Pyrococcus woesei, sp. nov., an ultra-thermophilic marine archaebacterium, representing a novel order, Thermococcales

System. Appl. Microbiol. 9, 62-70 (1987)

Pyrococcus woesei, sp. nov., an Ultra-Thermophilic Marine Archaebacterium, Representing a Novel Order, Thermococcales WOLFRAM ZILLIG, INGELORE HOLZ, HANS-PETER KLENK, JONATHAN TRENT, SIMON WUNDERL, DAVORIN JANEKOVIC, ERWIN IMSEL, and BIRGIT HAAS Max-Planck-Institur fur Biochemie, 8033 Martinsried, Federal Republic of Germany

Received May 5, 1986

Summary The anaero bic sulfur-reducing marine archaebacterium Pyrococcus iocesei is an "ultra-thermophile" growing optimall y between 100 and 103 °C at pH 6 to 6.5 and 30 gil NaC!. Growth proceeds, on solid supports or in suspension, by sulfur respiration of yeast extrac t or peprides, on yeast ext ract also without S° in the presence of H2, or on polysaccharid es in the presence of H! and 5". The generation time was as low as 35 minutes either on solid suppor ts or in suspension. The cells have a roughl y spherical, often elongated and constricted appearance, similar to Thermococcus celer. Frequently, they occur as diploforms. Cells grow n on solid supports have dense tufts of flagellae or pili atta ched to one pole. When P. woesei was grown by sulfur respiration on yeast extract or bactotr yptone in the presence of starch, complete lysis occurred after the peak of cell density had been reached. Concomitantly icosaedric particles of about 30 nm in diameter were liberated which showed a defined simple protein compositio n. P. woesei belongs to the Thermococcaceae as indicated by the immunochemical cross-reaction of its DNA-dependent RNA polymerase with the polymerases of T. celer and the isolate ANI from New Zealand. Quantit ative analysis of its phylogenetic position by DNA-rRNA cross-hvbridization places it at the end of a long branch of the Tbermococcaceae, whereas the isolate ANI ison '1 branch of intermediate length and T. celer on an extremely short branch. The phylogenetic depth of this group and its clear separation from the neighbour ing Thermoproteales and Methal1ococcales call for the intro duction of a separate order: Therntococcales , which represents a third major division of the archaebacteria between the Thermoproteales + Suljolobales and the methanogens + halophiles.

Key words: DNA-dep end ent RNA polymerase - DNA-rRNA-cross-h yb rid ization - Ev olution - Marine solfatara- Ph ylogeny - Taxonomy - Ultra-th erm ophil e

Introduction The urkingdom of the a rchaeba cteria ha s been divided into two major groups: (1) th e methan ogens plus extreme halophiles including the genus Thermoplasma and (2 ) the sulfur-depen dent archaebacter ia so fa r includi ng sulfurreducing Thermoproteales and th e sulfur-oxidizing Sul[olobales (Tu et aI., 1982; Waese et a I., 1984). The ph ylogenetic distance between Sulfo lob us and the Tb erm op roteales Desulfurococcus and Pyrodictium appears sma ller than the distance bet w een Thermoproteus and DesulA bbr eviatio ns: PEG

Polyethyleneglycol EDTA Ethylenedinitrotetra acetic acid PAGE Polyacrylamide gel electrophoresis

[urococcus within the Thermoproteales (Woese a nd Olsen, 1986). This implies th at the o rigin of the Sulfolobales w as within the Thermoproteales (Zillig et aI., 19 86 ). The sulfur-depende nt Thermococcus (Zillig et a l., 19 83 ), on th e other hand , appe a rs on a sho rt bran ch equally clo se to Thermoproteales and Methanococcales. Woese and Olsen (1986) therefore co ns ider it a primitive offshoot between the two m ajor groups of archaebacteria. Here we describe a no vel ultra-thermophilic marine arch aebacteriurn, Pyrococcus waesei, which belongs to th e Thermo coccaceae and upon induction produces a particle which appears to be a virus. On the basis of its phylogenetic relationship to Thermococcus celer and the isolate AN1

Pyrococcus woesei, sp. nov.

(Morgan and Daniel, 1982) we propose a separate order, the Thermococcales representing a third branch between the established major divisions of the kingdom of the archaebacteria. Materials and Methods Organisms Thermococcus celer, DSM 2476 and the Thermoproteales and Sulfolobales used for comparison were available in the laboratory. Isolate ANI from alkaline hot springs of New Zealand and Daniel, 1982) was obtained from Prof. H. W. Morgan.

