Three Isolates of Spherical Thermoacidophilic Archaebacteria (Strains TA- 1, TA-2 and N-8) Were Obtained from Acidic Hot Springs at Hakone and Noboribetsu in Japan

J. Gen. Appl. Microbiol., 41, 43-52 (1995)

CHARACTERIZATION AND IDENTIFICATION OF THERMOACIDOPHILIC ARCHAEBACTERIA ISOLATED IN JAPAN

NORIO KUROSAWA,* AKIHIKO SUGAI,' IKUKO FUKUDA,' TOSHIHIRO ITOH,' TADAO HORIUCHI, AND YUKO H. ITOH

Department of Bioengineering, Faculty of Engineering, Soka University, Hachioji 192, Japan 'Division of Chemistry, School of Liberal Arts and Sciences, Kitasato University, Sagamihara 228, Japan

(Received September 12, 1994; Accepted December 6, 1994)

Three isolates of spherical thermoacidophilic archaebacteria (strains TA- 1, TA-2 and N-8) were obtained from acidic hot springs at Hakone and Noboribetsu in Japan. We have studied the isolates using electron microscopy, Gram staining, sensitivity to lysozyme, growth conditions, lipid compositions, SDS-PAGE patterns of whole-cell proteins, DNA base composition and partial 16S rRNA sequences. These characteristics of the isolates were compared with those of the type strains, Sulfolobus acidocaldarius, Sulfolobus solfataricus, Acidianus brierleyi, Acidianus infernus, Metallosphaera sedula and Desulfurolobus ambivalens. Compara- tive studies indicated that the isolate TA-1 was novel species of either Sulfolobus or Acidianus, the isolate TA-2 belonged to the genus Metallo- sphaera, and the isolate N-8 belonged to the same species of S. acidocaldarius isolated from Yellowstone.

A comparative sequence study of the 16S rRNAs showed that living organisms can be divided into three kingdoms: the traditional eubacterial and eukaryotic kingdoms and the newly recognized archaebacterial kingdom (22,23). Although archaebacteria exhibit a prokaryotic cell structure and organization, they show distinguishing and unique features (1,10,11,14-16). From acidic hot springs in Japan, three isolates of spherical thermoacidophilic archaebacteria (strains TA-1, TA-2 and N-8) were obtained. Total lipid composition analysis and preliminary results of growth temperature and pH showed that these three strains belong to the order Sulfolobales (19, 20).

* Address reprint requests to: Dr . Norio Kurosawa, Department of Bioengineering, Faculty of Engineering, Soka University, 1-236 Tangi-cho, Hachioji 192, Japan.

43 44 KUROSAWA et al. VOL. 41

In sulfur-dependent thermophilic archaebacteria, the following five genera have been assigned to the order Sulfolobales: (i) the genus Sulfolobus, which is the type genus and includes the species S. acidocaldarius (3), S. solfataricus (24), S. shibatae (6) and S. metallicus (9), (ii) the genus Acidianus, which includes the species A. brierleyi (2,17) and A. infernus (17), (iii) the genus Metallosphaera, with one species, MMsedula (8), (iv) the genus Stygiolobus, with one species, S. azoricus (18), (v) the genus Desulfurolobus, with one species, D. ambivalens (25). All species of the order Sulfolobales are acidophilic cocci. Sulfolobus and Metallospha- era grow as aerobes. In contrast, Acidianus and Desulfurolobus are facultatively anaerobic organisms. Stygiolobus is an obligately anaerobic member of Sulfoloba- les. D, ambivalens has been suggested to belong to the genus Acidianus based on DNA-DNA hybridization experiments (18). This paper deals with the characterization and identification of the isolates TA- T, TA-2 and N-8 based on electron microscopy, Gram staining, sensitivity to lysozyme, growth conditions, core lipid compositions, SDS-PAGE patterns of whole-cell proteins, DNA base compositions and partial 16S rRNA sequences.

