International Journal of Systematic and Evolutionary Microbiology (2000), 50, 2093–2100 Printed in Great Britain

Ignicoccus gen. nov., a novel genus of hyperthermophilic, chemolithoautotrophic , represented by two new species, islandicus sp. nov. and Ignicoccus pacificus sp. nov.

Harald Huber, Siegfried Burggraf, Thomas Mayer, Irith Wyschkony, Reinhard Rachel and Karl O. Stetter

Author for correspondence: Karl O. Stetter. Tel: j49 941 943 3160. Fax: j49 941 943 2403. e-mail: Karl.Stetter!biologie.uni-regensburg.de

Lehrstuhl fu$ r Mikrobiologie Two species of novel, chemolithoautotrophic, sulfidogenic micro-organisms und Archaeenzentrum, were isolated from submarine hydrothermal systems in the Atlantic (at the Universita$ t Regensburg, Universita$ tsstrasse 31, Kolbeinsey Ridge north of Iceland) and in the Pacific (at 9 SN, 104 SW). The 93053 Regensburg, coccoid cells grew within a temperature range of 70–98 SC (optimum around Germany 90 SC). They gained energy by reduction of elemental using molecular hydrogen as the electron donor. 16S rDNA-based sequence comparisons revealed that the organisms are members of the crenarchaeal branch of the Archaea. They represent a new, deeply branching lineage within the family of the . In DNA–DNA hybridization experiments both strains exhibited low levels of hybridization to each other and to further representatives of this family. Therefore, they represent a new genus, for which the name Ignicoccus gen. nov. is proposed. At present it consists of two new species, Ignicoccus islandicus sp. nov. (type strain is Kol8T l DSM 13165T l ATCC 700957T ) and Ignicoccus pacificus sp. nov. (type strain is LPC33T l DSM 13166T l ATCC 700958T ).

Keywords: Archaea, , hyperthermophilic, marine, chemolithoautotrophic

INTRODUCTION consisting of lobed or coccoid-shaped thermoacido- philes, Thermoproteales, which harbours all rod- Within the Archaea, hyperthermophilic micro- shaped organisms of the kingdom, and the recently organisms growing optimally at temperatures between described order (Huber & Stetter, 80 and 110 mC are found in both kingdoms, the 2000). Desulfurococcales is composed of two families: Euryarchaeota and the Crenarchaeota (Stetter, 1988, the Pyrodictiaceae, members of which grow optimally 1996; Woese et al., 1990). Within the Euryarchaeota above 100 mC, and the representatives of the Desulfuro- they are represented by deep-branching organisms coccaceae, which exhibit optimal growth between 85 like Methanopyrus kandleri and members of the and 100 mC (Burggraf et al., 1997). The representatives genera Thermococcus, Pyrococcus, Methanothermus, of the Desulfurococcaceae are usually regular to Methanococcus and Archaeoglobus. So far, the cul- irregular cocci, which occur singly or in pairs. Some tivated Crenarchaeota are exclusively extremely species form chains or aggregates in addition. With the thermophilic or hyperthermophilic (Huber & Stetter, exception of the aerobic Aeropyrum pernix, all 1999). 16S rRNA-based sequence comparisons have members of the Desulfurococcaceae are obligate revealed that three orders are evident within the anaerobes, growing heterotrophically by sulfur res- Crenarchaeota (Burggraf et al., 1997): Sulfolobales, piration of various organic compounds (producing H#S) or by fermentation. In both cases, yeast extract, ...... peptides or sugars serve as substrates. Organic acids or The EMBL accession numbers for the 16S rDNA sequences of Ignicoccus alcohols are found as metabolic products (Huber & islandicus and Ignicoccus pacificus are X99562 and AJ271794, respectively. Stetter, 1999).

