Acidilobus Aceticus Gen. Nov., Sp. Nov., a Novel Anaerobic Thermoacidophilic Archaeon from Continental Hot Vents in Kamchatka

International Journal of Systematic and Evolutionary Microbiology (2000), 50, 2001–2008 Printed in Great Britain

Acidilobus aceticus gen. nov., sp. nov., a novel anaerobic thermoacidophilic archaeon from continental hot vents in Kamchatka

M. I. Prokofeva,1 M. L. Miroshnichenko,1 N. A. Kostrikina,1 N. A. Chernyh,1 B. B. Kuznetsov,2 T. P. Tourova1 and E. A. Bonch-Osmolovskaya1

Author for correspondence: E. A. Bonch-Osmolovskaya. Tel: j7 95 135 44 58. Fax: j7 95 135 65 30. e-mail: lbo!inmi.host.ru

1 Institute of Microbiology, New thermoacidophilic organisms that were able to grow anaerobically on Russian Academy of starch were isolated from the acidic hot springs of Kamchatka. Strain 1904T, Sciences, Prospect 60 Let Oktyabrya 7/2, 117811, isolated from a hot spring of the Moutnovski volcano, was characterized in Moscow, Russia detail. Its cells were regular or irregular cocci that were 1–2 µm in diameter, 2 Bioengineering Centre, non-motile, and had a cell envelope consisting of one layer of subunits. The Russian Academy of new organism was a hyperthermophile, growing in the temperature range Sciences, Prospect 60 Let 60–92 SC (with an optimum at 85 SC), an acidophile, having the pH range for Oktyabrya 7/1, 117811, Moscow, Russia growth of 20–60 (with an optimum at 38), and an obligate anaerobe. It fermented starch, forming acetate as the main growth product. Other growth substrates were yeast extract, beef extract and soya extract. Growth on yeast extract, beef extract and soya extract was stimulated by elemental sulfur,

which was reduced to H2S. Acetate, arabinose, cellulose, formate, fructose, galactose, glucose, glycine, guar gum, lichenan, malate, maltose, methanol, pectin, pyruvate, propionate, xylan, xylose or a mixture of amino acids failed to support growth both in the presence and the absence of sulfur. When starch was used as the growth substrate, yeast extract (100 mg lV1) was required as a growth factor. The GMC content of the DNA was found to be 538 mol%. Comparison of the complete 16S rDNA sequence with databases revealed that the new isolate belonged to the kingdom Crenarchaeota. It was not closely related to any described genera (showing sequence similarity below 908%) and formed a separate branch of the Crenarchaeota. On the basis of physiological differences and rRNA sequence data, a new genus – Acidilobus – is proposed, the type species being Acidilobus aceticus strain 1904T (l DSM 11585T).

Keywords: Acidilobus aceticus, Archaea, hyperthermophiles, anaerobes, acidophiles

INTRODUCTION and acidophiles. Alkaliphilic hyperthermophilic archaea are currently represented by a few species of Hyperthermophilic prokaryotes have a temperature the genus Thermococcus (Keller et al., 1995; Dirmeier optimum for growth of greater than 80 mC and are et al., 1998), which have a pH optimum for growth of represented by both the Bacteria and the Archaea 9n0. Thermoacidophilic isolates are much more nu- (Stetter, 1996). Although only neutrophilic species are merous and comprise mostly organisms with respir- known among hyperthermophilic bacteria, hyper- atory metabolism, i.e. obligate or facultative aerobes thermophilic archaea include neutrophiles, alkaliphiles (Scho$ nheit & Scha$ fer, 1995), and just one obligate anaerobe, Stigiolobus azoricus (Segerer et al., 1991), ...... which can grow at pH 1n0–5n5 by means of lithotrophic The GenBank accession number for the 16S rDNA sequence of strain 1904T sulfur respiration by utilizing molecular hydrogen as is AF191225. the sole growth substrate. Among the acidophiles,

