International Journal of Systematic and Evolutionary Microbiology (2000), 50, 997–1006 Printed in Great Britain

Ferroplasma acidiphilum gen. nov., sp. nov., an acidophilic, autotrophic, ferrous-iron-oxidizing, cell-wall-lacking, mesophilic member of the fam. nov., comprising a distinct lineage of the

Olga V. Golyshina,1,2 Tatiana A. Pivovarova,2 Grigory I. Karavaiko,2 Tamara F. Kondrat’eva,2 Edward R. B. Moore,1 Wolf-Rainer Abraham,1 Heinrich Lu$ nsdorf,1 Kenneth N. Timmis,1 Michail M. Yakimov1 and Peter N. Golyshin1

Author for correspondence: Peter N. Golyshin. Tel: j49 531 6181498. Fax: j49 531 6181411. e-mail: pgo!GBF.de

1 Division of Microbiology, An isolate of an acidophilic archaeon, strain YT, was obtained from a GBF National Research bioleaching pilot plant. The organism oxidizes ferrous iron as the sole energy Centre for Biotechnology, Mascheroder Weg 1, source and fixes inorganic carbon as the sole carbon source. The optimal pH for 38124 Braunschweig, growth is 17, although growth is observed in the range pH 13to22. The cells Germany are pleomorphic and without a cell wall. 16S rRNA gene sequence analysis 2 Institute of Microbiology, showed this strain to cluster phylogenetically within the order Russian Academy of ‘Thermoplasmales ’ sensu Woese, although with only 899 and 872% sequence Sciences, Prosp. 60-letiya Oktyabrya, Moscow, Russia identity, respectively, to its closest relatives, oshimae and Thermoplasma acidophilum. Other principal differences from described of the ‘Thermoplasmales ’ are autotrophy (strain YT is obligately autotrophic), the absence of lipid components typical of the ‘Thermoplasmales ’ (no detectable tetraethers) and a lower temperature range for growth (growth of strain YT occurs between 15 and 45 SC). None of the sugars, amino acids, organic acids or other organic compounds tested was utilized as a carbon source. On the basis of the information described above, the name acidiphilum gen. nov., sp. nov. is proposed for strain YT within a new family, the Ferroplasmaceae fam. nov. Strain YT is the type and only strain of F. acidiphilum. This is the first report of an autotrophic, ferrous-iron- oxidizing, cell-wall-lacking archaeon.

Keywords: Archaea,‘Thermoplasmales’, acidophilic, chemolithoautotrophic, ferrous- iron-oxidizing

INTRODUCTION proposed (Woese, 1987; Woese et al., 1990; Segerer & Stetter, 1992b), but has not been validly published, and Acidophilic aerobic or facultatively anaerobic Archaea the order Sulfolobales (Segerer & Stetter, 1992a). These that colonize biotopes such as ores, solfatara groups of acidophiles differ with respect to their fields etc., where sulfur and iron are typically in phenotypic properties; first of all, with respect to the reduced forms, generally represent two different phylo- carbon and energy sources utilized. Some repre- genetic groups of the Archaea, the order ‘Thermo- sentatives of the Sulfolobales, e.g. Acidianus brierleyi plasmatales’or‘Thermoplasmales’, which has been (Segerer & Stetter, 1992a), members of the

...... Metallosphaera (Fuchs et al., 1995; Huber et al., 1989) Abbreviations: CID, collision-induced dissociation; FAB, fast-atom bom- and Sulfolobus hakonensis (Takayanagi et al., 1996), bardment; MS, mass spectrometry. obtain energy by oxidizing sulfur, sulfide minerals and The EMBL accession number for the 16S rRNA gene sequence of Ferro- ferrous iron. Other species of the genus Sulfolobus, e.g. plasma acidiphilum strain YT is AJ224936. Sulfolobus acidocaldarius (Brock et al., 1972) and