MATERIALS AND METHODS

Bacterial strains. TA-1 and TA-2 were obtained from mud of an acidic hot spring (70°C, pH 3.0) of Ohwakudani, Hakone, Japan. N-8 was obtained from mud of an acidic hot spring (40°C, pH 3.5) of Jigokudani, Noboribetsu, Japan. The samples were transported at ambient temperature, and cultured at 70°C in the "TA" medium: ((NH 4)2SO4, 1.3 g; KH2PO4, 0.25 g; MgSOe 7H2O, 0.25 g; CaCl2 2H2O, 0.08 g/l), supplemented with 1.0 g of yeast extract, pH 3.0. They were purified by repeated serial dilution in the TA medium, and by single colony isolation on TA-plates solidified by 0.6% gellan gum (Merck, Darmstadt, Germany) at 70°C, pH 3.0. Sulfolobus acidocaldarius ATCC 33909 was obtained from the American Type Culture Collection. Sulfolobus solfataricus IFO 15331 (derived from DSM 1616), Acidianus brierleyi IFO 15269 (derived from DSM 1651), and Metallosphaera sedula IFO 15509 (derived from DSM 5348) were obtained from the Institute for Fermentation, Osaka, Osaka, Japan. Acidianus infernus DSM 3191 and Desulfurolobus ambivalens DSM 3772 were obtained from Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH. Growth conditions. TA-1, TA-2 and N-8 were grown in modified Brock's basal salts mixture: ((NH4)2SO4, 1.3 g; KH2PO4, 0.28 g; MgSO4.7H2O, 0.25 g; CaC12.2H2O, 0.07 g; FeC13.6H2O, 2.0 mg; MnC12.4H2O, 1.8 mg; Na2B40, • 10H2O, 4.5 mg; ZnSO4. 7H2O, 0.22 mg; CuC12.2H2O, 0.05 mg; NaMoO4.2H2O, 0.03 mg; VOSOr2H2O, 0.03 mg; CoSO4, 0.01 mg/l), supplemented with 1.0 g of yeast extract and 2.0 g of sucrose per liter, pH 2.5. They were grown at several temperatures (50-90°C). For determination of optimal pHs, they were cultured at optimal temperatures at several pHs (1.0-6.0). The original Brock's basal salts mixture contains 20 mg of FeC13.6H2O per liter (3). S. acidocaldarius, A. infernus and M. 1995 Characterization of Archaebacterial Isolates 45 sedula were grown in modified Brock's basal salts, supplemented with 1.0 g of yeast extract and 2.0 g of sucrose per liter, pH 2.5, at 80, 88 and 70°C, respectively. D. ambivalens was grown at 80°C in modified Brock's basal salts, supplemented with 0.2 g of yeast extract per liter, pH 2.5. A. brierleyi was grown at 69°C in IFO-286 medium ((NH4)2SO4i 3.0 g; K2HPO4, 0.5 g; MgSO4 7H2O, 0.5 g; KCI, 0.1 g; Ca(N03)2, 0.01 g/l) supplemented with 0.5 g of yeast extract and 2.Og of sucrose per liter, pH 2.0. All cells were grown aerobically in standing cultures of loosely capped glass tubes or long-necked conical flasks with stirring. The doubling times were calculated from the slopes of the growth curves. Anaerobic cultivations were performed with a BBL gaspack anaerobic system (BBL) according to the manufacturer's instructions. This system gives gas phase H2/C02 (9 :1). Modified Brock's medium for anaerobic cultivation was reduced by the addition of sodium sulfide (0.75 g/l), which was visualized by reduction of resazurin ( l mg/t). Electron microscopy. Thin sections of TA-1, TA-2 and N-8 were prepared by rapid-freezing and substitution fixation as previously described (5), and examined using a JEOL JEM-2000EX TEM at 100 kV. Gram-staining. Gram-staining tests of log-phase cells were carried out using the standard method. Escherichia coli LE392 and Bacillus subtilis 162 were used for negative and positive controls of staining, respectively. Sensitivity of lysozyme. Log-phase cells from 10-ml cultures were washed and resuspended in 3 ml of hypertonic solution (0.75 M sucrose in 10 mM Tris-HCI, pH 8.0). The following solutions were added to 1 ml each of the suspension at final concentrations of 100 mg/ml of lysozyme and 1 mM of EDTA: a) lysozyme, b) lysozyme and EDTA, c) no addition of lysozyme or EDTA. The reaction mixtures were incubated at 37°C for 30 min, and diluted with 4 volumes of distilled water. The decreases in turbidity were measured at 600 nm using a photometer (Milton Roy 21D). Lipid analysis. Total cellular lipids were extracted from stationary-phase cells as described in the previous report (19) and analyzed using a SHIMADZU FTIR- 8200 spectrophotometer. Core lipids of diglyceryl tetraether (DGTE) and caldityl glyceryl tetraether (CGTE) were prepared by acid methanolysis of total cellular lipids as described in a previous report (20). DGTE and CGTE were subjected to thin-layer chromatography with precoated plates (silica gel 60 HPTLC, Merck). The plates were developed by two ascending runs with the following solvent systems as: 1st step, chloroform : methanol : water=75 :25 : 2 (up to 2 cm from the origin); 2nd step, n-hexane : diethyl ether : acetic acid = 60 :40:2. Whole-cell SDS-PAGE protein patterns. Log-phase cells were suspended in a sample buffer (0.0625 M Tris-HCI, pH 6.8, 1% mercaptoethanol, 0.2% SDS, 0.001 % phenol-red, 10% glycerol, 0.002% bromophenol blue). Supernatants were recovered by centrifugation, heated at 100°C for 2 min and then applied on a 12% gel. Proteins were visualized by silver staining. DNA base composition. The G + C contents of DNAs were determined by reverse-phase HPLC of the DNAs digested with nuclease P1(21). 46 KUROSAWA et al. VOL. 41

