Stygiolobus Azoricus Gen

INTERNATIONAL JOURNALOF SYSTEMATIC BACTERIOLOGY,OCt. 1991, p. 495-501 Vol. 41, No. 4 0020-7713/91/040495-07$02.oo/o Copyright 0 1991, International Union of Microbiological Societies

Stygiolobus azoricus gen. nov., sp. nov. Represents a Novel Genus of Anaerobic, Extremely Thermoacidophilic Archaebacteria of the Order Suvolobales ANDREAS H. SEGERER,l ANTONIO TRINCONE,' MANFRED GAHRTZ,l AND KARL 0. STETTER1* Lehrstuhl fir Mikrobiologie, Universitat Regensburg, 0-8400 Regensburg, Federal Republic of Germany,' and Instituto per la Chimica di Molecole di Interesse Biologic0 de1 Consiglio Nazionale delle Ricerche, I-80072-Arc0 Felice, Naples, Italy2

On the basis of three isolates (strains FC6T [T = type strain], FC4, and RG1) of extremely thermophilic chemolithoautotrophic archaebacteria obtained from solfataric fields on Sao Miguel Island, Azores, the new genus Stygiolobus is described. These isolates grow obligately chemolithotrophically by reduction of So with H, (H,-SOlithotrophy) and are the first strictly anaerobic members of the order Sulfolobales. With a DNA G+C content of 38 mol%, the Stygiolobus isolates resemble Sulfolobus spp., which, however, are faculatively organotrophic and aerobic So oxidizers. The new isolates are also distinct from Acidianus spp., which resemble Stygiolobus by growing by H,-SO lithotrophy. However, Acidianus spp. can also grow aerobically by So oxidation and have G+C contents of 31 mol%. At this time, only one species of the genus Stygiolobus is known, Stygiolobus uzoricus sp. nov.; the type strain of S. uzoricus is strain FC6 (= DSM 6296).

The thermophilic sulfur-metabolizing archaea (archaebac- members of the Sulfolobales most likely belong to the same teria) (66; for reviews, see references 56 and 62) that occur in family, the Sulfolobaceae (29, 59). acid solfataric fields comprise the following three groups: (i) The ability to grow lithotrophically by So oxidation, ex- the order Sulfolobales (29, 59; for a review, see reference treme thermoacidophily, and the presence of unique lipid 51); (ii) the order Thermoproteales (24, 70; for a review, see compounds (e.g., glycerol-dibisphytanyl-nonitoltetraethers reference 22); and (iii) the genus Thermoplasma (9, 46, 55; and benzothiophenquinones) (10-12,38,63) are features that for reviews, see references 50 and 62), which because of its are characteristic of all members of the Sulfolobales which isolated phylogenetic position probably represents an order distinguish them phenotypically from both the genus Ther- that is distantly related to the methanogens (65, 67). The moplasma and the order Thermoproteales. Thermoplasma following four genera have been validly assigned to the order spp. are extreme acidophiles but only moderate thermo- Sulfolobales: (i) the genus Sulfolobus (7, 551, which is the philes. They are strictly heterotrophic facultatively aerobic type genus and currently includes the species Sulfolobus sulfur respirers that are incapable of So oxidation and lack a acidocaldarius (type species) (7,55), Sulfolobus solfataricus cell wall and envelope, a feature that is unique among the (23, 71), and Sulfolobus shibatae (16); (ii) the genus Metal- archaebacteria. The members of the Thermoproteales are losphaera, with one species, Metallosphaera sedula (18,29); strictly anaerobic, extremely thermophilic but almost neu- (iii) the genus Acidianus (47), which includes the species trophilic sulfur reducers which lack both the benzothiophen- Acidianus infernus (type species) and Acidianus brierleyi (5, quinone compounds and the nonitol tetraether lipids that are 47, 71); and (iv) the genus Desulfurolobus, with the single characteristic of members of the Suffolobales. In this paper, species Desulfurolobus ambivalens (26, 72). Suifolobus and we describe the genus Stygiolobus gen. nov., which com- Metallosphaera spp. are aerobic or facultatively anaerobic, prises strictly anaerobic, obligately chemolithoautotrophic facultatively organotrophic chemolithoautotrophs that are archaebacteria belonging to the order Sulfolobales. capable of oxidizing molecular sulfur, sulfide, or tetrathionate to H,SO, (6, 8, 18, 54, 68) and have relatively high DNA G+C contents (about 35 to 45 mol%). MATERIALS AND METHODS In contrast, Acidianus and Desulfurolobus spp. are facul- Bacterial strains. Sulfolobus acidocaldarius DSM 639T tatively anaerobic organisms that grow by either oxidation or (T = type strain), Sulfolobus solfataricus DSM 1616=, and reduction of elemental sulfur, forming H,SO, and H,S, A. brierleyi DSM 1651T were obtained from the Deutsche respectively (52). Relatively low G+C contents (about 31 Sammlung von Mikroorganismen, Braunschweig-Stock- mol%) are also characteristic of these organisms. The fea- heim, Federal Republic of Germany. D. ambivalens DSM tures that distinguish the genera Acidianus and Desulfurolo- 3772T and Sulfolobus shibatae DSM 5389T were kindly bus are not well defined. In fact, D. ambivalens closely provided by W. Zillig. M. sedula DSM 5348T, A. brierleyi resembles A. infernus in both phenotype and genotype (20, SP3dl (= DSM 6334) (53), A. infernus DSM 3191T, Pyro- 47, 72). A level of DNA homology of about 60% with A. baculum islandicum DSM 4184T (21, 27), Pyrodictium oc- infernus (20; this paper) indicates (45) that D. ambivalens is cultum DSM 2709T (25,60), and strains FC6T, FC4, and RG1 another species of the genus Acidianus. were isolates from our laboratory. As inferred from immunochemical features of their DNA- Culture conditions. All organisms except Pyrodictium oc- dependent RNA polymerases, all of the currently known cultum were grown in Allen medium (1); Pyrodictium occultum was grown in 0.5~SME medium (60). Sulfolobus spp., M. sedula, and A. brierleyi were grown aerobically at 75°C and pH 2.5 in the presence of 1 g of yeast extract (Difco * Corresponding author Laboratories, Detroit, Mich.) per liter. All other strains were