01229 # 2000 IUMS 997 O. V. Golyshina and others

Sulfolobus solfataricus (Brierley & Brierley, 1973), Anaerobic growth was assayed in closed vessels with or utilize sulfur and reduced sulfur compounds. Sul- without FeSO% in the presence of acetic acid (0n2%). The folobus metallicus (Huber & Stetter, 1991) exploits atmosphere consisted of 180 kPa CO# with or without the sulfidic ores, such as pyrite, sphalerite and chalco- addition of 40 kPa H#. Growth was monitored by the determination of the protein content of the culture using the pyrite, and elemental sulfur as energy sources. Al- # $ Bio-Rad protein assay. The concentrations of Fe + and Fe + though other members of the Sulfolobales are able to were determined by trilonometric titration (Reznikov et al., grow chemolithoautotrophically, S. metallicus (Huber 1970). Elemental sulfur and minerals containing reduced & Stetter, 1991) and Acidianus ambivalens (Fuchs et sulfur, Fe#S, ZnS, PbS and Sb#S$, were sterilized by al., 1996) are the only obligately chemolithoauto- autoclaving and added to the medium. trophic species known. In contrast, species of both Antibiotic-sensitivity analysis. The sensitivity of strain YT to genera of the order ‘Thermoplasmales’ described to antibiotics was determined by their addition in controlled date, Thermoplasma (Darland et al., 1970; Segerer et concentrations into cultures that had been pre-grown for al., 1988; Segerer & Stetter, 1992b) and Picrophilus one generation in the medium outlined above. (Schleper et al., 1995, 1996), are heterotrophic archaea that probably consume the decomposition products of Growth on organic substrates. The following organic com- the primary producers in solfatara fields and coal pounds were tested as possible substrates at concentrations of 0n1–0n2%, with or without the addition of FeSO%. Growth refuse piles, such as species of the genera Acidianus, was estimated, as described above, after incubation for 48 h. Thiobacillus and Sulfolobus. Sugars and related compounds: -arabinose, fructose, su- Here, we report the isolation, phylogenetic character- crose, -sorbitol, - and -glucose, glucose 1-phosphate, ization and phenotypic characteristics of strain YT, glucose 6-phosphate, -maltose, -xylose, -mannitol, lac- isolated from a pyrite-leaching pilot plant and repre- tose, cellobiose, -galactose, mannose, -fucose, gentio- biose, m-inositol, lactulose, -melibiose, β-methyl -gluco- senting a hitherto undescribed species of a new genus side, -psicose, raffinose, -rhamnose, -sorbitol, -trehal- that represents a new family within the order ‘Thermo- T ose, turanose, xylitol, cyclodextrin, dextrin, inosine, uridine, plasmales’. Strain Y represents the only strictly thymidine and glycogen. Organic acids and their salts: autotrophic, cell-wall-deficient archaeon described to aminobutyric acid, methyl pyruvate, monomethyl succinate, date. In recognition of its ability to oxidize ferrous iron acetic acid, cis-acetic acid, citric acid, formic acid, - and the absence of a distinct cell wall, together with the galactonic acid lactone, -galacturonic acid, -gluconic acid, acidic origin of isolation, the name Ferroplasma -glucosaminic acid, -glucuronic acid, α-hydroxybutyric acidiphilum gen. nov., sp. nov., within the family acid, β-hydroxybutyric acid, γ-hydroxybutyric acid, p-hy- Ferroplasmaceae fam. nov., is proposed, and strain YT droxyphenylacetic acid, itaconic acid, α-ketobutyric acid, α- ( DSM 12658T) is designated as the type strain. ketoglutaric acid, α-ketovaleric acid, -lactic acid, malonic l acid, propionic acid, quinic acid, -saccharic acid, sebacic acid, succinic acid, bromosuccinic acid, succinamic acid, METHODS urocanic acid and -pyroglutamic acid. Amino acids: glu- T curiamide, alaninamide, -alanine, -alanyl-glycine, -as- Isolation. Strain Y was isolated by serial dilution of the paragine, -aspartic acid, -glutamic acid, glycyl--aspartic aqueous phase of a bioreactor of a pilot plant (Tula, Russia), acid, glycyl--glutamic acid, -histidine, hydroxy--proline, which was bioleaching a gold-containing arsenopyrite\ -leucine, -ornithine, -phenylalanine, -proline, -serine, pyrite ore concentrate from Bakyrtchik (Kazakhstan), in a -serine, -threonine, -carnitine, putrescine and phenyl- modified 9K medium (see below). The temperature of the ethylamine. Alcohols: 2-aminoethanol, 2,3-butanediol, isolation source was 28–30 mC and the pH was 1n6–1n9. The glycerol, -α-glycerol phosphate, adonitol, -arabitol and purity of the culture and the absence of associated micro- i-erythritol. Others: Tweens 40 and 80, N-acetyl -galacto- organisms were controlled directly by phase-contrast mi- samine and N-acetyl -glucosamine. croscopy as well as by inoculation of heterotrophic liquid media. The purity of the culture was also estimated by (i) the Electron microscopy. Vegetative cells were fixed in 2n5% inability to obtain any bacterial PCR amplicons and (ii) the glutaraldehyde solution and absorbed to Formvar-coated homogeneity of sequences of PCR amplicons obtained by copper grids (300 square mesh) for 20–90 s, depending on using Archaea-specific oligonucleotide primers. the cell density, blotted with filter paper and air-dried. T Samples were shadowed unidirectionally with Pt\Cat15m Growth conditions. If not stated otherwise, strain Y was angle of elevation and a final thickness of 4 nm in an MED cultivated in 250 ml flasks with 100 ml modified medium " 020 evaporation unit (Baltec). Negative staining, embedding 9K (Silverman & Lundgren, 1959) containing (l− ): 04g n and ultrathin sectioning were done according to methods MgSO .7H O, 0 2 g (NH ) SO ,01 g KCl, 0 1g KHPO % # n % # % n n # % described previously (Yakimov et al., 1998). and 25 g FeSO%.7H#O. The medium was supplemented with 0n02% yeast extract (Difco) and trace elements, as described Incorporation of labelled CO2. Cells that had been pre-grown previously (Segerer & Stetter, 1992a). The pH of the medium for 3 d in modified medium 9K under the standard con- was adjusted to 1n7 by adding 10% (v\v) H#SO% and ditions described above were collected from 500 ml culture, measured with an InLab 416 electrode (Mettler Toledo). washed and resuspended in 1 ml of the same medium. Different dilutions of HCl served as references for low pH Aliquots of 0n25 ml were distributed into 2 ml micro- values. Strain YT was cultivated on a rotary shaker centrifuge tubes. Sterile, 0 3 ml glass conical inserts (glass n "% (150 r.p.m.) at 35 C. The 1000 vitamin stock solution inlets for HPLC; Supelco), containing 5 µlNa CO " m i & # $ contained (l− ): 100 mg biotin, 350 mg nicotinic acid amide, (Amersham), corresponding to 3 7 10 Bq (spec. act. 2 11 " n i n 300 mg thiamin\HCl, 200 mg p-aminobenzoic acid, 100 mg GBq mmol− ), at the bottom, were placed into these pyridoxal hydrochloride, 100 mg calcium pantothenate and microcentrifuge tubes. A 5 µl droplet of 20% (v\v) H#SO% 50 mg vitamin B"#. was then placed on the wall of the glass inserts, close to the