01268 # 2000 IUMS 2001 M. I. Prokofeva and others fermentative metabolism has been detected only in Thiobacillus ferrooxidans VKM B-458, after preliminary Thermoplasma acidophilum (Segerer et al., 1988), a incubation for 3 d at 80 mC and pH 2n5. facultative aerobe and moderate thermophile. Here we Determination of DNA GjC content. The GjC content of report the isolation of a new obligately anaerobic the genomic DNA was determined as reported earlier organism capable of fermentative growth at 85 mC and (Miroshnichenko et al., 1994). pH 3n8. Sequencing and analysis of 16S rDNA. The 16S rRNA gene was selectively amplified from the genomic DNA by a PCR using 5 -AGAGTTTGATCCTGGCTCAG-3 as the for- METHODS h h ward primer and 5h-TACGGTTACCTTGTTACGACTT-3h Sampling and enrichment. Samples of water, mud and soil as the reverse primer (Lane, 1991). The PCR reaction was were taken from solfataric fields (in Kamchatka) and were carried out in 100 µl reaction mixture containing 1 µg DNA shown to have pH values of 4n0 or less. The samples were put template, 200 µM (each) primer, 200 µM (each) dNTP and 3 in 100 ml flasks with screw caps, closed hermetically, U Tet-z polymerase (BioMaster) in reaction buffer (100 mM transported to the laboratory and used without delay for Tris\HCl pH 8n3, 500 mM KCl, 20 mM MgCl#). The tem- inoculation of the basal medium, which contained the perature regime involved 30 amplification cycles of 1 min at " following (mg lV ): KCl, 330; NH%Cl, 330; KH#PO%, 330; 94 mC, 1 min at 42 mC and 1 min at 72 mC. The final extension MgCl#.6H#O, 330; CaCl#.2H#O, 330; Na#S.9H#O, 500; was carried out at 72 mC for 6 min. The PCR products were starch, 5000; yeast extract (Difco), 100; resazurin, 1. Trace- purified using the PCR-prep kit (Promega) as recommended element (Kevbrin & Zavarzin, 1992) and vitamin (Wolin by the manufacturer. The 16S rRNA gene was sequenced in " et al., 1963) stock solutions were added at 1 ml lV . The both directions using the forward and reverse primers. The medium was prepared anaerobically under a flow of oxygen- DNA sequencing was performed by using USB Sequenase free CO . After the reduction of the medium by boiling and version 2 kit. # " consequent addition of Na#S.9H#O (700 mg lV ) and de- The 16S rDNA sequence was aligned with a representative colourization of resazurin, the pH was adjusted to 3.0 by the set of analogous sequences obtained from the latest versions addition of 5 M H#SO%. Portions of the medium (20 ml) were of the Ribosomal Database Project (RDP) or from GenBank dispensed into 50 ml bottles with screw caps, filled with CO# by using the  software (Corpet, 1988). Positions and sterilized at 110 mC. Each bottle was inoculated with 1 g that had not been sequenced in one or more reference of sample and incubated at 80 mC. Microbial growth was organisms were omitted and a total of 1247 nucleotides were monitored using light microscopy. used in the analysis. Pairwise evolutionary distances were Isolation of pure cultures. For isolation of colonies, flat computed by using the correction of Jukes & Cantor (1969). 100 ml bottles (Bellco) containing 3 ml of the same medium The phylogenetic tree was constructed by the neighbour- joining method (Saitou & Nei, 1987) with bootstrap analysis solidified with 0n8% (w\v) Gelrite and 0n05% (w\v) of 100 trees using the programs of the  package (Van MgSO%.7H#O were used. Individual colonies were picked up de Peer & De Wachter, 1994), with Methanococcus vannielii under a flow of oxygen-free CO# and transferred to the liquid medium. The purity of isolates was tested by using light as the outgroup. microscopy of cultures in different growth conditions. Nucleotide sequence accession numbers. The accession numbers of the sequences used as references are as follows: Morphology and ultrastructure studies. The morphology of Sulfurisphaera ohwakuensis TA1T, D85507; Sulfolobus acido- new isolates was examined using phase-contrast light mi- caldarius DSM 639T, D14053; Thermosphaera aggregans croscopy. The ultrastructure was studied in a JEM-100 M11TLT, X99556; Staphylothermus marinus F1 DSM 3639T, electron microscope, with samples prepared as described X99560; Hyperthermus butylicus DSM 5456T, X99553; elsewhere (Bonch-Osmolovskaya et al., 1990). Sulfophobococcus zilligii K1T, X98064; Stygiolobus azoricus " T T Metabolic studies. Potential growth substrates (5000 mg lV ) DSM 6296 , D85520; Acidianus infernus DSM 3191 , were added to the basal medium prepared without starch. D85505; Aeropyrum pernix K1T, D83259; Metallosphaera Organic acids were added as their sodium salts, and a sedula DSM 5348T, X90481; Stetteria hydrogenophila mixture of 20 amino acids was used (SAG-II). The headspace 4ABCT, Y07963; Pyrodictium occultum PL19, M21087; was filled with 100% CO#; when molecular hydrogen was Desulfurococcus mobilis, M36474; Thermofilum pendens tested as a growth substrate, it was used in a mixture with Hvv3T, X14835; Thermoproteus tenax, M35966; Pyro- T CO# (8:2, v\v). Growth was measured by direct cell counting baculum islandicum geo3 , L07511; Caldivirga maquilingen- under the light microscope. Growth products were detected sis IC-167T, AB013926; Thermocladium modestius IC-125T, using methods described earlier (Miroshnichenko et al., AB005296; and ‘Caldococcus noboribetus’ NC12, D85038. 1994). The temperature range and optimum for growth were determined on the medium with yeast extract and elemental RESULTS sulfur at pH 3n8. The pH range and optimum for growth were determined at 80 mC. Possible electron acceptors added Enrichment and isolation to the basal medium included elemental sulfur as sulfur " flower powder (10000 mg lV ), nitrate as the sodium salt, Samples obtained from acidic hot springs from dif- " (500, 1000 and 2500 mg lV ) and Fe(III) as amorphous ferric ferent areas in Kamchatka were used to inoculate oxide [90 mM Fe(III); Slobodkin et al., 1999] or Fe(III) anaerobic starch medium at pH 3n0. After 2–5 d citrate (20 mM). incubation at 80 mC, eight samples showed microbial Sensitivity to antibiotics. The influence of penicillin, strep- growth (Table 1). Cells of irregular coccoid shape were " tomycin (both at 500 mg lV ) and chloramphenicol observed in all cultures. When transferred to Gelrite- " (100 mg lV ) on the growth of the new isolate was tested. The solidified medium, smooth white colonies approximately 1 mm in diameter appeared after 7 d incu- stability of the antibiotics at high temperature and low pH T was checked by observing their effects on the growth of bation. By isolating single colonies, strains 1904 , 1919

