System. Appl. Microbiol. 23, 305-314 (2000) SYSTEM4T1C AND © Urban & Fischer Verlag _ht--,---tp:_Ilw_w_w_.ur_ba_nf_isc_h_er._de---,-/jo_u_rna_ls_/s_am ____________ APPLIED MICROBIOLOGY

Respiration of Arsenate and Selenate by Hyperthermophilic Archaea

1 Lehrstuhl fur Mikrobiologie und Archaeenzentrum, Universitat Regensburg, Germany 2 Betriebseinheit "Materialuntersuchung" - Fakultat fur Chemie und Pharmazie, Universitat Regensburg, Germany

Received May 30, 2000

Summary

A novel, strictly anaerobic, hyperthermophilic, facultative organotrophic archaeon was isolated from a hot spring at Pisciarelli Solfatara, Naples, Italy. The rod-shaped cells grew chemolithoautotrophically with carbon dioxide as carbon source, hydrogen as electron donor and arsenate, thiosulfate or elemental sulfur as electron acceptor. H2S was formed from sulfur or thiosulfate, arsenite from arsenate. Organ­otrophically, the new isolate grew optimally in the presence of an inorganic electron acceptor like sulfur, selenate or arsenate. Cultures, grown on arsenate and thiosulfate or arsenate and L-cysteine, precipitat­ed realgar (AS2S2). During growth on selenate, elemental selenium was produced. The G+C content of the DNA was 58.3 mol%. Due to 16S rRNA gene sequence analysis combined with physiological and morphological criteria, the new isolate belongs to the Thermoproteales order. It represents a new species within the genus Pyrobaculum, the type species of which we name Pyrobaculum arsenaticum (type strain PZ6\ DSM 13514, ATCC 700994).

Comparative studies with different Pyrobaculum-species showed, that Pyrobaculum aerophilum was also able to grow organotrophically under anaerobic culture conditions in the presence of arsenate, sele­nate and selenite. During growth on selenite, elemental selenium was formed as final product. In con­trast to P. arsenaticum, P. aerophilum could use selenate or arsenate for lithoautotrophic growth with carbon dioxide and hydrogen.

Key words: archaea - Pyrobaculum arsenaticum - hyperthermophile - solfataric - biogeochemistry-Re­algar - arsenic - arsenate respiration - selenate respiration

Introduction

Over the past years it was shown, that a number of mesophilic organisms within the domain Bacteria can use toxic metal ions as electron acceptors for anaerobic respi­ration, including arsenate, selenate or selenite (MACY et al., 1993; AHMANN et al., 1994; OREMLAND et al., 1994, MACY et al., 1996; NEWMAN et al., 1997a; SWITZER BLUM et al., 1998; STOLZ et al., 1999; STOLZ and OREMLAND, 1999; MACY et al., 2000). For arsenate reduction, Chrys­iogenes arsenatis is able to use acetate as the sole electron donor and carbon source (MACY et al., 1996). Desulfo­tomaculum auripigmentum, Bacillus arsenicoselenatis, Bacillus selenitireducens and Sulfurospirillum barnesii respire arsenate with lactate, forming acetate and CO2,

while Sulfurospirillum arsenophilum oxidized lactate completely to CO2 (LAVERMAN et al., 1995; NEWMAN et al., 1997a; SWITZER BLUM et al., 1998; STOLZ et al., 1999). Arsenite was identified as the final product formed during

dissimilatory growth on arsenate (STOLZ and OREMLAND, 1999). When grown on arsenate in the presence of cys­teine or sulfate, D. auripigmentum precipitates As2S3 both intra- and extracellularly (NEWMAN et al., 1997b).

Lactate is the preferred substrate for the respiration of selenate or selenite, with the production of acetate and CO2 , During growth on selenate, Thauera selenatis and B. arsenicoselenatis produce selenite, while S. barnesii re­duces selenate through selenite to elemental selenium (MACY et al., 1993; SWITZER BLUM et al., 1998; OREM­LAND et al., 1994). B. selenitireducens can respire selenite and selenium is formed as final product (SWITZER BLUM et al., 1998). Beside lactate, T. selenatis can use acetate as carbon source and electron donor for selenate respiration (MACY et al., 1993).

Within the domain Archaea, a diverse variety of hy­perthermophiles with unusual metabolic properties are

0723-2020100123/03-305 $ 15.00/0

306 R. HUBER et ai.

known (STETTER, 1995; STETTER, 1999a). Under chemo­litho autotrophic culture conditions, they gain energy by aerobic and anaerobic types of respiration with hydrogen as an important electron donor. Depending on the organ­ism, oxygen, nitrate, nitrite, sulfur, oxidized sulfur com­pounds, ferric iron and carbon dioxide are used as elec­tron acceptors (STETTER, 1999a; STETTER, 1999b).

Within the Crenarchaeota branch of the Archaea, the order Thermoproteales is represented by the Thermofi­laceae and Thermoproteaceae (BURGGRAF et aI., 1997). Within the Thermoproteaceae, members of the genus Py­robaculum exhibit an optimum growth temperature of 100°C (HUBER et aI., 1987; HUBER and STETTER, 1992; VOLKL et aI., 1993). To date, three different species are known (HUBER et aI., 1987; HUBER and STETTER, 1992; VOLKL et aI., 1993). Pyrobaculum islandicum and Py­robaculum organotrophum are strictly anaerobic. Both species can grow heterotrophically, using sulfur or oxi­dized sulfur compounds as electron acceptors, forming H2S and CO2 (HUBER et aI., 1987; SELIG and SCHONHEIT, 1994). Alternatively, P. islandicum can reduce ferric iron under organotrophic growth conditions (VARGAS et aI., 1998). Chemolithoautotrophically, P. islandicum grows with hydrogen, carbon dioxide and sulfur. The marine Pyrobaculum aerophilum is a facultative anaerobe, growing on a variety of organic compounds by aerobic respiration under microaerophilic conditions or by dis­similatory nitrate or nitrite reduction (VOLKL et aI., 1993). With hydrogen or thiosulfate as electron donor, P. aerophilum is able to grow chemolithoautotrophically by reduction of oxygen or nitrate (VOLKL et aI., 1993). Re­cently, a new Pyrobaculum species was isolated, which is able to reduce ferrous iron in order to form magnetite (STETTER, 1999b; STETTER, 1999c). By coexistence of anaerobic iron oxidizers like Ferroglobus placidus, a hot iron cycle was postulated, which may have existed al­ready within early life forms on earth (STETTER, 1999c).