998 International Journal of Systematic and Evolutionary Microbiology 50 fam. nov., gen. nov., sp. nov. top. The microfuge tube was closed and sealed with Parafilm. 50 ml cells from a late-exponential phase culture by using the A short run in the microcentrifuge was used to mix the CTAB miniprep protocol for bacterial genomic DNA "% droplets of Na# CO$ and H#SO% and to generate labelled preparations (Wilson, 1987). 16S rRNA genes were am- CO#, without direct contact between the cells and the sodium plified by PCR using the forward primer 16FpgA (5h- carbonate. Incubation was performed in an Eppendorf TCCGGTTGATCCTGCCGG-3h) and the reverse primer thermomixer at 35 mC. Samples of the cell suspension were 16RpgA (5h-TACGGYTACCTTGTTACGACTT-3h), cor- taken after 90 min, 3 h and 16 h incubation. Total radio- responding to positions 3–20 of the 16S rRNA of Haloferax activity incorporated into washed, hot-trichloroacetic-acid- volcanii (Gupta et al., 1983) and positions 1492–1513 of the precipitable material was determined using scintillation 16S rRNA gene of Escherichia coli (Brosius et al., 1981), cocktail and an LS 6500 scintillation counter (Beckman) respectively. Direct sequencing of the PCR-amplified DNA (Amaro et al., 1991). was carried out using an automated DNA sequencer and Polar lipid fatty acid analysis. Lipids were extracted using a Taq cycle-sequencing reactions, according to the protocols modified Bligh–Dyer procedure, as described previously of the manufacturer (Perkin-Elmer Applied Biosystems). (Bligh & Dyer, 1959; Vancanneyt et al., 1996). All solvents Sequence data were compared initially with 16S rRNA gene were freshly distilled and all glassware used was rinsed sequences using the electronic mail servers at the Ribosomal Database Project (RDP; Maidak et al., 1999) and  with dichloromethane. Wet cells (0n2 g) were suspended in 100 ml methanol\dichloromethane\phosphate buffer version 3.Ot71 (Pearson & Lipman, 1988) to search DNA sequence databases. Evolutionary distances and phylogen- (52n6:26n3:21n1), sonicated for 15 min (Labsonic U; Braun) and incubated overnight at room temperature. Additional etic relationships were estimated using the programmes of the Phylogeny Inference Package ( version 3.57c) and methanol\ dichloromethane\phosphate buffer (35n4:61:57) was added, followed by an additional 5 min ultrasonic dendrograms were derived using the additive tree model of treatment. The samples were centrifuged at 5860 g for 15 min the  program (Fitch–Margolish and least-sequences to separate the phases. The dichloromethane phase was distance methods) with random order input of sequence and filtered through dry sodium sulfate and a hydrophobic filter. the global rearrangement option (Felsenstein, 1989). The methanol\phosphate buffer phase was reextracted by the addition of 25 ml dichloromethane, followed by cen- RESULTS trifugation and filtration. This total lipid fraction was used for analysis by mass spectrometry. The total lipid fraction Morphology was reduced in volume by using a rotary evaporator and Vegetative cells of strain YT appeared irregular and further fractionated by column chromatography (B&J inert SPE, Silica; Burdick & Jackson). The column was con- pleomorphic by transmission electron microscopy ditioned by overnight heating at 100 mC and, after cooling to (Fig. 1). The irregular morphology of shadowed (Fig. room temperature, with 10 ml dichloromethane. The lipids 1d) and negatively stained (Fig. 1e, f) cells was ob- were fractionated by sequential elution with dichloro- served to resemble that of cells of Mycoplasma. The methane, acetone and methanol, which resulted in three cells ranged from 1n0to3n0 µm in length and from 0n3 fractions of different polarity: neutral, glyco- and phos- to 1n0 µm in width. The cytoplasm appeared homo- pholipids. The eluates were collected and dried under geneous and the chromosome was visualized as nitrogen. electron-translucent aggregates (Fig. 1b; asterisks). Fast-atom-bombardment mass spectrometry (FAB-MS). The cells did not exhibit a distinct cell wall, possessing FAB-MS was performed, in the negative mode with a only a cytoplasmic membrane as the peripheral barrier, mixture of triethanolamine and tetramethylurea (2:1, v\v) 4n1–6n8 nm thick, covered with a thin layer of amorph- as matrix, on the first of two mass spectrometers of a tandem ous, electron-dense material (Fig. 1b, c). A charac- high-resolution instrument of E1B1E2B2 configuration teristic feature of strain YT is the ability to form (JMS-HX\HX110A; JEOL) at 10 kV accelerating voltage. budding processes, which appeared tubular or ves- Resolution was set to 1:2000. The JEOL FAB gun was operated at 6 kV with xenon. icular in shape (Fig. 1a, d, f; filled arrows) and which tended to form septation annuli (Fig. 1e; open arrow- Tandem mass spectrometry. Negative daughter-ion spectra heads). The tubular extrusions were observed to range were recorded using all four sectors of the tandem mass from 85 to 142 nm in diameter and up to 1 µmin spectrometer. High-energy collision-induced dissociation length. The process of budding, or formation of (CID) took place in the third field free region. Helium served as the collision gas at a pressure sufficient to reduce the extrusions, is seen in Fig. 1(b), where the cell forms a precursor ion signal to 30% of the original value. The tip (open double arrows) as the initiation and a vesicle, collision cell was operated at ground potential in the negative which nearly terminates its budding (filled double mode. Resolution of MS2 was set to 1:1000. FAB-CID arrows). Different forms of ‘offspring’ are thereby spectra (linked scans of MS2 at constant B\E ratio) were released (Fig. 1d–f; indicated by an open arrow in recorded at 300 Hz filtering with a JEOL DA 7000 data Fig. 1d). system. DNA GjC content. The GjC content of genomic DNA T Relation to temperature and pH isolated from strain Y was determined directly by HPLC T with a Nucleosil 100-5 C-18 column (Macherey–Nagel), At the optimal pH for growth of 1n7, strain Y grew according to methods described previously (Mesbah et al., within a temperature range of 15–45 mC, having an 1989; Tamaoka & Komagata, 1984). Purified, non-meth- optimum at 35 mC (Fig. 2b). At the optimal tem- ylated lambda phage DNA (Sigma) was used as a control. perature, growth occurred within the pH range 1n3–2n2 16S rRNA gene sequence determination and analysis of (Fig. 2c). Cells were observed by phase-contrast phylogenetic relationships. Total DNA was isolated from microscopy to be osmotic and pH-sensitive.

