Archives of Arch Microbio! (1986) 144:324-333 fficrotnlggy © Springer-Verlag 1986 Thermotoga maritima sp. nov. represents a new genus of unique extremely thermophilic euhacteria growing up to 90° C * Robert Huber1, Thomas A. Langworthy2, Helmut König1, Michael Thomm1, Carl R. Woese3, Uwe B. Sleytr4, and Karl O. Stetter1 1 Lehrstuhl für Mikrobiologie, Universität Regensburg, D8400-Regensburg, Federal Republic of Germany 2 University of South Dakota, Vermillion, USA 3 University of Illinois, Urbana, USA 4 Universität für Bodenkultur, Wien, Austria Abstract. A novel type of bacterium has been isolated from the organisms were grown in "MMS"-medium, containing various geothermally heated locales on the sea floor. The (per litre): NaCl 6.93 g; MgS04 - 7H20 1.75 g; MgCl2 organisms are strictly anaerobic, rod-shaped, fermentative, • 6 H20 1.38 g; KCl 0.16 g; NaBr 25 mg; H3B03 7.5 mg; extremely thermophilic and grow between 55 and 90° C with SrCl2 • 6 H20 3.8 mg; KJ 0.025 mg; CaCl2 0.38 g; KH2P04 an optimum of around 80° C. Cells show a unique sheath• 0.5 g; Na2S 0.5 g; (NH4)2Ni(S04)2 2mg; trace minerals like structure and monotrichous flagellation. By 16S rRNA (Balch et al. 1979), 15 ml; resazurin, 1 mg; starch, 5 g; pH sequencing they clearly belong to the eubacteria, although 6.5 (adjusted with H2S04). In substrate utilization experi• no close relationship to any known group could be detected. ments, starch was omitted and substituted as described. The majority of their lipids appear to be unique in structure In order to determine optimal ionic conditions, inorganic among the eubacteria. Isolate MSB8 is described as components of "MMS"-medium were simultaneously in• Thermotoga maritima, representing the new genus creased or diminished with the same ratio as described for Thermotoga. NaCl. For growth on solid medium, 0.8% agar (Oxoid) was added to the medium. Reasonable growth of the isolates Key words: Evolution — Eubacteria — Thermophile - also occurred in "SME"-medium (Stetter et al. 1983) Anaerobe — Thermotoga maritima supplemented with 0.5% yeast extract, or in marine broth (Difco 2216). Ten milliliter cultures were grown in stoppered 28 ml serum tubes (Balch et al. 1979) pressurized with N2 (300 kPa) All extremely thermophilic bacteria isolated to date, growing and incubated without agitation. Large scale cultures were optimally between 80 and 105° C, belong to the archae- grown in a 1001 enamel-protected fermentor under N2 (2 1/ bacteria (Stetter et al. 1981; Zillig et al. 1981; Stetter 1982, min) with gentle stirring (200 rev/min). 1985; Stetter and Zillig 1985). Many eubacterial thermo• philic isolates with lower optimal growth temperatures, e.g. 50 to 70° C are known (Brock 1978). A few eubacterial Light microscopy isolates, e.g., Thermus thermophilus, Bacilluscaldolyticusand Cells were routinely observed with a Zeiss Standard phase Bacillus caldo tenax, do have temperature optima in the range contrast microscope with an oil immersion objective 100/ of 75° C with maxima at 80° C (Brock 1978). [A reported 1.3. For microscopy at 85° C, the slide was preheated to thermoadaptation of Bacillus caldolyticus to temperatures about 90° C, and a drop of still hot culture was placed on it above 80° C (Heinen and Lauwers 1981) could not be re• and the slide observed rapidly under phase contrast. produced (Huber and Stetter in preparation); we were also Micrographs were taken with a Leitz Ortholux II microscope unable to obtain the adapted cultures.] equipped with a vario-orthomat camera system (Leitz). This paper characterizes an anaerobic, extremely thermophilic eubacterium with an optimum growth temper• ature of 80° C, which has been isolated from geothermally Electron microscopy heated sea floors in Italy and the Azores. Logarithmic phase cells were fixed by addition of glutar- aldehyde (20 g/1, final concentration). For preparation of thin sections, cells were precipitated by centrifugation after Materials and methods fixation and further processed as previously described Culture conditions (Huber et al. 1982). Electron micrographs were taken with a JEOL JEM-100 C electron microscope. Freeze-etching of The new isolates were cultivated using the anaerobic tech• whole cells was performed with a Balzers Model 360 M nique of Balch and Wolfe (1976). Unless specified otherwise, (Balzer AG, Liechtenstein) or a Bioetch Model 2005 (Leybold-Heraeus, Köln, FRG) freeze-etching unit. The Offprint requests to: K. O. Stetter etching temperature was -100°C and the samples were * Dedicated to Otto Kandier on the occasion of his 65th birthday Present addresses: University of South Dakota, Vermillion, USA; etched for 2 min. After platinum-carbon shadowing and University of Illinois, Urbana, USA; Universität für Bodenkultur, carbon coating replicas were cleaned with 30% chromium Wien, Austria oxide and picked up on uncoated grids. Measurement of growth Hydrogen determination Bacterial growth was