Cloning and sequencing of the 16S rRNA genes. The fragments of partial 16S rDNAs from TA-1, TA-2, N-8, A. infernus and D, ambivalens were amplified by PCR with synthetic oligonucleotides (5'-CAG CAG CCG CGG TAA TAC and 5'- ACG GGC GGT GTG TGC). These sequences are universally found around positions 530 and 1400, respectively in the 16S rRNA genes not only in eubacteria but also in archaebacteria (7). The amplified fragments were subcloned into pBluescript SK- (Stratagene, La Jolla, U.S.A.). DNA sequence analysis was carried out by the dideoxynucleotide chain termination method with Fluorescein- isothiocyanate-labelled primers using a HITACHI SQ-3000 DNA sequencer. Bca- best DNA polymerase (Takara, Ohtsu, Japan) was used and dGTP was replaced with dITP in all four sequencing mixtures to avoid "peak compression." Nucleotide sequence accession numbers. The nucleotide sequence data re- ported in this paper will appear in the GSDB, DDBJ, EMBL and NCBI nucleotide sequence databases with the following accession numbers: D38773 /A. infernus, D38774 /D. ambivalens, D38775 /TA-1, D38776/TA-2 and D38777/N-8.

RESULTS

Morphology Under the phase contrast microscope, cells of the isolates TA-1, TA-2 and N- 8 appeared as round to slightly irregular cocci about 11am in width. Under the transmission electron microscope, thin sections of TA-2 revealed a surrounding envelope about 20 nm wide covering the cell membrane. Pili- or flagella-like structures were not observed (Fig. 1). Electron micrographs of TA-1 and N-8 were quite similar to that of TA-2 (not shown). All the isolates and S. acidocaldarius were Gram-negative and insensitive to 100 mg/ml of lysozyme even coexisting with 1 mM EDTA.

Fig. 1. Electron micrograph of a thin section of TA-2. Bar =0.5,tm. 1995 Characterization of Archaebacterial Isolates 47

Growth temperature and pH range Growth rates of the isolates at various temperatures and pHs are shown in Fig. 2. The optimal temperatures and pHs of the isolates and type strains are summarized in Table 1. The isolate TA-1 grew within the temperature range of 63 to 92 °C , and optimally at 84°C. TA-1 grew between pH 1.0 and 5.0, and optimally at pH 2.0. The isolate TA-2 grew within the temperature range of 50 to 82°C, and optimally at 75°C. No growth occurred at 85°C. TA-2 grew between pH 1.5 and 3.5, and optimally at pH 2.8. The isolate N-8 grew within the temperature range of 60 to 87°C, and optimally at 80°C. No growth occurred at 55 and at 90°C. N-8 grew between pH 1.5 and 5.0, and optimally at pH 3.0. The doubling time of TA- 1, TA-2 and N-8 were 5.9, 4.5 and 3.4 h, respectively, under optimal conditions.

Anaerobic cultivations Anaerobic cultivations were performed using a BBL gaspack anaerobic

Fig. 2. Growth rates of TA-1 (square), TA-2 (open circle) and N-8 (filled circle) at various temperatures (A) and pHs (B).

Table 1. Characteristics of the new isolates and type strains. 48 KUROSAWA et al. VOL. 41

system. This system gives gas phase H2/C02 (9: 1). Growth of TA-2, N-8 and S. acidocaldarius were not observed under this anaerobic condition. In contrast to these strains, TA-1 was able to grow under gas phase H2/C02 (9: 1) . The results indicated that TA-2 and N-8 grow as aerobes, and TA-1 is facultatively anaerobic.