(Morgan

Sampling Samples from the sediment of marine solfataras were aspirated into syringes through aluminium canulas (diameter 0.4 em, minimal length 60 em) which were directlv inserted into the sediments 10 to 30cm down. To avoid plugging, the sieve bottom of an inline filter was connected to the end of the canula. The samples were then injected into 100 ml serum bottles filled with C0 7 and containing 3 g of sulfur and 10 ul of a solution of 1 gil resazurin in water. Another sampling method involved the use of evacuated serum bottles closed with rubber stoppers and fastened to the end of a pole. By means of a string connected to a metal ring through the stopper, the bottles were opened at the sites to be sampled. When added resazurin (1 ug/ml) indicated the presence of oxygen, HzS water was injected into the closed bottles until complete decolorization, The samples were transportedat ambient temperature and stored at + 4 DC until enrichment.

Enrichment . Solid-suFport enrichments were done with water-glass (polysilicate) (4 Yo) or K9A40 gellan gum (0.8%) gels (Kelco, a division of Merck, Ltd.), The water-glass gels were poured in 19 ern diameter glass plates immediately after neutralizing the sodium silicate solution, The K9A40 gellan gum was melted in water. Gels were poured immediately after the addition of 0.05% CaS04 X 2H zO. For precipitation of colloidal sulfur in the gels these were first overlaid with 2 M sodium thiosulfate solution for 30 min, thoroughly rinsed with water and then overlaid with 1 M sulfuric acid for another 30 minutes. The plates were then equilibrated With the salt base of Brock's Sulfolobus medium (Brock et al., 1972), containing in addition 30 gil NaCl, 2 gil bactotryptone or yeast extract andlor 2 gil glycogen or starch. They were then covered with 300 ml each of the medium, inoculated under strictly anaerobic conditions, and incubated in a closed autoclave at 100 t? 105 DC under an atmosphere of usually 9 x 10 4 Pa CO" 9 X 10 Pa Hz and a trace of HzS and harvested after 15 to 36h.

Isolation of pure strains from colonies For plating colonies, K9A40 gellan gum gels were prepared in 9 cm diameter glass plates as above. They were, however, sulfumed by overlaying with 5 ml each of a solution of 500 g Soper liter 20% (NH 4hS solution for 20 seconds, and then, after a thorough rinse with water, with 1 M H zS04 for 5 min. After rinsing with water again, the gels were equilibrated in the salt base of Brock's Sulfolobus medium containing in addition 30 gil NaCl, 2 gil bactotryptone and 2 gil sucrose or starch at pH 6 and 1 mg/l resazurin as redox indicator. They were reduced in an atmosphere of 1 x 10 5 Pa of CO 2 containing 1 x 10 4 Pa H,S. For plating, the gels were overlaid with 1 ml each of a solution of 30 gil sodium silicate in the above medium which had been ad-

63

justed to pH 6.2 immediately before the addition of the diluted cells. The plates were incubated at 100 to 104°C under an atmosphere of 9 X 10 4 Pa CO 2 and 9 x 10" Pa H, with a trace of HzS for 24 to 48 h. The micro-colonies were surr-ounded by clear halos which could be recognized directly. They were usually picked under the dissection microscope. The recovery of clones grown from single colonies was 90%.

Liquid media Cultures in liquid media (composition described above but without the solid support) were shaken or stirred in 0.2 to 50 I glass bottles, continuously gassed with a mixture of 80% CO, and 20% H 2 at 96 to 97 DC, just below the ambient boiling temperature. Growth was inhibited by contact with stainless steel equipment.

DNA-dependent RNA polymerase preparation . The enzyme was prepared as described previously (Prangishet al., 1982; Zillig et al., 1983) by the polimin P method.

VIllI

Analyticaltechniques These were as described previously (Zillig et al., 1981).

