanosaeta concifii”) GP6 (= DSM 36711, Opfikon (= DSM 2139), FE (= DSM 3013), U,, and P,. These methanogens were exclusively acetotrophic and had a characteristic sheathed structure. The DNA base compositions of the strains which we studied ranged from 50.3 to 54.3 mol% guanine plus cytosine. The thermophilic strains often had phase-refractive gas vesicles inside their cells. Denaturing electrophoresis of proteins showed that the mesophilic and thermophilic Mefhanothrix strains formed two distinct groups and that there were differences in protein patterns between the groups. The difference between the thermophiles and mesophiles was also verified by comparing partial 16s rRNA sequences (ca. 30 base differences in ca. 540 bases). On the basis of our results, we propose the name Methanothrix thenophila for the three thermophilic strains. The type strain of M. thermophila is strain P, (= DSM 6194). We also propose that the name Methanothrix thenoacetophila (‘LMethanosaetathermoaceto- phila”), which was given to strain 2-517 (type strain), should be rejected because of its description, which was based on an enrichment culture, was inadequate.

Of the methanogenic microorganisms, members of the Patel and Sprott also proposed that Methanothrix ther- Methanothrix play a very important role as acetate moacetophila ,which was originally described by Nozhevni- consumers in anaerobic digestion of organic compounds (6). kova and Chudina (17), should be reclassified as Methano- Methanothrix spp. are sheathed, rod-shaped, aceticlastic saeta thermoacetophila (21). However, the description of methanogens, and the first Methanothrix strain was de- the type strain (strain 2-517) of “Methanothrix thermoace- scribed by Sohngen (24). Later, Barker described this organ- tophila ” (‘ ‘Methanosaeta thermoacetophila”) was not val- ism as Methanobacterium soehngenii (1). However, no idly published because it was not based on an axenic culture axenic Methanothrix cultures were obtained for many years (17), and the level of taxonomic relatedness between strains because of difficulties in cultivation. 2-517 and CALS-1 is not known. (Patel and Sprott have A Methanothrix strain was first isolated as an “acetate indicated that strain 2-517 has been purified and now is organism” (strain Opfikon) by Zehnder et al., and later these available as a pure culture [21], but no information about the authors described this strain as Methanothrix soehngenii (9, taxonomic characteristics of the pure culture has been 27). After this description, several mesophilic Methanothrix published.) Very recently, we isolated a new thermophilic strains were isolated or “highly purified” (8, 10, 18, 20, 26). Methanothrix (“Methanosaeta”) strain, strain PTT (T = A thermophilic Methanothrix strain was first enriched from type strain), from a thermophilic anaerobic digestor and thermal lake mud by Nozhevnikova and Chudina (17), and described its properties (11). In this study, we chemotaxo- these authors named their enriched organism (strain 2-517) nomically characterized three thermophilic Methanothrix Methanothrix thermoacetophila. Zinder et al. first obtained strains, including our isolate, and compared them with a pure culture of thermophilic Methanothrix sp. strain Methanothrix soehngenii (“”). We CALS-1 from a thermophilic anaerobic digestor (28). propose that a new , Methanothrix thermophila, Recently, Patel and Sprott proposed the genus Methuno- should be established and that the name “Methanothrix saeta (21). According to the proposal of these authors, thermoacetophila” (‘ ‘Methanosaeta thermoacetophila ”) Methanosaeta concilii is the only valid species which con- should be rejected. tains mesophiles, and the type strain is strain GP6, which was described as Methanothrix concilii by Patel (20). How- ever, there is controversy concerning the correct designation MATERIALS AND METHODS because Methanosaeta concilii, Methanothrix concilii, and Sources of microorganisms. Methanothrix soehngenii Methanothrix soehngenii are considered synonyms (3). (“Methanosaeta concilii”) Opfikon (= DSM 2139), Metha- nothrix soehngenii FE (= DSM 3013), Methanothrix soehn- genii GP6 (= DSM 3671), “Methanothrix thermoaceto- * Corresponding author. phila” (“Methanosaeta thennoacetophila”) 2-517 (= DSM

463 464 KAMAGATA ET AL. INT. J. SYST.BACTERIOL.

