Ingelore Holz

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.

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.

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.

Desulfurolobus ambivalens, gen. nov., sp. nov., an autotrophic Archaebacterium facultatively oxidizing or reducing sulfur

Summary The archaebacterium Desulfurolobus ambivalens gen. nov. sp. nov., family Sulfolobaceae , is an obligate chemolitho-autotroph, facultatively growing by the oxidation with O 2 or the reduction with H 2 of elemental sulfur with CO 2 as sole carbon source. D. ambivalens contains several plasmids, one of which, pSL10, is amplified during anaerobic growth. This amplification is accompanied by the appearance of virus-like particles resembling those described for Sulfolobus spec. B12. A subclone of D. ambivalens not containing the plasmid pSL10 is still able to grow by both sulfur oxidation and sulfur reduction indicating that the plasmid does not harbor genes for chemolithoautotrophic growth. The phylogenetic position of this organism within the Sulfolobaceae and the taxonomy of this group are discussed in the light of molecular evidence.

The genetic element pSSVx of the extremely thermophilic crenarchaeon Sulfolobus is a hybrid between a plasmid and a virus.

A new Sulfolobus islandicus strain, REY15/4, harboured both a novel fusellovirus, SSV2, and a small plasmid, pSSVx. The plasmid spread in S. solfataricus P1 together with the virus after infection with either the supernatant of a culture of REY15/4 or purified virus. Spreading of the plasmid required co‐transfection with either SSV2 or the related SSV1 as helpers. Virus purified from REY15/4 constituted a mixture of two sizes of particles, one with the dimensions of a normal fusellovirus and the other smaller. Cloned SSV2 produced only the larger particles and only SSV2 DNA, indicating that the smaller particles contained pSSVx packaged into capsids made up of SSV2 components. The 5.7 kb genome of pSSVx revealed regions of high sequence similarity to the cryptic Sulfolobales plasmids pRN1, pRN2 and pDL10. Thus, pSSVx belongs to the family of pRN plasmids that share a highly conserved region, which probably constitutes the minimal replicon. They also contain a variable region showing no sequence similarity. In pSSVx, this region contains three open reading frames (ORFs), two of which are juxtapositioned and show high sequence similarity to a tandem of ORFs in fusellovirus genomes. Neither pRN1 nor pRN2, which lack this tandem, spread in the presence of the fuselloviruses, which implies that the sequences of these ORFs enable pSSVx to use the packaging system of the viral helpers for spreading.

Relationships between fuselloviruses infecting the extremely thermophilic archaeon Sulfolobus: SSV1 and SSV2

The fusellovirus SSV2 from an Icelandic Sulfolobus strain was isolated, characterized and its complete genomic sequence determined. SSV2 is very similar in morphology, replication, genome size and number of open reading frames (ORFs) to the type virus of the family, SSV1 from Japan, except in its high level of uninduced virus production. The nucleotide sequences are, however, only 55% identical to each other, much less than related bacteriophage, related animal viruses and the rudiviruses of Sulfolobus, SIRV1 and SIRV2. Nevertheless the genome architecture is very similar between the two viruses, indicating that despite this genomic dissimilarity the virus genomes are mostly homologous. Unlike SSV1, the sequence of SSV2 indicates integration into a glycyl tRNA gene and is completely missing a DNA packaging gene. There is a unique, perfectly tandemly directly repeated sequence of 62 nucleotides in SSV2 that has no similarity to known sequences or structures. By comparison to the SSV2 genome, an integrated partial fusellovirus genome was found in the Sulfolobus solfataricus P2 genome further confirming the dynamism of the Sulfolobus genome. Clustering of cysteine codon containing ORFs both in SSV1 and SSV2 indicates that these Fuselloviridae arose from a genome fusion event.

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.