Huber, Harald

König, H.

Stetter, Karl Otto

Thies, G.

Thomm, Michael

English

University of Regensburg Publication Server

Arch Microbiol (1982) 132:47-50 Archives of Mhvobnloyy © Springer-Verlag 1982 Methanococcus Thermolithotrophicus, a Novel Thermophilic Lithotrophic Methanogen Harald Huber 1, Michael Thomm 1 , Helmut König 1 , Gesa Thies 2, and Karl O. Stetter1 1 Lehrstuhl für Mikrobiologie, Universität Regensburg, D-8400 Regensburg, Federal Republic of Germany 2 Lehrstuhl für Zoologie, Universität Regensburg, D-8400 Regensburg, Federal Republic of Germany Abstract. A n autotrophic thermophilic motile coccoid metha-nogen was isolated from geothermally heated sea Sediments close to Naples, Italy. Growth occurs on H 2 / C 0 2 and on formate between 30 and 70° C with an Optimum at 65° C. The optimal doubling time is only 55 min. The NaCl-concentration ranges from 1.3% to 8.3% with an Optimum around 4%. By its G + C-content of 31.3 mol %, its subunit envelope, and by D N A - R N A hybridization the new isolate is clearly defined to be a member of the genus Methanococcus. We name it Methanococcus thermolithotrophicus. Key words: Methanogens — Archaebacteria — Autotrophic — Thermophilic A l l thermophilic methanogens known so far are members of two Orders, the Methanobacteriales and the Methano-microbiales. The latter contains only one thermophilic strain, Methanosarcina TM-1 (Zinder and Mah 1979), which has been isolated from a sewage digestor and grows at a maximal temperature of 55° C. The Methanobacteriales contain the thermophilic Methanobacterium thermoautotrophicum, which occurs in thermal Springs and sewage digestors (Zeikus and Wolfe 1972; Zeikus et al. 1980), and grows between 37° C (Stetter unpublished) and 75° C (Zeikus and Wolfe 1973; Zeikus et al. 1980), and the extreme thermophilic species Methanothermus fervidus (Stetter et al. 1981), which was re-cently isolated from a hot spring in Iceland, and grows be-tween 65 and 97° C. In the order Methanococcales, 2 species, which can be cultivated only in the mesophilic temperature ränge, are known (Balch et al. 1979). Here we report on the Isolation and characterization of a new species of Methanococcus which grows at temperatures up to 70° C. Materials and Methods Strains Methanococcus voltae, D S M 1937, was obtained from the Deutsche Sammlung von Mikroorganismen, Göttingen. Culture Conditions The organisms were cultivated by using the technique de-scribed by Balch and Wolfe (1976). Methanococcus voltae was Abbreviations. G + C : Guanine 4- Cytosine; SDS: Sodium dodecylsul-fate (Sodium lauryl sulfate) Offprint requests to: K. O. Stetter grown in medium 3 of Balch et al. (1979), slightly modified (Wildgruber et al. 1982). For autotrophic growth of the new isolate, medium 3 of Balch et al. (1979), not containing sodium acetate, yeast extract, trypticase, and trace vitamines, pH 6.9 (adjusted with H 2 S 0 4 ) , was used ( " M G G medium"). Twenty milliliter cultures were grown in stoppered pres-surized 100 ml serum bottles (Bormioli, Italy) made of "type III"-glass by incubation in shakers (New Brunswick) at 140rpm, employing a glycerol bath. Enrichment and isolation medium ("supplemented sea water"): 750 ml sea water was supplemented with the follow-ingingredients:Na 2C0 3 1 g ; N H 4 H C 0 3 2 g; sodium formate 1 g; sodium acetate 5 g; yeast extract (Difco) 1 g; tryptic digest of casein (Merck) 2g; trace vitamines (Balch et al. 1979) 10ml; trace minerals (Balch et al. 1979) 10ml; 2 mercapto-ethanesulfonic acid 0.1mg; resazurin 1mg; L-cysteine hy-drochloride • H 2 0 0.5 g; Na 2 S • 9 H 2 0 0.5 g. Then, the medium was completed to 11 with H 2 0 dest. and the p H was adjusted to 7.5 (acetic acid). The media were sterilized for 20 min at 120°C. Light Microscopy The cells were observed and photographed with a Leitz Ortholux II microscope, equipped with a vario-orthomat camera System (Leitz). Fluorescence was observed in a Zeiss Standard fluorescence microscope with an excitation filter H436 and a selection filter LP470. Electron