TABLE 1. Properties of Methanothrix strains" DSM Optimum Growth Guanine-plus-cytosine Species Strain no. temp ("C)" factor(s)' content (mol%)d

~~~ ~~ Methanothrix soehngenii Opfikon' 2139 37 SFf 51.9 FE' 3013 35 NW 52.6 GP6 3671 35-40 SF, vitaminsh 50.3 U*" 37 ND 51.1 PM' 37 ND 50.9 Methanothhrix thennophila CALS-1 3870 60 CoM, biotin' 54.2 2-517 4774 55 ME, vitamins" 54.3 PTT 6194 55-60 SFf 52.7 The cells of all of the strains were sheathed rods, and all of the strains grew at a neutral pH (data from references 9 through 11, 17, 20, 21, and 26 through 28) and were not susceptible to lysis. " Data from references 9 through 11, 17, 20, 21, and 26 through 28. SF, sludge fluid; CoM, coenzyme M (mercaptoethane sulfonate); ME, manure extract. Data for strains Opfikon, FE, GP6, UA, P,, and PTT from references 10, 11, and 26. Highly purified culture. f Sludge fluid had a stimulatory effect but was not required (9, 11). 6 ND, not determined. Sludge fluid and vitamins had a stimulatory effect but were not required (20, 21). Coenzyme M had a stimulatory effect but was not required (28). j Manure extract and vitamins had a stimulatory effect but were not required (17, 21).

4774), and Methanothrix sp. strain CALS-1 (= DSM 3870) Osaka, Japan), as described by Tamaoka and Komagata were obtained from the Deutsche Sammlung von Mikroor- (25). Guanine-plus-cytosine contents were determined by ganismen und Zellkulturen GmbH (DSM), Braunschweig, reversed-phase high-performance liquid chromatography Germany. Methanothrix soehngenii UA and P, and Metha- (Shimadzu model LC-6A system). Separation was achieved nothrix sp. strain PTT (= DSM 6194T) were obtained in our at 40°C by using a flow rate of 1 ml/min, a column of laboratory (10, 11). The cultures of mesophilic strains Op- CLC-ODS (Shimadzu), and 5% methanol in 10 mM phos- fikon, FE, U,, and P, were highly purified but not axenic phate buffer (pH 3.5) as the mobile phase. Each deoxyribo- (10, 21, 26). However, these cultures were considered to be nucIeoside was detected by determining the A260,and an pure enough for analyses of protein patterns of whole cells, equimolar mixture of four deoxyribonucleosides was used as 16s rRNA sequences, and DNA base compositions, as well the standard. as the other physiological studies described below. 16s rRNA sequencing. We determined partial 16s rRNA Cultivation. Methanothrix soehngenii 0 fikon, FE, GP6, sequences for strains Opfikon, GP6, U,, P,, CALS-1, U,, and P, and Methanothrix sp. strain P,". were cultivated 2-517, and PTT. The RNA was extracted by using previously on DSM medium 334 (5). Methanothrix sp. strain CALS-1 described methods (13, 19). Partial 16s rRNA sequences and "Methanothrix thennoacetophila" 2-517 were culti- were determined by using the reverse transcriptase proce- vated on DSM medium 387 (5). Unless otherwise stated, all dure (13). In this procedure, we used three nucleotide of the strains were cultured at 37°C (strains Opfikon, FE, primers that were complementary to universally conserved GP6, U,, and P,) or 55°C (strains