International Journal of Systematic and Evolutionary Microbiology 50 999 O. V. Golyshina and others

(a) (b)

(c)

(d) (e) (f)

...... Fig. 1. Ultrastructure of vegetative cells. (a) Longitudinal ultrathin section showing a homogeneous cytoplasmic matrix; budding processes are indicated by arrows. (b) Detailed views of budding process as initial tip-formation (open double arrows) and almost-complete separation of vesicular offspring (filled double arrows); asterisks indicate the condensed bacterial chromosome. (c) High-magnification view of the cytoplasmic membrane, as indicated by opposing arrows. (d) Pt/C-shadow-cast bacterial cells. Open arrow indicates a tubular cellular offspring and filled arrows point to tubular extrusions; arrowhead gives shadowing direction. (e) Cell with bipolar budding processes; arrowheads indicate the septa. (f) Cellular extrusions of different sizes. Bars: 500 nm (a, d, e, f); 100 nm (b, c).

1000 International Journal of Systematic and Evolutionary Microbiology 50 Ferroplasma acidiphilum fam. nov., gen. nov., sp. nov.

100 60 (a) (b) 80

) 50 ) 80 –1 –1 60 40 60 30 40 40

20 oxidized (mM)

20 20 2+ Protein (mg l Protein (mg l

10 Fe 0 0 20406080100120 0 102030405060 Time (h) Temperature (°C)

70 70 60 (c) (d) ) 60 –1 50

40 ) 50 30 –1 40 20 Protein (mg l 10 30 0 0·5 1 1·5 2 2·5 3 Protein (mg l 20 pH Vitamins 10

0 0 0·002 0·02 0·2 0·4 Yeast extract (%)

...... T Fig. 2. Growth of Ferroplasma acidiphilum strain Y on modified 9K medium supplemented with FeSO4. (a) Growth curve of strain YT growing under conditions of optimal pH (1n7) and temperature (35 mC). (b) Growth of isolate YT at various − + temperatures, pH 1n7. $, Protein (mg l 1); , amount of Fe2 oxidized (mM); after 96 h growth. (c) Influence of pH on the growth of the isolate at 35 mC. (d) Effect of yeast extract and vitamin addition on biomass yield of a culture of strain + YT at pH 1n7 and 35 mC. Protein levels (b–d) and Fe2 concentrations (b) were measured after 96 h growth.

# Oxidation of inorganic substrates yeast extract alone in the absence of Fe +. A vitamin solution could be substituted for yeast extract, al- The growth curve of the isolate in the modified medium though the specific biomass yield after 96 h growth 9K at optimal pH and temperature is shown in Fig. " # decreased from 12 to 5 5 mg protein g− Fe +. 2(a). Growth of strain YT could be detected after n 24–35 h, when the ferrous iron started to be oxidized and the medium turned yellow. After 50–55 h cul- Fixation of inorganic carbon tivation, the medium became red and, after 80–90 h, Strain YT was observed to incorporate inorganic #+ "% reddish-brown, due to oxidation of Fe . Typically, carbon, added to the culture as CO . After 90 min, T "% # " strain Y oxidized ferrous FeSO% and pyrite (Fe#S). the radioactivity of incorporated C was 535 Bq mg− Sulfide minerals, such as sphalerite, galenite and "% T protein. Exposure of the biomass to CO# for longer antimonite, were not oxidized. Strain Y was capable times, 3 and 16 h, did not increase the incorporated " of growing on MnSO%, although it did not yield as radioactivity significantly (700 and 725 Bq mg− pro- much biomass as when grown in medium supple- #+ −" #+ tein, respectively). One possible explanation for this is mented with Fe (6n5 mg protein g Mn and 12 mg −" #+ the limitation of growth due to a lack of ferrous iron, protein g Fe ). Neither elemental sulfur in crys- which, after a short period of growth, possibly within talline or colloid form nor reduced compounds of a few minutes, was almost completely depleted by the sulfur, tetrathionate and thiosulfate, were oxidized. very high concentration of cells (residual concen- #+ trations of Fe were 23, 5 and !0n1 mM after 90 min, Growth on organic substrates 3 h and 16 h exposure). T Strain Y was not capable of growth on any of the Oxygen requirement organic substrates listed in Methods, although the addition of yeast extract was observed to be essential Strain YT was observed to grow strictly aerobically. for growth (Fig. 2d). Growth of strain YT was strongly No measurable growth occurred under anaerobic inhibited by the presence of yeast extract in amounts conditions in the presence of H#\CO#,CO# alone or #+ greater than 0n2% and growth was not detected on with formate or acetate, with or without Fe .

International Journal of Systematic and Evolutionary Microbiology 50 1001 O. V. Golyshina and others

Molecular phylogenetic analysis 805 T 100 The 16S rRNA gene sequence of strain Y was observed to cluster with those of organisms repre- senting the order ‘Thermoplasmales’ within the Eury- O archaeota (Fig. 4). Strain YT exhibited a relatively deep O branching, with uncertainty concerning its closest 80 OH O OH phylogenetic affiliation, although, according to the P O T O O– method of analysis used, strain Y was estimated to be phylogenetically most closely related to species of the genera Picrophilus and Thermoplasma, albeit with only 60 89n9 and 87n7% 16S rRNA sequence identity, re- spectively, to Picrophilus oshimae and Thermoplasma O acidophilum. O O 40 OH P O O– DISCUSSION

