Isolation and Characterization of a Thermophilic, Sulfate Reducing Archaebacterium, Archaeoglobus fulgidus Strain Z

System. Appl. Microbiol. 11, 151-160 (1989)

Isolation and Characterization of a Thermophilic, Sulfate Reducing Archaebacterium, Archaeoglobus fulgidus Strain Z GERHARD ZELLNER!, ERKO STACKEBRANDT2, HELMUT KNEIFEL3 , PAUL MESSNER\ u\VE B. SLEYTR\ EVERLY CONWAY DE MACARI05, HANS-PETER ZABEL!, KARL O. STETTER!, and JOSEF WINTERl 1 Department of Microbiology, University of Regensburg, D-8400 Regensburg, Federal Republic of Germany; 2 Institute of General Microbiology, Chrisrian-Albrechts-Universitat, D-2300 Kiel, Federal Republic of Germany; 3 Institute of Biotechnology III, Kernforschungsanlage Jiilich GmbH, D-5170 Jiilich, Federal Republic of Germany; 4Zentrum fUr Ultrastrukturforschung, Universitat fUr Bodenkultur, A-1180 Wien, Austria; sWadsworth Center for Laboratories and Research, New York State Department of Health, Albany, New York, USA

Received October 23, 1987

Summary An extremely thermophilic, strictly anaerobic sulfate reducing bacterium, strain Z, was isolated from sediments of a hydrothermal vent located near Mount Vulcanello, Vulcano, Italy. On the basis of 16S rRNA cataloguing, strain Z was shown to belong to the archaebacterial kingdom, representing another strain of Archaf!oglobus fulgidus. The weakly motile, irregular coccoid cells showed the fluorescence regarded as typical for methanogens, when viewed under the fluorescence microscope, although no relationship with methanogens was seen from comparative analysis of antigenic fingerprints with antibody S-probes. Furthermore, methane was not produced in significant amounts. Dissimilatory sulfate reduction was found with lactate, pyruvate and 2,3-butandjol as substrates. With thiosulfate as electron acceptor H z/C0 2, formate, lactate, pyruvate or fumarate were utilized as electron donors. Sulfur bloom was not used as electron acceptor. Growth was inhibited by molybdate, a typical inhibitor of sulfate reducers. Lactate was decarboxylated to acetate and part of the acetate was oxidized to COz with the reducing equivalents serving for sulfate reduction. The cell envelope consisted of a hexagonally arranged S-layer. The apparent molecular weight of its subunits, staining PAS-positive, was 132000. No significant amounts of polyamines were detected. The DNA polymerase was sensitive towards aphidicolin, a specific inhibitor of DNA polymerases type a of eucaryotes and of the DNA polymerase of Methanococcus vannielii. The DNA base composition was 45 mol% G+c. Archaeoglobus fulgidus strain Z has been deposited in the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH under number DSM 4139.

Key words: Archaebacteria - Thermophily - Sulfate reduction - Physiology - Acetate oxidation - S-layerPolyamines - 16 S rRNA - Aphidicolin - Glycoprotein

Introduction So far, with the exception of the extremely thermophilic Archaeoglobus fulgidus strain VC-16 (Achenbach-Richter et aI., 1987, Stetter et aI., 1987, Stetter, 1988), no sulfate reducing archaebacteria have been described. Extremely thermophilic eubacterial sulfate reducers are also rare. They are only represented by one species, Thermodesulfobacterium commune (Zeikus et aI., 1983). However, a 11 System. Appl. Microbiol. Vol. 1112

few moderately thermophilic sulfate reducers, e. g. Desulfovibrio thermophilicum (Rozanova and Khudyakova, 1974) and Desulfotomaculum nigrificans (Pfennig et aI., 1981) are known. We describe here the isolation and characterization of an additional strain of Archaeoglobus fulgidus, strain Z, isolated from volcanic environment in Vulcano, Italy.