CALS-1,2-517, and PTT) regions, TACCGCGGCGGCTGGC (position 520, reverse in 125-ml serum vials containing 30 ml of medium or in direction), CAATTCCTTTAAGTTTC (position 920, reverse 1,000-ml bottles containing 500 ml of medium under an direction), and ACGGGCGGTGTGTGC (position 1400, re- N,-C02 (80/20, vol/vol) atmosphere. The serum vials were verse direction) (E. coli 16s rRNA numbering). These prim- closed with butyl rubber stoppers that were sealed with ers were made by using an ABI model 381A DNA synthe- aluminum crimps. sizer. The DNA sequences were determined by the Electrophoresis. Sodium dodecyl sulfate (SDS)-polyacryl- dideoxynucleotide chain termination method (23). amide gel electrophoresis was performed as described by Lipid analysis. The membrane polar lipids of strain PTT Laemmli (12), using a gradient gel containing 4 to 20% were extracted and analyzed by using a previously described acrylamide. Portions (approximately 5 pg) of denatured method (16). protein from whole cells were loaded on the gel, and bovine Other methods. Physiological characteristics were deter- milk a-lactalbumin (molecular mass, 14 kDa), soybean mined by using the minimal standards described by Boone trypsin inhibitor (20 kDa), bovine erythrocyte carbonic and Whitman (4). anhydrase (30 kDa), egg white ovalbumin (43 ma), bovine serum albumin (67 kDa), and rabbit muscle phosphorylase b RESULTS AND DISCUSSION (94 kDa) were used as the M, standards. The gel was stained with Coomassie brilliant blue R-250. Morphological and physiological characteristics. Table 1 Determination of DNA base composition. We determined summarizes the morphological and physiological properties the DNA base compositions of strains 2-517 and CALS-1 in of three thermophilic and five mesophilic Methanothrix this study. DNA base composition data for the other strains strains. The data for optimum temperature, pH at which were obtained from previously published papers (10,11,26). growth occurred, and growth factors were obtained from DNA was extracted and purified by using the methods of previous reports (9-11, 17, 20, 21, 26-28). All of the strains Beji et al. (2) and Saito and Miura (22). The purified DNA had characteristic sheathed structures in which a number of was hydrolyzed with P1 nuclease (GC kit; Yamasa Shop cells were arranged. The cells were nonmotile straight rods Co., Chiba, Japan) and then with alkaline phosphatase from with flat ends and were gram negative. The mean dimensions Eschen'chia coli (Wako Pure Chemicals Industries, Ltd., of single cells were 0.8 to 1.3 by 2 to 6 pm, and cells were VOL.42, 1992 METHANOTHRIX THERMOPHILA SP. NOV. 465