Relative abundance (%) The order ‘’or‘Thermoplasmales’ 199 sensu Woese (Woese, 1987; Woese et al., 1990; Segerer & Stetter, 1992b; Schleper et al., 1996) is represented 20 by the facultatively anaerobic genus Thermoplasma 255 401 731 (Darland et al., 1970; Segerer et al., 1988; Segerer & 507 953 649 924 1024 1446 1608 Stetter, 1992b) and the strictly aerobic genus Picro- 1064 1373 1635 philus (Schleper et al., 1995, 1996), the species of which are thermoacidophilic, heterotrophic organisms. Phy- 500 1000 1500 2000 logenetically, these organisms are clustered within one m/z of the two main branches of the domain Archaea, the ...... , which also includes methanogenic and Fig. 3. (k)-FAB mass spectrum of the total lipid fraction of halophilic archaea (Woese et al., 1990). On the basis of Ferroplasma acidiphilum strain YT. Archaetidyl glycerol gave the 16S rRNA sequence comparisons, strain YT, isolated main ion at m/z 805, while the molecular ion of archaetidic acid is found at m/z 731. The ions of low intensity at m/z 1446 and from gold-containing pyrite ore concentrate, occupies 1608 were formed from the dimers of archaetidic acid and a distinct position between the genera Picrophilus and archaetidyl glycerol, respectively. The ion at m/z 507 is the Thermoplasma. result of the neutral loss of phytanol from archaetidyl glycerol. Other characteristic features of the ‘Thermoplasmales’ are the lack of a distinct cell wall in representatives of the genus Thermoplasma and the presence of an S-layer Antibiotic sensitivity in species of Picrophilus and a specific morphology: " Strain YT was resistant to ampicillin (50 µgml− ), cells of species of Thermoplasma have variously sized, which inhibits cell wall formation in bacteria, as well filamentous, coccoid-, disc- and club-shaped forms, " as to chloramphenicol ( 10 µgml− ), kanamycin that can be observed in the same culture, whereas cells " " " (50 µgml− ), rifampicin (25 µgml− ) and streptomycin of the Picrophilaceae are irregular cocci (Segerer & " (50 µgml− ). Strain YT was sensitive to tetracycline Stetter, 1992b; Schleper et al., 1995, 1996). The lack of " " (2 µgml− ) and gentamicin (2 µgml− ). a cell wall also confers a high osmotic and pH sensitivity upon strain YT, as well as insensitivity to ampicillin, as is the case for the ‘Thermoplasmales’. Analysis of cellular lipids The principal phenotypic characteristics of isolate YT, The phospholipids were purified by column chroma- apart from its acidic origin, lack of a cell wall and low tography or were determined in the extract of total GjC content, do not agree, however, with those of lipids. The structures of the individual phospholipids Thermoplas T other species belonging to the order ‘ - present in strain Y were identified by MS. The main males’ (Table 1). The most important difference from phospholipid was observed to be archaetidyl glycerol, other members of the ‘Thermoplasmales’ is the ob- although some archaetidic acid could also be detected ligate autotrophy of strain YT, which is the only (Fig. 3). Dimers of both ether lipids were observed in organism of this phylogenetic branch reported to date small amounts. No tetraethers were detected in the T T to fix inorganic carbon. In that strain Y assimilates total lipid extract of strain Y . CO and obtains energy at the expense of the oxidation # # # of Fe + and Mn +, it is metabolically similar to some representatives of the order Sulfolobales, e.g. S. metal- DNA GjC content licus and A. ambivalens, the only strict chemolitho- The GjC content of the genomic DNA was de- autotrophs of the order, as well as to A. brierleyi, termined to be 36n5mol%. and Metallosphaera prunae,

1002 International Journal of Systematic and Evolutionary Microbiology 50 Ferroplasma acidiphilum fam. nov., gen. nov., sp. nov.

Archaea (‘Thermoplasmales’) Bacteria Ferrroplasma acidiphilum strain YT

[Euryarchaeota] Thermoplasma acidophilum Picrophilus oshimae Escherichia coli

Halobacterium halobium

Methanobacterium formicicum

Archaeoglobus fulgidus Methanomicrobium mobile

Thermococcus celer

Pyrodictium occultum 0·1 Desulfurococcus mobilis Thermoproteus tenax

Sulfolobus acidocaldarius

[Crenarchaeota]

...... Fig. 4. The estimated phylogenetic position of Ferroplasma acidiphilum strain YT (l DSM 12658T), derived from 16S rRNA gene sequence comparisons, among the major evolutionary lineages of the Archaea. The sequence data for other organisms were obtained from the GenBank/EMBL databases under the following accession numbers: Archaeoglobus fulgidus, X05567; Desulfurococcus mobilis, M36474; Halobacterium halobium, AJ002949; Methanobacterium formicicum, M36508; Methanomicrobium mobile, M59142; Picrophilus oshimae, X84901; Pyrodictium occultum, M21087; Sulfolobus acidocaldarius, D14053; Thermococcus celer, M21529; Thermoplasma acidophilum, M32298; Thermoproteus tenax, M35966; Escherichia coli, J01695. The scale bar represents 10 substitutions per 100 nucleotide positions.

Table 1. Comparison of key characteristics of the archaea belonging to the order ‘Thermoplasmales’ ...... Data were taken from Schleper et al. (1995) (Picrophilus) and Darland et al. (1970), Segerer et al. (1988) and Segerer & Stetter (1992b) (Thermoplasma). j, Positive reaction or growth; k, negative reaction or growth.