495 496 SEGERER ET AL. INT. J. SYST.BACTERIOL. routinely grown anaerobically in the presence of 0.01% TABLE 1. Distribution and origin of the new isolates (wthol) yeast extract under an H,-CO, atmosphere (80:20, Positive samplesa vol/vol; 300 kPa). Isolates FC6T, FC4, and RG1 were grown No. of Source In situ In situ at 80°C (pH 3.0), A. infernus and D.ambivalens were grown samples No, at 85°C (pH 2.5), and Pyrodictium occultum and Pyrobacu- temp Strain(s) (“C) pH ium islandicum were grown at 100°C (pH 6.0). Anaerobic media were prepared by using the technique of Balch et al. Ribeira Quente 2 0 (3), as described elsewhere (22, 47, 51). Furnas Caldeiras 7 2 72, 80 2, 3 FC4, FC6T Large-scale cultivation was performed in enamel-pro- Caldeiras da Ribeira 1 1 80 4 RG1 tected fermentors (Bioengineering, Wald, Switzerland) with grande 100- and 300-liter working volumes. Caldeira da Velha lb 0

Bacterial growth. Cell concentrations were determined by (I Samples containing the new isolates. light microscopy, using a Thoma counting chamber (depth, Isolate HWl was obtained from this sample; however, this organism 0.02 mm). belongs to the genus Acidianus (47). In situ pH and temperature measurements. The in situ pH was estimated by using indicator paper (Acilit; Merck, Darmstadt, Federal Republic of Germany), and the temper- DNAs from other organisms (4,15,31) in 3~ SSC containing ature was determined with a NiCr-Ni model GTH 1200 10% (voVvol) formamide at 25°C below the melting point as temperature probe (Greisinger, Regenstauf, Federal Repub- described elsewhere (34, 52). lic of Germany). Electron microscopy. Thin sections were prepared and RESULTS AND DISCUSSION electron microscopy was performed as previously described (19). Enrichment and isolation. A total of 11 anaerobic (3, 58) Determination of growth temperatures. Growth tempera- samples from boiling solfataric springs, mud, and soils were tures were determined in water bath incubators by using taken on Sgo Miguel Island, Azores. The temperature and completely submerged culture vessels, as described else- pH ranges in situ were 72 to 102°C and pH 2.0 to 5.5, where (46). respectively. The samples were taken to the laboratory Organic substrates. Each of the following organic sub- without temperature and pH control. They were then inoc- strates was added to Allen mineral medium at a concentra- ulated into anaerobic (3) culture medium and incubated with tion of 1 g/liter under both aerobic growth conditions and shaking (150 rpm) at 85°C. Organisms became visible after 4 anaerobic growth conditions (atmosphere containing N, and days in only three of the inoculated bottles (Table 1) (a fourth CO, [80:20, voVvo1; 300 kPa]): 20 L-amino acids obtained culture, strain HW1, which was obtained from Caldeira da from Sigma Chemical Co., St. Louis, Mo.; D-(-)-ribose, Velha, later turned out to belong to the genus Acidianus D-(-)-fructose, D-(+)-glucose, D-( +)-xylose, L-( -)-sorbose, [47]). The final cell titer was about lo7 cells per ml in all three maltose, sucrose, starch, acetate, and meat extract, all of enrichments. After several transfers, cultures exhibited sig- which were obtained from Merck; and Casamino Acids, nificantly higher final cell titers (up to 8 x lo7 cells per ml). yeast extract, Bacto-Peptone, and