Here, we report for the first time on the ability of members of the archaeal domain to grow anaerobically by chemolithoautotrophic reduction of arsenate or sele­nate. They belong to the hyperthermophilic genus Pyro­baculum and may contribute significantly to the biogeo­chemical cycle of heavy metals like arsenic in high-tem­perature environments.

Materials and Methods

Strains and culture conditions Pyrobaculum islandicum DSM 4184 (HUBER et ai., 1987),

Pyrobaculum organotrophum DSM 4185 (HUBER et ai., 1987) and Pyrobaculum aerophilum DSM 7523 (VOLKL et ai., 1993) were obtained from the Lehrstuhl fiir Mikrobiologie, Regens­burg, culture collection.

P. aerophilum was grown anaerobically on BS medium (for litho trophic culture conditions) or on BSY medium (for organ­otrophic culture conditons) with a gas phase consisting of 300 kPa H2-C02 (80:20, v/v; VOLKL et ai., 1993). P. islandicum, P. organotrophum and the new isolate PZ6':- were cultivated in 1120 MG-CB-medium, containing the following (per liter of double-distilled H20): 17 mg of KCI, 1215 mg of MgCI2 x

6HzO, 12 mg of NH4CI, 7 mg of CaCI2 x 2H20, 7 mg of KzHP04 x 3HzO, 900 mg of NaC!, 50 mg of NaHC03, 50 mg of CaC03, 0.1 mg of (NH4hFe(S04h, 10 ml of a trace vita mine solution (BALCH et ai., 1979), and 10 ml of a trace mineral solu­tion. The mineral solution contained (per liter of double-dis­tilled H 20): 2475 mg of MgClz x 6H20, 527 mg of MnCl2 x 4HzO, 1000 mg of NaCl, 71.5 mg of FeClz x 4H20, 180 mg of CoCI2 x 6HzO, 100 mg of CaClz x 2H20, 85 mg of ZnClz, 6.8 mg of CuCl2 x 2H20, 3.15 mg of KCl, 5.6 mg of AlCl], 10 mg of H 3BOj, 10 mg of NazMo04 x 2HzO, 10 mg of NaZW04 x 2H20, 10 mg of NaZSe04' 140 mg of (NH4hNiClz x 6HzO. The pH was adjusted to 6.5-7 with HC!. In the metabol­ic studies, potential inorganic electron acceptors were added in the following final concentrations: Elemental sulfur (1 %, w/v), Na2SZ0j x 5HzO (0.1%, w/v), MgS04 (0.1%, w/v), NazSO] (0.01 or 0.1 %, w/v), L-cysteine (0.05%, w/v), KNO j (0.1 %, w/v), iron(lII) citrate (15 mM), SbCls (200 ]lM), NazHAs04 x 7HzO (10 mM), NaAsOz (10mM), NaZSe04 (20 or 50 mM) and NazSeO] (10 or 20 mM). To obtain organotrophic culture conditions, the medium was supplemented with 0.02 % yeast extract and 0.05% peptone for P. islandicum and P. organotro­phum, while PZ6" was grown on a mixture of organic matter consisting of yeast extract, peptone, meat extract and brain heart infusion (each at a final concentration of 0.01 %, w/v). For determination of salt dependence of growth, the salt con­centration in the medium was adjusted with NaCi. Strictly anaerobic culture media were obtained according to the anaero­bic technique of BALCH and WOLFE (1976). Oxygen was re­duced by adding 0.05% NazS x 7-9H20 with resazurin (5 ]lg/l) as the redox indicator. For growth studies on L-cysteine, NazS x 7-9H20 was omitted and the medium was reduced by addition of 0.05% L-cysteine. Prior to autoclaving, the medium was dispensed in 10 ml aliquots into 120 ml serum bottles, which were stoppered, and the gas phase was exchanged with the desired gas mixture. Unless indicated otherwise, the gas mixture consisted of 300 kPa H Z-C02 (80:20, v/v). All Pyro­baculum-species used in this study were grown in the dark at 95°C under shaking (45 rpm), if not mentioned otherwise.

Light microscopy Cells were routinely observed with a Zeiss (Oberkochen,

Germany) Standard phase contrast microscope with an oil im­mersion objective 100/1.3. Micrographs were taken by a Olym­pus BX60 (Hamburg, Germany) phase contrast microscope. For the visualization of PZ6 'f cells on solid surfaces, a DAPI flu­orescence staining method was employed (HUBER et ai., 1985).

XRD, SEM and EDS The precipitates formed during growth of P. aerophilum and

PZ6 ,- were analysed by X-ray diffraction (XRD), scanning elec­tron microscopy (SEM) and energy-dispersive X-ray spec­troscopy (EDS). The precipitates were collected from the culture medium by centrifugation for ten minutes, and dried overnight at room temperature. This procedure was carried out in an anaero­bic chamber. Afterwards, samples were dispersed in double-dis­tilled HzO and filtered through a polycarbonate filter with a pore size of 0.2 ]lm. For SEM, samples were gold-coated with a Sput­ter-Coater POLARON E 5200. For the analysis of the precipi­tates, a JEOL JSM-840 scanning electron microscope was used, operating at 25 kV and equipped with an EDAX PV 9100 ana­lyzer for EDS. The XRD spectrum (Philips PW 1729/1710) of a crystalline precipitate was identified by comparing this spectrum with files from the JCPDS data base.

Metal analysis The quantitative composition of the chemical elements in the

sediments was determined by ICP analysis (Inductively Coupled Plasma; JY 70 Plus, Jobin Yvon).

Analysis of metabolic products H 2S was determined as described by HUBER et al. (1986). Ar­

senate was determined indirectly by measuring the difference between the untreated and reduced samples, arsenite by mea­suring the difference between the oxidized and untreated sam­ples (JOHNSON and PILSON, 1972). Background phosphate (re­duced sample) was measured directly by the molybdenum blue spectrophotometric assay (JOHNSON and PILSON, 1972).

Determination of the G+C content The DNA was isolated by French press treatment of the

cells. The DNA was purified on hydroxyapatite (CASHION et aI., 1977) and hydrolyzed with PI nuclease. The nucleotides were de phosphorylated (MESBAH et aI., 1989) and analyzed by high­performance liquid chromatography (T AMAOKA and KOMA­GATA, 1984). The G+C content was calculated according to MESBAH et al. (1989) using non-methylated A-DNA (Sigma) as a standard (G+C content 49.858 mol%). The determination of the G+C content of the DNA was carried out by the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig. The calculated G+C content is the mean of three independent determinations.