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Materials and Methods Culture media and cultivation technique. Strain Z was cultured in standard medium containing the following components per I: KCI, 0.335 g; MgCI2 . 6H20, 2.75 g; MgS04· 7H 20, 3.45 g; Na2S04, 4.26 g; NH4CI, 0.25 g; KzHP0 4 , 0.14 g; sodium acetate trihydrate, }. g; NaCl, 18 g; CaCl z . 2H zO, 0.14 g; Fe(NH4h(S04h . 6H 20, 2 mg; Ni(NH4h(S04h . 6H 20, 2.4 mg; Naz W0 4 . 2H 20, 38 f.tg; resazurin, 1 mg; yeast extract (Merck No. 7213),2 g; peptone from casein, tryptically digested (Merck, No. 3753), 2 g; NaHC0 3 , 5 g; L-cysteine . HCI, 0.5 g; Na2S . 9H zO, 0.5 g; vitamin solution (Wolin et al., 1963), 10 ml; trace mineral solution (Wolin et al., 1963), 10 ml and L(+)-Iactate (sodium salt) 5 g. For testing the utilization of other electron donors, L( + )-Iactate was replaced by the substances listed in Table 1. They were added from anaerobic, sterile stock solutions (20% v/v or w/v) to a final concentration of 0.5% (v/v or w/v). To test for the utilization of electron acceptors other than sulfate, 30 mmo1!l thiosulfate, sulfite or nitrate or 1 gil sulfur was added to the standard medium while Na zS04 was omitted and MgS04 . 7H zO was replaced by the respective amount of MgCl z. The pH of all media was adjusted to 7.0. The gas phase was either HJCO z or N 2/C0 2 (80:20%, 300 kPa). The isolate was routinely grown at 78 DC on a rotary shaker at 100 rpm. Mass cultures growing on lactate/sulfate were obtained in three 10 I Biostat V fermenters (Braun Melsungen, FRG), serially flushed with H2/ COz (80: 20%, 30 IIh) to maintain anaerobic conditions and to strip toxic H 2S. Alternatively, strain Z was cultured in a 70 I New Brunswick fermenter. Methanogenic reference strains were cultured in modified medium I (Winter et al., 1984) or medium 3 (Balch et al., 1979) at 37 DC or at the temperatures stated. The anaerobic technique oLBalch et al. (1979) was applied, using a glove box (Aranki and Freter, 1972) for medium dispension and a gas station and disposable syringes for culture transfer (Balch et aI., 1979). Analytical and preparative methods. The optical density of cultures was measured at 578 nm with a Gilford Stasar II photometer (Oberlin, Ohio). Hz, COz and H 2S were analyzed with a Packard model 427 gas chromatograph equipped with a thermal conductivity detector as described by Winter et al. (1984). HzS standards were prepared by acidification of the respective amounts of NazS· hydrate in a serum bottle of 120 ml volume with hydrochloric acid. H 2S in the

gas phase of cultures was determined after acidification of the culture medium to pH 1. The portion of H 2S released from the reducing agent was quantified by subtraction of the amount of HzS released into the gas phase in sterile culture medium after acidification. No corrections were made for H 2S solubility. Volatile fatty acids, alcohols and methane were quantified with a Packard model 433 gas chromatograph, equipped with a flame ionization detector (Winter, 1980). Lactate and pyruvate were determined enzymatically (Horost, 1966). Sulfate was quantified according to Terho and Hartiala (1971). SDS-soluble whole cell extracts of strain Z were separated on 10% slab gels and stained either with Coomassie blue for proteins or periodic acid-Schiff (PAS)-reagent for carbohydrates as described by Messner et al. (1984). For the determination of the mol% G+C content, the DNA of strain Z was purified by CsCI gradient centrifugation. The mol% G+C was determined from the thermal denaturation in 0.1 x SSC as decribed by Marmur and Doty (1962). Calf thymus DNA served as a reference (42 mol% G+C). DNA-DNA-hybridization was carried out after radioactive labelling of the DNA (Kelly et aI., 1970) with a nick translation reagent kit (Bethesda Research Laboratories, USA) using the filter technique (Gillespie and Gillespie, 1971; Birnstiel et aI., 1972) as described by Konig (1984). The antigenic fingerprint of strain Z was determined using a panel of antibody S probes for reference methanogens as described by Macario and Conway de Macario (1983). Antigenic relatedness of strain Z with reference methanogens was measured by quantitative comparison of antigenic fingerprints applying published procedures (Macario and Conway de Macario, 1985). The 16S rRNA cataloguing approach, including 16S rRNA isolation and purification by SDS polyacrylamide gel electrophoresis, RNase Tl digestion, 5'labelling of oligonucleotides with y_32p_ATP, generation of a fingerprint and sequence analysis of isolated nucleotides by the mobility shift method followed published procedures (Stackebrandt et aI., 1982; Stackebrandt et aI., 1985). Similarity coefficients (SAB-values) were calculated as specified by Fox et al. (1977). The presence of polyamines was demonstrated after extraction, derivatization with dansyl chloride and separation by HPLC as described by Scherer and Kneifel (1983). The effect of aphidicolin on DNA-synthesis by crude extracts of strain Z and reference strains was tested by measuring the