Determination of the phylogenetic position by DNA-rRNA . cross-hybridization . The theoretical basis of the method and details of its applicanon have been described by Klenk (1986) and Klenk et al. (1987). DNA was purified and fixed on nitrocellulose filters according to Tu et al., 1982. The 30S- and 50S-ribosomal subunits were separated ov~r a sucrose gradient and extracted with phenol for the preparation of rRNA. 100 I-tg of rRNA were partially digested in a borate buffer and then terminally labeled with y31p-ATP as described (Silberklang et al., 1979). Smaller fragments were removed by sephadex G 100 column chromatography. Cross-hybridization was done with DNA and rRNA of 17 archaebacteria (see Fig. 9). For each rRNA a dilution series rangmg from 0.5 to 50 ug labeled rRNA in 1 ml 2 X SSC containing 20% formamide was prepared. The caps at each rRNA concentration contained nitrocellulose filters for each DNA. Hybridization was done at 50 DC for 48 h, followed by 3 RNase digestions With 15 ug/ml RNase A for 30 min at 37 "C. The nitrocellulose filters of each set were separated the RNA was determined bv scintillation counting and the DNA by a modified Burton Rea~­ tion (Meijs and Schilperoort, 1971). Values for the hybridization yield were taken from regression curves for each dilution series at a specific RNA concentration (l.l-tg RNA!~I). These values were expressed as fractional hybridizations, I. e. percentages of the corresponding self-hybridizations. Since the hybridization conditions were far from allowing saturation, the percent hybridization values and therefore the distance parameters derived from these solely depend on the hybridization velocity constants. The geometric mean of a pair of reciprocal fractional hybridizations is the hybridization homology normalized for different genome lengths and rRNA operon numbers. The homologies were transformed into sequence differences (corresponding to phylogenetic distances) using an empirical formula, distance (= sequence difference) = (10/ln2) X In(100/H), in which H is the percent hybridization homology. The formula was derived from the observation of Bonner et al. (1973) that the hybridization velocity constant is reduced by a factor of approximately 2 for each 10% sequence difference. The distances are additive over the whole range of the kingdom.

64

W. Zillig et al.

Results Sampling and enrichment Pyrococcus woesei was isolated from samples taken from marine solfataras at the northern beach of Porto Levante, Vulcano, Eolian, Islands, Italy. Interstitial water was collected about 20 em below the sediment-water interface in sandy to stony sediments at a total water depth of 70 to 150 em within solfataric vents, mainly releasing steam, CO 2, H 2 and some H 2S. The pH of this water was as low as 6.2 as compared to the neutral pH of the surrounding sea water. The NaCI concentration was only 3.0% as compared to the mediterranean 3.8%, possibly due to steam condensation. The temperature was maximally 102 to 103°C. Because we expected to sample mainly organisms attached to particles we used solid media for enrichment: polysilicate (water-glass) and K9A40 gellan gum gels containing colloidal sulfur. The gels were covered by liquid medium containing 3% NaCI and 2 gil yeast extract as carbon source, inoculated and then incubated under an atmosphere of 8 X 10 4 Pa CO 2 and 7 X 10 4 Pa HI> with a trace of H 2S, at temperatures between 100 and 105°C for 15 to 36 h. All plates inoculated with marine solfataric samples taken at 100°C or above were covered by flakes and lawns of primarily two types of organisms: dense mats of filaments lined by cells of Pyrodictium closely resembling but larger than Sulfolobus, and large clusters of irregularly spherical cells which were often elongated or in diploforms showing central constrictions of various degrees, sometimes merely thin threads-connecting two cells. Whereas Pyrodictium did not appear until 30 h after the start of enrichment, and required-yeast et.'tract for growth, these diploforms appeared as dense heap~ 'or mats within 15 h after inoculation on K9A40 gellangels even when yeast extract was omitted. In this case, these heaps of cells often appeared as dark colonies imbedded in the surface of the gels. Single colonies could be transferred from plate to plate allowing the isolation of pure strains by repeated transfer. In young cultures, e. g. after 15 h more than 90% of the cells were attached, the rest suspended in the overlying medium. Growth conditions With yeast extract or bactotryptone as carbon and hydrogen sources (on water-glass gels), the organism grew by