165 rRNA gene sequence analysis Isolation of nucleic acids, amplification of the 16S rRNA

genes and sequencing was carried out as described by EDER et al. (1999). The archaeal sequences were determined with the following forward and reverse primers: 8 aF (5' TCYGGTT GATCCTGCC 3'), 767 aR (5' TTCGYCCCTCACCGTCGG 3'), 1119 aR (5' GGYRSGGGTCTCGCTCGTT 3'), 1406 uR (5' ACGGGCGGTGTGTRCAA 3') and 1512 uR (5' ACGGH­TACCTTGTTACGACTT 3'). For the phylogenetic analyses, an alignment of approximately 10,500 homologous full and par­tial primary sequences available in public databases (ARB Pro­ject, LUDWIG and STRUNK, 1997; LUDWIG, 1995) was used. The new 16S rRNA gene sequences of PZ6" and WI]3 (1,418 and 1,409 nucleotides) were fitted in the 16S rRNA alignment by using the respective automated tools of the ARB software pack­age (LUDWIG and STRUNK, 1997; LUDWIG et aI., 1998). For tree reconstruction, maximum-parsimony, distance-matrix (Jukes and Cantor correction) and maximum-likelihood (fastDNAml) methods were applied as implemented in the ARB software package.

Nucleotide sequence accession number The 16S rRNA gene sequences of isolates PZ6" and WIJ3

were deposited in the EMBL Nucleotide Sequence Database under accession number A]277124 and accession number A]277125.

Results

Collection of samples

In the Pisciarelli Solfatara area near Naples, Italy (SEGERER et aI., 1985), acidic and neutral sediment sam­ples with original temperatures up to 100 QC were taken from different hot, strongly gassed water holes (samples PZ1-PZ7). During the joint multidisciplinary RV Sonne expedition SO 135 cruise (STOFFERS et aI., 1999a), four sediment samples WI]1-WI]4 with temperatures between 85 and 100 QC were taken on White Island Vulcano, Bay of Plenty, New Zealand.

The sample material was transferred into 100 ml bot-

Respiration of Arsenate and Selenate 307

tles (Schott), which were then tightly sealed with rubber stoppers. The samples were transported to the laboratory without temperature control.

Mineralogical and chemical analysis of original sample material from Pisciarelli Solfatara

The mineralogical analysis of the neutral greyish­black sediment sample PZ7 (original pH: 6.0; original temperature: 88 QC) revealed, that the main component is amorphous silica. In addition, alunite, pyrite, quartz, baryte and feldspar were detected.

ICP-analysis was performed with the neutral PZ7 sample in comparison with the acidic, greyish sediment sample PZ1 (original pH: 1.5; original temperature: 96 QC; Table 1). The analysis of PZ7 and PZ1 revealed high concentrations of arsenic between 0.2-0.4 gram per kg soil (Table 1).

Table 1. Chemical composition of hot sediments from Piscia­relli Solfatara, Naples, Italy (ICP analysis).

Trace element

Fe Mn Zn Hg Sb Au As Cd, Co, Cr, Cu, Mo, Ni, Se, Sn

Acidic soil PZ1 (mg/kg)

36000

40 5

400

Neutral soil PZ7 (mg/kg)

18000 20

100 5

80

200

- not present or below the detection limit of 0.5 mg per kg of sediment.

Enrichment and isolation

The high concentration of arsenic in the Pisciarelli Sol­fatara area prompted us to look for the possible exis­tence of hyperthermophiles able to use arsenic com­pounds in their metabolism. First cultivation experiments were carried out at 95 QC anaerobically with original sample PZ6'" (original pH: 6.0; original temperature: 92 QC), using arsenate under organotrophic culture con­ditions as possible electron acceptor. After two days incu­bation, about 107 rod-shaped cells became visible in the culture medium, which could be successfully transferred to the same medium. A pure culture was obtained by the selected cell cultivation technique, using the "optical tweezers trap" (HUBER et aI., 1995; HUBER, 1999). The isolate PZ6"", which was first obtained in pure culture, was studied in detail.

From the White Island sample WI] 3 (greyish-black sediment; orig. pH: 4.5; orig. temp: 85-98 QC), a further rod-shaped hyperthermophile was enriched anaerobical­ly at 90 QC in the presence of arsenate under organ­otrophic culture conditions after two days incubation. A

pure culture was obtained by the selected cell cultivation technique (HUBER et aI., 1995; HUBER, 1999).

Morphology

Cells of PZ6"· were cylinder-shaped rods with almost rectangular ends with an average length of about 4 ~m and a width of 0.7 ~m (Fig. 1a). Most cells were 3-7 ~m long. Rarely, cells with a length of up to 20 ~m were ob­served. During growth, the rods were often arranged in V-, X- and raft-shaped aggregates (Fig. 1a). True branched cells were not observed in cultures of PZ6':·. At the end of the exponential growth phase, about 1 % of the rods produced spherical bodies at the cell ends simi­lar to the "golf clubs" formed by different members of the Thermoproteales order (ZILLIG et aI., 1981; HUBER and STETTER, 1992). When PZ6" was grown organo­trophically in the presence of arsenate or on a mixture of arsenate and thiosulfate (gas phase: 300 kPa H2-C02;

80:20; v/v), small black particles of so far unknown com­position (diameter about 0.3-0.4 ~m) became visible on the cell surface in the stationary growth phase (Fig. 1 b; HUBER et aI., 2000). In contrast, no particles were ob­served, when nitrogen was used instead of hydrogen in the gas phase.

Metabolism

Under anaerobic culture conditions, PZ6" grew lithoautotrophically with CO2 as carbon source, using hydrogen as electron donor and arsenate, thiosulfate or elemental sulfur as electron acceptor. The stoichiometric reduction of arsenate to arsenite was directly coupled with exponential growth of PZ6" (doubling time: 270 min; Fig. 2). In control experiments without cells, only a slow abiotic reduction of arsenate was measured, con-

~

E ---... Q) .0 E :J c: Qi ()

101

lOS

Fig. 1. Phase contrast photomi­crographs of P. arsenaticum. Cells during the log-phase (A), grown organotrophically on 10 mM arsenate, and cells in the stationary phase (B), grown organotrophically on 20 mM arsenate and 0.1% thiosulfate. Cells are tightly covered with small particles. Bar: 10 pm (A, B).