Lipids analysis The infrared spectra of the total lipid extracts from the three isolates were characteristic, showing the presence of ether bonds at 1,100 cm-' and the total absence of fatty acid ester at 1,740-1,730 cm-'. These infrared spectroscopic analysis of the total cellular lipids of TA-1, TA-2 and N-8 indicated that the lipid constituents exclusively contained ether linkages. Thermoacidophilic archaebacteria of the order Sulfolobales have two types of core lipid structures; diglyceryl tetraether (DGTE) and caldityl glyceryl tetraether (CGTE) (4). The CGTE derivatives share 70% or more of the total lipids from cells (20) and are found in only Sulfolobales (4). These two core lipids can be obtained by acid methanolysis of the total lipids and are detected by thin-layer chromatography. The isolates TA-1 and N-8, S. acidocaldarius, A. brierleyi and MM sedula had both DGTE and CGTE. In contrast with these strains, the isolate TA- 2 had no CGTE (Fig. 3). An unidentified band was detected between the origin and CGTE in each lane of all the isolates and type strains.

Whole-cell SDS-PAGE protein patterns Whole cell SDS-PAGE protein patterns of TA-1, TA-2, N-8 and type strains are shown in Fig. 4. The pattern of TA-1 showed a unique profile. TA-2 and M.

I G. V `T .J V I V Fig. 3. Thin-layer chromatogram of core lipids from Acidianus brierleyi, lane 1; TA-1, lane 2; Metallosphaera sedula, lane 3; TA-2, lane 4; Sulfolobus acidocaldarius, lane 5; N-8, lane 6; standard of caldityl glyceryl tetraether (CGTE), lane 7 and diglyceryl tetraether (DGTE), lane 8. Solvent systems: 1st step, chloroform : methanol : water (75 : 25 : 2); 2nd step, n-hexane : diethyl ether : acetic acid (60 :40 : 2).

1995 Characterization of Archaebacterial Isolates 49

v . v V I Fig. 4. SDS-PAGE of the whole-cell proteins of TA-1, TA-2, N-8 and type strains. Lanes: 1, Desulfurolobus ambivalens; 2, Acidianus brierleyi; 3, TA-1 4, Metallosphaera sedula; 5, TA-2; 6, Sulfolobus acidocaldarius; 7, N-8.

Table 2. Comparison of the 16S rRNA sequences.

sedula exhibited very similar patterns except for a few bands around 70 kDa. N-8 and S. acidocaldarius exhibited very similar patterns. The patterns of D. ambivalens and A. brierleyi were not similar to those of the other type strains or isolates.

G+C content of DNAs and 16S rRNA sequences The G + C contents of TA-1, TA-2 and N-8 are summarized in Table 1 with type strains belonging to Sulfolobales. The G + C content of TA-1 was 32.9 mol%, 50 KUROSAWA et al. VOL. 41 which was identical to that of A.. infernus or D. ambivalens. The G + C content of TA-2 was 47.1 mol% which was similar to that of M sedula. N-8 had a 36.7 mol% G + C content which was identical to that of S. acidocaldarius. Comparison of the partial 16S rRNA sequences (about 860 bp) from our isolates and the type strains are summarized in Table 2. These sequences are located around position 500-1360 in the 16S rRNA gene. The 16S rRNA sequence of TA-1 showed 89 to 94% homologies with those of the other isolates or type strains, and were most identical to those of N-8 and S. acidocaldarius (93.7%). The 16S rRNA sequence of TA-2 was 99% identical (6 bases per 858 bases were different) to the corresponding sequence of MMsedula and showed 90 to 93% homology with those of TA-1 or other type strains. The 16S rRNA sequence of N- 8 was completely identical to the corresponding sequence of S. acidocaldarius and showed about 90% homology with those of TA-2 or other type strains. The homology between A. infernus and D. ambivalens was 99.7%.