template. Table 2 shows the levels of sequence homology and the numbers of base differences for the seven strains. Within the thermophilic group, the strain PTT sequence exhibited 100% similarity with the sequence of strain 2-517, while there were six base differences between strains PTT and CALS-1. Our comparative analysis of the 16s rRNAs revealed a considerable difference between the thermophiles and the mesophiles. Within the mesophilic group, the strains exhibited more than 99% similarity with each other, and this finding was in close agreement with the results of the DNA-DNA hybridization studies of Touzel et al. (26), who used strains FE, GP6, and Opfikon. However, the members of the thermophilic group exhibited less than 95% similarity (corresponding to 29 to 34 base differences) to the meso- philes, suggesting that the thermophilic group is quite distant from any of the mesophilic Methanothrix strains. Nomenclature of thermophilic strains belonging to the genus Methanothrix. The thermophilic Methanothrix strains form a taxon which is phenotypically and genotypically distinguish- able from the mesophilic group of the genus Methanothrix. However, as described above, the partial 16s rRNA se- quence of strain CALS-1 was different from the sequences of strains 2-517 and PTT at six positions, indicating that the thermophilic Methanothrix strains apparently should be sep- arated into two genogroups. In this study, the two genogroups could not be differenti- ated by any of conventional methods used for characteriza- FIG. 1. Protein patterns for three strains of Methanothe ther- tion (Table 1). In general, a species should be established mophila and five strains of Methanothrix soehngenii as determined by SDS-polyacrylamide gel electrophoresis. Electrophoresis was with phenotypic characteristics which apparently distinguish performed with a 4 to 20% acrylamide gel. Lane ST, M, standards. the species from allied species. Therefore, we are reluctant to separate the thermophilic Methanothrix strains into two species and propose that the two genogroups should be assigned to a single species. sometimes connected to each other. The sheathed filaments In the thermophilic Methanothrix group, “Methanothrix of the thermophiles were generally much shorter (length, 10 thermoacetophila” (‘ ‘Methanosaeta thermoacetophila ”) is to 100 pm) than those of the mesophiles (>lo0 pm). The the name that has been given to strain 2-517 (17, 21). cells were not susceptible to lysis by SDS or hypotonic However, the description of “Methanothrix thermoaceto- conditions at room temperature when we used the proce- phila” was inadequate because it was based on an enriched dures described by Boone and Whitman (4). culture (17), and the name is invalid according to the As described previously, one of the characteristics that International Code of Nomenclature of Bacteria (Rule 31a) differentiated tht; thermophiles from the mesophiles group (14). In this paper we propose the name Methanothrix was the fact that the thermophiles contained phase-refrac- thermophila sp. nov. for the thermophilic strains of the tive particles, probably gas vesicles, within their cells (11, genus Methanothrix. The type species is strain P, (= DSM 17, 28). These particles could be readily eliminated by 6194). Methanothrix themophila contains two genogroups centrifugation or sonication. However, in strain 2-517 we which differ in their 16s rRNA se uences. Genogroup 1 did not observe these particles, whereas Nozhevnikova and consists of strains PTT (= DSM 61949. ) and 2-517 (= DSM Chudina observed them in their original culture (17). 