tryptone, all of which This phenomenon may have been due to a selection process. were obtained from Difco Laboratories. Pure cultures were obtained by performing three serial Cell walls. Muramic acid and rneso-diaminopimelic acid dilutions. were analyzed as previously described (35, 44). Metabolism. The new isolates, including strain FUT,grew Lipids. Glycerol-dibisphytanyl-nonitoltetraether, caldari- chemolithoautotrophically on hydrogen by reducing molec- ellaquinone, and sulfolobusquinone were detected by thin- ular sulfur to H,S. Growth depended obligately on the layer chromatography on silica gel (Merck) after extraction presence of both H, and So, indicating that the organisms of lipids from freeze-dried cells as described previously (64). were H,-So lithoautotrophs (14). Sulfur could not be re- Quinones were eluted with n-hexane-ethylacetate (955). placed by sulfite, tetrathionate, L-(-)-cystine, or sulfate. Glycerol-dibisphytanyl-nonitol tetraethers were eluted with None of the organic compounds listed in Materials and CHC1,-methanol (9:l) after hydrolysis of total lipids as Methods could replace H, as a substrate. As reported described previously (64). Lipid extracts from Sulfolobus previously for other H,-So lithotrophic archaebacteria (e.g., solfataricus DSM 1616T, A. infernus DSM 3191T, and A. Acidianus spp. [47]), trace amounts (0.005 to 0.02%, wt/vol) brierleyi DSM 6334 which were prepared by using the of yeast extract, meat extract, tryptone, peptone, or procedures described above and authentic lipid compounds Casamino Acids raised the growth yield significantly (up to from Sulfolobus solfataricus MT4 (13, 71) were used as eight times). In contrast to the facultatively aerobic Acid- references. ianus spp., the new isolates, including strain FC6T, were not Immunological procedures. Polyclonal antibodies were able to grow aerobically or microaerobically, nor were they raised in rabbits by using the micromethod of Stetter (57). capable of oxidizing sulfur to H,SO, (Fig. 1). Even after 3 Immunodiffusion in gels was carried out by using standard weeks of (micro)aerobic incubation, no growth and no procedures (2, 41). acidification of the medium were detected. When the new DNA analyses. DNA was prepared by subjecting it to two organisms were inoculated into anaerobic medium (atmo- cycles of isopycnic centrifugation as described by Lauerer et sphere, N,-CO,) containing sulfur and either 0.1% (wt/vol) al. (39). DNA base composition was analyzed by the thermal Na,MoO,, 0.1% (wthol) KNO,, 0.1% (wthol) FeCl,, or denaturation method (40) in 0.1~SSC (IxSSC is 0.15 M 0.1% (wthol) KH,PO, as a possible electron acceptor, NaCl plus 0.015 M trisodium citrate). DNA from A. brierleyi neither growth nor production of H2S04 was detected. DSM 1651T (G+C content, 31 mol%) (71) was used as a Therefore, these organisms are not capable of growing by reference. The levels of DNA homology between organisms anaerobic So oxidation, in contrast to Sulfolobus and Acid- were detected by probing single-stranded filter-bound ge- ianus spp. (6, 8, 47). On the basis of the strictly anaerobic nomic DNA with nick-translated, 32P-labeled genomic mode of life, a distinct G+C content of about 38 mol%, and VOL. 41, 1991 STYGIOLOBUS AZORICUS GEN. NOV., SP. NOV. 497