Characteristic Picrophilus spp. Thermoplasma spp. Ferroplasma acidiphilum

Morphology Irregular cocci Pleomorphic Pleomorphic Flagella jjk Autotrophy kkj # Fe + oxidation kkj Aerobic growth jjj Anaerobic growth kjk Temperature for growth (mC): Optimum 60 60 35 Range 45–65 33–67 15–45 pH for growth: Optimum 0n7 1–2 1n7 Range 0n1–3n5 1–4 1n3–2n2 S-layer jkk DNA GjC content (mol%) 36 46 36n5

International Journal of Systematic and Evolutionary Microbiology 50 1003 O. V. Golyshina and others which, however, are also able to use a number of ferrooxidans, Thiobacillus thiooxidans,‘Leptospirillum organic compounds as sole carbon and energy sources ferrooxidans’, heterotrophic Acidophilium species and (Segerer et al., 1986; Huber et al., 1989; Huber & heterotrophic fungi. Stetter, 1991; Fuchs et al., 1995, 1996). In contrast to the Sulfolobales, isolate YT does not use elemental It is apparent from its phenotypic properties and the sulfur or its reduced forms. differences in 16S rRNA gene sequences that the iron- oxidizing, acidophilic archaeon, strain YT, isolated T Apart from the nutritional requirements, isolate Y from an arsenopyrite ore bioleaching reactor, cannot exhibits a marked difference from the ‘Thermo- be assigned to any previously recognized genus and plasmales’ in the composition of its cellular lipids. represents a phylogenetic lineage that corresponds to a Archaetidic acid and archaetidyl glycerol, which com- new species in a new genus, within a new family, under prise the majority of the total lipids of the organism, the epithet Ferroplasma acidiphilum fam. nov., gen. are commonly detected in organisms of the halophilic nov., sp. nov. and methanogenic lineages of the Euryarchaeota branch of the Archaea. Archaetidic acid was reported from Halobacterium cutirubrum and other halophilic Description of Ferroplasmaceae Golyshina et al. fam. archaea (Fredrickson et al., 1989; Lanzotti et al., 1989) nov. and from Methanobacterium thermoautotrophicum (Nishihara & Koga, 1990). Archaetidyl glycerol Ferroplasmaceae (Fer.ro.plas.mahce.ae. M.L. ferro has been reported from species of Halobacterium pertaining to ferrous iron; Gr. neut. n. plasma some- (Kushwaha et al., 1982), Methanosarcina barkeri thing shaped or moulded; L. -aceae ending denoting a (Nishihara & Koga, 1995), Methanospirillum hungatei family; M.L. Ferroplasmaceae a family of ferrous- (Kushwaha et al., 1981) and an unidentified, extremely iron-oxidizing forms). halophilic archaeon (Upasani et al., 1994). Lipids characteristic for the genera Thermoplasma and Picro- A family belonging to the order ‘Thermoplasmales’, philus, i.e. di-isopropanol 2,3-glycotetraether and bis- separate and distinct from the ‘Thermoplasmaceae’ phytanyltetraethers (Langworthy, 1985; Schleper et and the Picrophilaceae, which contains cell-wall- and al., 1995), were not found in strain YT. S-layer-lacking, ferrous-iron-oxidizing, chemolitho- autotrophic, acidophilic organisms. The segregation A significant feature that distinguished strain YT from of these organisms into a new family is justified (i) by other members of the ‘Thermoplasmales’, with which their distinct phylogenetic position (the 16S rRNA it clusters phylogenetically, and from the Sulfolobales, sequence is nearly equally distant, i.e. "10% dif- with representatives of which it shares a number of ference, from representatives of existing families, physiological traits, is the range of temperatures at Picrophilus oshimae and Thermoplasma acidophilum), which growth is observed. Strain YT has an optimum (ii) by obligate chemolithoautotrophy, whereas other temperature for growth of 35 mC and a maximum of members of the ‘Thermoplasmales’, the ‘Thermo- 45 mC, whereas the minimum temperature for growth plasmaceae’andthePicrophilaceae,areobligatehetero- of A. brierleyi and P. oshimae is 45 mC, and other trophs that are not able to grow autotrophically, (iii) thermoacidophilic archaea of the ‘Thermoplasmales’ by their mesophilic growth temperature range and (iv) and Sulfolobales exhibit minimum temperatures for by the dominance of archaetidic acid and archaetidyl growth that are 10–15 mC higher (Segerer & Stetter, glycerol as membrane lipids and the complete absence 1992a, b; Schleper et al., 1995). Thus, the first part of of tetraether lipids, which are predominant in the the descriptive adjective thermoacidophilic is not ‘Thermoplasmaceae’ and the Picrophilaceae. applicable to isolate YT, which conforms to the ranks of mesophilic prokaryotes and is, by this criterion, similar to the autotrophic species of the bacterial Description of Ferroplasma Golyshina et al. gen. nov. genera Thiobacillus and ‘Leptospirillum’, which also oxidize ferrous iron chemolithoautotrophically Ferroplasma (Fer.ro.plashma. M.L. ferro pertaining to (Temple & Colmer, 1951; Markosyan, 1972). ferrous iron; Gr. neut. n. plasma something shaped or moulded; M.L. Ferroplasma a ferrous-iron-oxidizing A recent 16S rRNA gene sequence analysis of a total form). DNA extract from a stable microbial consortium of a copper-leaching reactor reported the detection of an Cells are irregular cocci, varying from spherical to archaeon that probably represented a novel family filamentous, forming duplex and triplex forms. Gram- within the ‘Thermoplasmales’(Va! squez et al., 1999). negative. Strict aerobes. Cell wall and S-layer The archaeon, morphologically similar to species of are absent. Acidophilic. Strictly chemolithoauto- Thermoplasma, exhibited only two nucleotide dif- trophic; no organic compounds have been found that #+ ferences from the 16S rRNA gene sequence of strain are used as carbon sources. Oxidizes Fe from FeSO% T #+ Y over 912 homologous nucleotide positions. The and pyrite (Fe#S); oxidizes Mn from MnSO%. authors, however, failed to obtain a pure culture of the Mesophilic. Principal lipids are archaetidic acid and organism, which was presumed to be dependent on archaetidyl glycerol. The type and only species of the associated autotrophic bacteria such as Thiobacillus genus is Ferroplasma acidiphilum.