4774), and genogroup 2 contains strain CALS-1 (= DSM DNA base composition. The guanine-plus-cytosine con- 3870). tents of the DNAs of strains CALS-1 and 2-517 were Description of Methanothrix thermophila sp. nov. Methan- calculated to be 54.2 and 54.3 mol%, respectively. The othrix thermophila (ther. mo‘ phi. la. Gr. adj. thermos, hot; values for the other strains were obtained from previous Gr. adj. philos, loving; Gr. adj. thermophila, heat loving) is reports (10, 11, 26) (Table 1). a thermophilic (optimum growth occurs at 55 to 60”C), Protein patterns of whole cells. The protein patterns for the rod-shaped, sheathed, strictly anaerobic, aceticlastic meth- whole cells of the three thermophiles, as determined by anogen that belongs to the genus Methanothrix (“Methano- SDS-polyacrylamide gel electrophoresis, were almost iden- saeta ”). Methanothrix thermophila is apparently distinct tical (Fig. l), and these patterns were quite distinct from from Methanothrix soehngenii (‘ ‘Methanosaeta concilii’ ’) those of the mesophiles. because of its thermophily and 16s rRNA sequence. Cells Partial 16s rRNA sequences. Figure 2 shows an alignment are nonmotile, strictly anaerobic rods with flat ends and are of the partial 16s rRNA sequences obtained for three ther- gram negative. The mean dimensions of single cells are 0.8 to mophiles and four mesophiles. Sequence information was 1.3 by 2 to 6 pm. Cells are enclosed inside an annular, obtained for approximately 540 bases (ca. 37% of all of the striated sheathed structure and are separated by partitions 16s rRNA bases). The alignment was optimized by using the that have a concentric structure. Cells grow in the form of secondary structure of Methanobacteriumformicicum DSM loose sediment, and culture broth exhibits opalescent turbid- 1312 (15). The sequence for strain Opfikon determined by us ity after gentle shaking. Most of the sheathed filaments are was identical to that determined previously by Eggen et al. less than 100 pm long, and they do not form bundles or the (7), suggesting a high fidelity of sequencing from the RNA cottonlike flocs which are common in Methanothrix soeh- m 281 291 301 31 1 1. M. fomiciMcm DSM1312 C;GGUUGUGAG AGCAAGAGCC CGGAGAUGGA ACCUGAGACA AaCCAGG 2. Opfikon C(XUUGUGAG AGCAAGAGCC CGGAGAUGGA WCUGAGACA CGAAUCCAGG 3. GP6 GCGUUGUGAG AGCAAGAGCC CGGAGAUGGA WCUGAGACA CGAAUCCAffi 4. UA GCGUUGUGAG AGCAAGAGCC CCXAGAUGGA WCUGAGACA CGAAUCCAGG 5. PM GGGUUGUCAG AGCAAGAGCC CGGAGAUGGA WCUGAGACA CGAAUCCAGG 6. CALS-1 CGGUUGUGAG AGCAAGAGCC CGGAGAUGGA WCUGAGACAYGAAUCCAGG 7.2517 GGGUUGUGAG AGCAAGAGCC CGGAGAUGGA WCUGAGACA UGAAUCCAGG 8. Pr GCGUUGUGAG AGCAAGAGCC CGGAGAUGGA WCUGAGACA UGAAUCCAGG