CO 2

CO 2

SO

CO 2

H2

growth with N2 CO 2

H2

SO

so

sulfur respiration. With yeast extract, but not with bactotryptone, it also grew in the absence of So, provided H 2 was available. No purely chemolithoautotrophic growth, with CO 2 as sole carbon source and H 2 + So as energy source, was observed. With polysaccharides like glycogen, starch or K9A40 gellan gum, little, though significant growth occurred by sulfur respiration but this was greatly stimulated by the addition of H 2 • Though the production of H 2S was less pronounced than with Thermoproteales like Thermoproteus and Desulfurococcus, no fermentative growth was found with any of the mentioned carbon sources (Table 1). The maximal growth temperature was 104.8°C. Most rapid growth was observed over a temperature range between 100 and 103°C. At 100°C, the organism multiplied 5 times faster than at 95 0C. The optimal NaCI concentration was 3% (Fig. 1). Pyrococcus woesei colonies could be grown on the surface of K9A40 gellan gum gels containing colloidal sulfur. Visible, ochre-coloured micro-colonies grew with a plating efficiency of more than 90% within 24 to 48 h at 100 to 103°C under an atmosphere of 9 x 10 4 Pa CO 2 , 9 X 10 4 Pa H 2 and a trace of H 2S. After a lag phase of several hours, growth by sulfur respiration also proceeded in liquid media, in magnetically stirred glass bottles gassed with CO 2 alone or CO 2 and H 2, in the absence of a support, except suspended So, e. g. with yeast extract or bactotryptone (2 gil each) as carbon sources. It was stimulated by the addition of 2 gil glycogen, starch or sucrose. Even at 97°C (just below the boiling point under atmospheric pressure) generation times as low as 35 min were observed (Fig. 2). Growth was almost completely inhibited in V4A steel fermenters. In an aluminum fermenter gassed continously with CO 2 and a trace of H 2 at a pressure of 1.8 X 105 Pa and stirred with a teflon bar, growth was observed at 102°C but not 103 0C.

Morphology In stationary cultures, the cells appear more or less spherical and highly refractile in the light microscope. In exponentially growing cultures, they are usually elongated and often constricted to various extents (Fig. 3). Sometimes, cell doublets are merely connected by short thin threads. When the cells had been grown on solid supports, attached bundles of filaments could be recognized even in the light microscope. In electron microscopy, these ap-

N2 H2

N2 Hz S"

carbon sources (except CO 2) none yeast extract bactotryptone starch K9A40 gellan

Table 1. Requirements for growth of Pyrococcus woesei on waterglass gels. Cells were counted after homgeneous suspension in overlaid medium. Cells/ ml: ++++ = >2 X 10 7, +++ = 1- 2 X 10 7, ++ = 4 - 8 X 10 6, + = 2 X 10 6, + - = < 2 X 10 6, = no growth, n. d. = not determined.

a

0

+++

0

0 0

0

++++ ++++ ++-

0

+++

0 0 0

0

++++ ++++ +++ +++

n.d.

n.d.

0

0 0

n.d, ++++ ++++ +++

n.d.

n.d.

n.d.

++++ ++++

+

Pyrococcus uioesei, sp. nov.

65

O

5x1d

,. ,

~

"E

"E. ~

c.

,

.

,

~

Ei

~

:>

10

3x10

0

=

, •

-

~ ~

0 ~

a;

s,

Fig. 3. Micrographs of exponential P. woesei culture. Magnification 1560 fold. Dark strip on left interference contrast, right phase contrast.

10

1x10

Fig. 1. Salt concentration dependence of growth of P. uoesei. Cells were grown for 15 h at 103 DC on sulfurized gelrite plates covered with 300 ml medium containing 0.2';', veast extract in an atmosphere of 1 x 105 Pa CO, and 7 X 104 Pa H, as described in Materials and Methods. Cell lawns were removed from surface with a rubber spatula, homogenouslv suspended in overlaid medium and counted.

peared as dense beards of smoothly curved fimbria, flagellae or possibly pili, emerging from one pole of the cell (Fig. 4). They were absent when the cells had been grown in liquid culture. Isolated fimbria yielded one protein band of about 20 Kdaltons in PAGE (not shown).

12 0 0

170 0 hours

22 0 0

30 0

Fig. 2. Growth and lysis of liquid culture of P. u/oesei WIth O.2'X, least extract + 0.2% starch and 1% S" at 96.3°C and pH 6.J gassed with 80% CO 2 and 20% H 2• The generation time in the exponential growth phase was between 35 and 40 minutes. 5 System. App'. Microbia!' Vo!' 9/1-2

Thin sections show the cytoplasmic membrane covered by a two-layered envelope about 33 nm thick in total (Fig. 5 c). Many of the sections exhibit lattices of bundled filaments in various orientations. These bundles show alternating dark and light cross-striation (Fig. 5 a). Arrays of ring-like structures could be cross-sections of these bundles (Fig. 5 b). These lattices were found to be composed mainly of a 60 Kdalton protein which constitutes approximately 10% of the total soluble proteins of P. woesei (not shown). The dense clump of globular particles slightly smaller than ribosomes just beside the cross section of a bundle in Fig. 5 b is another peculiar structure often seen.

