System. Appl. Microbiol. 14, 397-404 (1991) © Gustav Fischer Verlag, Stuttgart/New York

Hyperthermophilic and Bacteria Occurring within Indonesian Hydrothermal Areas

G. HUBER, R. HUBER, B. E. JONES1, G. LAUERER, A. NEUNER, A. SEGERER, K. O. STETTER*,

and E. T. DEGENS2

Lehrstuhl für Mikrobiologie, Universität Regensburg, 8400 Regensburg, Germany ' Gist-Brocades, Delft, The Netherlands 2 previously in: Geologisch-Paläontologisches Institut, Universität Hamburg, 2000 Hamburg, Germany

Received February 20, 1991

Summary

From 85 samples taken during cruise 45B of the R/V SONNE within the Sunda Arc subduction zone and from solfatara fields in Java, thermophilic and hyperthermophilic archaea and bacteria were isolated. The archaea were found to belong to the genera Methanol? act er tum, Methanolobus, Methajtosarcina, , Thermoproteus, Desulfurococcus, Thermoplasma, Thermococcus and a so far unknown ther- moacidophilic continental metal mobilizer. All 7 Thermoplasma isolates were found to represent a new genotype so far only found in Indonesia. The extremely thermophilic bacterial isolates are a new of

Thermotoga and a novel strict anaerobe, thriving by H2/NOj~ autotrophy.

Key words: Archaea - Thermoplasma - Thermophilic - Solfataras - Nitrate.