2002 International Journal of Systematic and Evolutionary Microbiology 50 Acidilobus aceticus gen. nov., sp. nov.

Table 1. Characteristics of the Kamchatka sampling sites used for enrichment of anaerobic organotrophic thermoacidophiles

Sample no. Location Description Temp. (mC) pH

1904T Mutnovski volcano Hot spring with ferric iron deposits 87 4n0 1919 Mutnovski volcano Hot well surrounded with grass 90 3n0 1920 Mutnovski volcano Hot spring with ferric iron deposits 90 3n0 301 Geyser valley Black mud near the Giant geyser 91 4n5 310 Geyser valley Hot well near the Giant geyser 80 4n5 321 Uzon caldera, Orange thermal ﬁeld Mud hole 70 4n0 322 Uzon caldera, Orange thermal ﬁeld Mud hole 87 4n5 345 Moutnovski volcano Mud hole 82 4n0

(a)

(b)

S ol (c)

...... Fig. 2. Electron micrographs of ultrathin sections of isolate 1904T cells in the late-exponential phase of growth. Abbreviations: s, subunit; cm, cytoplasmic membrane; ol, osmiophilic layer. Bars, 0n5 µm.

Morphology and ultrastructure Cells of isolate 1904T were regular to irregular cocci ...... approximately 1–2 µm in diameter (Fig. 1, top). Active Fig. 1. Electron micrographs of isolate 1904T whole cells in the motility was never observed in light microscope, and late-exponential phase of growth. Bars, 0n5 µm. no ﬂagella were ever seen on the electron micrographs of the whole cells. Double or triple cells were frequently observed in young cultures (Fig. 1, middle and bot- and 1920 were obtained. Isolate 1904T, obtained from tom). Electron microscopy of thin sections revealed the Moutnovski volcano sample, showed the best that the cell envelope comprised an S-layer attached to growth and was selected for further experiments. the cytoplasmic membrane (Fig. 2, top). The S-layer

International Journal of Systematic and Evolutionary Microbiology 50 2003 M. I. Prokofeva and others

