Viruses, plasmids and other genetic elements of thermophilic and hyperthermophilic Archaea.

We review and update the work on genetic elements, e.g., viruses and plasmids (exluding IS elements and transposons) in the kingdom Crenarchaeota (Thermoproteales and Sulfolobales) and the orders Thermococcales and Thermoplasmales in the kingdom Euryarchaeota of the archael domain, including unpublished data from our laboratory. The viruses of Crenarchaeota represent four novel virus families. The Fuselloviridae represented by SSVI of S. shibatae and relatives in other Sulfolobus strains have the form of a tailed spindle. The envelope is highly hydrophobic. The DNA is double-stranded and circular. Members of this group have also been found in Methanococcus and Haloarcula. The Lipothrivciridae (e.g., T TV1 to 3) have the form of flexible filaments. They have a core containing linear double-stranded DNA and DNA-binding proteins which is wrapped into a lipid membrane. The "Bacilloviridae" (e.g., TTV4 and SIRV) are stiff rods lacking this membrane, but also featuring linear double-stranded DNA and DNA-binding proteins. Both virus types carry on both ends structures involved in the attachment to receptors. Both types are represented in Thermoproteus and Sulfolobus. The droplet-formed novel Sulfolobus virus SNDV represents the "Guttaviridae" containing circular double-stranded DNA. Though head and tail viruses distantly resembling T phages or lambdoid phages were seen electronmicroscopically in solfataric water samples, no such virus has so far been isolated. SSV1 is temperate, TTV1 causes lysis after induction, the other viruses found so far exist in carrier states. The hosts of all but TTV1 survive virus production. We discuss the implications of the nature of these viruses for understanding virus evolution. The plasmids found so far range in size from 4.5 kb to about 40 kb. Most of them occur in high copy number, probably due to the way of their detection. Most are cryptic, pNOB8 is conjugative, the widespread pDL10 alleviates in an unknown way autotrophic growth of its host Desulfurolobus by sulfur reduction. The plasmid pTIK4 appears to encode a killer function. pNOB8 has been used as a vector for the transfer of the lac S (beta-galactosidase) gene into a mutant of S. solfataricus.

Picrophilus gen. nov., fam. nov.: a novel aerobic, heterotrophic, thermoacidophilic genus and family comprising archaea capable of growth around pH 0.

Two species belonging to a novel genus of archaea, designated Picrophilus oshimae and Picrophilus torridus, have been isolated from two different solfataric locations in northern Japan. One habitat harboring both organisms was a dry, extremely acidic soil (pH < 0.5) that was heated by solfataric gases to about 55 degrees C. In the laboratory both species grew heterotrophically on yeast extract and poorly on tryptone under aerobic conditions at temperatures between 45 and 65 degrees C; they grew optimally at 60 degrees C. The pH optimum was 0.7, but growth occurred even around pH 0. Under optimal conditions, the generation time was about 6 h, yielding densities of up to 10(10) cells per ml. The cells were surrounded by a highly filigreed regular tetragonal S-layer, and the core lipids of the membrane were mainly bis-phytanyltetraethers. The 16S rRNA sequences of the two species were about 3% different. The complete 16S rRNA sequence of P. oshimae was 9.3% different from that of the closest relative, Thermoplasma acidophilum. The morphology and physiological properties of the two species characterize Picrophilus as a novel genus that is a member of a novel family within the order Thermoplasmales.

A multicopy plasmid of the extremely thermophilic archaeon Sulfolobus effects its transfer to recipients by mating.