followed by cell counting in a Hydrogen was analyzed in a Packard model 427 gas "Neubauer" counting chamber (depth: 0.02 mm). Chromatograph equipped with a thermal conductivity detec• tor and a 2m Teflon column packed with Porapac N (Supelco). The oven temperature was 70° C. DNA isolation DNA was isolated as described (Stetter et al. 1981). Analyses of organic fermentation products Glucose, acetate, formate, citrate, L(4-)- and D(~)-lactate, DNA base composition L(+)-malate and ethanol were determined enzymatically (Bergmeyer 1974). Methanol, ethanol, isopropanol, «-pro• The GC-content of DNA was determined by melting point panol, n-butanol, iso- and w-pentanol and acetone were de• analysis (Marmur and Doty 1962), using calf thymus DNA termined by gas chromatography, as were acetate, pro• (42 mol% GC) as a reference. Direct analysis of DNA base pionate, iso- and n-butyrate, and iso- and ^-valerate after composition was performed by high performance liquid acidification of the suspension (Zabel et al. 1985). Sorbitol chromatography after digestion of the DNA with nuclease and mannitol were similarly determined, as butylboronate PI (Zillig et al. 1980). derivatives (Eisenberg 1974) on column 1% GE SE-30 (Perkin-Elmer). The unknown product "A" was detected as a radioactive Lipids spot by thin layer chromatography (DC-cellulose, Merck; Lipids were extracted from freeze-dried cells and frac• solvent system I: isopropylether, water-saturated:formic tionated into neutral and polar lipid by silicic acid column acid = 65:35 v/v). The unidentified product "B" was found chromatography as described elsewhere (Langworthy 1982; as a radioactive spot after thin layer chromatography (silica Langworthy et al. 1983). Polar lipids were hydrolyzed in plates, Merck; solvent system II: 2-propanol:ethylacetate anhydrous 1 M methanolic-HCl to cleave polar head groups :H20 = 83:ll:6v/v/v). and the fl-hexane-soluble apolar residues were separated by thin layer chromatography developed in the solvent «-hex- H S determination ane-diethyl ether-acetic acid (70:30:1, by vol.). Individual 2 components were examined by gas chromatography, in• H2S was detected qualitatively by addition of 10 ul of frared spectrometry, and by gas chromatography/mass saturated lead acetate solution to 1 mi samples. A dark spectrometry employing instrumentation and conditions de• brown precipitate demonstrated its presence. For quan• scribed previously (Langworthy et al. 1983). titative analysis, H2S was determined by titration (Williams 1979). Fermentation by cell suspensions Identification of muramic acid All procedures were performed anaerobically under a C02- atmosphere. A cell culture (51) growing exponentially Muramic acid was detected in cell hydrolysates (4N HCl; (3xl07/ml) in "MMS"-medium supplemented with 0.4% 16 h; 100°C) with an amino acid analyzer (LC 5000, Bio- glucose and 0.05% yeast extract (Difco) was harvested tronik; program: "Bacterial hydrolysates") and by paper by centrifugation (8,000 rev/min; 20 min; 5°C; rotor chromatography (Schleifer and Kandier 1972). The isolated 889 WKF). The supernatant was discarded and the cells compound was further identified as a methyl ester derivative washed 3 times with a 0.1 M Sörensen phosphate buffer, (3N methanolic HCl; 30 min; 110°C; Albersheim et al. pH 6.3 (Rauen 1964), each wash followed by centrifugation 1967) by gas liquid chromatography (Hewlett Packard 5880 (20,000 rev/min; 1.5 min; 5°C; rotor JA 20 Beckman). A). The alditol acetate derivative was separated on a 3 ft. Finally, the pellet was suspended in 10 ml of the same buffer. metal column packed with 3% SP-2340 on 100/120 To 5 ml of this suspension, 20 mg of 14C-glucose (2 uCi; Supelcoport (Supelco). uniformly labelled) were added and, after removal of C02 by flushing with N2, the mixture was incubated at 85°C in closed serum tubes under a nitrogen atmosphere (300 kPa). Test for diaminopimelic acid A second 5 ml suspension without glucose was treated the Diaminopimelic acid was assayed chromatographically same way, as a control. After 2 h, the suspensions were (Rhuland et al. 1955). cooled down to 4° C and samples of the gas phase were taken by a syringe fitted with a gas lock (Hamilton) and analyzed Isolation of soluble murein immediately for H2 by gas chromatography. The pH was then adjusted to 9 (100 jil 4 N NaOH) and the mixtures Cells were disrupted by sonification (3 min; 70 W; B 12, shaken for 90 min at 4°C. In order to analyze the radioactive Branson) and then incubated in the presence of 0.5% (v/v) C0 , 2 ml of the suspension were transferred to a closed 2 Triton X-100 (60° C; 30 min). The envelopes were sedimented Warburg vessel; C0 was liberated by addition of 200 ul of 2 by ultracentrifugation (40,000 rev/min; 45 min; rotor 50 Ti; 5% (v/v) sulfuric acid to the suspension and trapped on a Beckman L5-50) and then washed 3 times with H20, each filter paper soaked with 100 ul of 20% (w/v) KOH.