321 331 341 351 361 371 381 1. CCCUACGGGG CGCAGCAGGC GCGAAACCUC CGCAAUGCAC GAAAGUGCGA -C CCAAGUmCA 2. CXXUAC(XGG UGCAGCAGGC GCGAAAACUU UACAAUGCUG GCAACAGCGA UAAGGGAACC UCGAGUGCCA 3. CXXUACGGGG UGCAGCAGGC GCGAAAACUU UACAAUGCUG GCAACAGCGA UAAGGGAACC UCGAGUGCCA 4. CCCUACGGGG UGCAGCAGGC GCGAAAACUU UACAAUGCUG GCAACAGCGA UAAGGGAACC UCGAGUGCCA 5. CCCUACC;GGG UGCAGCAGGC GCGAAAACUU UACAAUGCUG GCAACAGCGA UAAGc;GAACC UCGAGUGCCA 6. CXXUACGGGG UGCAGCAW GCGAAAACUU UACAAUGCS GCAAC-SGA UAAGGGQICC UCGAGUa- 7. CCCUACGGGG UGCAGCAGGC GCGAAAACUU UACAAUGCGG GCAAC-A UAAVCC UCGAGU- 8. CCCUACGGGG UGCAGCAGGC GCGAAAACUU UACAAUGCS GCAAC-GA UAAGGGQlCC UCGAGU-

391 401 41 1 421 1. CUCUUAACGG GGUGGcUUUU CUUAAGUGUA AAAAGCUUUU 2. G-GWACNNA UCUGGCUGUC GANAUGCCUA AAAAGCAUUU 3. G-GWACAAA UCUGGCUGUC GANAUGCCUA AAAAGCAUUU 4. G-GWACNNA UCUGGCUGUC GANAUGCCUA AAAAGCAUUU 5. GGWACNNA UCUGGCUGUC GANAUGCCUA AAAAGCAUUU 6. G-GUUACAAA -ccvcC~GGDGCCUA AAAAGCAGUC 7. G-GUUACAAA pGCUGW GGGGUGCCUA AAAAGCAGUC 8. G-GWACAAA WGGCUGW -_CGCCUA AAMGCAGUC

641 651 661 671 681 691 701 1. AUAAUCCCGG GAGGACCACC UGUGGCGAAG GCGGCUAACU GGMCGGGCC UGACGGUGAG UAACGAAAGC 2. GUAAUCCUUG AAGGACCACC AGUGGCGAAG GCGUCUCACC AGAACGGAAC UGACGGCAAG GGACGAAAGC 3. GUAAUCCUUG AAGGACCACC AGUGGCGAAG GCGUCUCACC AGAACGGAAC UGACGGCAAG GGACGAAAGC 4. GUAAUCCUUG AAGGACCACC AGUGGCGAAG GCGUCUCACC AGAACGGAAC UGACGGGUG GGACGAAAGC 5. GUAAUCCUUG AAGGACCACC AGUGGCGAAG GCGUCUCACC AGAACGGAAC UGACGGCAAG GGACGAAAGC 6. GUAAUCCUUG AGGGACCACC AGUGGCGAAG GCGUCUCACC AGAACGGAUC CGACGGCAAG GGACGAAAGC 7. GUAAUCCUCG A-mCCACC AGUGGCGAAG GCGUCUCACC AGAACGGAUC CGACGGCAAG GGACGAAAGC 8. GUAAUCCUCG A-CCACC AGUGGCGAAG GCGUCUCACC AGAACGGAUC CGACGGCAAG GGACGAAAGC 7ll 721 731 741 751 761 771 1. CAGGGKGCG AACCGGAUUA GAUACCCGGG UAGUCCUGGC CGUAAACGAU GUGGACUUGG UGUUGGGAUG 2. UAGGGGCACG AACCGGAWA GAUACCCGGG UAGUCCUAGC CGUAAACGAU ACUCGCUAGG UGUCGGC - AC 3. UAGGGGCACG AACCGGAUUA GAUAKCGGG UAGUCCUAGC CGUAAACGAU ACUCGCUAGG UGUCGGC - AC 4. UAGGGGCACG AACCGGAUUA GAUACCCGGG UAGUCCUAGC CGUAAACGAU ACUCGCUAGG UGUCGGC - AC 5. UAGGGGCACG AACCGGAUUA GAUACCCGGG UAGUCCUAGC CGUAAACGAU ACUCGCUAGG UGUCGGC - AC 6. UAGGGGCACG AACCGGAUUA GAUACCCGGG UAGUCCUAGC CGUAAACGAU ACUCGCUAGG UGUCGGC-AC 7. UAGGGGCACG AACCGGAWA GAUACCCGGG UAGUCCUAGC CGUAAACGAU ACUCGCUAGG UGUCGGC - AC 8. UAGGGGCACG AACCGGAUUA GAUACCCGGG UAGUCCUAGC CGUAAACGAU ACUCGCUAGG UGUCGGC - AC 781 791 801 81 1 821 831 1. GCUCCGAGCU GCCCCAGUGC CGAAGGGAAG CUGWAAGUC CACCGCCUGG GAAGUACG 2. GGUGCGACCG WGUCGGUGC CGUAGGGAAG CCGUGAAGCG AGCCACCUGG GAAGU 3. GGUGCGACCGUUGUCGGUGC CGUAGGGAAG CCGUGAAGCG AGCCACCUGG GAAGU 4. GGUGCGACCG WGUCGGUGC CGUAGGGAAG CCGUGAAGCG AGCCACCUGG GAAGU 5. CZUGCGACCGUUGUCGGUGC CGUAGGGAAG CCGUGAAGCG AGCCACCUGG GAAGU 6. GGUGCGACCG WGUC~XGC CGUAGGGAAG CCGUGAAGCG AGCCACCUGG GAAGU 7. GGUNNUCCG UUGUCGGUGC CGUAGGGAAG CCGUGAAGCG AGCCACCUGG GAAGU 8. OCUGCGACCGUUGUCGGUGC CGUAGGGAAG CCGUGMGCG AGCCACCUGG GAAGU FIG. 2. Partial 16s rRNA sequences for three strains of Methanothrix themophila and five strains of Methanothrix soehngenii. The sequences are aligned with the known complete sequence for Methanobacterium fomicicum (15). A, adenine; U, uracil; C, cytosine; G, guanine; N, ambiguous nucleotide. The dashes indicate positions that were deleted in the sequences. The underlined bases are bases that were different from those of Methanothrix soehngenii Opfikon.