Fig. 1. Electron micrograph of a negatively stained preparation of a monopolarly, polytrichously flagellated cell of Archaeoglobus ~ fulgidus strain Z, showing the presence of 4 flagella. Bar, 1 f.tm. Fig. 2. Negatively stained preparation showing the irregularly shaped, coccoid cell body of Archaeoglobus fulgidus strain Z, which is completely covered by the hexagonal S-Iayer lattice. The insertion area of the flagella is marked with an arrow. Bar, 0.5 !-1m. Fig. 3. Freeze-etched preparation of intact cells of Archaeoglobus fulgidus strain Z showing the hexagonal S-Iayer lattice and numerous flagella collapsed onto the S-Iayer surface. Bar, 0,2 !-1m. Fig. 4. Electron micrograph of an ultrathin section of whole cells of Archaeoglobus fulgidus strain Z demonstrating the dense packing after centrifugation. The boxed area corresponds to the arrangement of the cells shown in Fig. 5. Bar, 0.2 !-1m. Fig. 5. Electron micrograph of a fractured, deep etched preparation of Archaeoglobus fulgidus strain Z revealing the characteristic ultrastructure of the hexagonal lattice. Bar, 0.2 f.tm . Fig. 6. Cell envelope profile of a thin sectioned cell of Archaeoglobus fulgidus strain Z. S = surface layer; CM = cytoplasmic membrane. Fig. 7. SDS-polyacrylamide electrophoresis of whole cell extracts of Archaeoglobus fulgidus strain Z. Lane a, molecular weight standard; lane b, Coomassie blue staining; lane c, PAS staining. The band characteristic for the S-Iayer (s) gives a positive reaction with both staining methods.

Thermophilic, Archaebacterial Sulfate Reducer

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154

G. Zellner et al.

incorporation of (methyPH)thymidine 5'-triphosphate into DNA as described (Zabel et aI., 1985). Incorporation of 63Ni2+, 185wol- and 2- 14C-acetate into cells of strain Z was studied as described recently (Zellner and Winter, 1987; Zellner et aI., 1987). Radioactivity was counted with Bray's scintillation cocktail (Bray, 1960) with an efficiency of 86%. Methyl-CoM was prepared from CoM (~-mercap­ toethanesulfonic acid) according to Romesser and Balch (1980). Microscopy. A Zeiss Standard 18 microscope, equipped with a G 436, FT 510, LP 520 filter combination was used for epifluorescence microscopy. Micrographs of thin sections, negatively stained, or freeze-etched cell preparations were taken on a Philips EM 301 electron microscope at 80 kV as described (Messner et aI., 1986). Chemicals and radio chemicals. All chemicals were of analytical grade and were purchas\;d from Merck (Darmstadt, FRG). L( +)-lactic acid was from Fluka (Neu-Ulm, FRG) and was neutralized with NaOH before lIse. 2-Mercaptoethanesulfonic acid (CoM, sodium salt) was obtained from Sigma (Deisenhofen, FRG). L-LDH from rabbit muscle and D-LDH from Lactobacillus leichmannii were purchased from Boehringer (Mannheim, FRG). All gases and gas mixtures were obtained from Linde AG (Miinchen, FRG). y- 32 p_ATP was gurchased from New England Nuclear (NEN, Dreieich, FRG), 3NiC12, Na2185W04, (Methyl3H)thymidine-5'-triphosphate and 2- 14 C-acetate (58.3 mCiI mmol) were obtained from Amersham (Braunschweig, FRG).