10

8

-<>-- Cell number 2

-<>-- As (III) with inoculum -+- As (V) with inoculum

o -A- As (V) without inoculum 10s+-~----r-------r-~~--r-----~

o 10 20 30 40

Time (hours)

Fig. 2. Growth of P. arsenaticum under chemolithoautotrophic culture conditions at 95°C with arsenate as electron acceptor and hydrogen as electron donor (300 kPa H2-C02; 80:20; v/v). The results are the means of triplicate determinations.

firming the lithotrophic growth of PZ6* on arsenate (Fig. 2). No growth was observed, when hydrogen or ar­senate was omitted from the medium or when hydrogen was replaced by acetate or L( + )-lactate as energy source.

Under organotrophic culture conditions, PZ6" was able to grow without an additional electron acceptor (gas phase: 300 kPa H2-C02 or 300 kPa N2-C02; 80:20; v/v). However, in the presence of nitrogen, higher final cell densities and higher growth rates were determined, indi-

Respiration of Arsenate and Selenate 309

Table 2. Organotrophic growth of Pyrobaculum arsenaticum and Pyrobaculum aerophilum at 95°C in the presence of different in­organic electron acceptors (gas phase Hz-COz, 80:20, v/v).

Electron acceptor Pyrobaculum arsenaticum

Metabolic product

Pyrobaculum Metabolic aerophilum product

Arsenate Arsenate and Sodium thiosulfate Arsenate and L-cysteine Selenate

+++

+++ +++

+++

ND Realgar-precipitate! Realgar-precipitate l

Precipitate of hexagonal selenium!

+++ ND +++ No precipitate visible +++ No precipitate visible +++ No precipitate visible

Selenite

Oxygen Nitrate Nitrite Sodium thiosulfate Elemental sulfur No electron acceptor added

ND +++ +++

+

Sulfide Sulfide ND

+++ Precipitate of hexagonal selenium!

+++

+++ NitriteZ, DinitrogenZ

+++ DinitrogenZ

++ Sulfide

ND

+++ growth with final cell densities of about 1 x 108 cells per ml (after two days of incubation) ++ growth with final cell densities of 1 x 107 cells per ml (after two days of incubation) + growth with final cell densities of 1 x 107 cells per ml (after three days of incubation) - no growth; ND, not determined. 1 _ identification by XRD, SEM and EDS; Z - data from VOLKL et ai., 1993

eating a growth inhibition by hydrogen. This inhibition could be overcome by addition of thiosulfate, sulfur, ar­senate or selenate (Table 2). If PZ6" was grown under a gas phase of H2-C02 or N z-C02 with sulfur or thiosul­fate, sulfide was formed as metabolic product. When grown on hydrogen and sulfur or thiosulfate, the genera­tion time of PZ6':' decreased by about 50% in compari­son to nitrogen (data not shown). However, similar final cell densities were obtained. Hydrogen inhibition could

10'~------'-------'-------'--------r10

8

10' E }> ........ 6 (/I ... S Q) .D E '3 ::J c: 4 ~ Qi (J 10e

I -0- Cell number 2

-+- As (Y) WIth inoculum As (Y) without inoculum

105 0 0 5 10 15 20

Time (hours)

not be overcome by the addition of nitrate, sulfate, or an­timony(V) chloride. Growth of PZ6" was even prevent­ed, when arsenite, selenite, sulfite, or iron (III) citrate was added to the medium. No growth of PZ6" was also ob­served organotrophically under microaerophilic culture conditions with oxygen (1-3% O 2 tested).

During growth of PZ6" on selenate at 95°C, the medium turned black in the late logarithmic to the sta­tionary growth phase. Large black precipitates were

®

8

~ 10' I }>

6 (/I ... S Q)

.D E ....... ::J 3 c: 4 ~ Qi

........

(J 10e

2 -0- Cell number -+- As (Y) WIth inoculum --4.-- As (Y) without inoculum

105 0 0 5 10 15 20

Time (hours)

Fig. 3. Growth of P. arsenaticum under organotrophic culture conditions at 95°C with arsenate (A), 300 kPa H 2-C02; 80:20; v/v; (B), 300 kPa NZ-C02 ; 80:20; v/v. The results are the means of triplicate determinations.

310 R. HUBER et al.

formed, identified as elemental, crystalline, hexagonal se­lenium (Table 2). This biomineralization occurred under a H2-C02 as well as under a N2-C02 gas phase. In con­trast, when PZ6" was grown at 75°C or 85 °C, the medium turned red during the logarithmic growth phase, indicating a primary formation of elemental red seleni­um. Prolonged incubation at both temperatures resulted finally in the transformation into a black precipitate, most likely representing elemental black selenium.

When PZ6" was grown organotrophic ally at 95°C on arsenate, similar final cell densities but different doubling times were determined under a gas phase of H2-C02

(doubling time: 81 min) or N 2-COz (doubling time: 116 min) (Fig. 3). With hydrogen, arsenate reduction was stimulated (Fig. 3). Under both gas phases, no significant abiotic arsenate reduction was observed in control exper­iments during the incubation period of almost twenty hours (Fig. 3).

Formation of arsenic sulfides

When cells of PZ6<' were cultured organotrophically under a H2-C02 gas phase and a combination of arsen­ate and thiosulfate, large amounts of a yellow precipi­tate became visible in the medium. The formation of the precipitate occurred, when different concentrations of arsenate (5 mM, 10 mM, 20 mM, 30 mM) were com­bined with a constant kept concentration of 0.1 % thio­sulfate. Doubling times and final cell densities were the same with 5 mM, 10 mM or 20 mM arsenate and there­fore not strictly correlated with the initial arsenate con­centration.

Also the combination of varying thiosulfate concentra­tions (0.02%,0.05%,0.1 %, 0.2%, 0.3%) with 10 mM arsenate resulted in each experiment in the production of a yellow precipitate. Further detailed experiments with 10 mM arsenate and 0.1% thiosulfate showed, that strong, visible precipitation took place after only four-

teen hours incubation (1 % inoculum) at the beginning of the stationary growth phase (doubling time: 106 min). The precipitate occurred in a crystalline form and was identified as realgar (As252; Table 2). Phase contrast mi­croscopy revealed, that the realgar particles were tightly covered with cells of PZ6"', sticking to the surface (Fig. 4). In contrast, growth on arsenate and thiosulfate under a N2-C02 gas phase did not result in the formation of a visible precipitate. The calculated doubling time under these conditions was 122 min.