01444 # 2000 IUMS 2093 H. Huber and others

In this study we describe the isolation and properties For quantitative determinations, H#S was analysed by of novel, chemolithoautotrophic members of the titration (Williams et al., 1979). Desulfurococcaceae which grow by sulfur reduction Lipid analysis. Core lipids were analysed according to with molecular hydrogen as electron donor. Trincone et al. (1992). DNA isolation and DNA base composition. DNAs were METHODS prepared as described previously (Wildgruber et al., 1982). The GjC content of genomic DNAs was determined by Sources of samples. At the Kolbeinsey Ridge, north of melting point analysis (Marmur & Doty, 1962) and by direct Iceland, eight samples of submarine sandy sediments and analysis of the nucleotides after digestion of the DNA with venting water (original temperatures around 90 mC) were nuclease P1 and separation by HPLC chromatography taken by the research submersible ‘Geo’ at depths between (Vo$ lkl et al., 1993). Calf thymus DNA was used as reference. 103 and 106 m (Fricke et al., 1989; Burggraf et al., 1990). Furthermore, black smoker samples were obtained during DNA–DNA hybridization. DNA–DNA hybridization was dive 3072 of the submersible ‘Alvin’ at the East Pacific carried out using the filter technique (Gillespie & Gillespie, Rise at 9m N, 104m W at a depth of 2500 m. The samples 1971; Birnstiel et al., 1972) as described by Pley et al. (1991). were brought to our laboratory anaerobically without Nucleic acids were hybridized under optimal conditions temperature control. (25 mC below TM in 3iSSC buffer at 65 mC) (Marmur & Doty, 1961; Brenner, 1973; Meyer & Schleifer, 1978) using Strains and culture conditions. The new isolates were the DNAs of Kol8T and LPC33T as probes. enriched and cultivated in strictly anaerobic half-strength SME medium (Stetter et al., 1983; Pley et al., 1991), prepared 16S rRNA analysis. The nearly complete 16S rRNA genes of according to Balch & Wolfe (1976). The medium contains the new isolates were PCR-amplified (Saiki et al., 1985, −" the following components (l ): NaCl, 13n85 g; MgSO%; 1988). The primers used in the amplification corresponded 7H O, 3 5 g; MgCl ;6H O, 2 75 g; KH PO ,05g; to positions 8–23 (TCYGGTTGATCCTGCC), and # n # # n # % n T CaCl#;2H#O, 0n38 g; KCl, 0n33 g; (NH%)#SO%,0n25 g; 1512–1492 (ACGGHTACCTTGTTACGACTT) for Kol8 NaBr, 0 05 g; H BO ,0015 g; SrCl ;6H O, 7 5mg; KI or 1406–1390 (ACGGGCGGTGTGTRCAA) for LPC n " $ $ n # # n (1 mg ml− ), 25 µl; elemental sulfur, 5n0 g. Reduction of the strains (Escherichia coli 16S rRNA numbering; Brosius et al., medium was carried out by addition of 20 ml Na#S(2n5%, 1981). Both strands of the PCR products were directly w\v); afterwards, the pH was adjusted at room temperature sequenced as described by Burggraf et al. (1997). The T T to 5n5 with sulfuric acid. The organisms were grown routinely sequences (Kol8 , 1465 bases; LPC33 , 1311 bases; LPC37, in 120 ml serum bottles containing 20 ml medium pres- 1297 bases) were aligned with a set of representative archaeal surized with H#\CO# (80:20, v\v; 250 kPa). Incubation was sequences using the ARB program (W. Ludwig & O. Strunk, carried out at 90 mC with shaking (100 r.p.m.). Heterotrophic 1998, http:\\www.mikro.biologie.tu-muenchen.de\pub\ growth was tested under a gas phase of N#\CO# (80:20, v\v; ARB\). Dendrograms were computed with the neighbour- 200 kPa). joining, maximum-parsimony and maximum-likelihood methods included in the ARB package. Unless otherwise stated, organic substrates and alternative electron acceptors (thiosulfate, sulfite, sulfate, nitrate and nitrite) were added at final concentrations of 0n1%. Batch RESULTS cultures were grown in a 300 l enamel-protected fermenter (HTE Bioengineering) at 90 mC with stirring (100 r.p.m.) and Enrichment and isolation −" gassing with H#\CO# (80:20; 2 l min ). To enrich chemolithoautotrophic, sulfur-reducing Light and electron microscopy. Cells were routinely observed hyperthermophiles, serum bottles with 20 ml half- with an Olympus BX 60 phase-contrast microscope with an strength SME medium supplemented with 1% (w\v) oil immersion objective, UPlanFl 100\1n3. Bacterial growth sulfur (gas phase H#\CO#) were inoculated with about was followed by direct cell counting using a Thoma chamber 1 g of the sandy sediments from the Kolbeinsey Ridge (depth 0n02 mm). Electron microscopy was performed as follows. For direct visualization, cells were chemically fixed or rocky black smoker material obtained from the by adding glutaraldehyde (2%, v\v, final concn) to the deep sea of the East Pacific Rise. The enrichment culture medium, concentrated by centrifugation, applied attempts were incubated with shaking at 90 mC. After onto a carbon-coated copper grid and shadowed with 2 d, irregular cocci had grown in 3 out of 10 samples T T 1nmPt\C (angle 15m). For ultrathin sections, cells were (Kol8 , LPC33 and LPC37) and large amounts of cultivated in cellulose capillary tubes, high-pressure frozen, H#S could be detected qualitatively in the culture freeze-substituted in acetone containing 1% OsO% and medium. The enrichment cultures were successfully embedded in Epon\Araldite (Rieger et al., 1997). Sections transferred into fresh medium and cells were first were stained with uranyl acetate and lead citrate. For freeze- purified by serial dilution carried out three times using etching, a concentrated cell suspension was frozen in liquid the same medium. In addition, the ‘optical tweezer’ nitrogen, freeze-etched for 4 min at 97 C and shadowed k m et al with 1 nm Pt\C (angle 45m) and with 10 nm C (angle 90m). technique was used for final purification (Huber ., Replicas were cleaned overnight on sulfuric acid (70%, 1995). The isolates were designated as the samples. w\v). Electron micrographs were taken on a model CM12 transmission electron microscope (Philips) with an acceler- Morphology ation voltage of 120 kV. H S determination. The formation of H#S was qualitatively Cells of both new isolates were irregular cocci usually 2 occurring singly or in pairs. They exhibited cell monitored by addition of 20 µl saturated lead acetate T solution to 0n5 ml samples, yielding a dark brown precipitate. diameters between 1n2 and 3 µm (Kol8 ), and 1 and