Virus-like particles In liquid cultures containing yeast extract or bactotryptone plus starch (2 gil each) and S" (5 to 10 gil) and gassed with CO 2 plus H 2 , a period of fast growth up to a density of about 2-4 X 10° cells per ml was followed by more or less complete lysis of the cells (Fig. 5) accompanied by the liberation of spherical to icosahedral particles of about 30 nm diameter (Fig. 6 a). Distorted hexagonally denseh packed crystals of particles of the same size and such particles aligned in tubules were also found in cross-sections of unlysed cells from lysing cultures (Fig. 6 b, c). Lysis was less distinct when starch was replaced by sucrose. Five of eight clones derived from single colonies showed fast lysis accompanied by the formation of more than 10 II! particles per ml. Three reached stationary state at around 2 x 10 s cells per ml forming less than I ()C) particles per rnl. The particles were unstable in the slightly acidic (pH 5.5) lysate which contained Mg+- and Ca"" ions, but could be stabilized bv addition of a small excess of EDT A and the adjustment to pH 6.5. After removal of sulfur and residual cells the particles were purified by precipitation with 12% PEG followed by repeated banding in a CsCI gradient at an average density of 1.25 g/m!. The particles were stable in TE buffer (0.01 M Tris HC], 0.001 M EDTA), also with 0.05 M Mg h , in 0.1 'Yo triton X 100, or at pH 9.5.

66

W. Zillig et al.

0.5pm

, .

1.0pm

1------.:---11

C

Fig. 4. Electron micrographs of P. iooesei grown on sulfurized gelrite plates (see Materials and Meth ods ). a) and b) negatively stain ed, c) rotatio n sha dowed with Pt.

Thus far we were unable to isolate nucleic acids from the se particles. However, in SDS PAGE they exhibited a defined pattern of proteins (Fig. 7). By treatment with phenol, a group of bands with high molecular weights was tr ansformed into two band s of 82 and 41 K daltons, the latter being the major component, where as the bands with 26 and 25 K daltons remained un changed.

DNA-dependent RNA polymerase

Q

c

b

The DNA-dependent RN A polymer ase of P. woesei was isolated by the standard procedure (Prangishvilli et al., 1982). As typical for RNA polymerases of archaebacteria, it wa s insensitive to rifampicin and streptolydigin. Its PAGE component pattern (Fig. 7) wa s of the same type as those of sulfur-dependent archaebacteria i. e. Thermoproteales and Sulfolobales (Schnabel et al., 1983) and of T. celer (Zillig et al., 1983). In Ouchterlony's gel-immunodiffusion test, it showed no cross-reaction with the polymerase of H. acidocaldarius (not shown), but incomplete immunochemical cross-reaction with the polymerases from Tbermococcus celer (Zillig et al., 198 3) and the isolate ANI from New Zealand employing antisera directed again st both the T. celer and the ANI enzyme (Fig. 8). Th e latter two enzymes exhibited complete cross-reaction with each other with both antisera. Thus, P. woesei along with the other two organisms belongs to the Thermococcaceae, but it is less closely related to T. celer and ANI than the se are to each other.

DNA-rRNA hybridization

Fig. 5. Electron micrographs of thin sections of P. tooesei showing a) cross-striated bundles of filament s; b) possible cross section of a bundle; c) constricted cell exhibiti ng two-layered envelope and containing virus-like particles within tubular structures.

A recentl y developed method allows the calculation of phylogenetic distances from hybridization homologies (Klenk, 1986). These hom ologies were derived from the kinetics of cross hybridi zation of total rRNAs with filterbound DNAs by normalization for genome length and numb er of rRNA operons per genome and were then tr ansformed into sequence difference values (corresponding to phylogenetic distances) by an empirical formula (Klenk, 1986; Bonner et al., 1973). The phylogenetic tree

Pyrococcus woesei, sp.nov.

67

Suliolobales and of the methanogens + halophiles, between which its branching point from the rest of the archaebacteria is situated.

Q

0.1 )Jm

--

122 101

44

33 26

17.5

b

0.2).Jm

I

13.8 I

2 3 4

KD

Fig. 7. SDS PAGE of virus-like particles and DNA-dependent RNA polymerase of P. woesei. (1) Virus-like particle without and (2) with phenol treatment; (3) RNAP of Sulfolobus acidocaldarius DSM 639; (4) RNAP of P. woesei.

AN1

C.W.