Furthermore, realgar formation also occurred under a H2-C02 gas phase, when arsenate and L-cysteine were supplied simultaneously to the culture medium (Table 2).

Physiological characterization

PZ6* grew at temperatures between 68°C and 100°C. No growth was observed at 65°C and 102°C. Growth of PZ6" took place at salt concentrations be­tween 0% and 3% NaCI. No growth was observed in the presence of 3.3% NaCI.

DNA base composition

Total DNA of isolate PZ6" had a G+C content of 58.3 mol% as calculated by direct analysis of the mono­nucleosides.

Taxonomy and 165 rONA phylogeny

By 165 rRNA sequence comparisons, PZ6" was affili­ated within the Crenarchaeota with closest relationship to members of the genus Pyrobaculum. The phylogenetic position was supported by all tree construction methods. The evolutionary distances between PZ6* and Pyrobacu­lum islandicum, Pyrobaculum aerophilum, Pyrobaculum sp. WI]3 and Thermoproteus tenax were 1.4%, 1.3%, 1,4% and 3.2% (without correction), respectively.

Fig. 4. Photomicrographs of P. arsenaticum, grown organotrophically with arsenate and thiosulfate. Phase contrast photomicro­graph of a realgar particle (A). Cells of P. arsenaticum stained with DAPI (B), showing that P. arsenaticum is attached to the solid surface of realgar (identical microscopic field as in A). Bar: 10 pm (A, B).

Metabolic studies with pyrobaculum organotrophum, P. islandicum and P. aerophilum

Under organotrophic culture conditions, P. organotro­phum was unable to grow in the presence of selenate or arsenate. P. islandicum exhibited weak growth with final cell titers up to about 107 cells/ml, when arsenate was present in the medium. No growth was observed with se­lenate.

In contrast, P. aerophilum grew organotrophically in the presence of arsenate, selenate or selenite with final cell densities of about lOX cells/ml (Table 2). A black pre­cipitate was formed during growth on selenite, but not on selenate (Table 2). The precipitate was identified as el­emental, crystalline, hexagonal selenium (Table 2). With thiosulfate, lower cell densities of about 1 x 107 cells/ml were observed. Growth on thiosulfate resulted in the for­mation of sulfide as metabolic product. When grown on a combination of arsenate and L-cysteine or arsenate and thiosulfate, no visible precipitate was formed by P. aerophilum.

Similar to PZ6"", P. aerophilum was able to grow chemolithoautotrophically with hydrogen as electron donor and arsenate as electron acceptor. However, beside arsenate, P. aerophilum was able to perform chemolitho­autotrophic growth with selenate as electron acceptor in the presence of hydrogen.

Discussion

A variety of hyperthermophilic archaea are known, which can use CO2 as single carbon source for growth (STETTER, 1995; STETTER, 1999a). In their energy-yield­ing reactions, hydrogen is the preferred electron donor, while different inorganic compounds serve as electron

Respiration of Arsenate and Selenate 311

acceptors (STETTER, 1999b). This study reports on new modes of chemolithoautotrophy in hyperthermophilic archaea, which can use heavy metals as electron accep­tors for anaerobic respiration.

The first microorganism identified to grow chemo­lithoautotrophically on arsenate as electron acceptor with hydrogen is the hyperthermophilic isolate PZ6"", performing a true arsenate respiration (Fig. 1). However, under organotrophic culture conditions, hydrogen is in­hibitory on growth. This inhibition can be overcome in the presence of arsenate (Fig. 2). Therefore, an arsenate respiration on organic matter performed by PZ6"- seems unlikely and a kind of "hydrogen-detoxification" reac­tion is more probable. So far, the elimination of inhibito­ry hydrogen by the use of an external electron acceptor (e.g. sulfur) was reported only for strictly organotrophic hyperthermophiles and occurs within the order Thermo­togales and Thermococcales (HUBER et aI., 1986; FIALA et aI., 1986; HUBER and STETTER, 1999). For this reaction, a hydrogen-dependent sulfur reductase activity was identi­fied in Pyrococcus furio5us, and termed sulfhydrogenase (MA et aI., 1993). An enzyme with a similar dual-func­tion may be also present in PZ6", reducing arsenate in a hydrogen-stimulated way.

Based on 165 rRNA gene sequence comparisons, PZ6"- is a member of the Crenarchaeota with closest re­lationship to members of the genus Pyrobaculum (Fig. 5). However, from the described Pyrobaculum species, PZ6"- differs significantly in its metabolic properties, in­cluding heavy metal respiration (see below; Table 2). Therefore, we propose, that PZ6 ,'- represents a new species within the genus Pyrobaculum, which we name Pyrobaculum arsenaticum.

Arsenic is ubiquitous, being highest in marine shale materials, magmatic sulfides and iron ores (NEWMAN et aI., 1998). In geothermal settings, it is an important com-

,--------------- Halobacterium salinarum, M38280

'----- Methanothermus fervidus, M32222

Fig. 5. 16S rRNA gene based phylogenetic tree showing the po­sitions of Pyrobaculum arsen­aticum PZ6" and Pyrobaculum sp. WIJ3. The topology of the tree is based on results of a maximum­parsimony analysis. Reference se­quences were chosen to represent the broadest diversity of Archaea. Only sequence positions that share identical residues of 50% or more of all available crenarchaeal 16S rRNA gene sequences were included for tree construction (Bar: 10% estimated difference in nucleotide sequences). 0.10