2094 International Journal of Systematic and Evolutionary Microbiology 50 Ignicoccus gen. nov.

2 µm (LPC33T). The organisms stained Gram-nega- Optimal growth conditions tive. They were both motile, possessing one bundle of T up to nine flagella (for isolate Kol8T; Fig. 1a). The cell Isolate Kol8 grew between 70 and 98 mC. No growth was observed at 65 mC or below, nor at 100 mC or above architecture of the isolates was studied by sectioning T cells after high-pressure freezing and freeze-substi- (Fig. 2). Isolates LPC33 and LPC37 were able to grow tution (Fig. 1b) and by the freeze-etch\freeze-fracture between 75 and 98 mC with an optimum at 90 mC (Fig. technique (Fig. 1c). The cytoplasm was densely 2). When the medium was supplemented with 0n1% meat extract, the shortest doubling times of 1n4 and packed and surrounded by a membrane (approx. T T 6–8 nm wide). The periplasm had a variable width, 0n8 h were observed for isolates Kol8 and LPC33 at ranging between 20 nm and 350 nm. It contained 90 mC, respectively (Fig. 2). Under chemolithoauto- numerous, round or elongated vesicles, 50–60 nm in trophic conditions, the shortest doubling times were 1n7 and 1n6 h at 90 mC, respectively (data not shown). diameter and up to 300 nm in length, each surrounded T Kol8 grew at NaCl concentrations between 0n3 and by a membrane (Fig. 1b). Rarely, these periplasmic T vesicles were in close contact with the cytoplasmic 5n5% (w\v) (Fig. 3), while strain LPC33 exhibited a membrane. In sections, the outermost part of the cell minimum NaCl requirement of 1n0% and a maximum envelope occasionally had a weak double-layer ap- of 5n0% (Fig. 3). For all strains the optimal NaCl concentration was around 2% and cells lysed at NaCl pearance. This ‘sheath’ was frequently found to be T fractured into two leaflets in freeze-etch\freeze-frac- concentrations above 6n0% in the medium. Kol8 grew between pH 3n8 and 6n5 with an optimum around ture experiments (Fig. 1c) and, therefore, most likely T represented a (lipid) membrane. Close inspection of pH 5n8 (Fig. 4). The optimal pH for growth of LPC33 the fracture planes revealed that this membrane was was 6n0 with a minimum at pH 4n5 and a maximum at tightly packed with particles, most likely proteins. A pH 7n0 (Fig. 4). two-dimensional crystalline arrangement, as observed in S-layer sheets of most Archaea and many Bacteria, T was never observed. The core lipids of isolate Kol8 Storage are acyclic 2,3-di-O-phytanyl-sn-glycerol and glycerol- dialkyl glycerol tetraether in a relative ratio of about Stock cultures, containing 5% (v\v) dimethylsulfoxide 1:1 (A.Gambacorta, personal communication). and stored at k140 mC over liquid nitrogen served as viable inocula for at least 3 years.