Results and Discussion Enrichment and isolation Samples were anaerobically drawn in the coastal region close to mount Vulcanello on Vulcano island in Italy and transferred with syringes into prereduced medium 1 and 3 of Balch et aI. (1979) in serum bottles under a gas atmosphere of H 2/C02 (80: 20%, 300 kPa). Inoculated media were incubated at temperatures between 50°C and 70 0c. In the samples incubated in medium 3 at 70°C, some very small, fluorescent, coccoid bacteria were observed among other non-fluorescent, coccoid and rod-like bacteria. No enrichment was achieved in the presence of H 2/C0 2 , formate, methanol, methylamines and acetate. After transfer into standard medium that contained lactate and sulfate, growth of the fluorescent organisms was greatly improved. After enrichment, a pure culture (strain Z) was obtained by repeated application of the serial dilution technique. Purity was checked by inoculation of strain Z into a rich, glucose, peptone and yeast extract containing medium and microscopical control after growth. Characteristics described in the following paragraphs justified an assignment of strain Z as Archaeoglobus fulgidus (Stetter, 1988). Strain Z was deposited in the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, D-3300 Braunschweig under No.DSM 4139. Cell morphology A. fulgidus strain Z is an irregularly shaped, coccoid bacterium of 1.0-1.5 !-tm in diameter, motile by the aid of four flagella (Fig. 1,2). Cells stained Gram-negative. Similar to strain VC-16, A. fulgidus strain Z revealed the greenish fluorescence restricted to methanogens (Doddema and Vogels, 1978), Streptomyces griseus (Eker et aI.,

1980) and several other Actinomycetes (Daniels et aI., 1985). The fluorescent compound might be related to coenzyme F420 found in all methanogens and in strain VC16 (Stetter et aI., 1987). The cell envelope consisted of an S-layer with hexagonally arranged subunits, staining PAS-positive (Fig. 2,3,5). The center-to-center spacings of the morphological units were ca. 17.5 nm. A similar architecture was found for Slayers of most archaebacteria (Messner et aI., 1986; Sleytr et ai. 1986) whereas in eubacteria square and oblique lattices have frequently been observed (Sleytr and Messner, 1983). Centrifugation for pelleted samples used for thin sections and freeze-etching yielded closely packed cells with characteristic fractures (Fig. 5). Only surface arrays adjacent to the intercellular space (Fig. 4, boxed area) became visible during etching. Thin sections showed no rigid cell wall component, but an S-layer as the only envelope component outside the cytoplasmic membrane (Fig. 6). The Slayer was soluble in 0.1 % SDS. SDS-PAGE of SDS-soluble whole cell extracts revealed a broad spectrum of cellular proteins, but only one band was stainable by the PASprocedure for carbohydrates. Presumably, this band with an apparent molecular weight of 132000 represented the S-layer glycoprotein (Fig. 7). Antigenic fingerprinting The antigenic fingerprint of strain Z was determined at positions 2, 3, 11, 12, 13, 14, 15, 17, 18,20,23,24,26, 28 and 29 (sequence defined by the S probes for the following reference methanogens (Macario and Conway de Macario, 1983, 1985): Methanobacterium formicicum (MF), Methanosarcina barkeri (MS), Methanobacterium thermoautotrophicum (GC1), Methanobacterium thermoautotrophicum (~H), Methanococcus vannielii (SB), Methanococcus voltae (PSv), Methanogenium marisnigri GRlm), Methanogenium cariaci GRlc), Methanosarcina mazei (S6), Methanosarcina thermophila (TM1), Methanomicrobium mobile (BP), Methanothermus fervidus (V24S), Methanococcus maripaludis OJ), Methanoplanus limicola (M3), Methanococcus thermolithotrophicus (SN1), respectively). Comparison of the antigenic fingerprints of strain Z with reference methanogens showed no relationship. Therefore, immunologically, strain Z was not found to be related to any of the tested methanogenic reference strains recognized by the antibody probes used. Comparison of oligonucleotides of the 16S rRNA The oligonucleotide catalogue of the 16S rRNA of strain Z was compared with that of other bacteria. Strain Z shared each of the four universal archaebacterial oligonucleotides (CCCUACG, CUCCUUG, CACACACCG, AAACUUAAAG; Mc Gill et aI., 1986). Similarity coefficients (SAB values), calculated for the catalogue of strain Z, a variety of catalogues from eubacteria and all published catalogues for archaebacteria ranged between 0.05 to 0.16 and 0.22 to 0.40, respectively. A similarity coefficient SAB of 0.92 was found with