7 30 3

25 2·5 6 ) –1

) 20 2·0 –1 5 ml mol ml –6 µ

10 15 1·5 S ( 2 i 4

Cells ( 10 1·0 Doubling time (h) Acetate, H

3 5 0·5

2 0 70 75 80 85 90 95 100 12345678 Temperature (°C) Time (d) ...... T Fig. 5. Production of H2S( ) and acetate (>), and growth ($), Fig. 3. Inﬂuence of temperature on the growth of isolate 1904 T 0 on the medium with yeast extract and elemental sulfur at pH of isolate 1904 on the medium with yeast extract and S . 3n8.

was 2n5 h. The maximum cell concentration achieved ( " T was 2–2n5i10 cells mlV . Isolate 1904 was found to 13 be a strict anaerobe unable to grow without pre- reduction of the medium. 11 Isolate 1904T was found to grow on starch, yeast extract, beef extract and soya extract. None of the 9 following supported growth: acetate, arabinose, cellulose, formate, fructose, galactose, glucose, glycine, guar gum, lichenan, malate, maltose, methanol, pectin, 7 pyruvate, propionate, xylan, xylose or a mixture of 20 " amino acids. Yeast extract (100 mg lV ) was required 5 as a growth factor when starch was used as the growth Doubling time (h) substrate. T 3 Growth of isolate 1904 on a medium with yeast extract, beef extract and soya extract as the growth substrates was stimulated by elemental sulfur, which 1 123456 was reduced to hydrogen sulﬁde. On a medium with pH starch, the stimulatory eﬀect of sulfur was less promi-

...... nent. Nitrate, at all concentrations tested, did not Fig. 4. Inﬂuence of pH on the growth of isolate 1904T on the inﬂuence growth on either medium. Fe(III), added as medium with yeast extract and elemental sulfur (incubation ferric oxide or ferric citrate, did inhibit growth. temperature, 80 mC). The main metabolic product of isolate 1904T, both on yeast extract (Fig. 5) and on starch, was acetate. Neither H# nor ethanol was formed in the course of T was composed of a single layer of subunits covered growth on any of the substrates used. Isolate 1904 with a thin osmophilic layer that was probably was shown to be resistant to penicillin, streptomycin proteinaceous in nature (Fig. 2, bottom). and chloramphenicol.

Physiology The GjC content of the DNA T T Isolate 1904 grew at temperatures between 60 and The GjC content of the total DNA of isolate 1904 92 mC, with an optimum at 85 mC (Fig. 3), and within a was 53n8 mol%. Similar values were obtained for other pH range of 2n0–6n0, with an optimum at 3n8 (Fig. 4). isolates: 54n5 mol% (isolate 1919), 55n1 mol% (isolate The minimum doubling time under optimal conditions 1920).