AN1

Fig. 6. Negatively stained virus-like particles from lysate of P. iooesei concentrated 800 fold by centrifugation (a) and virus-like particles in thin sections of P. iooesei cells: particles in pack of tube-like structures (b); distorted crystal (c).

of the archaebacteria constructed in this way corresponds closely to that obtained from 165 rRNA total sequence data (Waese and Olsen, 1986, showing additivity of distances over the whole range. The analysis of the phylogenetic position of P. woesei in relation to T. celer, ANI and a number of Thermoproteales, Sulioiobales. methanogens and H. halobium done in this way (Fig. 9) proves that P. woesei belongs to the Thermococcaceae but is at a distance from T. celer and ANI which justifies placing it into another genus of this family. Its distance from the other isolates confers a phylogenetic depth to this group comparable to those of the groups of the Thermoproteales +

T.e.

r.e .

r.c.

c.w. AN1

Fig. 8. Comparison of DNA-dependent RNA polymerases of P. woesei (c. w.) Thermococcus celer (T. c.) and the isolate AN1 from New Zealand by Ouchterlany's gel immunodiffusion test. Antiserum directed against ANI RNAP (left) and T. celer RNAP (right).

Discussion Besides Pyrodictium occultum, Pyrococcus woesei is the only extreme thermophile known to date to grow optimally above I 00 "C, In nature, it occurs in the same environmental niche as Pyradictium. Relative to other sulfur-dependent archaebacteria it grows fast, with a generation time as low as 35 min and reaches a higher yield approaching I gil. The DNA-dependent RNA polymerase (V. Gawantka, unpublished) and the glyceraldehyde phosphate dehydrogenase (R. Hensel, pers. comm.) exhibit extreme thermostabilities, the former being stable up to

68

W. Zillig et al.

/THERMOCOCCALESI ITHERMOPLASMALESI Ihermoplus acidophilum

IMETHANOM ICROBI ALES I Methanolobus lindarius Pyrococcus woesei

[SULFOLOBALES I

Fig. 9. Phylogenetic tree of representative species of archaebacteria derived from DNA/rRNA cross-hybridization velocities. Distances correspondto calculated sequence differences. For detailssee Materials and Methods.

HA LOBACTER IALE S Sulfolobus sol falaricus Pl

!METHANOCOCCAL@j Melhanococcus vannielii

Sulfolobus brierlsyi

Desulfuralobus ambivalens

EUBACTERIA Escherichia coli Melhanolhermus fervidus =------," Methanoboclerium thermoautol roph icu m

IMETHANOBACTERIALES

I

ARCHAEBACTERIA

Desulfurococcus mobilis

Thermoproleus lena x

[ffiERMOPROTEALES I 105°C, the latter up to 100 "C. During growth, the organism produces little H 2S and much less of the offensive odor typical of T. celer and Desul{urococcus mucosus. It thus appears suited for mass production. The isolation procedure accounted for the preference of the organism to grow attached to solid supports. P. woesei can grow by sulfur respiration, preferably with yeast extract or bactotryptone as carbon sources. It grows poorly on polysaccharides, unless Hz + S° are available, probably as energy sources. In the absence of S°, only yeast extract served as an effective carbon source. Hz is required in this case, possibly for energy generation with an electron acceptor present in the yeast extract. The dense tuft of fimbria 12 nm in diameter, tound attached to one pole of the cell during growth on solid supports does not serve for locomotion since it is absent when P. woesei grows in liquid media. Possibly it effects attachment to the surface and/or transport of sulfur or acts in the mats as in a ciliated epithelium. The upper temperature limit for growth is almost 3°C higher in the attached than in the free state, suggesting a role of the filaments in thermotolerance. The function of the striated lattices of thin filaments seen in the thin sections remains unknown. Even its crystalline nature might be an artifact due to the high concentration of its main protein component in the cell and to the special conditions of preparation. Final proof for the viral nature of the icosahedral particles is lacking. Yet, their liberation by cell lysis, their high titer in the lysates and their defined protein composition are suggestive. The tubular particle-containing structures

seen in thin sections resemble those found in plant cells infected with several different icosaedric RNA viruses (Conti and Lovisolo, 1971; Hitchborn and Hills, 1968; Martelli and Russo, 1977). The mechanism of lysis induction is not understood but is in some way related to nutrient consumption. Before the discovery of P. woesei, the sulfur-dependent archaebacterium T. celer (Zillig et al., 1983) appeared phylogenetically in a unique and isolated position (Woese and Olsen, 1986), on a short branch close to the origin of the kingdom and more or less between its major branches. As shown by cross-reaction in Ouchterlony's immunodiffusion test, the genus Pyrococcus, the isolate ANI from New Zealand and T. celer belong to the same family, the Thermococcaceae. As indicated by the distance between Pyrococcus, Thermococcus and ANI (Fig.9), the phylogenetic depth of this family and its separation from the Thermoproteales and the Methanococcales are large enough to call for the introduction of a separate order, the Thermococcales, which forms a third major division of the kingdom between those of the Thermoproteales + Sul[olobales on one side and the methanogens + halophiles on the other. The other "ultra-thermophile", P. occultum, is phylogenetically closely related to Desul{urococcus and Sul[olobus, which are both significantly less thermotolerant. T. celer is a "normal" extreme thermophile (temperature optimum 90°C) like most Thermoproteales, and AN 1 is even less thermoresistant (temperature optimum 75°C). Ultra- in contrast to extreme thermophilia thus appears as a special adaptation rather than a primeval feature.