Introduction

The most extremely thermophilic organisms known up nesia, Yellowstone National Park (USA), and New Zea• to now belong to the Archaea (previously: archaebac- land {Brock, 1978; Stetter and Zillig, 1985; Stetter, 1986; teria), the third domain of life besides the Bacteria and the Stetter et al., 1987; Huber et al., 1990). Since hydrother• Eucarya (Woese et al., 1978; Woese et al., 1990). These mal biotopes are widely dispersed and hyperthermophiles hyperthermophiles are the archaeal sulfur metabolizers are unable to grow at normal environmental temperatures, and some methanogens, growing optimally at tem• the question concerning dispersion arises. Although very peratures above 80 °C {Stetter, 1982; Stetter, 1986; Burg• few systematic studies on the distribution of hyperther• graf et al., 1990; Stetter et al., 1990). Thermotoga mariti• mophiles have been carried out up to now, there is evi• ma and the closely related Thermotoga neapolitana are the dence for the existence of an endemic group: The only bacterial hyperthermophilic species known with opti• Methanothermaceae have so far only been found in the mal growth at 80 °C (Huber et al., 1986 b; Jannasch et al., southwest of Iceland (Lauerer et al., 1986). During the 1988; Windberger et al., 1989). Thermotoga represents R/V SONNE 45B cruise in Indonesia within marine areas the deepest phylogenetic branch-off within the bacterial of active volcanism along the Sunda Arc subduction zone domain {Huber et al., 1986 b; Woese, 1987; Woese et al., and at solfatara fields in Bali and Java, attempts were 1990). Due to their exceptional position in the evolution made to address the following points: (a) the existence of of life and their outstanding cellular properties, hyperther• novel hyperthermophilic archaea and bacteria in In• mophiles are of great interest for microbiology and donesia, (b) the distribution of hyperthermophilic archaea biotechnology. within Indonesian hydrothermal systems and solfatara Different hyperthermophilic archaea have been isolated fields, (c) the dissemination of hyperthermophilic archaea from continental and submarine hydrothermal systems at between different hydrothermal systems through cold sea the Azores, East Pacific Rise, Iceland, Italy, Japan, Poly- water, (d) the survival of hyperthermophiles in cold deep- sea sediments. Here we report on the first results of our * Corresponding author screening program. Materials and Methods were deoxygenated as described for the sediments. In another experiment, 3—5 1 of the anaerobic water samples Strains. (DSM 3191), Pyrococcus furiosus were passed through millipore ultrafilters (pore width: 0.4 (DSM 3638), Thermococcus celer (DSM 2476), Thermococcus urn) in order to concentrate the microorganisms. The filter litoralis (DSM 5473), Thermoplasma acidophilum (DSM 1728), concentrates were then stored anaerobically in tightly Thermoplasma volcanium (DSM 4299), Thermotoga maritima closed 20 ml storage tubes. (DSM 3109), and Thermotoga neapolitana (DSM 4359) were During land expeditions, anaerobic and aerobic water obtained from the Deutsche Sammlung von Mikroorganismen and mud samples were taken from the following places und Zellkulturen (DSM, Braunschweig-Stockheim, FRG). (Table 1): Lake Batur, Bali: Toye Bungkah Hot Springs Culture conditions. Strictly anaerobic organisms were cultured by following the anaerobic technique described by Balch et al. (TB). Dieng Plateau, Java: Kawah Sikidang Crater (KS), (1979). Methanobacterium, Methanosarcina and Methanolobus Kawah Candradimuka Crater (KC), and Kawah Sileri Hot isolates were obtained after enrichment and plating on media Lake (SL). Tangkuban Prahu, Bandung, Java: Kawah described (Balch et al., 1979; König and Stetter, 1982). For plat• Domas Crater (KD; altitude: 1500 m above sea level), ing, the media were solidified by 2% (w/v) agar. If not mentioned Kawah Badak Crater (KB), and Kawah Djarian Crater otherwise, Thermococcus celer, Thermococcus litoralis, Pyrococ• (KDj). Ciater, Bandung, Java: Ciater Hot Springs (C). cus furiosus and Thermococcus-Wkc isolates were grown on Ganung Gede, Java: Hot Waterfall (GG). All samples were "SME" medium supplemented with 0.1% (w/v) yeast extract and 2.5% (w/v) S° (Stetter et al., 1983). Thermotoga maritima, Ther• motoga neapolitana and the Thermotoga-\'\ke isolates were grown as described (Huber et al., 1986 b; Windbergcr ct al., Table 1. Samples taken from hot springs and continental solfat• 1989). Acidianus infernus and AcidianusAikc isolates were ara fields in Bali and Java grown anaerobically at pH 2 in the presence of H2 and S° or Abbreviations: TB = Toye Bunkah; KS = Kawah Sikidang; KC aerobically on S° in Allen's medium (Allen, 1959; Segcrer et al., = Kawah Candradimuka; SL = Kawah Sileri; KD = Kawah 1986 a). SulfolobusAike isolates and the metal mobilizers were Domas; KB = Kawah Badak; KDj = Kawah Dj arian; C = Cia- enriched and isolated aerobically at pH 2 in Allen's medium (Al• ter; GG = Ganung Gede. len, 1959), supplemented with S° or ore mixture Gl (Huber et al., 1986 a). Thermoproteus and Desulfurococcus-Wke isolates were Designation Orig inal Designation Original enriched and grown anaerobically at pH 5.5 in Allen's medium PH Temp PH Temp (Allen, 1959) supplemented with 0.1% (w/v) yeast extract, 0.1% (°C) (°Q (w/v) peptone and 2.5% (w/v) S°. Thermoplasma acidophilum, Thermoplasma volcanium and the Thermoplasma isolates were TB 1 7 42 KD 1 3 91 cultivated, enriched, and cloned by platingas described by Seger- 2 7 39 2 2 94 er ct al. (1988). 