DISCUSSION

In this study, Stygiolobus azoricus which is a strict anaerobe was not used for comparison, because all of our isolates were known to grow under aerobic conditions by preliminary experiments. The lipid constituents of both strains exclusively contained ether linkages. This is strong evidence that the isolates TA-1, TA-2 and N-8 belong to archaebacteria. D, ambivalens has been suggested to belong to the genus Acidianus based on experiments of DNA-DNA hybridization (18). We found that the homology of the 16S rRNA between A. infernus and D. ambivalens is 99.7% (Table 2). These results indicate that D. ambivalens belongs to the genus Acidianus. Acidianus species are the only facultative anaerobic members in the order Sulfolobales while Sulfolo- bus and Metallosphaera are aerobic and Stygiolobus is strictly anaerobic. The isolate TA-1 is a facultative anaerobe, and the G + C content of TA-1 is almost the same as those of A. infernus or D. ambivalens. In contrast with these results, the 16S rRNA of TA-1 is more similar to those of Sulfolobus strains than those of Acidianus strains. Because of these characteristics, it appears that the isolate TA-1 is a novel species of either Sulfolobus orAcidianus. We should perform other experiments such as DNA-DNA hybridization for the final assignment of taxonomic status of TA-1. The isolate TA-2 is a Gram-negative cocci and grows as an aerobe. The optimal temperature and pH for growth, doubling time, and G + C content of TA- 2 are almost the same as those of M. sedula. The whole-cell proteins of TA-2 and MMsedula exhibit the same ladder in SDS-PAGE except for several bands. The 16S rRNA sequence of TA-2 is 99% identical to that of MMsedula. In contrast with these characters, their lipid compositions are quite different. TA-2 has only DGTE as the tetraether-type lipid while MMsedula has both DGTE and CGTE. This is a 1995 Characterization of Archaebacterial Isolates 51 very special feature of TA-2, because the CGTE is a major core lipid and its derivatives share 70% or more of the total lipids of sulfur-dependent thermoacidophilic archaebacteria (20). The isolate TA-2 is the first example of sulfur- dependent thermoacidophilic archaebacteria which lacks a CGTE. These results suggest that the isolate TA-2 belongs to the genus Metallosphaera and CGTE may not be essential for survival of sulfur-dependent thermoacidophilic archaebacteria. The isolate N-8 is a Gram-negative cocci and grows as an aerobe. The optimal temperature and pH for growth, doubling time, and G + C content of N-8 are almost the same as those of S. acidocaldarius. Furthermore ,, the whole-cell protein pattern and the 16S rRNA sequence of N-8 are identical to those of S. acidocaldarius. These results indicate that the isolate N-8 obtained from Noboribetsu belongs to the same species of S. acidocaldarius from Yellowstone.

We expressour thanksto Dr. Y. Futaesakuand Mr. T. Nishikawa,Laboratory of Ultrastructure Research,Faculty of Bioscience,Kitasato University, Sagamihara, Kanagawa, for preparationof the thin sectionsand electronmicrographs.

REFERENCES

1) Bock, A. and Kandler, 0., Antibiotic sensitivity of archaebacteria. In The Bacteria, Vol. VIII: Archaebacteria, ed. by Woese, C. R. and Wolfe, R. S., Academic Press, New York (1985), p. 525- 544. 2) Brierley, C. L. and Brierley, J. A., A chemoautotrophic and thermophilic microorganism isolated from an acid hot spring. Can. J. Microbiol., 19, 183-188 (1973). 3) Brock, T. D., Brock, K. M., Belly, R. T., and Weiss, R. L., Sulfolobus: A new genus of sulfur-oxidizing bacteria living at low pH and high temperature. Arch. Mikrobiol., 84, 54-68 (1972). 4) De Rosa, M. and Gambacorta, A., The lipids of archaebacteria. Frog. Lipid Res., 27, 153-157 (1988). 5) Dempsey, G. P. and Bullivant, S., A copper block method for freezing non-cryoprotected tissue to produce ice-crystal-free regions for electron microscopy. J. Microsc., 106, 251-260 (1976). 6) Grogan, D., Palm, P., and Zillig, W., Isolate B12, which harbors a virus-like element, represents a new species of the archaebacterial genus Sulfolobus, Sulfolobus shibatae, sp. nov. Arch. Microbiol., 154, 594-599 (1990). 7) Hoshina, S., Kahn, S. M., Jiang, W., Green, P. H. R., Neu, H. C., Chin, N., Morotomi, M., LoGerfo, P., and Weinstein, I. B., Direct detection and amplification of Helicobacter pylori ribosomal l6S gene segments from gastric endoscopic biopsies. Diagn. Microbiol. Infect. Dis., 13, 473-479 (1990). 8) Huber, G., Spinnler, C., Gambacorta, A., and Stetter, K. 0., Metallosphaera sedula gen. and sp. nov. represents a new genus of aerobic, metal-mobilizing, thermoacidophilic archaebacteria. Syst. App!. Microbiol., 12, 38-47 (1989). 9) Huber, G. and Stetter, K. 0., Sulfolobus metallicus, sp. nov., a novel strictly chemolithoautotro- phic thermophilic archaeal species of metal-mobilizers. Syst. App!. Microbiol., 14, 372-378 (1991). 10) Huet, J., Schnabel, R., Sentenac, A., and Zillig, W., Archaebacteria and eukaryotes posses DNA-dependent RNA polymerases of a common type. EMBO J., 2, 1291-1294 (1983). 11) Kjems, J. and Garrett, R. A., An intron in the 23S ribosomal RNA gene of the archaebacterium Desulfurococcus mobilis. Nature, 318, 675-677 (1985). 12) Kurosawa, N. and Itoh, Y. H., Nucleotide sequence of the 16S rRNA gene from thermo- 52 KUROSAWA et al. VOL. 41