Methanococcus jannaschii, M59126

Sulfolobus solfataricus, X90478

Thermosphaera aggregans, X99556

Pyrodictium occultum, M21087

Pyrobaculum arsenaticum PZ6*, AJ277124

Pyrobaculum is/andicum, L07511

Pyrobaculum aerophilum, L07510

Pyrobaculum sp. WIJ3, AJ277125

Thermoproteus tenax, M35966

Caldivirga maquilingensis, AB013926

Thermocladium modestius, AB005296

Thermofilum pendens, X14835

312 R. HUBER et al.

ponent and high concentrations of arsenic are present in continental solfatara fields and in shallow marine and abyssal hydrothermal systems (Table 1; SARANO et aI., 1989; FOUQUET et aI., 1991; EARY, 1992; STOFFERS et aI., 1999b). In these high temperature habitats, hyperther­mophilic arsenate reducers may be widely distributed on earth. From samples, taken recently on White Island Vol­cano during the joint multidisciplinary RV Sonne expedi­tion SO 135 cruise (STOFFERS et aI., 1999a), another, strictly anaerobic arsenate reducer was isolated, most likely representing an additional Pyrobaculum species (Pyrobaculum sp. WIJ3 in Fig. 5). White Island is a pas­sively degassing arc volcano, where large fluxes of heavy metals like arsenic are a common feature (STOFFERS et aI., 1999b). Beside their widespread occurrence, hyperther­mophilic arsenate reducers can thrive in significant cell numbers in volcanic areas. At least 107 metabolically ac­tive cells per gram sediment were determined in three dif­ferent neutral sites from Pisciarelli Solfatara, Naples. Furthermore, due to their broad salt range suitable for growth, hyperthermophiles like P. arsenaticum and Pyro­baculum aerophilum (see below) can use arsenate active­ly in metabolism in low salt continental solfatara fields the same as in marine hydrothermal areas.

The most common As-bearing minerals found in geothermal systems include orpigment (AszS3 ), realgar (AszSz), arsenopyrite (FeAsS), loellingite (FeAs) and na­tive arsenic (EARY, 1992). So far, formation of arsenic­sulfur compounds in these high-temperature environ­ments was attributed to chemical processes (EARY, 1992; MIGDISOV and BYCHKov, 1998). P. arsenaticum is the first example, that precipitation of realgar can be gener­ated biologically. Necessary for this formation are arsen­ate and a sulfur-containing compound like thiosulfate, one of the dominating sulfur-bearing species in volcanic environments (MIGDISOV and BYCHKOV, 1998). P. arsen­aticum can precipitate realgar over its temperature range of growth. Interestingly, these results fit well with geo­logical data from Uzon caldera, Kamchatka. In this solfa­tara field, realgar was the only sulfur-bearing mineral, found in the temperature zone between 70 and 95°C (MIGDISOV and BYCHKov, 1998). All these results indi­cate, that microbial activities of hyperthermophiles in volcanic areas playa major role in the biogeochemical cycle of arsenic and it is not simply chemistry that deter­mines the speciation of arsenic in high-temperature envi­ronments.

Instead of arsenate, P. arsenaticum can grow organ­otrophically in the presence of the heavy metal selenate (Table 2). First growth studies at different temperatures resulted in the formation of elemental red or black seleni­um. This may indicate, that P. arsenaticum is able to pro­duce different modifications of elemental selenium. However, it can not be excluded, that red selenium is formed intermediary at all temperatures, and turns black afterwards due to thermodynamic reactions. The black modification is energetically favoured at high tempera­tures (WIBERG, 1985). Therefore, at 95°C, the conver­sion of selenium may be so fast, that the red form does not become visible during growth.

Metabolic studies with different Pyrobaculum species showed, that P. aerophilum is also able to respire arsen­ate chemolithoautotrophically with hydrogen. However, significant differences in selenate and selenite metabolism between P. arsenaticum and P. aerophilum are evident (Table 2). A unique feature of P. aerophilum is its ability to grow autotrophically with hydrogen as electron donor and selenate as electron acceptor. These different modes of selenium respiration indicate the participation of a dif­ferent set of enzymes during growth on different seleni­um-bearing species.

Description of pyrobaculum arsenaticum sp. nov.

(ar.se.na'ti.cum M.L. neutr. adj. arsenaticum, describ­ing the ability of the organism to grow autotrophic ally on arsenate) Cylinder-shaped rods, usually between 3 and 7 ).lm long and 0.7 ).lm wide. Motile by flagellation. Rods often arranged in V-, X- and raft-shaped aggre­gates. No true branching. Cells with terminal spheres occur in the stationary phase. When grown organotroph­ically on arsenate or on arsenate and thiosulfate with hy­drogen, small black particles (width: 0.3-0.4 ).lm) occur on the cell surface in the stationary phase. Packed cell masses exhibit a greyish-black colour. Growth between 68 and 100°C and at 0 to 3.0% NaCI. No growth at 65 and 102°C. No growth at 3.3% NaC!. Optimally doubling time, 81 min. Organotrophic growth on a mix­ture of yeast extract, meat extract, peptone and brain heart infusion. Organotrophically, hydrogen inhibits growth. Hydrogen inhibition can be overcome by addi­tion of arsenate, selenate, sulfur or thiosulfate. Forma­tion of realgar in the presence of arsenate and thiosulfate or arsenate and L-cysteine (Hz-CO z gas phase). Forma­tion of elemental selenium during organotrophic growth on selenate (Hz-COz or Nz-CO z gas phase). Lithoauto­trophic growth by reduction of arsenate, elemental sulfur or thiosulfate with hydrogen as electron donor. HzS is formed from elemental sulfur or thiosulfate, arsenite from arsenate. DNA base composition, 58.3 mol% G+c. By 16S rDNA gene sequence comparisons, 1.4% evolu­tionary distance to P. islandicum, 1.3 % to P. aerophilum, 1.4% to Pyrobaculum sp. WIJ3 and 3.2% to Thermo­proteus tenax. Type strain: Pyrobaculum arsenaticum PZ6\ DSM 13514, Braunschweig, Germany and ATCC 700994, Maryland, USA (isolated from Pisciarelli Solfa­tara, Naples, Italy).

Acknowledgements We are grateful to K.O. STETTER for stimulating and critical

discussions. We furthermore thank W. EDER for phylogenetic analysis and M. BOCK and I. HALLER for excellent technical help. The highly valuable help of the Geological Institute of the University of Kiel, of P. STOFFERS, is appreciated. We thank Master H. ANDERSEN and crew of RV Sonne (SO 135 cruise) for their expert technical assistance. Furthermore, we are indebted to the New Zealand Government for a sampling permit. This work was financially supported by the Deutsche Forschungsge­meinschaft (Ste 297110), the BMBF (grant no. 03G0135A to PS) and by the Fonds der Chemischen Industrie (to KOS).

References AHMANN, D., ROBERTS, A. L., KRUMHOLZ, L. R., MOREL, E M.

M.: Microbe grows by reducing arsenic. Nature 371, 750 (1994).

BALCH, W. E., WOLFE, R. S.: New approach to the cultivation of methanogenic bacteria: 2-mercaptoethanesulfonic acid (HS­CoM)-dependent growth of Methanobacterium ruminan­tium in a pressurized atmosphere. Appl. Environ. Microbiol. 32,781-791 (1976).