Pyrococcus tooesei, sp.nov.

Description of a novel order, family, genus and species Order Thermococcales, Zillig ord. nov. Ther.mo.coc.ca'les M.L.fem.pl.n. Thermococcaceae type family of order; -ales ending to denote order. M.L.fem.pl.n . Thermococcales the Thermococcaceae order. Cells spherical to elongated, often constricted or in diploforms, gram negat ive, ana erobi c, utilize yeast extract, peptides (bactotryptone), polysaccharides with production of CO 2 and reduction of sulfur to H2S . Occur in the sediments of marine solfataras or in continent al solfata ras with elevated NaCI conte nt. Divide by constriction. Cell envelope 5 layer, no murein sacculus. Lipids contai n glycerol ethers of phytanol. RNA polymerase rifamp icin and streptolydigin resistant. RNA polymerase component patt ern of the same type as in Thermoproteales and Suliolobales. Cnicbterlony ingel-immunodiffusion cross-reaction among RNA polymerases within the or der but not with enzymes from Thermoproteales or Suliolobales. Related to each other and distant from Thermoproteales and Sullolobales on grounds of rRNA DNA cross-hybr idization. On grounds of the nature of thei r envelopes, lipids, RNA polymerases, 165 rR NA sequence and their insensitivity to vanco mycin, streptomycin and chloramphenicol the Thermococcales are archaebacteria.

69

Ultr a-thermophili c anaerobes. Existing in marin e solfatar as at temperatures around 100 °C. Grow optimal y between 100 and 103 °C in the presence of 30 gil Nael. Upper temperatur e limit 104 .8 °C. Yeast extr act and peptid es (bacto trypto ne) and , barely polysaccharide s, utilized by sulfur respir ation, yeast extra ct also in the absence of sulfur and in the pr esence of H 2, and polysaccharides efficiently in the presence of H 1 and S°. No fermenta tion observed. Envelope S layer devoid of murei n. Oth er features as described for the order. G + C cont ent of the DN A 37 .5 mol% . Type strain DSM 3773. Isolat ed fro m 102 to 103 °C hot sediments in vents of marin e solfataras at the beach of Vulcano island, Italy. Acknowledgement. We thank Michael Rettenberger for measuring the G+ C ratio by the hydrolysis-HPLC method. Note added in proof. After this paper had been submitted, Fiala and Stetter (198 6) described an apparently similar though metabolically different organism, Pyrococcus [uriosus sp. nov., Fiala and Stetter, 1986. Comparison of restriction patterns of the DNAs of both organisms revealed striking similarities, though some significant differences were seen. Accounting for the similarities but also the differences, we now propose that our isolate represents a second species of the same genus.

References Thermococcaceae, Zillig, [am. nov. Ther.mo. coc.ca'ce.ae. M.L.m asc.n. Th ermococcus type genus of the family; -aceae ending to denote a family. M.L.f em.pl.n. Thermococcaceae the Thermococcus family. Onl y one family Thermococcaceae is known in the order Thermococcales. Cell appea rance an d metabolism and other featur es as described for the or der. Th e family Thermococcaceae contai ns two genera : The first, Thermococcus (Z illig et aI., 1983), is the type genus. The second is Pyrococcus, Stette r and Fiala, gen. nov., described by Fiala and Stetter (1 986). Th e genus Pyrococcus contains two sl' l'\:it''' . Th e first is Pvrococcus [uriosus, Steu er :111\ 1 lt.i l.t, sp. nov. , described by Fiala and Stetter , 1') :-)(, ) . Th e second is Py roCOCCl/S tooesi, Zillig, Sl'. 1WI .

Pyrococcus tooesei, Zillig (sp. nov.) woe'se.i of Woese, named for Carl R. Woese, who recognized the archaeb acteria and their testimony for phylogeny. Roughly spherical to elongated, often constricted, cells of 0.5 to Zum diameter, frequently linked to doubl ets by short thin thr eads, gram neagative with large bundl es of smoothly bent filaments (flagellae?) att ached to one pole when cells grew on solid supp orts.