Microscopy For platinum shadowing, the cells were fixed on collodium coated grids and shadowed with platinum-iridium under an angle of 7°. To prepare thin sections, cells were centrifuged and then fixed in MGG-medium with 20 g glutaraldehyde/1 for 2 h and postfixed with 10 g Os0 4 / l for 1 h. Durcupan (Fluka) epoxy resin was used for embedding and thin sections were contrasted with lead citrate (5 min), uranylacetate (5 min) and again with lead citrate (3 min). Electron micrographs were taken with a J E O L J E M 100 C electron microscope at 80 kV and with a 40 um objective aperture. Isolation of DNA The D N A ' s of Methanococcus voltae and the new isolate were prepared as described elsewhere (Wildgruber et al. 1982). 0302-8933/82/0132/0047/S01.00 Analysis ofthe Cell Wall The existence of a cell wall sacculus was checked for as described (Stetter et al. 1981). Muramic acid and glycoprotein were analyzed as de-scribed (König and Stetter, in preparation). Methane Detection Methane was determined with a Hewlett Packard gas Chro-matograph, model 5880 A , using a 6 foot glass column filled with carbosieve S (Supeico) at 70° C isothermal. Temperature Measurement The temperature of the sea sediments was measured with an electronic thermometer (Metratherm 1200 d, BBC Metra-watt, Germany) equipped with a T126 electrode. Results Collection of the Sample The sample SN 1 was taken from the sandy geothermally heated sea floor of the beach at Stufe di Nerone close to Naples (Italy)vabout 3 m away from the shore in about 0.5 m depth. The original temperature of the sediment was 50° C, the p H 6.5 and the conductivity 7.5 mS, which is that of normal sea water. The sample was immediately filled into a sterile 100 ml storage bottle, which was sealed with a rubber stopper after the addition of 0.1 ml of resazurin (0.1 % w/v in water). The redox potential was then lowered as described (Wildgruber et al. 1982). The sample was carried to the laboratory at room temperature (around 20° C). Enrichment and Isolation In a freter type anaerobic Chamber (Aranki and Freter 1972), a 100 ml serum bottle containing 20 ml "sea water sup-plemented" and 50 \ig vancomycin/ml and 30 |ig penicillin/ml was inoculated with 1 ml of sample SN 1. After sealing with a stopper, the bottle was pressurized (300 kPa H 2 : C 0 2 = 80:20; Balch and Wolfe 1976) and then incubated in a shaker (New Brunswick) at 50° C. After 4 days, the medium became turbid and methane could be detected in the gas phase. In the fluorescence microscope, masses of strongly green fluorescing cocci were visible. One milliliter culture was transferred into the same medium, and incubated at 70° C. Next day the new organism, called SN 1, was grown without visible infection. This enrichment culture was streaked onto agar plates (2 % agar) prepared with "supplemented sea water". After 1 week, round convex smooth yellow colonies, about 0.5 — 1 mm in diameter became visible. Culture and Storage Cultures were transferred each day into fresh MGG-medium (2% inoculation). For a long time preservation, the strain SN 1 was grown for 6 h and then, after renewing the gas atmosphere, it was stored at 4°C, from where it can serve at least 6 months as inoculum. 0 25 30 35 40 45 50 55 6 0 66 70 tCC) »» Fig. 1. Optimal growth temperature. x x Methanococcus voltae; 0 ^ Methanococcus thermolithotrophicus. Growth was determined several times during the exponential phase by O.D.578-measurement. The hours/doubling were calculated from the slopes of the growth curves (not shown) 0 1 2 3 4 5 6 7 8 NaCl [%] Fig. 2. Influence of NaCl-concentration on growth of Methanococcus thermolithotrophicus. The NaCl was added to a MGG-medium, not supplemented with NaCl. This basal medium already contains 0.33% NaCl, which is not considered in the diagram Optimal Growth Temperatures The isolate SN 1 shows a broad temperature ränge of growth between 30° C and 70° C (Fig. 1). At 75° C (data not shown) the O . D . 