BALCH, W. E., Fox, G. E., MAGRUM, L. J., WOESE, C. R., WOLFE, R. S.: Methanogens: Reevaluation of a unique biological group. Microbiol. Rev. 43, 260-296 (1979).

BURGGRAF, S., HUBER, H., STETTER, K. 0.: Reclassification of the crenarchaeal orders and families in accordance with 16S ribosomal RNA sequence data. Int. ]. Syst. Bacteriol. 47, 657-660 (1997).

CASHION, P., HOLDER-FRANKLIN, M. A., MCCULLY, ]., FRANKLIN, M.: A rapid method for the base ratio determination of bac­terial DNA. Anal. Biochem. 81,461-466 (1977).

EARY, L. E.: The solubility of amorphous As2SJ from 25 to 90°C. Geochim. Cosmochim. Acta 56, 2267-2280 (1992).

EDER, W., LUDWIG, W., HUBER, R.: Novel 16S rRNA gene se­quences retrieved from highly saline brine sediments of Kebrit Deep, Red Sea. Arch. Microbiol. 172, 213-218 (1999).

FIALA, G., STETTER, K. 0.: Pyrococcus furiosus, sp. nov. repre­sents a novel genus of marine heterotrophic archae bacteria growing optimally at 100°C. Arch. Microbiol. 145, 56-61 (1986).

FOUQUET, Y., VON STACKELBERG, U., CHARLOU,]. L., DONVAL,]. P., ERZINGER, J., FOUCHER, ]. P., HERZIG, P., MUHE, R., SOAKAI, S., WIEDICKE, M., WHITE CHURCH, H.: Hydrothermal activity and metallogenesis in the Lau back-arc basin. Nature 349,778-781 (1991).

HUBER, H., HUBER, G., STETTER, K. 0.: A modified 4',6'-di­amidino-2-phenylindole fluorescence staining procedure suit­able for the visualization of lithotrophic bacteria. System. Appl. Microbiol. 6, 105-106 (1985).

HUBER, R., LANGWORTHY, T. A., KONIG, H., THOMM, M., WOESE, C. R., SLEYTR, U. B., STETTER, K. 0.: Thermotoga maritima sp. nov. represents a new genus of unique extreme­ly thermophilic eubacteria growing up to 90°C. Arch. Mi­crobiol. 144,324-333 (1986).

HUBER, R., KRIST]ANSSON, ]. K., STETTER, K. 0.: Pyrobaculum gen. nov., a new genus of neutrophilic, rod-shaped archae­bacteria growing optimally at 100°C. Arch. Microbiol. 149, 95-101 (1987).

HUBER, R., STETTER, K. 0.: The order Thermoproteales, pp. 677-683. In: The Prokaryotes (BALOWS, A., TRUPER, H. G., DWORKIN, M., HARDER, W., and SCHLEIFER, K. H., eds.) 2nd ed., Berlin, Heidelberg, New York, Springer 1992.

HUBER, R., BURGGRAF, S., MAYER, T., BARNS, S. M., ROSSNAGEL, P., STETTER, K. 0.: Isolation of a hyperthermophilic ar­chaeum predicted by in situ RNA analysis. Nature 376, 57-58 (1995).

HUBER, R., STETTER, K. 0.: Thermotogales. In: Embryonic En­cyclopedia of Life Sciences. London: Nature Publishing Group. (http://www.els.net) (1999).

HUBER, R.: Die Laserpinzette als Basis fur Einzelzellkultivierun­gen. BIOspektrum 5, 289-291 (1999).

HUBER, R., HUBER, H., STETTER, K. 0.: Microbial high tempera­ture ecosystems. FEMS Microbiol. Rev. (submitted) (2000).

JOHNSON, D. L., PILSON, M. E. Q.: Spectrophotometric determi­nation of arsenite, arsenate and phosphate in natural waters. Anal. Chern. Acta 58, 289-299 (1972).

LAVERMAN, A. M., SWITZER BLUM, ]., SCHAEFER, ]. K., PHILLIPS,

Respiration of Arsenate and Selenate 313

E. ]. P., LOVLEY, D. R., OREMLAND, R. S.: Growth of strain SES-3 with arsenate and other diverse electron acceptors. Appl. Environ. Microbiol. 61, 3556-3561 (1995).

LUDWIG, W.: Sequence databases (3.3.5), pp. 1-22. In: Molecu­lar microbial ecology manual (AKKERMANS, A. D. L., VAN ELSAS, ]. D., and DE BRUUN, E ], eds.) Dordrecht, Kluwer 1995.

LUDWIG, W., STRUNK, 0.: ARB: a software environment for se­quence data. http://www.mikro.biologie.tu-muenchen.de/ pub/ ARB/documentation/arb. ps (1997).

LUDWIG, W., STRUNK, 0., KLUGBAUER, S., KLUGBAUER, N., WEIZENEGGER, M., NEUMAIER, J., BACH LEITNER, M., SCHLEI­FER, K. H.: Bacterial phylogeny based on comparative se­quence analysis. Electrophoresis 19, 554-568 (1998).

MA, K., SCHICHO, R. N., KELLY, R. M., ADAMS, M. W. W.: Hy­drogenase of the hyperthermophilic Pyrococcus furiosus is an elemental sulfur reductase or sulfhydrogenase: Evidence for a sulfur-reducing hydrogenase ancestor. Proc. Natl. Acad. Sci. USA 90, 5341-5344 (1993).

MACY, ]. M., RECH, S., AULING, G., DORSCH, M., STACKE­BRANDT, E., SLY, L. I.: Thauera selenatis gen. nov., sp. nov., a member of the beta subclass of Proteobacteria with a novel type of anaerobic respiration. Int. ]. Syst. Bacteriol. 43, 135-142 (1993).

MACY,]. M., NUNAN, K., HAGEN, K. D., DIXON, D. R., HARBOUR, P.]., CAHILL, M., SLY, L. I.: Chrysiogenes arsenatis gen. nov., a new arsenate-respiring bacterium isolated from gold mine wastewater. Int.]. Syst. Bacteriol. 46,1153-1157 (1996).

MACY,]. M., SANTINI, ]. M., PAULING, B. V., O'NEILL, A. H., SLY, L. I.: Two new arsenate/sulfate-reducing bacteria: mech­anisms of arsenate reduction. Arch. Microbiol. 173, 49-57 (2000).