1004 International Journal of Systematic and Evolutionary Microbiology 50 Ferroplasma acidiphilum fam. nov., gen. nov., sp. nov.

Description of Ferroplasma acidiphilum Golyshina et (1995). Metallosphaera prunae, sp. nov., a novel metal- al. sp. nov. mobilizing, thermoacidophilic Archaeum, isolated from a uranium mine in Germany. Syst Appl Microbiol 18, 560–566. Ferroplasma acidiphilum (a.ci.dihphi.lum. M.L. neut. n. Fuchs, T., Huber, H., Burggraf, S. & Stetter, K. O. (1996). 16S acidum an acid; Gr. adj. philos loving; M.L. neut. adj. rDNA-based phylogeny of the archaeal order Sulfolobales and acidiphilum acid-loving). reclassification of Desulfurolobus ambivalens as Acidianus ambi- Morphology and nutritional requirements are as valens comb. nov. Syst Appl Microbiol 19, 56–60. described for the genus. GjC content of DNA is Gupta, R., Lanter, J. M. & Woese, C. R. (1983). Sequence of the 36n5 mol%. Growth occurs between temperatures of 16S ribosomal RNA from Halobacterium volcanii, an archae- 20 and 45 mC with an optimum at 35 mC and at pH bacterium. Science 221, 656–659. 1n3–2n2 with an optimum at pH 1n7. Huber, G. & Stetter, K. O. (1991). Sulfolobus metallicus, sp. nov., T a novel strictly chemolithoautotrophic thermophilic archaeal Strain Y is the only and type strain of Ferroplasma species of metal-mobilizers. Syst Appl Microbiol 14, 372–378. acidiphilum. Ferroplasma acidiphilum strain YT has T Huber, G., Spinnler, C., Gambacorta, A. & Stetter, K. O. (1989). been deposited in the DSMZ as strain DSM 12658 . Metallaosphaera sedula gen. nov. and sp. nov. represents a new genus of aerobic, metal-mobilizing, thermoacidophilic archae- bacteria. Syst Appl Microbiol 12, 38–47. ACKNOWLEDGEMENTS Kushwaha, S. C., Kates, M., Sprott, G. D. & Smith, I. C. P. (1981). We gratefully acknowledge Michael Seeger and Carlos Jeres Novel polar lipids from the methanogen Methanospirillum for fruitful discussions. We thank Peter Wolff and Carsten hungatei GP1. Biochim Biophys Acta 664, 156–173. Stroempl for their excellent technical assistance in chemical Kushwaha, S. C., Perez, J. G., Rodriguez, V.-F., Kates, M. & analysis. Ruprecht Christ is thanked for his skilful work at Kushner, D. J. (1982). Survey of lipids of a new group of the tandem mass spectrometer. This work was supported by extremely halophilic bacteria from salt ponds in Spain. Can J a grant from the German Federal Ministry for Science, Microbiol 28, 1365–1372. Education and Research (project no. 0319433C). T.A.P., T.G.K. and G.I.K. acknowledge the support of the Russian Langworthy, T. A. (1985). Lipids of archaebacteria. In The Foundation for Fundamental Research (grant N 96-04- Bacteria, pp. 459–497. Edited by C. R. Woese & R. S. Wolfe. 48287) and the State Program ‘Novel Methods in Bio- Orlando, FL: Academic Press. engineering’. K.N.T. gratefully acknowledges the generous Lanzotti, V., Nicolaus, B., Trincone, A., De Rosa, M., Grant, W. & support of the Fonds der Chemischen Industrie. We wish to Gambacorta, A. (1989). An isoprenoid ether analog of phos- thank Hans Tru$ per (Universitaet Bonn) and Brian Tindall phatidic acid from a halophilic archaebacterium. Biochim (DSMZ, Braunschweig) for advice and corrections of Latin Biophys Acta 1002, 398–400. names. Maidak, B. L., Cole, J. R., Parker, C. T., Jr and 11 other authors (1999). A new version of the RDP (Ribosomal Database REFERENCES Project). Nucleic Acids Res 27, 171–173. Markosyan, G. E. (1972). Leptospirillum ferrooxidans gen. nov., Amaro, A. M., Chamorro, D., Seeger, M., Arredondo, R., Peirano, sp. nov., a new iron-oxidizing bacterium. Biol J Armenia 25, I. & Jerez, C. A. (1991). Effect of external pH perturbations on in 26–29. vivo protein synthesis by the acidophilic bacterium Thiobacillus ferrooxidans. J Bacteriol 173, 910–915. Mesbah, M., Premachandran, U. & Whitman, W. B. (1989). Precise measurement of the GjC content of deoxyribonucleic acid by Bligh, E. G. & Dyer, W. J. (1959). A rapid method for total lipid high-performance liquid chromatography. Int J Syst Bacteriol extraction and purification. Can J Biochem Physiol 37, 911–917. 39, 159–167. Brierley, C. L. & Brierley, J. A. (1973). A chemoautotrophic and Nishihara, M. & Koga, Y. (1990). Natural occurrence of archae- thermophilic microorganism isolated from an acid hot spring. tidic acid and caldarchaetidic acid (di- and tetra-ether analogs Can J Microbiol 19, 183–188. of phosphatidic acid) in the archaebacterium Methanobacterium Brock, T. D., Brock, K. M., Belly, R. T. & Weiss, R. L. (1972). thermoautotrophicum. Biochem Cell Biol 68, 91–95. Sulfolobus: a new genus of sulfur-oxidizing bacteria living at Nishihara, M. & Koga, Y. (1995). Two new phospholipids, low pH and high temperature. Arch Mikrobiol 84, 54–68. hydroxyarchaetidylglycerol and hydroxyarchaetidylethanol- Brosius, J., Dull, T. J., Sleeter, D. D. & Noller, H. F. (1981). Gene amine, from the archaea Methanosarcina barkeri. Biochim organization and primary structure of a ribosomal RNA operon Biophys Acta 1254, 155–160. from Escherichia coli. J Mol Biol 148, 107–127. Pearson, W. R. & Lipman, D. J. (1988). Improved tools for Darland, G., Brock, T. D., Samsonoff, W. & Conti, S. F. (1970). A biological sequence comparison. Proc Natl Acad Sci USA 85, thermophilic, acidophilic Mycoplasma isolated from a coal 2444–2448. refuse pile. Science 170, 1416–1418. Reznikov, A., Mulikovskaja, E. P. & Sokolov, N. U. (1970). Felsenstein, J. (1989).  – Phylogeny Inference Package Methods of Analysis of Natural Waters. Moscow: Gosgeol- (version 3.2). Cladistics 5, 164–166. techizdat (in Russian). Fredrickson, H. L., De Leeuw, J. W., Tas, A. C., Van der Greef, J., Schleper, C., Puehler, G., Holz, I., Gambacorta, A., Janekovic, D., LaVos, G. F. & Boon, J. J. (1989). Fast atom bombardment Santarius, U., Klenk, H.-P. & Zillig, W. (1995). Picrophilus gen. (tandem) mass spectrometric analysis of intact polar ether lipids nov., fam. nov.: a novel aerobic, heterotrophic, thermo- extractable from the extremely halophilic archaebacterium acidophilic genus and family comprising archaea capable of Halobacterium cutirubrum. Biomed Environ Mass Spectrom 18, growth around pH 0. J Bacteriol 177, 7050–7059. $ 96–105. Schleper, C., Puhler, G., Klenk, H.-P. & Zillig, W. (1996). Picrophilus Fuchs, T., Huber, H., Teiner, K., Burggraf, S. & Stetter, K. O. oshimae and Picrophilus torridus fam. nov., gen. nov., sp. nov.,

