D. Janekovic

Screening for Sulfolobales, their Plasmids and their Viruses in Icelandic Solfataras

Summary We have sampled acidic springs, water and mud holes of 14 major solfataric fields of Iceland in order to isolate both heterotrophic and autotrophic members of the order Sulfolobales and to find affiliated genetic elements e.g. plasmids and viruses. The diverstiy of 120 isolates was analysed by comparing DNA restriction fragment patterns. The 44 heterotrophic isolates belonged to only two types, the solfataricus (S) type and the islandicus (I) type as judged by the restriction patterns of their DNAs. None of the heterotrophic isolates was able to grow autotrophically. The more than 70 autotrophic isolates belonged to only three types, the Desulfurolobus (D) type, the closely related A type and, rarely, the B type. None of the autotrophic isolates was able to grow heterotrophically. Primary heterotrophic colonies often gave rise to obligately autotrophic isolates when submitted to autotrophic selection, probably because they constituted synthrophic associatons. Inversely, primary autotrophic colonies sometimes yielded obligately heterotrophic isolates when submitted to heterotrophic selection. Cell-free filtrates of the field samples precipitated with PEG 6000 yielded different types of viruslike particles as visualized by electron microscopy. Some of these were probably Thermoproteus viruses. No infectious or even lytic virus was obtained from these samples. Four different multicopy plasmids, three, pRN1, pRN2 and pHE7 from heterotrophic hosts and one, pDL10, originally found in Desulfurolobus ambivalens DSM 3772, occurring in all but two autotrophs of the D type, were characterized and used for developing cloning vectors. All 18 representatives of the heterotrophic S type and none of the I type were lysogenic for the virus SSV1 originally discovered in S. shibatae. Different lysogens exhibited different ratios of free circular and linearly integrated viral DNA. The I type isolate KVEM10H1 multiplied SSV 1 but did not integrate its genome into the chromosome. One heterotrophic I type and one autotrophic D type isolate carried doublestranded DNA viruses, SIRV and DAFV. SIRV is a stiff rod, 0.95 μm long and 0.026 μm in diameter, consisting of 33 kbpairs of linear double stranded DNA, a strongly basic DNA binding protein and terminal tentacles involved in attachment to thin filaments, most probably pili. It lacks a membrane or hydrophobic coat and represents a novel virus tpye. The flexible filamentous DAFV, 2.3 μm long and 0.027 μm in diameter, containing 56 kbpairs of linear double-stranded DNA, appears to be enwrapped in a membrane. It resembles representatives of the lipotrixviridae, most closely the virus TTV2.

The Archaebacterium Thermococcus celer Represents, a Novel Genus within the Thermophilic Branch of the Archaebacteria

Thermococcus celer, isolated from a solfataric marine water hole on a beach of Vulcano, Italy, is a spheric organism of about 1 μm diameter, during multiplication often constricted to diploforms. The organism utilizes peptides and protein, which are oxidized to CO(2) by sulfur respiration. Alternatively, though less efficiently, it can exist by an unknown type of fermentation. The optimal growth temperature is 88 °C, the optimal pH 5.8, the optimal NaCl concentration 3.8 g/l. Under these conditions with yeast extract (2 g/l) as carbon source and in the presence of finely distributed sulfur (10 g/1), the generation time is about 50 min. The envelope consists of subunits in two dimensional hexagonal dense packing. The absence of murein, the presence of polyisopranyl alcohols in the membrane, the component pattern and the rifampicin resistance of the DNA dependent RNA polymerase and the insensitivity of the organism towards the antibiotics streptomycin and vancomycin prove the archaebacterial nature of Thermococcus celer. The component pattern of the DNA dependent RNA polymerase conforms with the type pattern of RNA polymerases from thermoacidophilic archaebacteria. The absence of an immunochemical cross-reaction of the enzyme from Thermococcus with those from Thermoproteus, Desulfurococcus, Sulfolobus and Thermoplasma and the extent of cross-hybridization of the 16S rRNA with DNAs of other thermoacidophiles place it into the thermoacidophilic branch of the archaebacteria as a novel isolated genus.