466 VOL. 42, 1992 METX4NOTHRLX THEMOPHILA SP. NOV. 467

1141 1151 1161 1171 1181 1191 lzol 1. UAGGUCCGUA UGCCCCGAAU CCCNGGGCU ACA-GGGC UACAAUGGUU AGGAWUGG GUUCCGACAC 2. UAGGUCAGUA UGCCCCGANN AUCCCGGGCU ACACGCGAGC UACAAUGGUU GGUACAAUGG GUAUCUACCC 3. UAGGUCAGUA UGCCCCGAAU AUCCCGGGCLJ ACACGCGACX UACAAUGGUU GGUACAAUGG GUAUCUACCC 4. UAGGUCAGUA UGCCCCGA" AUCCCGGGCU ACACGCGAGC UACAAUGGUU GGUACAAUGG GUAUCUACCC 5. UAGGUCAGUA UGCCCCGA" ALJCCCGGGCU ACACGCGAGC UACAAUGGUU GGUACAAUGG GUAUCUACCC 6. UAGGUCAGUA UGCmCGANU EQGGGCU ACACGCGGGC UAClUUGGK GGQCAAUGG GUAUCQMXC 7. UAGGUCAGUA UCCCCCGAAU ECCCGGGCU ACACGCGGGC UACAAUGGUC GGUACAAW GUAUCaCCC 8. UAGGUCAGUA UGccCcGAAU CCCCGGGCU ACAC- UACAAUGGUC GGUACAAUGG GUAUCWCCC

1211 1221 1231 1241 1251 1261 127l 1. UGAAAGGUGG AGGUAAUCUC CUMCCUGG CCUUAGUUCG GAWGAGGGC UGUAACUCGC CCUCAUGAAG 2. CGAAAGCXGA CGGGAAUCUC CUAAAACCAA UCUUAGUUCG GAWGAGGGC UGCAACUCGC CCUCAUGAAG 3. CGAAA(%%GA CGCGAAUCUC CUAAAACCAA UCUUAGUUCG GAWGAGGGC UGCAACUCGC CCUCAUGAAG 4. CGAMGGCXA CGGGAAUCUC CUAAAACCAA UCUUAGUUCG GAWGAGGGC UGCAACUCGC CCUCAUGAAG 5. CGAAAGGGGA OGGGAAUCUC CUAAAACCAA UCUUAGUUCG GAWGAGGGC UGCAACUCGC CCUCAUGAAG 6. -mGGGGy 4GGQiAUCcC CUMSCGA UCWAGUUCG GAWGAGGGC UG&UCUCGC CCUCAUGAAG 7. CGMiA- &XQlAUCcC CUAAAACCEA UC_cvAcvvcc GAUUGAGGGC UG&UiCUCGC CCUCAUGAAG 8. CGAAA- 4wUCcC CUAAAACCEA UCQJAGUUCG GAWGAGGGC UG&UCUCGC CCUCAUGAAG

1281 1291 1301 131 1 1321 1. CUGGAAUGCG UAGUAAUCGC GUGUCAUAAC CGCGCGGLJGA AUACGUCCCU 2. CUGGAAUCCG UAGUAAUCGC GUUUCAACAG AACGCGGUGA AUACGU 3. CUGGAAUCCG UAGUAAUCGC GUUUCAACAG AACGCGGUGA AUACGU 4. CUGGAAUCCG UAGUMUCGC GUUUCAACAG AACGCGGUGA AUACGU 5. CUGGAAUCCG UAGUAAUCGC GUUUCAACAG AACGCGGUGA AUACGU 6. CUGGAAUCCG UAGUAAUCGC GUUUCAACAG AACGCGGUGA AUACGU 7. CUGGAAUCCG UAGUAAUCGC GUUUCAACAG AACGCGGUGA AUACGU 8. CUGGAAUCCG UAGUAAUCGC GUUUCAACAG AACGCGGUGA AUACGU FIG. 2-Continued. ngenii. Colonies are sometimes observed in acetate agar (dibiphytanediyl-dig1 cerol tetraether; caldarchaeol) of lipid medium when high cell concentrations are present (11, 28). is found in strain P, T. Gas vesicles are often observed inside the cells. Cells are not The optimum temperature for growth and methanogenesis susceptible to lysis by SDS or hypotonic conditions at room is 55 to 60"C, depending on the strain (11, 17, 21, 28). The temperature, but cells are lysed by SDS at 60°C. optimum pH is around 7. Acetate is the only substrate used The intensity of autofluorescence (405-nm excitation) is for growth and methanogenesis (11, 17, 28). Neither metha- higher than that for Methanothrix soehngenii because of a nogenesis nor growth is observed when H,-CO,, formate, higher content of coenzyme