MESBAH, M., PREMACHANDRA, U., WHITEMAN, W. B.: Precise measurement of the G+C content of desoxynucleic acid by high-performance liquid chromatography. Int. ]. Syst. Bacte­riol. 39,159-167 (1989).

MIGDISOV, A. A., BYCHKOV, A. Y.: The behaviour of metals and sulfur during the formation of hydrothermal mercury-anti­mony-arsenic mineralization, Uzon caldera, Kamchatka, Russia.]. Volcanol. Geotherm. Res. 84, 153-171 (1998).

NEWMAN, D. K., KENNEDY, E. K., COATES,]. D., AHMANN, D., ELLIS, D. ]., LOVLEY, D. R., MOREL, E M. M.: Dissimilatory arsenate and sulfate reduction in Desulfotomaculum auripig­mentum, sp. nov .. Arch. Microbiol. 168,380-388 (1997a).

NEWMAN, D. K., BEVERIDGE, T. J., MOREL, E M. M.: Precipita­tion of arsenic trisulfide by Desulfotomaculum auripigmen­tum. Appl. Environ. Microbiol. 63,2022-2028 (1997b).

NEWMAN, D. K., AHMANN, D., MOREL, E M. M.: A brief review of microbial arsenate respiration. Geomicrobiology 15, 255-268 (1998).

OREMLAND, R. S., SWITZER BLUM, J., CULBERTSON, C. W., VISS­CHER, P. T., MILLER, L. G., DOWDLE, P., STROHMAIER, E E.: Isolation, growth, and metabolism of an obligately anaero­bic, selenate-respiring bacterium, strain SES-3. Appl. Envi­ron. Microbiol. 60, 3011-3019 (1994).

SARANO, E, MURPHY, R. c., HOUGHTON, B. E, HEDENQUIST, ]. W.: Preliminary observations of submarine geothermal activi­ty in the vicinity of White Island Volcano, Taupo Volcanic Zone, New Zealand.]. Royal Soc. N.Z. 19,449-459 (1989).

SELIG, M., SCHONHEIT, P.: Oxidation of organic compounds to CO2 with sulfur or thiosulfate as electron acceptor in the anaerobic hyperthermophilic archaea Thermoproteus tenax and Pyrobaculum islandicum proceeds via the citric acid cycle. Arch. Microbiol. 162,286-294 (1994).

SEGERER, A., STETTER, K. 0., KLINK, E: Two contrary modes of chemolithotrophy in the same archaebacterium. Nature 313, 787-789 (1985).

314 R. HUBER et al.

STETIER, K. 0.: Microbial life in hyperthermal environments. ASM News 61, 285-290 (1995).

STETIER, K. 0 .: Hyperthermophiles: Isolation, classification, and properties, pp. 1-24. In: EXTREMOPHILES Microbial life in extreme environments (HORIKOSHI, K., GRANT, W. D., eds.) New York, John Wiley & Sons, Inc. 1999a.

STETIER, K. 0.: Extremophiles and their adaption to hot envi­ronments. FEBS Lett. 452, 22-25 (1999b).

STETIER, K. 0.: Microorganisms in high-temperature sulfur en­vironments. In: Embryonic Encyclopedia of Life Sciences. London: Nature Publishing Group. (http://www.els.net) (1999c).

STOFFERS, P., WRIGHT, I. c., DE RONDE, c., HANNINGTON, M., HERZIG, P., VILLINGER, H. and the Shipboard Scientific Party: Longitudinal transect of the Kermadec-Havre Arc-Back Arc­System: Initial results of R/V Sonne Cruise SO-135. Inter­Ridge News 8,45-50 (1999a).

STOFFERS, P., HANNINGTON, M ., WRIGHT, I. c., HERZIG, P., DE RONDE, C. and the Shipboard Scientific Party: Elemental mercury at submarine hydrothermal vents in the Bay of Plenty, Taupo volcanic zone, New Zealand. Geology 27, 931-934 (1999b).

STOLZ, J. E, OREMLAND, R. S.: Bacterial respiration of arsenic and selenium. FEMS Microbiol. Rev. 23, 615-627 (1999).

STOLZ, J. E, ELLIS, D. J., SWITZER BLUM, J., AHMANN, D., Lov­LEY, D. R., OREMLAND, R. S.: Sulfurospirillum barnesii sp. nov. and Sulfurospirillum arsenophilum sp. nov., new mem­bers of the Sulfurospirillum clade of the £ Proteobacteria. Int. ]. Syst. Bacteriol. 49,1177-1180 (1999).

SWITZER BLUM, J., BURNS BIND!, A., BUZZELLI, J., STOLZ, J. E, OREMLAND, R. S.: Bacillus arsenicoselenatis, sp. nov., and Bacillus selenitireducens, sp. nov.: two haloalkaliphiles from Mono Lake, California that respire oxyanions of selenium and arsenic. Arch. Microbiol. 171, 19-30 (1998).

TAMAOKA, J., KOMAGATA, K.: Determination of DNA base com­position by reversed-phase high-performance liquid chro­matography. FEMS Microbiol. Lett. 25, 135-128 (1984).

VARGAS, M., KASHEF!, K., BLUNT-HARRIS, E. L., LOVLEY, D. R.: Microbiological evidence for Fe(III}reduction on early Earth. Nature 395, 65-67 (1998).

VOLKL, P., HUBER, R., DROBNER, E., RACHEL, R., BURGGRAF, S., TRINCONE, A., STETIER, K. 0.: Pyrobaculum aerophilum sp. nov., a novel nitrate-reducing hyperthermophilic archaeum. Appl. Environ. Microbiol. 59, 2918-2926 (1993).

WIBERG, N., ed.: Lehrbuch der Anorganischen Chemie 33rd ed., Berlin, Walter de Gruyter & Co. 1985.

ZILLIG, W., STETIER, K. 0., SCHAFER, W., ]ANEKOVIC, D., WUN­DERL, S., HOLZ, I.: Thermoproteales: a novel type of extreme­ly thermoacidophilic anaerobic archae bacteria isolated from icelandic solfataras. Zbl. Bakt. Hyg., I. Orig. C2 , 205-227 (1981).

Corresponding author: ROBERT HUBER, Lehrstuhl fur Mikrobiologie, Universitat Re­gensburg, Universitatsstr. 31, 93053 Regensburg, Germany Tel: ++49/(0}941/943-3182; Fax: ++49/(0}941/943-2403; e-mail: robert.huber@biologie.uni-regensburg.de