Thermoproteales: A novel type of extremely thermoacidophilic anaerobic archaebacteria isolated from Icelandic solfataras

Summary Two types of anaerobic extremely thermoacidophilic bacteria have been isolated from more than 50% of the samples collected in solfataric fields throughout Iceland. They both possess highly stable cell envelopes with a characteristic subunit structure, are usually rods of variable length (about 1 to more than 50 μ ) without septa, but can give rise to spheric bodies which exist either attached to the rods or in free state. Under certain conditions, truly branched filaments are frequently observed. The similarities in morphology and growth characteristics indicate that the two types are related to each other. In view of its variable appearance the larger organism (diameter about 0.4 μ m) was called Thermoproteus tenax . Thermoproteus tenax forms H 2 S and CO 2 from elemental sulfur and organic substrates, like glucose, ethanol, malate or formamide. The absence of murein, the presence of polyisoprenoid ether lipids, the complete resistance against the antibiotics rifampicin, streptomycin, vancomycin and chloramphenicol and the component pattern and rifampicin insensitivity of the DNA-dependent RNA polymerase are evidence that Thermoproteus tenax belongs to the archaebacteria. It represents a novel order of the thermoacidophilic branch of this urkingdom.

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

Summary The anaerobic sulfur-reducing marine archaebacterium Pyrococcus woesei is an “ultra-thermophile” growing optimally between 100 and 103°C at pH 6 to 6.5 and 30 g/l NaCl. Growth proceeds, on solid supports or in suspension, by sulfur respiration of yeast extract or peptides, on yeast extract also without So in the presence of H2, or on polysaccharides in the presence of H2 and So. The generation time was as low as 35 minutes either on solid supports or in suspension. The cells have a roughly spherical, often elongated and constricted appearance, similar to Thermococcus celer. Frequently, they occur as diploforms. Cells grown on solid supports have dense tufts of flagellae or pili attached to one pole. When P. woesei was grown by sulfur respiration on yeast extract or bactotryptone 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 composition. 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 AN1 from New Zealand. Quantitative analysis of its phylogenetic position by DNA-rRNA cross-hybridization places it at the end of a long branch of the Thermococcaceae, whereas the isolate AN1 is on a branch of intermediate length and T. celer on an extremely short branch. The phylogenetic depth of this group and its clear separation from the neighbouring Thermoproteales and Methanococcales call for the introduction of a separate order: Thermococcales, which represents a third major division of the archaebacteria between the Thermoproteales + Sulfolobales and the methanogens + halophiles.

SAV 1, a temperate u.v.-inducible DNA virus-like particle from the archaebacterium Sulfolobus acidocaldarius isolate B12.

Sulfolobus acidocaldarius, strain B12, which harbours a double‐stranded DNA species both as a plasmid and in a linear form, which is integrated at a specific site of the chromosome, produces virus‐like particles upon u.v. irradiation. These particles contain the same circular DNA and a number of coat proteins and are probably surrounded by a lipid membrane. They are lemon shaped, 100 x 60 nm in size and carry tail structures at one pole. The host cell recovers and remains lysogenic after virus production. Though a large fraction of liberated particles is found attached to structures derived from the cells, neither adsorption nor infection of a number of Sulfolobus isolates has so far been observed.

The Archaebacterium Thermofilum pendens Represents, a Novel Genus of the Thermophilic, Anaerobic Sulfur Respiring Thermoproteales.

Thermofilum pendens, an anaerobic, sulfur respiring archaebacterium representing a novel genus, possibly even a novel family, of the extremely thermophilic mildly acidophilic Thermoproteales, has been isolated from an Icelandic solfataric hot spring. The growth of the organism requires peptides, sulfur and H(2)S and, in addition, a fraction of the polar lipids of the distantly related archaebacterium Thermoproteus tenax devoid of phosphate. This fraction cannot be replaced by an analogous fraction from Thermoplasma acidophilum.