Metabolism DNA base composition T The new isolates were obligate anaerobes, growing The GjC content of genomic DNA of isolate Kol8 chemolithoautotrophically by sulfur reduction using was 41 mol%, calculated by melting point analysis and molecular hydrogen as electron donor (final cell by direct analysis of the mononucleotides. Isolates ( −" T concentrations up to 4i10 cells ml ). Up to 25 µmol LPC33 and LPC37 exhibited 45 mol% GjC in their −" H#S (ml culture medium) was formed by isolate genomic DNA. Kol8T at the end of the exponential growth phase. In the presence of H# and Sm, growth of all isolates was stimulated by the addition of meat extract, tryptone DNA–DNA similarity and glycogen (each 0 1%; final cell densities around ( ( n " 6 10 –8 10 cells ml− ). The addition of yeast DNA–DNA hybridization experiments revealed no i i T extract (0 1%) resulted in shorter doubling times for significant hybridization signals between isolate Kol8 n T both strains, although higher final cell densities were and isolates LPC33 and LPC37 (similarity 11 and only observed for LPC33T. No effect on growth or 8%, respectively). In contrast, a DNA–DNA simi- T final cell concentrations was obtained in the presence larity of 76% was obtained between LPC33 and of Casamino acids, casein, starch, gelatin, maltose or LPC37. The DNA of each of the new isolates yielded glucose (each 0n1%) for all strains. The addition of no significant hybridization signal (similarity between formate or acetate (0n05%) decreased final cell 5 and 9%) with DNA of the following Crenarchaeota ( −" densities to about 1i10 cells ml . No growth was and Euryarchaeota: Staphylothermus marinus, observed on organic substrates like meat extract, yeast Desulfurococcus mobilis, Thermococcus celer and extract, peptone, Casamino acids, gelatin, starch, Pyrococcus furiosus. formate, acetate or glucose when cultures were pressurized with hydrogen-free gas (N#\CO#, 80:20; 200 kPa). In a similar way, sulfur could not be replaced Phylogenetic analysis by oxygen (0n5–5% v\v), thiosulfate, tetrathionate, sulfite, sulfate or nitrate (each 0n1%) as electron Comparison of 16S rDNA sequences between the new acceptors. Ampicillin, rifampicin and vancomycin isolates and representatives of the Crenarchaeota and " (final concn 50 µgml− ) were not inhibitory to growth Euryarchaeota using all three major approaches for of the strains. tree reconstruction clearly indicated that the new

International Journal of Systematic and Evolutionary Microbiology 50 2095 H. Huber and others

(a) (b)

(c)

...... Fig. 1. For legend see facing page. 2096 International Journal of Systematic and Evolutionary Microbiology 50 Ignicoccus gen. nov.