acidophilic archaea Sulfolobus acidocaldarius ATCC33909. Nucleic Acids Res., 21, 357 (1993), DDBJ accession number: D14876. 13) Kurosawa, N., Ohkura, K., Horiuchi, T., and Itoh, Y. H., Characterization of the 16S ribosomal RNA genes and phylogenetic relationships of sulfur-dependent thermoacidophilic archaebacteria. J. Gen. Appl. Microbiol., 41, 75-81 (1995), Accession numbers for GSDB, DDBJ, EMBL and NCBI nucleotide sequence databases: D26490/S. solfataricus, D26489/A. brierleyi and D26491/ M. sedula. 14) Langworthy, T. A., Lipids of archaebacteria. In The Bacteria, Vol. VIII: Archaebacteria, ed. by Woese, C. R. and Wolfe, R. S., Academic Press, New York (1985), p. 459-497. 15) Matheson, A. T., Ribosomes of archaebacteria. In The Bacteria, Vol. VIII: Archaebacteria, ed. by Woese, C. R. and Wolfe, R. S., Academic Press, New York (1985), p. 345-377. 16) Rivera, M. C. and Lake, J. A., Evidence that eukaryotes and eocyte prokaryotes are immediate relatives. Science, 257, 74-76 (1992). 17) Segerer, A., Neuner, A., Kristjansson, J. K., and Stetter, K. 0., Acidianus infernus gen. nov., sp. nov., and Acidianus brierleyi comb. nov.: Facultatively aerobic, extremely acidophilic thermophilic sulfur-metabolizing archaebacteria. Int. J. Syst. Bacteriol., 36, 559-564 (1986). 18) Segerer, A., Trincone, A., Gahrtz, M., and Stetter, K. 0., Stygiolobus azoricus gen. nov., sp. nov. represents a novel genus of anaerobic, extremely thermoacidophilic archaebacteria of the order Sulfolobales. Int. J. Syst. Bacterial., 41, 495-501 (1991). 19) Sugai, A., Itoh, T., Kon, K., and Ando, S., Analysis of diglyceryl-trialkyl-tetraether from thermoacidophilic archaebacteria: Sulfolobus (in Japanese). Shishitsu-Seikagaku-Kenkyu, 31, 263-266 (1989). 20) Sugai, A., Itoh, T., Kon, K., and Ando, S., Diversity of core lipids from extremely thermophilic acidophilic archaebacterium; strain N-8 (in Japanese). Shishitsu-Seikagaku-Kenkyu, 33, 267-270 (1991). 21) Tamaoka, J. and Komagata, K., Determination of DNA base composition by reversed phase high-performance liquid chromatography. FEMS Microbiol. Lett., 25, 125-128 (1984). 22) Woese, C. R. and Fox, G. E., Phylogenetic structure of the prokaryotic domain: The primary kingdoms. Proc. Natl. Acad. Sci. U.S.A., 74, 5088-5090 (1977). 23) Woese, C. R., Kandler, 0., and Wheelis, M. L., Towards a natural system of organisms; Proposals for the domains Archaea, Bacteria and Eucarya. Proc. Natl. Acad. Sci. U.S.A., 87, 4576-4579 (1990). 24) Zillig, W., Stetter, K. 0., Wunderl, S., Schulz, W., Priess, H., and Schulz, I., The Sulfolobus- "Caldariella" group: Taxonomy on the basis of the structure of DNA -dependent RNA polymerases. Arch. Mikrobiol., 125, 259-269 (1980). 25) Zillig, W., Yeats, S., Holz, I., Bock, A., Rittenberger, M., Gropp, F., and Simon, G., Desulfuro- lobus ambivalens, gen. nov., sp. nov., an autotrophic archaebacterium facultatively oxidizing or reducing sulfur. Syst. Appl. Mikrobiol., 8, 197-203 (1986).