Desulfurococcaceae, the Second Family of the Extremely Thermophilic, Anaerobic, Sulfur-Respiring Thermoproteales*

Summary Two novel species of anaerobic thermophilic archaebacteria isolated from acidic hot springs of Iceland, Desulfurococcus mucosus and Desulfurococcus mobilis , representing a second family, termed Desulfurococcaceae , of the order Thermoproteales are described. They utilize yeast extract or casein or its tryptic digest, but not casamino acids, as carbon sources, by sulfur respiration with the production of H 2 S and CO 2 , or by fermentation. The pH optimum of growth is pH 5.5 to 6, the temperature optimum 85 °C. The archaebacterial nature of the Desulfurococcaceae is evident from their insensitivity towards vancomycin, streptomycin and chloramphenicol, the lack of a murein cell wall, the presence of phytanol and polyisoprenoid dialcohols in the lipids, and the composition and the properties of the DNA dependent RNA polymerase. They are closely related to the recently described anaerobic thermoacidophilic sulfur-respiring Thermoproteus tenax representing the first family, Thermoproteaceae of the Thermoproteales . Of the other divisions of archaebacteria, Sulfolobus is the nearest relative. Desulfurococcus mucosus has a slimy polymer attached to its envelope. Desulfurococcus mobilis possesses flagellae in monopolar polytrichous arrangement.

TTV1, TTV2 and TTV3, a family of viruses of the extremely thermophilic, anaerobic, sulfur reducing archaebacterium Thermoproteus tenax

SummaryThree different temperent viruses of the extremely thermophilic chemolithoautotrophic archaebacterium, Thermoproteus tenax, TTV1, TTV2 and TTV3, each contain linear, double-stranded DNA, TTV1 and TTV2 of 16 kb, TTV3 of 27 kb. They are oblong and each consists of an outer envelope and an inner core associated with the DNA.TTV1 contains four major proteins, an envelope of unknown nature and non-protein material linked to two of the proteins in non-covalent manner. The 5′-ends of the DNA are protected by hydrophobic ligands. The viruses have neither homologies with each other nor with the host.Lysogens are induced upon sulfur depletion during autotrophic growth. Alternatively, sensitive, non-lysogenic cells allow lytic multiplication.

Structure and function of the DNA dependent RNA polymerase of the Archaebacterium Thermoplasma acidophilum

Summary DNA dependent RNA polymerase from Thermoplasma acidophilum was isolated by a procedure involving precipitation by polymin P, elution from the sediment, DEAE chromatography, heparin cellulose chromatography, sucrose glycerol gradient centrifugation and DNA cellulose chromatography. This technique has proved to be generally suitable for the isolation of RNA polymerase from Eubacteria and Archaebacteria and is probably also useful for Eukaryotes. The purified enzyme consists of 7 components with the molecular weights 135000 108000, 56000, 35000, 22000, 13500 and 11500. The components with 56000 and 22000 daltons are absent from an incomplete inactive particle which can be separated from active enzyme by sucrose glycerol gradient centrifugation or DNA cellulose chromatography at low glycerol concentration. The component with 35000 daltons can be partially removed by DNA cellulose chromatography at high glycerol concentration and is not required for activity on poly [d(A – T) · d(A – T)]. The optimal conditions for the transcription of poly [d(A – T) · d(A–T)] and native phage DNA were determined. The activity on native phage DNA is only a few percent of that on poly [d(A – T) · d(A – T)]. It is stimulated by the addition of Mn ++ instead of Mg ++ ions and by removal of the component with the molecular weight 35000. The subunit pattern and composition are similar to those of the RNA polymerases of other Archaebacteria. The resistance against rifampicin, streptolydigine and α-amanitine is shared with all other known archaebacterial RNA polymerases.