2004 International Journal of Systematic and Evolutionary Microbiology 50 Acidilobus aceticus gen. nov., sp. nov.

compared with the corresponding sequence data from the RDP. This analysis revealed that new isolate 1904T was a member of the kingdom Crenarchaeota of the domain Archaea. Additional sequence alignments and phylogenetic analyses performed with type species of the validly published genera of this kingdom showed that strain 1904T was not closely related to any reference organism (82n5–90n8% sequence similarity). In the phylogenetic tree (Fig. 6), strain 1904T formed a single cluster with Aeropyrum pernix (90n8% sequence similarity), but the bootstrap probability of this branching point was not high (80%). More detailed analysis of the 16S rRNA gene of strain 1904T revealed unique deviation from the known crenarchaeotal sequences at positions where all other Crenarchaeota exhibited conserved bases (Table 2). Of these changes, the deviations at positions 592:647, 1308:1329 and 1310:1327 occurred (using the inferred secondary structure) in helixes and involved paired bases (com- ...... pensatory changes). When the 16S rRNA sequence of T Fig. 6. Phylogenetic tree showing the relationship between isolate 1904 was compared with all available se- novel strain 1904T and other archaea, based on a comparison of quences, it exhibited a high degree of 16S rRNA 16S rDNA sequences. sequence similarity (98n3%) to crenarchaeotal strain NC12 (‘Caldococcus noboribetus’) and had the same signature nucleotides. Table 2. Sequence signatures of the 16S rRNA from isolate 1904T DISCUSSION Sequence Corresponding bases† position* Hot springs with low-pH water are quite common in Isolate 1904T Common to different volcanic areas of the world (Brock, 1978; crenarchaeotal species‡ Stetter et al., 1990). The first representative of extremely thermophilic archaea, Sulfolobus solfataricus, 321:332 C:G A:G was isolated in 1972 and was a thermoacidophile 592:647 C:G G (u,a):C (a,u) growing optimally at 70–75 mC and pH 2n0–3n0 (Brock 678 C U et al., 1972). More recently, significant progress has 913 U A been made in investigations of both the phenotypic 1302 C U (a) and the phylogenetic diversity of hyperthermophiles 1308:1329 U:A C:G (Blo$ chl et al., 1995; Stetter, 1996); 10 genera of 1310:1327 G:C A:U thermoacidophilic archaea have been described (Table 1335 C G 3). Most of the genera belong to the Crenarchaeota, 1393 U C with the exception of Thermoplasma (Segerer et al., 1414 U C 1988) and Picrophilus (Schleper et al., 1995), which belong to the order Thermoplasmales of the kingdom * Numbering according to Escherichia coli nomenclature. Euryarchaeota. The majority of the thermoacidophilic † Base pairing was deduced from secondary-structure assign- archaea are aerobes capable of lithotrophic growth ment. with elemental sulfur, sulfides or molecular hydrogen ‡ All of the crenarchaeotal 16S rRNA sequences were from the as electron donors (Stetter, 1996; Scho$ nheit & Scha$ fer, Ribosomal Database Project; lower-case letters indicate bases 1995). Some of them, like Acidianus infernus (Segerer found in less than 15% of assayable cases. et al., 1986), are facultative anaerobes and are capable of anaerobic growth with molecular hydrogen by using elemental sulfur as an alternative electron acceptor. Analysis of 16S rDNA The metabolism of Thermoproteus tenax (Zillig et al., 1981) and Stigiolobus azoricus (Segerer et al., 1991), The almost complete (1411-nucleotide) sequence of the which are obligate anaerobes, is also based on this 16S rRNA gene (corresponding to positions 27–1479 reaction. Whilst most of the organisms mentioned using Escherichia coli numbering) of isolate 1904T was above have coccoid or flat irregular cells, repre- determined. It was found to have a high GjC content sentatives of the genus Thermoproteus have rod-shaped (67n3 mol%), like the 16S rRNA genes of other cells (Zillig et al., 1981). In this genus, it is only the type thermophilic prokaryotic organisms. In an initial species, Thermoproteus tenax, that is moderately acido- T analysis, the 16S rDNA sequence of strain 1904 was philic, growing at pH values in the range 2n5–6n0 (with

International Journal of Systematic and Evolutionary Microbiology 50 2005 M. I. Prokofeva and others

Table 3. Characteristics of thermoacidophilic archaea ...... Abbreviations: R, respiration; F, fermentation; Org, organic substrates.

Genus/type species Topt. (mC) pHopt. Type of Electron Electron Reference metabolism donors acceptors

! Sulfolobus\S. acidocaldarius 70–75 2–3 R H#, org, S , O# Brock et al. (1972) # # Fe V,S%O'V ! Thermoproteus\T. tenax 90 5 R H#,org S Zillig et al. (1981) ! # # Sulfurococcus\S. mirabilis 70–75 2–3 R Org, S ,FeV,S%O'V O# Golovacheva et al. (1985) ! # ! Acidianus\A. infernus 90 2 R S ,FeV,H# S ,O# Segerer et al. (1986) Thermoplasma\T. acidophilum 59 1–2 F\R Org O# Segerer et al. (1988) ! # Metallosphaera\M. sedula 75 1–4n5R H#, org, S ,S%O'V O# Huber et al. (1989) ! Stigiolobus\S. azoricus 80 2n5–3 R H# S Segerer et al. (1991) Picrophilus\P. oshimae 60 0n7 R Org O# Schleper et al. (1995) ! ! Sulfurisphaera\S. ohwakuensis 84 2n0R H#, org, S S ,O# Kurosawa et al. (1998) ! # # Thermocladium\T. modestius 75 4n0 R Org S ,SO%V,S#O$V Itoh et al. (1998) -cystine ! Isolate 1904T 85 3n8 F(R?) Org S This work