I rOI !o 0 10 20 30 LO 50 60 70 TIME (h) FIG. 1. Growth of (0 and 0) and acidification of medium by (H and 0)cells of isolate FC6T (0 and D) and A. infprnus DSM 3191T (0 and B) under aerobic growth conditions.

FIG. 2. Phase-contrast micrograph of cells of Stygiolobus atori- the lack of a significant level of DNA homology with any cus FChT. Bar = 10 pm. member of the Sulfolobales (Table 2), we describe the new genus Stygiolobus below; Stygiolobus azoricus is the type species of this genus, and strain FC6 (= DSM 6296) is the fractions produce incomplete immunochemical cross-reac- type strain of Stygiolobus azoricus. tions with polyclonal antibodies against the native DNA- Description of Stygiolobus gen. nov. Stygiolobus (Sty.gi. dependent RNA polymerase from Sulfolobus acidocaldarius o.lo’bus. L. masc. adj. stygius, stygian, from hell; Gr. masc. DSM 639T (Fig. 5a) and the native (NiFe) hydrogen uptake n. lobos, lobe; M.L. masc. n. Stygiolobus, lobed organism hydrogenase from A. brierleyi DSM 6334 (53) (Fig. Sb), and from Hades, referring to its biotope, in which the gate to hell fractions enriched for acid-soluble proteins produce com- was located in Dante’s Divina Cornrnedia) cells are gram plete immunochemical cross-reactions with polyclonal anti- negative, coccoid, and highly irregular in shape (Fig. 2 and bodies against histone-like proteins HSNP c’ and DBNP B 3). They are strongly lobed or have sharp edges and bends. from Sulfolobus acidoculdurius DSM 639T (36, 43). Thin sections reveal a surrounding envelope covering the The purified DNA has a G+C content of about 38 mol%. cell membrane (Fig. 4), which is composed of subunits in a There is no significant DNA homology with other members hexagonal array (42). of the Sulfolobales (Table 2). Cells are strictly anaerobic, chemolithotrophic thermoacid- The organisms occur in acidic solfataric fields. ophiles that thrive by means of So reduction with H,. The type species is Stygiolobus azoricus. Neither muramic acid nor rneso-diaminopimelic acid is Description of Stygiolobus azoricus sp. nov. Stygiolobus present, indicating the absence of murein (30). azoricus (a.zo’ri.cus. M.L. masc. adj. azoricus, from the Elongation factor EF-G is sensitive to adenosine diphos- Azores, referring to the place of isolation) exponentially phate ribosylation by diphtheria toxin (32, 33). growing cells are about 0.5 to 1.8 pm wide and occur almost Cells contain glycerol-dibisphytanyl-nonitol tetraether lip- exclusively singly under optimal culture conditions. Under ids and sulfolobusquinone. No caldariellaquinone (11) has suboptimal conditions, especially at an elevated pH of 24, been detected. aggregates of 2 to 50 cells occur commonly. Cells are The organisms are resistant to vancomycin, ampicillin, frequently surrounded by pilus- or fimbrialike appendages and kanamycin at concentrations of 150 kg/ml. Protein (Fig. 3). No flagellation has been detected. No motility is

TABLE 2. Levels of DNA homology for the new isolates and type strains of species belonging to the Sulfolobales % Homology with 32P-labeled DNA from:

Sulfolobus Sulfolobus Source of filter-bound DNA Isolate acidoca,darius solfataricus A. brierleyi A. infernus D. ambivalens FC6T DSM 1616T DSM 16.511 DSM 3191T DSM 3772T DSM 639= Isolate FC6T 100 13 11 20 10 8 Isolate FC4 98 ND“ ND ND ND ND Isolate RG1 101 ND ND ND ND ND Sulfolobus acidocaldarius DSM 639T 12 100 13 16 7 ND Sulfolobus solfataricus DSM 1616T 4 11 100 22 6 ND Sulfolobus shibatae DSM 5389T 5 ND ND ND ND ND M. sedula DSM 534gT 7 9 11 12 5 ND A. brierleyi DSM 1651T 17 6 10 100 7 ND A. infernus DSM 3191T 13 7 11 16 100 61 D. ambivalens DSM 3772T 10 9 13 19 59 100 ND, not determined. 498 SEGERER ET AL. INT. J. SYST.BACTERIOL.