3 5 67 Light microscopy and ultrastructure. Cultures were inspected KS 1 1.5 93 4 2 46 by light microscopy as described by Burggraf et al. (1990). Elec• 2 1.5 92 5 2 86 tron microscopy was performed as described previously (König 3* 3 22 6* 1.5 32 and Stetter, 1982). 4* 3 92 7 2 70 DNA preparation, base composition and homology. The DNA 5 1.5 50 8 4 94 was isolated as described by Lauerer et al. (1986). The GC-con- 6* 2 24 9 3 50 tent of the DNA and DNA-DNA homology were determined as 7* 3 27 10 3 50 described by Segerer et al. (1988). 8 3.5 51 11 3 58 9 7 92 12 3 93 10* 3 50 13 3 92 Results and Discussion 11 3 50 12 2.5 92 KB 1* 1 90 Sampling 13 3.5 22 2 1.5 68 During cruise SO-45B, 10 anaerobic samples (each 100 14* 3.5 22 3 2 94 15 .5 92 ml) of shallow (depth: 30 to 82 m; T = 16 to 20°C) and 16* 3 30 KDj 1* 3 92 deep sea (depth: 1283 to 1743 m; T = 2.9 to 3.7°C) 17* 2 30 2 3 93 sediments were taken from box grabs, piston- and box 3 5.5 92 cores at different stations north, east and south of Sumba- KC 1 7.5 88 4 1.5 92 wa and in the Komodo-Rinja region. Thirteen additional 2 6 90 5 3.5 94 sediment samples were taken by a syringe (Stetter, 1982) 3 5 60 6 1.5 90 within the Satonda Crater lake (depth = 0.6 to 2 m; T = 4 7.5 80 32 °C) and from shallow submarine hot springs (depth: 0 C 1 3 42 to 2 m; T = 75 to 96°C) at the beach of Sangeang Island, SL 1* 6.5 . 55 2 3 43 2 6.5 62 about 7 km to the north of Kelapa village. The freshly 3* 6 55 GG 1 6.5 56 taken samples were collected in 100 ml storage bottles. 4* 6.5 40 2 6.5 56 Oxygen was reduced immediately by the addition of 5* 3.5 32 sodium dithionite and sodium sulfide until the redox indi• 6 5.8 72 cator resazurin added before became colorless (Stetter, 7 3.5 32 1982). The bottles were tightly stoppered and stored at 4°C. Eight samples of sea water (depth: 14 to 2000 m) * aerobic sample carried back to the laboratory by airplane without temper• in marine sediments. A novel highly irregular ("leach"- ature control. Growth experiments were performed at 20, shaped) methanogen from Lake Satonda was up to now 37, 60, 85, 100 and 110°C and at pH 2, 5.5, 7 and 8.5 obtained only in syntrophic mixed culture with Clostridia. with various organic and anorganic substrates (e. g. Fiala and Stetter, 1986). Hyperthermophilic archaeal and bacterial isolates from the shallow submarine hot springs at the beach of Archaeal isolates from sea sediments and sea water Sangeang Island samples from the Sumbawa and Komodo-Rinja areas From sample SG7 (original temp. == 90 °C), a new coc- In order to detect hyperthermophiles possibly existing coid strictly anaerobic heterotrophic hyperthermophilic and spreading between zones of active submarine volcan- archacon was isolated under anaerobic growth conditions ism, water and sediment samples with low ambient tem• at 90°C in artificial sea water (SME; Stetter et al., 1983) peratures were incubated anaerobically at high tem• supplemented with yeast extract (0.1% w/v) and elemental peratures in the presence of substrates suitable for such sulfur. The coccoid cells of the isolate measure about 0.5 organisms. No hyperthermophiles could be enriched. Al• to 2 urn in diameter, occur mainly in pairs (Fig. 1) and are though only relatively few sarnples were taken, this result suggests that (a) these sediments did not contain signifi• cant amounts of hyperthermophiles which survived at low temperatures in the resting state and (b) the surrounding water contains less than 1 cell of hyperthermophiles per 5 liters. Probably much larger quantities of sea water within Éw'iÍTTiaririu::. hydrothermally active areas have to be filtered (e. g. cubicmeters) in order to detect spreading cells of hyper• thermophiles. Archaeal hyperthermophiles in concentrations of up to lOVml sea water have recently been discovered within the open sea plume of an erupting seamount (lluber ct al., 1990). When samples from the Sumbawa and Komodo- Rinja areas were incubated at 30 °C, mesophilic strictly anaerobic mcthanogenic archaea were enriched from some sediments and were purified by plating (Table 2). The phy• siological properties and morphology suggest that these mcthanogenic isolates belong to the genera Methanosarci• na, Methanobacterium, and Methanolobus (Balch et al., 1979; König and Stetter, 1982). Methanolobus is an obli• gate methylotroph which has so far been isolated only from a black marine sediment in Tindari, Sicily (König and Fig. 1. Dividing cell of the hyperthermophilic archaeal isolate Stetter, 1982). Our results suggest that it may be common SG7. EM micrograph, negative staining.

