Wolfram Zillig

The Sulfolobus-“Caldariella” group: Taxonomy on the basis of the structure of DNA-dependent RNA polymerases

The similarity of the morphology and of DNA composition, the homology of the component patterns of DNA-dependent RNA polymerases and their immunochemical crossreactivity support the conclusion that several extreme thermoacidophiles are related to each other. We name two new species of the genus Sulfolobus. The first, Sulfolobus solfataricus (DSM 1616 and DSM 1617) has the same GC content in its DNA and the same general properties as S. acidocaldarius, but differs significantly from the latter species in the molecular weights of the 11 components of its RNA polymerase and in the salt requirements of this enzyme. The second, Sulfolobus brierleyi, DSM 1651, differs from S. acidocaldarius in several respects. The cells show much less stability at neutral pH. The GC content is significantly lower. The RNA polymerase lacks two components present in the enzymes from the other species. The residual 9 components show larger size differences from the homologous subunits of the S. acidocaldarius enzyme.Like the enzyme from S. solfataricus, the polymerase from S. brierleyi yields an incomplete immunochemical crossreaction with an antibody against the RNA polymerase from S. acidocaldarius.The isolates DSM 1616 and DSM 1617 of Sulfolobus solfataricus are probably identical with or similar to the “Caldariella” strains MT 3 and MT 4, isolated by de Rosa et al. (1975).Like all other known archaebacterial RNA polymerases the enzymes from these species are insensitive to rifampicin and streptolydigin.

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.

Electron Tomography of Ice-Embedded Prokaryotic Cells

Whole cells of archaea were embedded in vitreous ice by plunge freezing and investigated by automated energy-filtered electron tomography at 120 kV. The embedded cells were between 300 and 750 nm thick, and their structures were reconstructed to a resolution of 20-40 nm from tilt series comprising 50-140 images. The dose was kept within tolerable limits. A resolution of 20 nm allowed visualization of the individual stalks of the S-layer of Pyrobaculum aerophilum cells, which had undergone partial lysis, in three dimensions. The attainable resolution for low-dose electron tomography under different experimental conditions was theoretically investigated in terms of the specimen thickness. To obtain 2-nm resolution at 120 kV (300 kV), the specimen must not be thicker than 100 nm (150 nm). For a resolution of 10 nm, the maximum thickness is 450 nm (700 nm). An accelerating voltage of 300 kV is advantageous, mainly for specimens thicker than 100 nm. Experimental investigations so far have resulted in a resolution that is worse by a factor of 2-5 as compared to theory.

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.

Complete nucleotide sequence of the virus SSV1 of the archaebacterium Sulfolobus shibatae.

The DNA sequence of the Sulfolobus shibatae virus SSV1 is the first complete sequence of an archaebacterial virus genome. The viral DNA is a closed double-stranded DNA circle of 15465 bp. The features of the sequence, the positions of all 11 transcripts, the three characterized proteins, and the open reading frames are described.

Genetic elements in the extremely thermophilic archaeon Sulfolobus.

Abstract This minireview summarizes what is known about genetic elements in the archaeal crenarchaeotal genus Sulfolobus, including recent work on viruses, cryptic plasmids, a novel type of virus satellite plasmids or satellite viruses, and conjugative plasmids (CPs), mostly from our laboratory. It does not discuss IS elements and transposons.

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.

Methanothermus fervidus, sp. nov., a novel extremely thermophilic methanogen isolated from an Icelandic hot spring

Summary A rod-shaped extremely thermophilic methanogen is described, growing between 65 and 97 °C with an optimal temperature around 83 °C and a doubling time of 170 min. The GC-content of its DNA is 33 mol %. The isolated cell wall sacculus contains pseudomurein. The complex cell envelope exhibits two layers, each about 12 nm thick; the inner represents the pseudomurein sacculus and the outer a protein envelope. An enriched fraction of RNA polymerase does not react with antiserum against RNA polymerase from Metbanobacterium thermoautotrophicum, indicating that the isolate belongs to a new family, the Methanothermaceae, within the order Methanobacteriales. The new organism is named Methanothermus fervidus.