Thermophilic, Archaebacterial Sulfate Reducer

A. fulgidus strain VC-16. Strain Z is therefore considered to be an additional strain of A. fulgidus.

155

Glucose was not utilized by either of the two strains in the rich, yeast extract and peptone containing standard medium with sulfate as electron acceptor (Table 1). Strain VC-16 has been reported (Stetter et aI., 1987) to grow on glucose/SO~- and starch/SO~- in a mineral medium (Stetter et aI., 1987; Stetter, 1988); while strain Z was not able to grow on glucose/SOl- in standard medium from which lactate was omitted. Lactate utilization and product formation by strain Z is shown in Fig. 8. Fourty-four mmolll L( + )-lactate were converted to 12 mmolll acetate and to CO 2 (not quantitatively determined) under the formation of 40 mmolll sulfide. Growth proceeded to an optical density of E578 = 1.2. From the decarboxylated lactate, 44 mmolll acetate and 22 mmolll sulfide should have been generated by a pathway similar to that of many sulfate reducers. However, most of the acetate (30 mmolll) was probably oxidized to CO 2, while little may have served for cell synthesis. Since 44 mmollliactate was fermented to 12 mmolll acetate and 108 mmolll CO 2, reducing equivalents for the formati~n of 54 mmolll sulfide should have been available, of which

Substrates and metabolism

Strain Z used sulfate, thiosulfate or sulfite (only with fumarate as electron donor) as electron acceptors (Table 1). Nitrate or molecular sulfur were not utilized. Growth to high final optical densities was achieved with L( + )lactate, D( - )-lactate and pyruvate in the presence of sulfate or thiosulfate. Growth on H 2/C0 2, formate and fumarate was found only in the presence of thiosulfate, but was less effective (Table 1). A variety of other typical electron donors for dissimilatory sulfate reduction could not be used by strain Z (Table 1). The substrate spectrum of strain VC-16, grown in parallel under identical growth conditions, is also included in Table 1. Strain VC-16 was able to grow with formate/sulfate or formate/thiosulfate to high optical densities, while only little growth of strain Z on formate/thiosulfate was found. Repeated transfers of strain Z in the latter medium did not result in better growth.

Table 1. Growth of Archaeoglobus fulgidus strains Z and VC-16 in the presence of different electron acceptors (30 mmol/l) and electron donors (0.5% w/v or v/v) expressed as maximal optical densities (E578 ) Electron donors

Electron acceptors

Strain VC-16

Strain Z Na2S203

Na2S03

HiC0 2 * H 2/C0 2 **

0.16 0.16

nd

0.13 nd

Formate Acetate Propionate n-Butyrate

0.10

0.39

0.76

Methanol Ethanol I-Propanol 2-Propanol I-Butanol 2-Butanol Glycerol 2,3-Butandiol L(+) or D(-)-Lactate Pyruvate Fumarate Malate Succinate Choline Citrate Glucose * * * Fructose Ribose

0.08 0.15 0.10

0.10

nd

0.92 0.74

0.48 0.41 0.10

nd

0.09

nd 0.67 0.53

0.73 0.67

nd nd

nd nd

0.07

Cultures were grown at 80 DC in standard medium (MgS0 4 • 7H2 0 replaced by MgCh) with electron donors and acceptors as indicated. N/ CO2 (80:20%, 300 kPa) was the gas phase. nd = not detemined, - = no growth. Growth in standard medium on either yeast extract or peptone reached E578 of 0.05 WIth SO~- and E578 of 0.15 with S20~- and was subtracted. * H 2/C0 2 , 80:20%,300 kPa; ** H/C0 2, 80:20%, 300 kPa plus 10-7 M WOh * * * grown at 66°C. No growth was found with So or in the absence of an electron acceptor on H 2 /C0 2, formate, methanol, ethanol, lactate, pyruvate, fumarate, glucose, fructose and ribose. Other electron donors not tested.

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G. Zellner et al.

40 mmolll was actually detected in the gas phase. The difference between expectation and actual finding may be due to H 2S solubility in the acidified medium. In a medium containing 8 J.Lmol/ml acetate, 62%, and in a medium containing only 0.6 nmollml acetate, 75% of 40

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the acetate, derived from lactate decarboxylation, were oxidized to CO 2 (Table 2). In the medium with a very low acetate concentration but with yeast extract and peptone, much better growth was obtained (Table 2). Only minor amounts of acetate were incorporated into the cells. Lactate utilization by strain Z was slightly reduced in the presence of H2 in the gas phase, while it was hardly affected by H2 in cultures of strain VC-16 (Table 3). Cultures of strain VC-16, grown in the presence of 10, 20,30 and 40 J.Lmollmllactate oxidized the supplied lactate completely to CO 2 , Strain Z, grown in parallel, always released between 24 - 35% of the acetate, generated during lactate decarboxylation, into the medium (data not shown) . The stoichiometry of lactatelsulfate utilization by A. futgidus strain Z was in part in accordance with the incomplete oxidation of lactate during dissimilatory sulfate reduction, according to equation I: I) 2 CH3 CHOHCOOH + SO/- ~ 2 CH3 COOH + 2 CO 2 + S2- + 2 H 2 0 ~Go , = -160.3 kJ/mol (Thauer et al., 1977)