5 7 8 of the culture doubles within 5 h, followed by a period of cell lysis within the next 6 h. The shortest doubling time (Fig. 1) is only 55min at 65°C. For comparison, Methanococcus voltae shows (Fig. 1) a temperature Optimum around 40° C, under the same conditions. Substrates and Organic Components of the Medium H 2 and formate serve as Substrates for methane formation, No organic components are required nor do they stimulate growth significantly (data not shown). Optimal Salt Conccntration The isolatc S N 1 grows in the presence of 1.3 — 8.3% N a C l (Fig . 2). The optimal conccntration is around 4 " u N a C l . Optimal pH The new organism grows in a p H ränge from 6 to 8 with an Optimum around 7 (data not shown). Morphology In the light microscope, regulär to irregulär highly mottle eoeei about 1.5 um in diameter, occurring singly and in pairs, can he seen (Fig. 3a). The eells are Gram-negative. In the presence of 2 % S D S (w/v) the eells are immediately lysed. As seen in the electron microscope, a tuft of flagella is present, which is inserted in a distinet area on the cell surface (Fig. 3b). Fach of the about 20 flagella of a cell is up to 3 um long and 20nm thick. In thin sections a cell envelope can be seen (Fig. 3c), which consists of subunits, most likely of protein (Fig. 3d), similar to other Methanococci (Jones et al. 1977). N o muramic acid and no glycoproteins could be detected in preparations of the cell envelope. DNA Base Composition The D N A eontains 31.3 mol % G + C as determined by the melting point in 1 x S S C (Marmur and Doty 1962) using D N A of Methanococcus voltae as a reference (30.7 mol % ( i l - C). A second analysis of the D N A base composition per-formed by H P L C chromatography of a nuclease PI hy-drolysate (Zi l l ig et al. 1980) yielded at G + C-content of 31.6 mol % for the isolate S N 1. Discussion The isolate S N 1 is the First thermophilie Methanococcus. Its growth temperature Optimum at 65"C is similar to that of Methanobacterium thermoautotrophicum (Zeikuset al. 1980). In contrast to the other coeeoid methanogens described (Balch et al. 1979), yeast extract does not stimulate growth. With a doubling time of only 55 min, the new isolate is the fastest growing archaebacterium known to date, even faster than Methanobacterium thermoautotrophicum, strain Marburg , which has an optimal doubling time of 1.6 h (Brandis et al. 1981). This rapid growth is similar to that of fast growing cubactcrial anacrobians, e.g. Lactohacilli (K. O. Stetter, unpublished). Due to its vigorous growth and its autotrophy, the new isolate may be well suited for biochemi-cal studies. The new organism SN 1 is clearly determined to be a Methanococcus (Balch et al. 1979) by its coeeoid shape, its Substrate spectrum, its subunit cell envelope, and its D N A with a G + C-content of 31.3 mol%. In D N A - R N A hy-bridization experiments (Tu et al. 1982), very stable hybrids (fractional stability: 0.94) between the D N A of the isolate SN 1 and the labelled R N A of Methanococcus voltae were obtained, indicating a relatively close relationship with this species and confirming the affiliation to the Methano coccales. Judging its place of Isolation, its thermophily, its litho-trophic growth and its salt Optimum, the isolate SN 1 seems to be distributed in volcanic areas on the sea floor. It is named Methanococcus thermolithotrophicus. Description and Classification of the Methanococcus thermolithotrophicus Order Methanococcales (Balch and Wolfe 1979) Family Methanococcaceae (Balch and Wolfe 1979) Genus Methanococcus (Barker 1936) Methanococcus thermolithotrophicus', Huber, Thomm, and Stetter (sp. nov.) ther.mo.li.tho.tro'phi.cus. Gr. fem. n. therme heat; Gr. masc. n. lithos stone; Gr. masc. n. trophos one who feeds; M . L. masc. adj. thermolithotrophicus thermophilic and litho-trophic. Regulär to irregulär cocci, about 1.5 um in diameter, occurring singly or in pairs. On agar, round, convex, smooth, yellowish colonies, around 1 mm in diameter are formed. About 20 flagella are inserted at a distinet area on the cell surface. The cells are Gram-negative, and