an optimum at pH 5n0). Other species, as well as come from surrounding areas with abundant vege- representatives of the genus Pyrobaculum (Huber et tation. Isolate 1904T, described in this work, is a true al., 1987), grow optimally at pH 6n0–7n0. Members of hyperthermophile, having a temperature optimum for these two genera also have a respiratory metabolism growth of 85 mC, and is a true acidophile, having a pH T and are capable of lithotrophic or organotrophic optimum for growth of 3n8. Isolate 1904 produced growth via sulfur respiration (Scho$ nheit & Scha$ fer, significant growth on organic substrates in the absence 1995) or, in the case of Pyrobaculum aerophilum (Vo$ lkl of elemental sulfur; acetate was the main metabolic et al., 1993), via aerobic respiration or nitrate re- product. The metabolism of this isolate, therefore, duction. The fermentative growth capacity was could be characterized as fermentative. The growth- observed only with Thermoproteus uzoniensis (Bonch- stimulating action of sulfur could be explained by the Osmolovskaya et al., 1991), which is an obligate need for an additional electron sink for the ferm- anaerobe and neutrophile. This type of metabolism is entation of some of the yeast-extract components. This quite common among representatives of the neutro- phenomenon was shown for several organotrophic, philic, hyperthermophilic archaea (Scho$ nheit & hyperthermophilic archaea (Fiala & Stetter, 1986; Scha$ fer, 1995). In the Euryarchaeota kingdom, they Bonch-Osmolovskaya & Miroshnichenko, 1994). are represented by the genera Thermococcus (Zillig However, the possibility of respiratory metabolism in et al., 1983) and Pyrococcus (Fiala & Stetter, 1986). In this organism cannot be excluded until additional the Crenarchaeota, obligately anaerobic fermentative experiments have been performed. Three other strains, micro-organisms belong to the genera Desulfurococcus isolated from different terrestrial thermal habitats of (Zillig et al., 1982), Staphylothermus (Fiala et al., low pH, phenotypically resemble isolate 1904T and 1986), Hyperthermus (Zillig et al., 1990), Thermo- have similar GjC DNA contents. All of the isolates sphaera (Huber et al., 1998), Sulphophobococcus probably represent the same species. (Hensel et al., 1997) and Pyrodictium (Pley et al., 1991). Comparison of an almost complete 16S rDNA se- All these organisms are capable of fermenting peptides T and\or polysaccharides and produce volatile fatty quence from isolate 1904 with nucleotide sequences acids, hydrogen and CO#. Stetteria hydrogenophila of reference micro-organisms showed that it does not belong to any validly described Crenarchaeota genera. (Jochimsen et al., 1997) requires molecular hydrogen T and elemental sulfur for its organotrophic growth and Isolate 1904 exhibited a high degree of 16S rDNA thus was considered to possess a respiratory type of sequence similarity (98n3%) and shared signature metabolism. All these organisms are extreme thermo- nucleotides only with crenarchaeotal strain NC12 (‘C. philes or hyperthermophiles, and neutrophiles. noboribetus’; Aoshima et al., 1996). The description of this organism, however, has never been published and Our findings show that obligately anaerobic organisms strain NC12 was not available for the comparison. Of with fermentative metabolism are widespread in the representatives of the validated Crenarchaeota Kamchatka hot springs with low-pH water. Organic taxa, the closest to our isolate was A. pernix (90n8% substrates for their growth might be synthesized by similarity). This organism is a neutrophile and an lithotrophic components of the same microbial comm- aerobe (Sako et al., 1996). On the basis of the unity (Bonch-Osmolovskaya et al., 1999) or might differentiating phenotypic and genomic features of