50 60 Time (hI

Fig. 8. Kinetics of L( + )-lactate/sulfate conversion and acetate, CO 2 and sulfide formation by Archaeoglobus fulgidus strain Z. Cultures were grown in standard medium at 78 °C and 100 rpm on a shaker. • = Optical density (E578 ) ; X = L(+)-lactate;. = Acetate; .... = Sulfide.

However, a great proportion of the acetate (65% or more) was completely oxidized to CO 2 (Fig. 8, Table 2), probably in accordance with equation II: II) CHrCOOH + SO/- ~ 2 CO 2 + S2- + 2H2 0 ~Go, = - 47.3 kJ/mol (Thauer et al., 1977)

Table 2 , Decarboxylation of lactate and total oxidation of part of the acetate by strain Z Modification of the medium'

L( + )-lactate consumption !-tmol/ml

Optical density E578

Omission of yeast extract and peptone. 8 !-tmol/ml 2- 14C-acetate (195 Bq/!-tmol) added

0.29

8.4

0.6 nmol/ml 2- l4 C-acetate (2740 Bq/nmol) added

0.68 3

37.4

14COzl

2- 14 C-acetate initially Bq/ml !-tmol/ml

finallr Bq/ml !-tmol/ml

Bq/ml

14C in cells Bq total

8.0

1560

11.2

931

625

4.4

0.0006

1640

9.2

404

1191

50.4

Cultures were grown in duplicate in modified standard medium, containing 40 !-tmol/ml L( + )-lactate, 40 !-tmol/ml NazS04 and labelled acetate as indicated. Incubation was at 78°C on a shaker (100 rpm) for 2 days. I Determined by subtraction of the label in acidified culture supernatant from initial 14C-activity. 2 Radioactivity in culture supernatant after 14C02-extrusion by acidification with H 2S0 4 , 3 Growth in the presence of yeast extract was better, resulting in an almost quantitative utilization of lactate. a

Strain

Gas phase

Optical density ES78 1

L(+ )-lactate consumed !-tmol/ml

Acetate produced !-tmol/ml

Sulfate consumed !-tmol/ml

Methane produced !-tmol/ml

VC-16

N 2/C0 2 H z/CO 2

0.50 0.50

23.0 20

0.0 0.0

27.7 22.4

0.20 0.11

Z

N /C0 2 Hz/CO z

0.56 0.51

35 25

8.3 8.6

34.4 27.6

0.13 0.08

Cultures were grown in standard medium without acetate in duplicate for 52 h at 80°C. 1 Optical density values corrected for growth in standard medium without lactate/sulfate (control).

Table 3. Stoichiometry of lactatel sulfate utilization by Archaeoglobus fulgidus strains VC-16 and Z grown on L( + )-lactate/S O~-

Thermophilic, Archaebacterial Sulfate Reducer

The fermentation stoichiometry of strain Z may thus be represented by equation III: III) 2 CH3 CHOHCOOH + 2 50/- ~ CH3COOH CO 2 + 2 S2- + 4 H 2 0 LlGo, == -: 207.6 kJ/mol