Table 2. Mesophilic methanogenic archaea isolated from sea sediments with low original temperatures of the Sumbawa and Komodo Rinja areas

Sample Station Water Original Description Isolate depth (m) temperature (°C)

FS-1 101-2 1735 2.9 greyish-green Methanosarcina sp. sediment FS-6 107-2 82 16 greyish sediment Methanolobus sp. with concretions FS-7 113-1 45 19 greyish sediment Methanolobus sp. banded hematite FS-14 Satonda Crater Lake, 0 27 black mud in spring Methanolobus sp. Stat. 16 FS-15 Satonda Crater Lake, 2 32 black sediment Methanolobus sp. embarkation site

FS-15a Satonda Crater Lake 60 29 " black sediment, Methanosarcina sp.

bottom contains H2S enrichment culture: novel irregular leach-shaped methanogen motile by polar monotrichous flagellation. Complex or• strains, the Indonesian isolates grow in aggregates (up to ganic nutrients like yeast extract, peptone, meat extract 15 cells) and can therefore be easily distinguished. and tryptone serve as energy source for growth. Elemental sulfur is not essential for growth. However, in the presence Mcthanogenic isolates from the Toye Bungkah hot

of S°, H2S is formed instead of H2 (up to 3 umolcs/ml springs, Bali culture medium). The new isolate grows between 46 and 96 °C with an optimum at around 88 °C. Growth occurs in From anaerobic samples taken from the black mud of the presence of 0.7 to 8% NaCl at a pH between 5.0 and the two ponds at the hot spring of Toye Bungkah, Lake 8.0. The DNA of isolate SG7 shows a GC-contcnt of 40 Batur, Bali (TB 1, 2; Table 1), a member of the mol% and is therefore similar to Thermococcus litoralis Methanobacterium (Balch ct al., 1979) was isolated from (38 mol% GC; Neuner et al., 1990) and different to Ther• the pond close to the origin of the spring (TB 1). From the mococcus celer (56 mol% GC; Zillig et al., 1983) and second pond (TB 2), a Methanobacterium sp. similar to Pyrococcus furiosus (37 mol% GC; Piala and Stetter, TB 1 and a Methanosarcina sp. were isolated. The deter• 1986). By DNA-DNA hybridization (Table 3) a specific mination of their exact taxonomic position is in progress. phylogenetic relationship of SG7 with Thermococcus litoralis was detected, indicating that the Sangeang isolate Plyp er thermophilic and thermophilic archaea and bac• belongs to the same species which has so far been found teria isolated froni solfatara fields at the Dieng Plateau only in Italy (Neuner et al., 1990). and Tangkuban Prahu, Java In Java, hot springs and solfatara fields at the Dieng Plateau, Tangkuban Prahu, Ciater, and Ganung Gede Table 3. DNA homology (%) between the new archaeal isolate (Table 1) were investigated for thermophilic anaerobic and SG7, Thermococcus celer, Thermococcus litoralis, and Pyrococ• aerobic archaea and bacteria (Table 5). At the Dieng cus furiosus Plateau, samples were taken from three different types of Source of filter-bound % Homology with the following solfatara fields: (a) Kawah Sikidang with strongly acidic DNA sources of uP-labelcd DNA water- and mudholes of up to boiling temperatures, (b) Kawah Candradimuka with neutral to slightly acidic al• Tc. Tc. Pc. Isolate most boiling hot springs, and (c) Kawah Sileri with neutral celer litoralis furiosus SG7 to slightly acidic muddy blackish water with temperatures around 65 °C. Within the Tangkuban Prahu area, samples Thermococcus celer (100) 2 6 5 were collected at three different solfatara fields mainly Thermococcus litoralis 6 (100) 2 85 Pyrococcus furiosus 3 4 (100) 7 with strongly acidic pH and very high temperatures: (a) Isolate SG7 6 88 12 (100) Kawah Domas with many strongly gassed water- and mudholes, (b) Kawah Badak with many fumaroles and very few tiny watcrholes, and (c) Kawah Djarian situated within a rain forest with trees decomposing within boiling From samples SGI and SG7 (both with original tem• holes of sulfur mud. In addition, samples were taken from peratures of 90 °C), rod-shaped cells with a characteristic an acidic warm spring in Ciater and from a hot waterfall outer sheath could be enriched and isolated anaerobically with neutral pH at Ganung Gede. at 85 °C in Thermotoga medium (Huber et al., 1986 b). No archaeal isolates were obtained from the samples of Both isolates turned out to be bacterial hyperthermophiles Ciater and Ganung Gede. From the acidic hot springs and belonging to the genus Thermotoga. They represent a new mudholes at.the Dieng Plateau and at Tangkuban Prahu, species as indicated by the low DNA homology with the members of the genus Acidianus were isolated in Allen's type strain Thermotoga maritima DSM 3109 and with medium (Allen, 1959; Table 5). These are chemolitho- some other still undescribed Thermotoga isolates from autotrophic facultative aerobes, growing by oxidation or elsewhere (Table 4). In contrast to the other Thermotoga reduction of elemental sulfur, depending on the redox po• tential (Segerer et al., 1985; Segerer et al., 1986a). All isolates grow at temperatures of up to 96 °C and are by Table 4. DNA homology (%) between Thermotoga maritima, this feature similar to the type species Acidianus infernas Thermotoga neapolitana, and some recent Thermotoga isolates (isolated in Italy). Again from the acidic hot springs of this and isolates SGI and SG7 from Sangeang area, many enrichment cultures could be ob• tained aerobically on S° and yeast extract (data not Source of filter-bound % Homology with the following shown), which were not further characterized up to now. DNA sources of -12P-labcled DNA From a hot acidic waterhole (KB 1; Table 5) in Kawah Isolate SGI Isolate SG7 Badak, a novel coccoid Sulfolobus-shaped (Brock, 1978) archaeal metal mobilizer was isolated, suitable for micro• Thermotoga maritima 15 13 bial leaching of sulfidic ores at high temperatures. The Thermotoga neapolitana 55 40 organism grows chemolithoautotrophically at temper• Isolate RQ 2 (Azores) 12 15 atures up to 80 °C with pyrite, chalcopyrite and sphalerite Isolate RQ 7 (Azores) 55 38 as energy sources. After 1 week at 80 °C, cell densities of Sangeang isolate SGI (100) 82 108/ml are obtained in the laboratory, indicating very vig- Table 5. Hyperthermophilic and Area Name of solfatara Isolates from Genus Growth thermophilic archaeal and bacte• rial isolates from hot springs and field samples up to (°C) mudholes in Java Kawah Sikidang KS 1, 2, 12, 15 Acidianus infernus (?) 96 KS 9, 15 Thermoproteus sp. 96 KS 9, 15 Desulfurococcus sp. 96 KS 5, 8, 11 Thermoplasma sp. 67