F,,, (11). Coenzyme F,, with methylamines, or methanol is used for the substrate (11, 17, four glutamyl residues on the side chain is more abundant 28). Formate is not split into H, and CO, (11,28). NaCl is not than the other coenzyme F,,, types, although the total required for growth (11). Coenzyme M (mercaptoethane coenzyme F,, contents are much lower than the contents in sulfonate), vitamins, manure extract, or sludge fluid stimu- other hydrogenotrophic methanogens (11). A tetraether type lates growth (11, 17, 28). Chloramphenicol (5 pg/ml), baci- tracin (10 pg/ml), neomycin (10 pg/ml), tetracycline (100 pg/ml), and kanamycin (100 pg/ml) inhibit growth and meth- anogenesis (11). TABLE 2. Levels of 16s rRNA sequence homology for four The DNA base composition ranges from 52.7 to 54.3 mol% mesophilic and three thermophilic Methanothrix strains guanine plus cytosine as determined by high-performance No. of base differences or % homology with liquid chromatography (Table 1). The 16s rRNA sequence is straina: quite distinct from that of Methanothrix soehngenii (level of Strain F similarity, less than 95%). The type strain of Methanothrix themophila is strain P, (= DSM 6194). Methanothrix ther- mophila contains two genogroups. Genogroup 1 consists of strains PTT (= DSM 6194T) and 2-517 (= DSM 4774), and Opfikon (= DSM 2139) 0 1 0 33 29 29 genogroup 2 contains strain CALS-1 (= DSM 3870). The GP6 (= DSM 3671) 100 1 0 33 29 29 partial 16s rRNA sequences described above have been UA >99 >99 1 34 30 30 submitted to the National Institute of Genetics, Shizuoka, PM 100 100 >99 33 29 29 CALS-1 (= DSM3870) 94 94 94 94 66 Japan. 2-517 (= DSM 4774) 95 95 94 95 99 0 PTT (= DSM 6194=) 95 95 94 95 99 100 ACKNOWLEDGMENTS

a The values on the upper right are the numbers of base differences, and the We thank Kazuo Komagata (Tokyo University of Agriculture), values on the lower left are the percentages of homology (calculated from the Tsuyoshi Morinaga (Daicel Chemical Industries, Ltd.), Ken-ichiro data in Fig. 2). Suzuki (Japan Collection of Microorganisms, Institute of Physical 468 KAMAGATA ET AL. INT.J. SYST.BACTERIOL. and Chemical Research, RIKEN), and KO Imai (Institute for Fer- H. P. R. Seeliger, and W. A. Clark (ed.). 1975. International mentation, Osaka, Japan) for their helpful suggestions. code of nomenclature of bacteria. 1976 Revision. Amzrican This work was supported by a grant from the New Energy and Society for Microbiology, Washington, D.C. Industrial Technology Development Organization (Research and 15. Lechner, K., G. Wich, and A. Boeck. 1985. 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