1:10

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A. fulgidus strain VC-16 was apparently able to oxidize lactate completely to COz (Table 3). Although oxidation of acetate per se is endergonic and would not proceed in the absence of an electron acceptor, under conditions of sulfate reduction acetate oxidation becomes favourable. Thus, sulfate reducers can gain more energy for growth by sulfate respiration according to equation III than to equation I. The best energy balance is obtained by strain VC16, when acetate is totally oxidized. Small amounts of methane were detected in cultures of strain VC-16 and strain Z (Table 3). Addition of methyl coenzyme M, however, did not stimulate methane production. The maximum methane generation of both strains was considerably lower than for instance in Methanocorpusculum parvum (Zellner et aI., 1987), but higher than the so-called mini-methane-production of some sulfate reducers (Postgate, 1969). Vice versa, methanogens were shown to produce HzS by reduction of molecular sulfur (Stetter and Gaag, 1983). However, energy conservation has not been demonstrated for either methane production by sulfate reducers, or sulfide production from sulfur by methanogens. Whenever sulfate reduction by methanogens has been demonstratea, for instance in Methanococcus thermolithotrophicus (Daniels et aI., 1986), it served assimilatory purposes. The methane produced during the mini-methane-production of eubacterial sulfate reducers has been shown to be derived from the methyl-group of pyruvate or methionine (Postgate, 1969) and not from COz-reduction or reduction of a methyl-moiety as in methanogens. Archaebacterial sulfate reducers may produce small amounts of methane in a manner similar to eubacterial sulfate reducers. Addition of 0.1 mmolll sodium molybdate, instead of the 0.4 [lmolll normally present in the medium, completely prevented growth of strain Z (Fig. 9a,b). Molybdate probably acts in a similar manner as in eubacterial sulfate reducers (Huisingh and Matrone, 1972). Tungstate, an antagonist of molybdate did not prevent the inhibition by molybdate (Fig. 9a). It remains to be proven, whether in cells of strain Z molybdate inhibited also the first enzyme of sulfate reduction, ATP-sulfurylase, as in eubacterial sulfate reducers.

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Temperature and pH optimum A. fulgidus strain Z grew in a temperature range of > 60°C and < 90 °C with the optimum between 75 and 80°C (Fig. 10), while the type strain VC-16 has been reported to grow optimally at a temperature of 83°C (Stetter, 1988). The shortest generation time for strain Z growing on lactate/sulfate was 3.5 h, while in the same medium strain VC-16 had a generation time of 5 h. The shortest generation time of strain VC-16 with formate/sulfate was 4 h and with formate/thiosulfate it was 5.5 h, while strain

157

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80

Tempera ture

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90

Fig. 10. Optimal temperature for growth of Archaeoglobus fulgidus strain Z with lactate/sulfate. Average of triplicate cultures in standard medium. No growth at 60°C and 90 DC, respectively.

158

G. Zellner et al.

Z did not or only very poorly grow under these conditions (Table 1). The optimal pH for growth of strain Z was around 7.0. Inr;arporation of 63Ni and 185W into cells of strain Z

During growth of strain Z on lactate/sulfate, nickel and tungstate were incorporated into cells (Table 4). Nickel may be essential for the hydrogenase and carbon monoxide dehydrogenase, while tungstate may have been incorporated into formate dehydrogenase. Polyamine content of strain Z

While significant amounts of one or more polyamines were detected in members of several different thermophilic archaebacteria (Kneifel et aI., 1986), strain Z contained

only minor amounts of polyamines (Table 5). A different polyamine pattern was found for autotrophically and heterotrophically grown cells (Table 5). The dis.tribution pattern of polyamines was shown to be a useful marker for the chemotaxonomy of archaebacteria, for instance of methanogens (Scherer and Kneifel, 1983; Kneifel et aI., 1986). Similar to the members of the order Methanomicrobiales, little homospermidine was found in cells of strain Z. There was also a certain similarity with the Thermoplasmales, but in contrast to the Sulfolabales and Thermoproteales, strain Z did not contain norspermidine and norspermine (Table 5). Inhibition of DNA-synthesis byaphidicalin

DNA synthesis of strain Z was strongly inhibited by 30 11M aphidicolin. A similar inhibition was observed for

Radioactive isotope

Optical density Em

Dry weight of cells mg/m!

Amount of radioactivity incorporated added Bq/mg Bq/m! Bq/ml cells

Isotope incorporation ng/mg % of cells supply

63Ni 185W

0.82 0.80

0.40 0.40

90 685

0.14 4.8

25 180

63 450

28 27

Table 4. Incorporation of 63Ni and 185W into cells of Archaeoglobus fulgidus strain Z. Cultures were grown in the presence of 0.5% L( + )-lactate and 30 mmolll NaZS04 at 78°C on a shaker at 100 rpm for 36h

Table 5. Comparison of the polyamine content of Archaeoglobus fulgidus strain Z with that of other thermophilic archaebacteria Organisms

DSM-No.

Methanobacteriales Mb. thermoautotrophicum]W 501 Methanobacterium wolfeih Methanobacterium wolfeia Methanothermus fervidus*

Growth temperature DC DAP

SPM

0.07

0.36

4.35

10.6 1.22

1.72

2036 2970 . 2970 2088

60 60 60 85

0.03 n.d.