Kawah Candra- KG 1, 2, 4 Thermoproteus 96 ; Dieng ditnuka KG 1, 2 Desulfurococcus 96 Plateau KG 4 novel unnamed H2/NO;T- 80 autotrophic bacterium

Kawah Silcri SL 7 Thcrmoplasma sp. 67 SL 3 vibrio-shaped new bac• 50 terial metal mobilizer

Kawah Dornas KD 1, 2, 3, 5 Acidianus infernus (?) 96 KD 3, 5, 7 Thcrmoplasma sp. 67

Kawah Badak KB 3 Acidianus infernus (?) 96 angkuban 4 ^ . , , , , n , KB 1 Goccoid novel archaeaI 80 Pralui . .... metal mobilizer

Kawah Djarian KDj 3 Acidianus infernus (?) 96 KD] 3, 5 Thermoproteus 96

orous growth on ores compared to other thermophilic ore only within smoldering coal refuse piles (Darland et al., leachers (Huber et al, 1986 a). A moderately thermophilic 1970; Brock, 1978). so far unknown acidophilic vibrio-shaped ore leaching Similar to other Thermoplasma isolates which we had bacterium was isolated from sample SL3 (table 5). It grows obtained recently from solfataric fields in Italy, Yellow• optimally at 45 °C (5 hours doubling time) in the presence stone National Park, the Azores, and Iceland, the Indone• of sphalerite and pitch blend or, alternatively, yeast ex• sian isolates are different from the type species Thermo• tract (£. Drobner and K. O. Stetter, unpublished). plasma acidophilum and exhibit a GC-content of their The less acidic to neutral hot samples contained DNA of 40 mol% (instead of 46 mol% for T.a.; Segerer et anaerobic, sulfur respiring, rod shaped organisms, grow• al., 1988). In DNA-DNA hybridization experiments all 7 ing at 96 °C in Allen's medium (Allen, 1959) in the pre• Indonesian isolates turned out to be genetically identical sence of yeast extract, peptone and elemental sulfur. Ele• (100% DNA homology; data not shown), while they exhi- mental sulfur was replaceable by thiosulfate (0.1% w/v). The rods exhibit true branching and terminal spherical bodies ("golf clubs") which are typical for members of the genus Thermoproteus {Zillig et al., 1981). In the same enrichment cultures, also coccoid archaea were found, thriving organotrophically by S°-respiration at tem• peratures up to 96 °C (Table 5). Most likely, they belong to the genus Desulfurococcus (Zillig et al., 1982). From locations with strongly acidic pH and moderately hot temperatures (50 °C), cell wall-less (Fig. 2) highly irre• gular coccoid thermoacidophilic archaea growing up to 67°C could be enriched in Darland's medium (Darland et al., 1970). They were cloned by plating on medium sol• idified by 10% starch. After 4 days of incubation at 60 °C