Bonner, T. I., Brenner, D. J., Neujield, B. R., Britten, R. J.: Reduction in the rate of DNA reassociation by sequence divergence. ]. Molec. Bio!. 81, 123- 135 (1973) Brock, T. D., Brock, K. M., Belly, R. T., Weiss, R. L.: Sulfolobus: a new genus of sulfur-oxidizing bacteria living at low pH and high temperature. Arch. Mikrobio!. 84, 54- 68 (1 972) Conti, M., Louisolo. 0 .: Tubular structures associated with maize rough dwarf virus particles in crude extracts: Electron microscopy study. ]. gen. Viral. 13, 173-176 (1971) Fiala, G., Stetter, K. 0 .: Pyrococcus [uriosus sp. nov. represents a novel genus of marine heterotrophic archaebacreria growing optimally at 100°C. Arch. Microbial. 145,5 6-61 (1 986) Hitchborn, j. H ., Hills, G. [.. A study of tubes produced in plants infected with a strain of turnip yellow mosaic virus. Virology 35,50-70 (1968) Klenk , H.-P.: Diploma Thesis, Eberhard-Karls-Universirat, Tiibingen (1 986) Klenk, H.-P., Haas, B., Schwass, V., Zillig, W.: Hybridization homology: A new parameter for the analysis of phylogenetic relations, demonstrated with the urkingdom of the archaebacteria. ]. Molec. Evo!. (1987), in press Martelli, G. P., Russo, M.: Plant virus inclusion bodies. In: Advances in Virus Research, Vol. 21 (M. A. Lauffer, F. B. Bang, K. Maramorosch and K. M. Smith, eds.), pp. 175-266. New York, Adacemic Press 1977 Meijs, H. W., Schilperoort, R. A.: Determination of the amount of DNA on nictrocellulose membrane filters. FEBS Lett. 12, 166-168 (1971) Morgan, H. W., Daniel, R. M.: Proceedings of the XlII International Congress of Microbiology, Boston. Mass.. USA, August 1982

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Prangishuilli, D., Zillig, W., Gierl, A., Biesert, 1., Holz, I.: DNAdependent RNA polymerases of thermoacidophilic archaebacteria. Europ. ]. Biochem. 122, 471-477 (1982)

Schnabel, R., Thomm, M., Gerardy-Schabn, R., Zillig, W., Stetter, K. 0., Huet, H.: Structural homology between different archaebacterial DNA-dependent RNA polymerases analyzed by immunological comparison of their components. EMBO ]. 2, 751-755 (1983) Silberklang, H., Gillum, A. M., Raibhandary, Y. 1. : Use of in vitro 32p labeling in the sequence analysis of nonradioactive tRNAs. Meth, Enzymo!. 59G, 58-1 09 (1979)

Tu, j., Prangishuilli, D., Huber, H., Wildgruber, G., Zillig, W., Stetter, K. 0 .: Taxonomic relations between archaebacteria including 6 novel genera examined by cross hybridization of DNAs and 16S rRNAs. ]. Molec, Evo!. 18,109-114 (1982) Woese, C. R., Gupta, R., Hahn, C. M., Zillig, W., Tu, j.: The phylogenetic relationship of three sulfur dependent archaebacteria. System. App!. Microbiol, 5, 97- 105 (1984)

Woese, C. R., Olsen, G.]. : Archaebacterial phylogeny: Perspectives on the urkingdoms. System. App!. Microbiol, 7, 161-1 77 (1986)

Zillig, W., Stetter, K. 0 ., Scharer, W., [anekouic, D., Wunderl, S., Holz, I., Palm, P.: Thermoproteales: A novel type of extremely thermoacidophilic anaerobic archaebacteria isolated from Icelandic solfataras, Zb!. Bakt, Hyg., 1. Abt, Orig. C 2, 205-227 (1981)

Zillig, W., Holz, I., [anekovic, D., Schafer, W., Reiter, W. D.: The archaebacterium Thermococcus celer represents a novel genus within the thermophilic branch of the archaebacteria. System. App!. Microbiol. 4, 88- 94 (1983) Zillig, W., Yeats, S., Holz, I., Bock, A., Rettenberger, M., Gropp, P., Simon, G.: Desulfurolobus ambivalens gen. nov., sp. nov., an autotrop hic archaebacterium facultatively oxidizing or reducing sulfur. System. App!. Microbio!. 8, 197-203 (1986).

Professor Dr. Wolfram Zillig, Max-Planck-Institur fur Biochemie, D-8033 Martinsried