0.13

Methanococcales Methanococcus thermolithotrophicush Methanococcus jannaschiih

2095 2661

60 80

0.03

0.24

Methanomicrobiales Methanogenium thermophilum h Strain Ratisbona Methanosarcina thermophila h

2640 1825

60 55

0.03

5.00 0.39

0.24

15.5 8.57

0.15

24.5

0.18 0.10

9.00

2.81

4.90 0.07 12.0 0.07 0.32 0.35

0.50 0.10 2.57 0.10 0.03

Thermoplasmales Thermoplasma spec. *

70

Sulfolobales Sulfolobus acidocaldarius' Thermoproteales Thermoproteus tenax* Thermofilum pendens' Desulfurococcus spec. • Thermococcus spec.' Thermodiscus maritimus' Pyrodictium occultum* Archaeoglobus fulgidus strain Zb Archaeoglobus fulgidus stfain

za

* Kneifel et al. (1986);

n.d.

Polyamines (Ilmoilg cell dry weight)' HSPD NSPM PUT NSPD SPD

a

639

70

0.47

0.82

3.28

19.8

2078 2475

0.36 0.66 0.29 0.16

0.07 0.30 0.18 0.14 0.06 3.10

0.22 0.03 0.64 0.06 0.05 0.08

6.50 0.63 3.90 0.10 0.12 1.87

0.29 0.04

2709

70 70 70 70 85 105

4139 4139

78 78

0.03 0.14

0.25 0.39

0.41

0.07

autotrophic growth (yeast extract and peptone omitted); h heterotrophic growth

0.06

2.70

0.39 0.10

Thermophilic, Archaebacterial Sulfate Reducer

Halobacterium halobium and representatives of the Methanococcaceae, while most members of the Methanomicrobiaceae were only slightly or not at all sensitive towards aphidicolin (Table 6). Aphidicolin is a specific inhibitor of eucaryotic DNApolymerases type a (Hubermann, 1981) and of some archaebacterial DNA-polymerases (Forterre et al., 1984; Zabel et al., 1985). Like in eucaryotes, aphidicolin competitively interacted with the dCTP binding site of the DNA-polymerase of Methanococcus vannielii (Zabel et al., 1987) and it might be speculated, that the same mechanism of interaction is prevalent in A. fulgidus strain

Z.

Table 6. Effect of aphidicolin (30 fAmol/l) on DNA-synthesis in crude extracts. Numbers represent % activity of DNA-polymerase of a non-inhibited control. Assay as reported by Zabel et al. (1987) Crude extract of

DSMNo. of strain

% DNApolymerase activity

Thermoplasma acidophilum

25905 1

100

639

100

Methanobacterium formicium Methanobacterium thermoautotrophicum Methanobacterium wolfei Methanothermus fervidus

1535 1053 2970 2088

100 100 100 100

Methanospirillum hungatei Methanosarcina barkeri Methanogenium tationis Methanocorpusculum parvum

864 800 2702 3823

100 94 90 75

Methanococcus vannielii Methanococcus voltae Methanococcus thermolithotrophicus Methanococcus ;annaschii

1224 1537 2095 2661

32 25 39 59

Sulfolobus acidocaldarius

Halobacterium halobium Archaeoglobus fulgidus strain Z

Mol% G

+

670

60

4139

49

C of the DNA and DNA-DNA-homology

The G+C content of the DNA of A . fulgidus strain Z was 45 mol%, compared to 46 mol% of strain VC-16 (Stetter, 1988). DNA-DNA-homology of both strains (3 determinations) was between 95 and 98%, indicating that both strains belong to the same species. Acknowledgement. This work was supported by grants of the Deutsche Forschungsgemeinschaft (DFG) to J. W. (Wi-52417-7) and to E. S. (Sta 184/6-1). Immunological studies were supported by grant No. DE-FG02-84R13197 from the US Department of Energy to E.C. de M. We thank A. j. L. Macario for his input in the immunological work. Polyamines were determined with expertise by Mrs. E. Schoelgens, KFA Jiilich. S-layer studies were supported by a grant from the Osterreichisches Bundesministerium fur Wissenschaft und Forschung to U.B.S.

159

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Dr. G. Zellner and Professor Dr. j. Winter, Institute of Microbiology, University of Regensburg, Universitiitsstr. 31, D-8400 Regensburg