in the presence of a C02/air atmosphere (50:50), small (0.2 mm 0) "friedegg"-shaped colonies became visible (Fig. 3). Very surprisingly, cell extracts of the isolates showed serological cross-reaction with antibodies pre• pared against the histone-like protein of Thcrmoplasma acidophilum (DSM 1728), indicating a phylogenetic rela• tionship of the isolates to the genus Thermoplasma (Stein Fig. 2. Cell of the Thermoplasma isolate KD3. EM micrograph, and Searcy, 1978). This genus had been described to exist ultrathin section.

27 System. Appl. Microbiol. Vol. 14/4 type species T. acidophilum also by physiological proper- tics, for example their lower minimal and higher maximal growth temperatures and their higher salt tolerance (Table 6). However, in contrast to their genetic difference, no separating features were found so far between them and some of the Thermoplasma isolates from other sol• fataric fields which had been described as Thermoplasma volcanium (Table 6; Segerer et al., 1988). Similar to both type strains, the Indonesian Thermoplasma isolates are organotrophic facultative anaerobes (Segerer et al., 1986 b). The recent isolation of members of Thermoplas• ma in Indonesia and other countries demonstrates, that solfatara fields are the (most likely primary) biotope of this group of archaea (Segerer et al., 1988). The ability to Fig. 3. Colonies of the Thermoplasma isolate KD3 on a starch grow within the mesophilic temperature range and the plate. isolation of strain SL 7 from a tropical swamp (original temperature: 32 °C) in Kawah Sileri show that tropical swamps and similar biotopes, e.g. self-heating organic hired no significant homology (7 to 12%; Segerer ct al., waste, may be further^ biotopes for these acidophilic ar• 1988) with Thermoplasma acidophilum and the isolates chaea. from the other solfataric fields. Therefore, the Indonesian From the hot mud sample KC 4, a novel rod-shaped isolates represent a so far unknown genotype of Thermo• flagellated Gram-negative bacterium (Fig. 4) was obtained plasma. Phenotypically, they can be distinguished from the on medium 1 (Balch et al., 1979) at 75 °C. The isolate (KC

Table 6. Physiological properties of Thermoplasma acidophilum (DSM 1728), Thermoplasma volcanium (DSM 4299), and the Indonesian Thermoplasma isolate KD3

Strain Growth temperature (°C) pH for growth NaCl requirement (%) GC content min opt max min opt max min opt max (mol%)