10 6

8 5

4 6 3 4 2 Doubling time (h)

2 Doubling time (h) 1 0 6070 80 90 100 110 0 2468 ° Temperature ( C) pH ...... Fig. 2. Optimal growth temperature of Ignicoccus islandicus Fig. 4. Effect of pH on growth of Ignicoccus islandicus strain strain Kol8T ( ) and Ignicoccus pacificus strain LPC33T ( ) 4 Kol8T (4) and Ignicoccus pacificus strain LPC33T ( )at90mC during growth on H and sulfur at pH 5 5 and 2% NaCl. 2 n and 2% NaCl. Doubling times were calculated as described in Doubling times were calculated from the slopes of the growth the legend to Fig. 2. curves (not shown).

DISCUSSION 10 The novel hyperthermophilic isolates Kol8T, LPC33T and LPC37 represent the first obligate chemolitho- 8 autotrophic sulfur reducers within the crenarchaeal family of the Desulfurococcaceae. They are repre- 6 sentatives of this family due to their coccoid cell shape, the lack of a cell sacculus, their negative Gram 4 reaction, their optimal growth temperature of around 90 mC and their 16S rDNA sequences (Huber & Stetter, Doubling time (h) 2 2000). They can be differentiated from the other members of the Desulfurococcales by their unique cell 0 0246wall architecture (Baumeister & Lembcke, 1992) and the extremely limited spectrum of utilized electron NaCl (%) donors and acceptors: molecular hydrogen is the only ...... electron donor and no acceptors other than elemental Fig. 3. Effect of NaCl concentration on growth of Ignicoccus sulfur can be used. Therefore, the organisms are T T islandicus strain Kol8 (4) and Ignicoccus pacificus strain LPC33 obligate hydrogen-sulfur autotrophs, producing H#S. ( )at90mC and pH 5n5. Doubling times were calculated as described in the legend to Fig. 2. Some organic components stimulate growth by decreasing the shortest doubling times and yielding higher final cell densities. However, they cannot be used as sole energy sources. In contrast, all other anaerobic members of the Desulfurococcaceae known strains belong to the crenarchaeal branch of the so far grow organotrophically. In addition to organic archaeal domain (Fig. 5). In all calculations they are acids or alcohols, several representatives produce H#S members of the new order Desulfurococcales, repre- from elemental sulfur (e.g. Desulfurococcus, Staphylo- senting a deep branch within the Desulfurococcaceae thermus,‘Thermodiscus’orStetteria), while the (Fig. 5). They exhibit sequence differences between 6 members of the genera Sulfophobococcus and Thermo- and 10% to any of the other members of this family sphaera are inhibited in the presence of elemental (data not shown). In contrast, the differences among sulfur (Huber & Stetter, 1999). This physiological T the new isolates were between 0n2 (LPC33 and LPC37) separateness of the new isolates is confirmed by 16S T and 2n0% (Kol8 and LPC strains). rDNA sequence analyses, where the new isolates

Fig. 1. (a) Electron micrograph of a cell of isolate Kol8T, exhibiting numerous flagella. The sample was fixed with 2% glutaraldehyde and shadowed with Pt/C. Bar, 1 µm. (b) Electron micrograph of a thin section of a freeze-substituted cell of isolate Kol8T. C, cytoplasm; CM, cytoplasmic membrane; P, periplasm; OS, outer sheath; white arrowhead, contact site of a periplasmic vesicle with the cytoplasmic membrane; black arrows, areas of the outer sheath with double-layer appearance. Bar, 0n5 µm. (c) Electron micrograph of freeze-etched cells of isolate Kol8T. C, cytoplasm; CM, cytoplasmic membrane; P, periplasm; FF, fracture face of the outer sheath; OS, outer sheath. Bar, 0n5 µm.