are lysed by 2% SDS; the cell envelope consists of subunits. Optimal growth is at 65° C with a doubling time of 55 min. Growth takes place between 30 and 70°C. The optimal pH is 6.5 — 7.5. Culture is possible in the presence of 1.3-8.3% NaCl with an optimal growth at 4%. Methane is only formed from H 2 / C 0 2 and from formate. Organic material does not stimulate growth. The D N A base composition is 31.3 mol % G + C. In D N A -R N A hybridization, the D N A of the isolate forms hybrids with R N A of Methanococcus voltae with a fractional stability of 0.94. The type strain is D S M 2095 Göttingen, F R G . Acknowledgements. The excellent technical assistance of Petra Frischeisen and Elisabeth Kleemeier is acknowledged. Thanks are due to Dr. Jurek Madon for HPLC analysis. Furthermore, we greatly appreciate the support in electron microscopy by Dr. Kurt Ober and by Davorin Janecovic. This work was supported by a grant of the Deutsche Forschungs-gemeinschaft to K. O. Stetter. References Aranki A, Freter R(1972) Use of anaerobic gloveboxes for cultivation of strictly anaerobic bacteria. Am J Clin Nutr 25:1329—1334 Balch WE, Fox G E , Magrum LJ, Woese CR, Wolfe RS (1979) Methanogens: Reevatuation of a unique biological group. Microbiol Rev 43:260-296 Balch WE, Wolfe RS (1976) New approach to the cultivation of methanogenic bacteria: 2-mercaptoethansulfonic acid (HS-CoM)-dependent growth of Methanohacterium rwninantium in a pres-surized atmosphere. Appl Environ Microbiol 32:781 —791 Barker HA (1936) Studies on methane producing bacteria. Arch Mikrobiol 7:420-438 Brandis A, Thauer RK, Stetter K O (1981) Relatedness of strains AU and Marburg of Methanohacterium thermoautotrophicum. Zbl Bakt Hyg, I Abt Orig C 2:311-317 Jones JB, Bowers B, Stadtman T C (1977) Methanococcus vannielii: Ultrastructure and sensitivity to detergents and antibiotics. J Bacteriol 130:1357-1363 Marmur J, Doty P (1962) Determination of the base composition of deoxyribonucleie acid from its thermal denaturation temperature. J Mol Biol 5:109-118 Stetter K O , Thomm M , Winter J, Wildgruber G , Huber H ? Zillig W, Janecovic D, König H , Palm P, Wunderl S (1981) Methanothermus fervidus, sp. nov., a novel extremely thermophilic methanogen isolated from an Icelandic hot spring. Zbl Bakt Hyg, I Abt Orig C 2:166-178 Tu J, Prangishvilli D, Huber H , Wildgruber G, Zillig W, Stetter K O (1982) Taxonomic relations between archaebacteria including 6 novel genera examined by cross hybridization of DNAs and 16s rRNA. J Mol Evol (in press) Wildgruber G , Thomm M , König H, Ober K, Ricchiuto T, Stetter K O (1982) Methanoplanus limicola, a plate-shaped methanogen repre-senting a novel family, the Methanoplanaceae. Arch Microbiol in press) Zeikus JG, Arie Ben-Bassat, Hegger PW (1980) Microbiology of methanogenesis in thermal, volcanic environments. J Bacteriol 143:432-440 Zeikus JG, Wolfe RS (1972) Methanohacterium thermoautotrophicum sp. n., an anaerobic, autotrophic, extreme thermophile. J Bacteriol 109:707-713 Zeikus JG, Wolfe RS (1973) Finestructure of Methanohacterium ther-moautotrophicum. Effect of growth temperature on morphology and ultrastructure. J Bacteriol 113:461—476 Zillig W, Stetter K O , Wunderl S, Schulz W, Priess H , Scholz J (1980) The iSw(/b/0&M.?-"Caldariella" group: Taxonomy on the basis of the strueture of DNA-dependent R N A Polymerase. Arch Microbiol 125:259-269 Zinder SH, Mah RA (1979) Isolation and characterization of a ther-mophilic strain of Methanosarcina unable to use H 2 - C 0 2 for methanogenesis. Appl Environ Microbiol 38:996—1008 Received March 26, 1982/Accepted April 24, 1982 

Finestructure of Methanohacterium thermoautotrophicum. Effect of growth temperature on morphology and ultrastructure.

Freter R(1972) Use of anaerobic gloveboxes for cultivation of strictly anaerobic bacteria.

Methanococcus thermolithotrophicus, a novel thermophilic lithotrophic methanogen

Methanococcus thermolithotrophicus, a novel thermophilic lithotrophic methanogen

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