2006 International Journal of Systematic and Evolutionary Microbiology 50 Acidilobus aceticus gen. nov., sp. nov. isolate 1904T, we propose that this isolate should be nov. – a new thermophilic sulfur-reducing eubacterium. Arch assigned to a new genus – Acidilobus – the type species Microbiol 153, 151–155. being Acidilobus aceticus. Bonch-Osmolovskaya, E. A., Miroshnichenko, M. L., Kostrikina, N. A., Chernyh, N. A. & Zavarzin, G. A. (1991). Thermoproteus uzoniensis sp. nov., a new extremely thermophilic archae- Description of Acidilobus gen. nov. bacterium from Kamchatka continental hot springs. Arch Acidilobus (A.ci.di.lohbus. L. masc. adj. acidus acid; Microbiol 154, 556–559. Gr. masc. n. lobos lobe; M.L. masc. n. Acidilobus acid Bonch-Osmolovskaya, E. A. & Miroshnichenko, M. L. (1994). The lobe). influence of molecular hydrogen and elemental sulfur on the metabolism of extremely thermophilic archaea of genus Thermo- Cells are regular to irregular cocci. The cell envelope coccus. Microbiology (English translation of Mikrobiologiya) consists of an S-layer attached to the cytoplasmic 63, 777–782. membrane. Archaeon. Hyperthermophile. Acidophile. Bonch-Osmolovskaya, E. A., Miroshnichenko, M. L., Slobodkin, Obligate anaerobe. Organotroph. Peptides and poly- A. I. & 7 other authors (1999). Biodiversity of anaerobic saccharides serve as energy and carbon sources. lithotrophic prokaryotes in terrestrial hot springs of Kam- Acetate is the major growth product. Elemental sulfur chatka. Microbiology (English translation of Mikrobiologiya) stimulates growth and is reduced to hydrogen sulfide. 68, 398–406. Resistant to antibiotics. The type species is Acidilobus Brock, T. D. (1978). Thermophilic Microorganisms and Life at aceticus. The habitat is terrestrial acidic hot springs. High Temperatures. New York: Springer. Brock, T. D., Brock, K. M., Belly, R. T. & Wiess, R. L. (1972). Description of Acidilobus aceticus sp. nov. Sulfolobus: a novel genus of sulfur-oxidizing bacteria living at low pH and high temperature. Arch Microbiol 84, 54–68. Acidilobus aceticus (a.cehti.cus. L. masc. adj. aceticus Corpet, F. (1988). Multiple sequence alignment with hierarchical producing acetate). clustering. Nucleic Acids Res 16, 10881–10890. Cells are non-motile, regular to irregular cocci approxi- Dirmeier, R., Keller, M., Hafenbradl, D., Braun, F.-J., Rachel, R., mately 1–2 µm in diameter. The cell envelope consists Burggraf, S. & Stetter, K. O. (1998). Thermococcus acidamino- of an S-layer attached to the cytoplasmic membrane. vorans sp. nov., a new hyperthermophilic alkalophilic archaeon growing on amino acids. Extremophiles 2, 109–114. Growth occurs in the temperature range 60–92 mC (optimum at 85 mC) and in the pH range 2n0–6n0 Fiala, G. & Stetter, K. O. (1986). Pyrococcus furiosus sp. nov. (optimum at 3n8). Strictly anaerobic. Heterotrophic. represents a novel genus of marine heterotrophic archaebacteria Yeast extract, beef extract, soya extract and starch growing optimally at 100 mC. Arch Microbiol 45, 56–61. may serve as growth substrates. Elemental sulfur Fiala, G., Stetter, K. O., Jannasch, H. W., Langworthy, T. H. & stimulates growth on yeast extract but is not obligately Madon, J. (1986). Staphylothermus marinus sp. nov. represents a required. Growth products are acetate and, in the novel genus of thermophilic submarine organotrophic archae- ! bacteria growing up to 98 mC. Syst Appl Microbiol 8, 106–113. presence of S ,H#S. Resistant to penicillin, streptomycin and chloramphenicol. The source of isolation Golovacheva, R. S., Val’ekho-Roman, K. M. & Troitskii, A. V. was a solfataric field near the Moutnovski volcano, (1985). Sulfurococcus mirabilis gen. nov., sp. nov., a new thermophilic archaebacterium with the ability to oxidize sulfur. Kamchatka. The GjC content of the DNA is T Microbiology (English translation of Mikrobiologiya) 56, 53n8 mol%. The type strain is Acidilobus aceticus 1904 100–107. ( DSM 11585T). l Hensel, R., Matussek, K., Michalke, K., Tacke, L., Tindall, B. J., Kohlhoff, M., Siebers, B. & Dielenschneider, J. (1997). Sulpho- ACKNOWLEDGEMENTS phobococcus zilligii gen. nov., sp. nov., a novel hyperthermophilic archaeum isolated from hot alkaline spring of Iceland. This work was supported by the Russian Foundation for Syst Appl Microbiol 20, 102–110. Basic Research, grants no. 96-04-49463 and 99-04-48360 and by the ‘Biodiversity Programme’ of the Russian Huber, R., Kristiansson, J. K. & Stetter, K. O. (1987). Pyrobaculum Ministry of Science and Technology. We also thank T. 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