Therm op las n la acid op b Hum 45 59 63 0.8 2 4 0.01 0.25 2 46 DSM 1728

Thermoplasma volcanium 32 59 67 0.8 2 4 0.01 0.25 2 38 DSM 4299 *

Isolate KD3 DSM 4300 32 59 67 0.8 2 4 0.01 0.25 4 40

* isolated from Vulcano, Italy

Fig. 4. Cells of isolate KC4. EM micrograph, negative staining. 4; Table 5) is a strictly chemolithoautotrophic anaerobe, Köllig, II., Stetter, K. ().: isolation and characterization of growing in mineral medium in the presence of molecular Methanolohus tindarius sp. nov., a coccoid methanogen grow• ing only on methanol and methylamines. Zbl. Bakt. Hyg., I. hydrogen, nitrate (0.1% w/v) and C02 at neutral pH and temperatures of up to 80 °C. Stoichiometric amounts of Abt. Orig. C 3, 478-490 (1982) Lauerer, G., Kristjansson, J. K., Langivorthy, T. A., König, R., ammonia are formed during growth (not shown), indicat• Stetter, K. O.: Methanololms sociabilis sp. nov., a second ing that the new organism is gaining energy by the so far species within the Methanothermaceae growing at 97 °C. Sys• unknown H2/N03~ chemolithoautotrophy. This way of tem. Appl. Microbiol. S, 100-105 (1986) nutrition is made possible by the relatively high nitrate Neuner, A., Jannasch, R. W., Belkin, S., Stetter, K. O.: Ther• content of up to 5 ttmoles/1 present in this type of hot mococcus litoralis sp. nov.: A new species of extremely ther• springs (G. Liehezeit, pers. comm.). Batch cultures (50 1) mophilic marine archaebacteria. Arch. Microbiol. 253, of isolate KC 4 were grown in an enamel-protected fcr- 205-207 (1990) mcntor (yield: 0.5 g wet weight/1). Phylogcnetically the Segerer, A., Stetter, K. O., Klink, F.: Two contrary modes of novel isolate trees deeply among the bacteria and does not chemolithotrophy in the same archaebacterium. Nature 313, 787-789 (1985) seem to belong to any known phylum as indicated by 16S Segerer, A., Neuner, A., Kristjansson, J. K., Stetter, K. O.: rRNA sequence comparisons (C. R. Woese, pers. comm.). Acidianus infernas gen. nov., sp. nov., and Acidianus brierleyi comb, nov.: Facultatively aerobic, extremely acidophilic ther• Acknowledgements. We wish to thank D. Fjsma, W. v. Raug- mophilic sulfur-metabolizing archaebacteria. Int. J. System. ii'itz, S. Kempe, A. Masduki, J. Schilling and A. Zänker I for Bact. 36, 559-564 (1986a) essential assistance in obtaining samples and G. Liehezeit for Segerer, A., Stetter, K. O., Klink, F.: Novel facultatively aerobic water analyses. This work was supported by grants from the sulfur-dependent archaebacteria, p. 430. In: Archaebacteria Bundesministerium für Forschung und Technologie, Bonn, to \85 (O. Kandier and W. Zilling, eds.), Stuttgart-New York, G. F..T.D. and from the Deutsche Forschungsgesellschaft, Schwer• Fischer Verlag, 1986 b punkt: Mcthanogene Bakterien to K.O.S. and from Gist Segerer, A., Langivorthy, T. A., Stetter, K. O.: Thermoplasma Brocades, Delft to B.E.J, and K.O.S. acidophilum and Thermoplasma volcanium sp. nov. from sol• fatara fields. System. Appl. Microbiol. 10, 161-171 (1988) Stein, /). B., Searcy, D. G.: Physiologically important stabiliza• tion of DNA by a prokaryotic histone-like protein. Science 202, 219-221 (1978) References Stetter, K. O.: Ultrathin mycclia-forming organisms from sub• marine volcanic areas having an optimum growth temperature Allen, M. ß.: Studies with Cyanidium caldarium, an anomalously of I05°C. Nature 300, 258-260 (1982) pigmented chlorophyte. Arch. Mikrobiol. 