FIG. 3. Electron micrograph (Pt shadowed) of a cell of Stygiofobus azoricus FC6T showing fimbrialike appendages. Bar = 0.5 km. evident in a microscopic chamber either at room tempera- acidic geothermal springs on S5o Miguel Island, Azores ture or at 75°C. The envelope surrounding the cells is about (Table 1). 22 nm wide. Packed cells are black. The type strain is isolate FC6 (= DSM 6296). Growth is strictly chemolithotrophic by means of H,-So The new organisms described above are archaebacteria on lithoautotrophy (14). Trace amounts of yeast extract (0.005 the basis of the occurrence of isopranyl ether lipids in their to 0.02%) stimulate growth. Cells grow at temperatures cells (12, 37), their resistance to cell wall and protein ranging from about 57 to 89"C, with optimal growth occur- synthesis antibiotics (17), their lack of murein (30), and the ring around 80°C (Fig. 6). No growth is detected at 50°C presence of elongation factor EF-G which is sensitive to (incubation time 3 weeks) and 89°C. The pH suitable for diphtheria toxin (32). They resemble chemolithotrophic growth ranges from 1.0 to 5.5, with optimum growth around members of the Thermoproteales (24, 70) in that they grow pH 2.5 to 3 (data not shown). No growth is detected at pH by means of H,-S* lithotrophy, but strongly differ from these 0.8 or 6.0. organisms (and from all other members of the Thermopro- Cultures of isolate FC6T can be used for at least 1 year as teales [for details see reference 221) in their shape, their inocula when they are stored anaerobically at 4°C after the extreme acidophily, and their much lower G+C contents. pH is increased to 5.5 by adding sterile CaCO,. Cells lyse at Although they are strict anaerobes that are incapable of So pH values above 7.5 to 8. oxidation, they are members of the Sulfolobales (29, 59), as Isolates were obtained from hot water, mud, and soil at indicated by their morphology, their extreme thermoacidophily, their relatively low G+C contents, the presence of characteristic lipid compounds (10-12), their strongly acidic biotope, and the immunochemical cross-reactions of their DNA-dependent RNA polymerases , hydrogenases (53), and histonelike proteins with antibodies against the respective enzymes and proteins from members of the Sulfolobales. None of those cross-reactions occurs with representative type species belonging to the Thermoproteales, Pyrobacu- lum islandicum DSM 4184= (21, 27), and Pyrodictium occultum DSM 2709T (25, 60) (Fig. 5) (36). Usually, DNA- dependent RNA polymerases reveal immunochemical cross- reactions only within members of the same family (61). On the basis of this criterion, isolate FC6T belongs to the family Sulfolobaceae (29, 59). On the basis of their strictly anaerobic mode of life, the lack of significant DNA homology with other members of the Sulfolobales (Table 2), and their G+C FIG. 4. Ultrathin section through a cell of Stygiolobus azoricus contents of about 38 mol%, the new organisms are distinct FC6=. Electron micrograph. Bar = 0.5 Lm. from the four previously described genera in this order. VOL. 41. 1991 STYGIOLOBUS AZORICUS GEN. NOV., SP. NOV. 499