International Journal of Systematic and Evolutionary Microbiology 50 1005 O. V. Golyshina and others two species of hyperacidophilic, thermophilic, heterotrophic, Temple, K. L. & Colmer, A. R. (1951). The autotrophic oxidation aerobic archaea. Int J Syst Bacteriol 46, 814–816. of iron by a new bacterium Thiobacillus ferrooxidans. J Bacteriol Segerer, A. H. & Stetter, K. O. (1992a). The order Sulfolobales.In 62, 605–611. The Prokaryotes, 2nd edn, pp. 684–701. Edited by A. Balows, Upasani, V. N., Desai, S. G., Moldoveanu, N. & Kates, M. (1994). H. G. Tru$ per, M. Dvorkin, W. Harder & K.-H. Schleifer. New Lipids of extremely halophilic archaeobacteria from saline York: Springer. environments in India: a novel glycolipid in Natronobacterium strains. Microbiology 140, 1959–1966. Segerer, A. H. & Stetter, K. O. (1992b). The genus Thermoplasma. In The Prokaryotes, 2nd edn, pp. 712–718. Edited by A. Vancanneyt, M., Witt, S., Abraham, W.-R., Kersters, K. & Balows, H. G. Tru$ per, M. Dvorkin, W. Harder & K.-H. Fredrickson, H. L. (1996). Fatty acid content in whole-cell Schleifer. New York: Springer. hydrolysates and phospholipid fractions of pseudomonads: a taxonomic evaluation. Syst Appl Microbiol 19, 528–540. Segerer, A., Neuner, A., Kristjansson, J. K. & Stetter, K. O. (1986). ! Acidianus infernus gen. nov., sp. nov., and Acidianus brierleyi Vasquez, M., Moore, E. R. B. & Espejo, R. T. (1999). Detection by comb. nov.: facultatively aerobic, extremely acidophilic thermo- polymerase chain reaction-amplification and sequencing of an archaeon in a commercial-scale copper bioleaching plant. philic sulfur-metabolizing archaebacteria. Int J Syst Bacteriol FEMS Microbiol Lett 173, 183–187. 36, 559–564. Wilson, K. (1987). Preparation of genomic DNA from bacteria. Segerer, A., Langworthy, T. A. & Stetter, K. O. (1988). Thermo- In Current Protocols in Molecular Biology, pp. 2.4.1–2.4.2. plasma acidophilum Thermoplasma volcanium and sp. nov. from Edited by F. M. Ausubel, R. Brent, R. E. Kingston, D. D. solfatara fields. Syst Appl Microbiol 10, 161–171. Moore, J. G. Seidman, J. A. Smith & K. Struhl. New York: Silverman, M. P. & Lundgren, D. G. (1959). Studies on the Wiley. chemoautotrophic iron bacterium Ferrobacillus ferrooxidans.1. Woese, C. R. (1987). Bacterial evolution. Microbiol Rev 51, An improved medium and harvesting procedure for securing 221–271. high cell yields. J Bacteriol 77, 642–647. Woese, C. R., Kandler, O. & Wheelis, M. L. (1990). Towards a Takayanagi, S., Kawasaki, H., Sugimori, K., Yamada, T., Sugai, A., natural system of organisms: proposal for the domains Archaea, Ito, T., Yamasato, K. & Shioda, M. (1996). Sulfolobus hakonensis Bacteria, and Eucarya. Proc Natl Acad Sci USA 87, 4576–4579. sp. nov., a novel species of acidothermophilic archaeon. Int J Yakimov, M. M., Golyshin, P. N., Lang, S., Moore, E. R. B., Syst Bacteriol 46, 377–382. $ Abraham, W.-R., Lunsdorf, H. & Timmis, K. N. (1998). Alcanivorax Tamaoka, J. & Komagata, K. (1984). Determination of DNA base borkumensis gen. nov., sp. nov., a new, hydrocarbon-degrading composition by reversed-phase high-performance liquid and surfactant-producing marine bacterium. Int J Syst Bacteriol chromatography. FEMS Microbiol Lett 25, 125–128. 48, 339–348.

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