A plasmid of 45 kb, designated pNOB8, was found in high copy number in a new heterotrophic Sulfolobus isolate, NOB8H2, from Japan. Dissemination of the plasmid occurred in six cultures of nine different Sulfolobus strains when small amounts of the donor were added. These mixed cultures exhibited a high average copy number of the plasmid, between 20 and 40 per chromosome, and showed a marked growth retardation. Horizontal transfer of pNOB8 was proved by isolating transcipients from mating mixtures via single colonies. In these isolates, the copy number of the plasmid appeared to be subject to a control mechanism. Cell-free filtrates of donor cultures did not transmit the plasmid, and plating of the donor on lawns of recipients did not result in plaque formation, suggesting that the transfer was not mediated by a virus. Rapid formation of cell-to-cell contacts between differently stained donor and recipient partners was demonstrated after the two strains were mixed. Electron microscopic analysis of mating mixtures revealed many cell aggregates made up of 2 to 30 cells and intercellular cytoplasmic bridges connecting two or more cells. Cells that had been transformed with purified plasmid DNA as well as transcipients isolated from mating mixtures were shown to serve as donors for further transmission of pNOB8. The plasmid undergoes extensive genetic variations, since deletions and insertions were frequently observed in plasmid preparations from the donor strain and from mating mixtures.

Hyperthermus butylicus, a hyperthermophilic sulfur-reducing archaebacterium that ferments peptides.

The hyperthermophilic peptide-fermenting sulfur archaebacterium Hyperthermus butylicus was isolated from the sea floor of a solfataric habitat with temperatures of up to 112 degrees C on the coast of the island of São Miguel, Azores. The organism grows at up to 108 degrees C, grows optimally between 95 and 106 degrees C at 17 g of NaCl per liter and pH 7.0, utilizes peptide mixtures as carbon and energy sources, and forms H2S from elemental sulfur and molecular hydrogen as a growth-stimulating accessory energy source but not by sulfur respiration. The same fermentation products, CO2, 1-butanol, acetic acid, phenylacetic acid, and a trace of hydroxyphenylacetic acid, are formed both with and without of S0 and H2. Its ether lipids, the absence of a mureine sacculus, the nature of the DNA-dependent RNA polymerase, and phylogenetic classification by DNA-rRNA cross-hybridization characterize H. butylicus as part of a novel genus of the major branch of archaebacteria comprising the orders Thermoproteales and Sulfolobales, representing a particularly long lineage bifurcating with the order Sulfolobales above the branching off of the genus Thermoproteus and distinct from the genera Desulfurococcus and Pyrodictium.

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.

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.

DNA-dependent RNA polymerase from the archaebacterium Sulfolobus acidocaldarius.

Purified DNA-dependent RNA polymerase from Sulfolobus acidocaldarius is composed of 10 different subunits, one of which is present as four copies. Their molecular weights are 122 000, 101 000, 44 000, 32 000, 24 000, 17 500, 13 800, 11 800 (four copies), 11 200, 10 800, summing up to a total Mr of 423 500. The sedimentation velocity is 13.5 S, indicating that at 0.5 M NH4Cl the enzyme exists in the monomeric form. At pH 9.2 in cellogel electrophoresis two of the subunits migrate towards the cathode. The composition is quite different from that of a typical eubacterial RNA polymerase. Its complexity reminds one of eucaryotic RNA polymerase. Maximal transcription of DNA from a Halobacterium halobium phage øH (øH DNA) proceeds at pH 8.5 AND 75 DEGREES C. The enzyme is stable up to 75 degrees C and strictly requires a DNA template. øH DNA and poly[d(A-T) . d(A-T)] are the most efficient. The temperature dependence of the transcription rate is characteristic for the template. Actinomycin D and heparin prevent transcription, while rifampicin, streptolydigin and alpha-amanitin have no effect. During storage, even at -- 70 degrees C, the enzyme loses its activity to transcribe øH DNA, whereas transcription of poly[d(A-T) . D(A-6)] remains unaffected.

In vivo and in vitro phosphorylation of DNA-dependent RNA polymerase of Escherichia coli by bacteriophage-T7-induced protein kinase.

After infection with bacteriophage T7 the beta' and to a lesser extent the beta subunits of E. coli DNA-dependent RNA polymerase (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) are phosphorylated by a phage-gene-encoded protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37). The phosphorylation occurs on threonine residues and appears site-specific. It is probably the molecular basis of the early transcriptional control.