range as the G+C content of Stygiolobus azoricus. How- ever, Sulfolobus spp. are facultatively organotrophic So oxidizers that are incapable of H,-So lithotrophy (16,48,51). Acidianus and Desulfurolobus spp. can grow by means of H,-So lithotrophy, but they are facultatively aerobic So oxidizers that have distinctly lower G+C contents (about 31 mol%) (26, 47, 49, 72). Stygiolobus azoricus represents the first obligately anaerobic member of the Sulfofobafes.This organism is incapable of oxidation of sulfur compounds. Previously, its mode of energy conservation was considered to be characteristic of the order Thermoproteales (24, 69, 70). Therefore, the Sulfolobales appear to be metabolically more versatile than the Thermoproteales (22, 51). Since probably no significant amounts of oxygen were present on the archaean earth, it is tempting to speculate that the strictly anaerobic organism Stygiolobus azoricus might reflect the most ancient phenotype of the Sulfolobales and might even represent a missing link between the Sulfolobales and the Thermoproteales. The distribution of the genus Stygiolobus is unknown. Strains have been obtained only from solfataras on S5o Miguel Island. We were unable to isolate it from solfataric samp!es obtained from Yellowstone National Park (United States), Iceland, Southern Italy (Naples and Vulcano Is- land), Indonesia, the Southern Kurils (USSR), and Hok- kaido (Japan) (51). This could be'explained by the presence of Acidianus strains in the samples, which may have over- grown Stygiolobus strains in the enrichment cultures. On the other hand, it may be that the genus Stygiolobus is a very rare and possibly even endemic genus in the Azores. Other possibly endemic archaebacteria are the anaerobic Methan- FIG. 5. Ouchterlony in-gel immunodiffusion test of protein sam- othermus ples (crude extracts; about 200 pg of protein per well) from various spp., which have been found so far only in distinct members of the Sulfolobales, Pyrobaculum islandicum DSM 4184T, Icelandic solafatara fields (39, 61), and the genetically unre- and Pyrodictium occultum DSM 2709T. (a) Polyclonal antibodies lated strains of Thermoplasma volcanium (28), which occur against DNA-dependent RNA polymerase from Sulfolobus aci- only on Vulcano Island and Java (Indonesia) (46,50). A final docaldarius DSM 639T. The center well contained antiserum. Ex- decision about the distribution of the genus Stygiolobus has tract 1, Sulfolobus acidocaldurius DSM 639T; extract 2, A. infernus to await further ecological studies. DSM 3191T; extracts 3 and 6, Stygiolobus uzoricus FC6T; extract 4, Pyrobaculum islandicum Pyrodictium occul- DSM 4184T; extract 5, ACKNOWLEDGMENTS tum DSM 2709T. (b) Polyclonal antibodies against the membrane- associated (NiFe) hydrogen uptake hydrogenase from A. brierleyi We thank Reinhard Rachel (Regensburg, Federal Republic of DSM 6334 (53). The center well contained antiserum. Extract 1, A. Germany) for electron microscopy, Friedrich Klink (Kiel, Federal brierleyi DSM 1651T; extract 2, Stygiolobus azoricus FC6T; extracts Republic of Germany) for communicating unpublished data, 3 and 5, Pyrodictium occulturn DSM 2709=; extract 4, Pyrobaculum Michael Thomrn (Regensburg) for providing RNA polymerase anti- islandicum DSM 4184T; extract 6, A. brierleyi DSM 6334. bodies, and Wolfram Zillig (Martinsried, Federal Republic of Ger- many) for providing cultures of Sulfolobus shibatae and D. ambiv- dens. The excellent assistance of Ludwig Deml, Konrad Eichinger, Hans Peter Hummel, Gerhard Neugebaur, and Monika Poignee is M. sedula (18, 29) has a G+C content of 45 mol%, which is highly appreciated. the highest value in the Sulfolobales. This organism occurs This work was supported by grants from the Deutsche Forschungs- as round, regular cocci and is a strict aerobe. The G+C gemeinschaft and the Fonds der Chemischen Industrie (to K.O.S.). contents of Sulfolobus spp. (7, 16, 23, 55, 71) are in the same REFERENCES 1. Allen, M. B. 1959. Studies with Cyanidium caldarium, an anomalously pigmented chlorophyte. Arch. Mikrobiol. 32:27& 0.25 277. 2. Bailey, G. S. 1984. Immunodiffusion in gels, p. 301-310. In J. M. Walker (ed.), Methods in molecular biology, vol. 1. Proteins. Humana Press, Clifton, N.J. 3. Balch, W. E., G. E. Fox, L. J. Magrum, C. R. Woese, and R. S. Wolfe. 1979. Methanogens: reevaluation of a unique biological group. Microbiol. Rev. 43:26&296. 3 Birnstiel, M. B. H. Sells, and I. F. Purdom. EO 05 4. L., 1962. Kinetic complexity of RNA molecules. J. Mol. Biol. 63:21-39.

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