32, 270-277 (1959) Stetter, K. O.: Diversity of extremely thermophilic archaebac• Balch, W. /:., Fox, G. E., Magrum, L.J., Woese, C. R., Wolfe, R. teria, pp. 39-74. In: Thcrmophiles: General, Molecular and $.: Mcthanogens: Recvaluation of a unique biological group. Applied Microbiology (T. D. Brock, ed.), New York-London- Microbiol. Rev. 43, 260-296 (1979) Sydney-Toronto, J. Wiley & Sons, Inc., 1986 Brock, T. D.: Thermophilic microorganisms and life at high tem• Stetter, K. O., Zillig, W.: Thermoplasma and the thermophilic peratures. Berlin-Heidelberg-New York, Springer-Verlag, sulfur-dependent archaebacteria, pp. 85-170. In: The Bac• 1978 teria, Vol. VIII (R. S. Wolfe and C. R. Woese, eds.). New York, Burggraf, S., Fricke, IL, Neuner, A., Kristjansson, ]., Rouvier, P., Academic Press, 1985 Mandelko, L., Woese, C. R., Stetter, K. O.: Methanococcus Stetter, K. O., König, R., Stackebrandt, E.: Pyrodictium gen. igneus sp. nov., a novel hyperthermophilic methanogen from a nov., a new genus of submarine disc-shaped sulphur reducing shallow submarine hydrothermal system. Appl. Microbiol. 13, archaebacteria growing optimally at 105 °C. System. Appl. 263-269 (1990) Microbiol. 4, 535-55\ (1983) Darland, G., Brock, T. D., Samsonoff, W., Conti, S. P.: A ther• Stetter, K. O., Lauerer, G., Thomm, M., Neuner, A.: Isolation of mophilic, acidophilic Mycoplasma isolated from a coal refuse extremely thermophilic sulfate reducers: Evidence for a novel pile. Science 170, 1416-1418 (1970) branch of archaebacteria. Science 236, 822-824 (1987) Fiala, G.y Stetter, K. O.: Pyrococcus furiosus sp. nov. represents a Stetter, K. O., Fiala, G., Huber, G., Ruber, R., Segerer, A.: Hy• novel genus of marine heterotrophic archaebacteria growing perthermophilic microorganisms. FEMS Microbiol. Rev. 75, optimally at 100°C. Arch. Microbiol. 145, 56-61 (1986) 117-124 (1990) Ruber, G., Huber, R., Stetter, K. O.: isolation and characteriza• Windberger, E., Huber, R., Trincone, A., Fricke, R., Stetter, K. tion of new metal-mobilizing bacteria. Biotech. Biocng. Symp. ().: Thermotoga thermarum sp. nov. and Thermotoga 16, 239-251 (1986 a) neapolitana occurring in African continental solfataric springs. Huber, R., Langivorthy, T. A., König, R., Thomm, M., Woese, Arch. Microbiol. 151, 506-512 (1989) C. R., Slcytr, U. B., Stetter, K. O.: Thermotoga maritima sp. Woese, C. R.: Bacterial evolution. Microbiol. Rev. 51, 221-271 nov. represents a new genus of unique extremely thermophilic (1987) eubacteria growing up to 90°C. Arch. Microbiol. 144, Woese, C. R., Magrum, L. ]., Fox, G. E.: Archaebacteria. J. 324-333 (1986 b) Molcc. Evol. 11, 245-252 (1978) Ruber, R., Stoffers, P., Ghcminee, ]. L., Richnow, R H., Stetter, Woese, C. R., Kandier, O., Wheelis, M. L.: Towards a natural K. O.: Hyperthermophilic archaebacteria within the crater and system of organisms: Proposal for the domains archaea, bac• open-sea plume of erupting Macdonald seamount. Nature teria and eucarya. Proc. Natl. Acad. Sei. USA 87, 4576-4579 345, 179-182 (1990) (1990) Jannasch, R. W., Ruber, R., Belkin, S., Stetter, K. ().: Thermoto• Zillig, W., Stetter, K. O., Schäfer, W., Janecovic, D., Wunderl, S., ga neapolitana sp. nov. of the extremely thermophilic, cubacte- Holz, L, Palm, P.: Thcrmopr ote ales: A novel type of extremely rial genus Thermotoga. Arch. Microbiol. 150, 103-104 (1988) thcrmoacidophilic anaerobic archaebacteria isolated from Icelandic solfataras. Zbl. Bakt. Hyg., I. Abt. Orig. C 2, Zillig, W., Holz, l.,)anecovic, D„ Schäfer, D., Reiter, W. D.:The 205-227(1981) archaebacterium Jhertnococcus celer represents a novel genus Z/7%, W., Steuer, K. CK Vrangisbvillu IX, Schäfer, W., Wunder!, within the thermophilic branch of the archaebactefia. System.

S., Janecouic, /)., Holz, /., Palm, P.: l)esulfurococcaceacy the Appl. Microbiol. 4, 88-94 (1983) second family of the extremely thermophilic, anaerobic, sulfur- respiring Thermoprotcales. Zbl. Bakt. Hyg., 1. Abt. Orig. C 3, 304-317 (1982)

Professor Dr. Karl O. Stettcr, Universität Regensburg, Universitätsstr. 31, D-8400 Rcgcnsbiirg