International Journal of Systematic and Evolutionary Microbiology 50 2097 H. Huber and others

‘ ‘

...... Fig. 5. Phylogenetic position of Ignicoccus species (isolates Kol8T, LPC33T and LPC37), calculated by the neighbour-joining method using Jukes & Cantor correction. Bar, 10 estimated substitutions per 100 nt.

exhibit sequence differences of at least 6% to any other found in hydrothermal fluids (Jannasch & Mottl, representative of the Desulfurococcaceae but only 1985), representatives of Ignicoccus may be widely about 2% to each other. This indicates that the new distributed in submarine hydrothermal systems. Due isolates belong to the same new genus, for which we to their chemolithoautotrophic mode of life, they are propose the name Ignicoccus gen. nov., the ‘fireball’, probably important primary producers of organic expressing the extremely high temperature optimum of matter in these biotopes and are essential for the all strains. The first isolate, Kol8T, can be distinguished growth of organotrophic hyperthermophiles, like from the two LPC strains by a generally bigger cell the other marine representatives of the Desulfuro- diameter, a lower minimum growth temperature of coccaceae. 70 mC, growth at lower NaCl concentrations (0n3% in contrast to 1n0%), a low DNA–DNA similarity to these two organisms and a 4 mol% lower genomic Description of Ignicoccus gen. nov. (Huber, Burggraf DNA GjC content. It represents the type species of and Stetter) the new genus, which we have named Ignicoccus T T islandicus (type strain is Kol8 l DSM 13165 l Ignicoccus (Ighni.cochcus. L. masc. n. ignis fire; Gr. ATCC 700957T), referring to the location of its masc. n. coccos berry; M.L. masc. n. Ignicoccus the isolation. berry of the fire). Isolates LPC33T and LPC37 show similar physio- Irregular cocci, about 1–3 µm in diameter, monopolar logical and molecular characteristics. Furthermore, in polytrichous flagella. Gram-negative. Occurring singly DNA–DNA hybridization experiments, a similarity of and in pairs. Cell envelope consists of a cytoplasmic 76% was obtained for these two organisms. This membrane, a periplasm (20–350 nm wide) and a sheath indicates that they belong to the same second species, resembling an outer membrane. Cells contain phytanyl which we have named Ignicoccus pacificus, referring to di- and tetraether lipids. Growth between 70 and the location of its isolation. The type strain is LPC33T 98 mC, pH optimum around 6. Strictly anaerobic. T T (l DSM 13166 l ATCC 700958 ). Chemolithoautotrophic growth in the presence of H# and CO# with sulfur as electron acceptor. Sulfate, Ignicoccus islandicus and Ignicoccus pacificus were sulfite, thiosulfate, tetrathionate, nitrate and oxygen isolated from hot sediments at the Kolbeinsey Ridge, are not used as electron acceptors. H#S formed during north of Iceland, and from active black smoker walls growth. Ampicillin-, rifampicin- and vancomycin- from the East Pacific, respectively. These biotopes are resistant. DNA base composition between 41 and located far from each other and represent different 45 mol% GjC. Based on 16S rDNA sequence com- geological environments. Therefore, since their sub- parison, the genus is a member of the family Desulfuro- strates (hydrogen, CO# and sulfur) are commonly coccaceae, within the kingdom Crenarchaeota. Type

2098 International Journal of Systematic and Evolutionary Microbiology 50 Ignicoccus gen. nov.

T T species is Ignicoccus islandicus (Kol8 l DSM 13165 Manfred Biebl for practical work on strain LPC37 and T l ATCC 700957 ). Agata Gambacorta for lipid analysis. This work was supported by grants of the Deutsche Forschungsgemein- schaft (STE 297\10–3) and the Fonds der Chemischen Description of Ignicoccus islandicus sp. nov. (Huber, Industrie. Burggraf, Mayer and Stetter) Ignicoccus islandicus (is.lanhdi.cus. M.L. masc. adj. islandicus Icelandic, pertaining to the location of its REFERENCES first isolation). Balch, W. E. & Wolfe, R. S. (1976). New approach to the Slightly irregular cocci, about 1n2–3 µm in diameter, cultivation of methanogenic bacteria: 2-mercaptoethane- monopolar polytrichous flagella. Gram-negative. Oc- sulfonic acid (HS-CoM)-dependent growth of Methano- curring singly and in pairs. No evidence for a regular bacterium ruminantium in a pressurized atmosphere, Appl arrayed surface protein. 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