Wolf-Dieter Reiter

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.

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.

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.

Glycogen in thermoacidophilic archaebacteria of the genera Sulfolobus, Thermoproteus, Desulfurococcus and Thermococcus

Glycogen has been found in thermoacidophilic archaebacteria of the genera Sulfolobus, Thermoproteus, Desulfurococcus and Thermococcus. Thermoplasma acidophilum yielded a related, though less defined compound.Glycogen was identified by elementary analysis, infrared spectroscopy, the nature of the hydrolysis products, the iodine reaction, and the nature of the products of periodate oxydation and reduction. The average chain length was 7.From crude extracts of Sulfolobus and Thermoproteus complexes of glycogen with 4 respectively 2 proteins have been isolated by CsCl density gradient centrifugation. In either case, one of the proteins was identified as glucosyl transferase.The glucosyl transferase of Sulfolobus acidocaldarius strain B 12 utilizes UDP-glucose as well as ADP-glucose as substrates, with Km values of 0.42 and 0.2 mM respectively and turnover numbers of 4.6 and 5.2 per second respectively.In electron micrographs the isolated glycogen protein complex appears as scale like aggregates, whereas in cell sections amorphous bodies fill large portions of the cells.

Gene expression in archaebacteria: physical mapping of constitutive and UV-inducible transcripts from the Sulfolobus virus-like particle SSV1.

SummaryThe transcription of the genome of the UV-inducible Sulfolobus virus-like particle SSV1 was studied. Eight different transcripts could be distinguished by Northern analysis that were present in uninduced cells and that coordinately increased in amount after UV induction of SSV1. Using single-stranded DNA probes from different parts of the genome, the approximate map positions of these RNAs and the directions of transcription were determined. In two cases, terminator read-through resulted in the formation of more than one RNA species from a single 5′ end and therefore the eight different RNAs corresponded to only five different transcriptional starts. Two RNAs sharing a common 5′ end encode SSV1 structural proteins. The 5′ end of these transcripts was determined by S1 nuclease analysis. About 20 nucleotides upstream of the transcriptional start of these RNAs, there is an AT-rich region resembling putative promoter sequences which have been found at a similar distance 5′ to the genes encoding stable RNAs in Thermoproteus. In addition to the eight constitutive transcripts, a UV-inducible RNA of 0.3 kb was mapped on the SSV1 genome. In contrast to all other RNAs, it was not detectable in uninduced cells and it is expressed shortly before the amplification and packaging of the SSV1 genome commences.

Identification and characterization of the genes encoding three structural proteins of the Sulfolobus virus-like particle SSV1

SummaryThree structural proteins, VP1, VP2 and VP3, of the virus-like particle SSV1 of the thermoacidophilic archaebacterium Sulfolobus sp. strain B12 were purified. VP1 and VP3 are very hydrophobic and show a high degree of homology. They consist of 73 and 92 amino acid residues, respectively. The third protein, VP2, is extremely basic containing 29 basic amino acids but only 4 acidic ones in a total of 74 amino acid residues. The genes encoding these three proteins were mapped within the genome by comparison of N-terminal amino acid sequences with the SSV1 DNA sequence. The three genes are closely linked in the order VP1-VP3-VP2 and the coding strand is the same in all three genes. Ten nucleotides separate the stop codon for VP1 from the initiation codon for VP3 and one nucleotide separates the genes encoding VP3 and VP2. Duplicate putative ribosome binding sites are found upstream of the initiation codons for VP2 and VP3. The major coat protein VP1 does not start with a methionine residue and appears to be the result of proteolytic cleavage of a precursor molecule. Transcription of the region encoding VP1, VP2 and VP3 results in the formation of two mRNAs of 0.5 kb and 1.0 kb, the shorter one only encoding VP1, the longer one spanning all three genes. A 61 bp sequence encoding part of VP1 is exactly repeated within the gene for VP3 and these identical sequences are translated into stretches of identical amino acids in the two proteins. A function of this repeated DNA sequence beyond its coding properties is very likely.

Viruses of Archaebacteria

The archaebacteria constitute the third distinct urkingdom of life, beside eubacteria and eucytes (eukaryotic nucleus and cytoplasm) (Woese and Fox, 1977; Woese et al., 1978; Fox et al., 1980). They exhibit a characteristic mosaic of features, some of them—e.g., their lipids—unique to the group (for review see Langworthy, 1985); others—e.g., the organization of genes in operons (Konheiser et al., 1984; Hamilton and Reeve, 1985; Reeve et al., 1986; Reiter et al., 1987a) and the existence of ribosome-binding sites in mRNAs (Reiter et al., 1987a, and literature cited therein)—of eubacterial quality; and a third type—e.g., the ADP ribosylatability of their EFIIs by diphtheria toxin (Kessel and Klink, 1982)—of eukaryotic quality. Most interestingly, features of a fourth group—e.g., the structures of 5S rRNAs, initiator tRNAs, and DNA-dependent RNA polymerases and the occurrence of introns in tRNA genes—are highly divergent in different archaebacteria (Zillig et al., 1985a). Phylogenetically, the archaebacterial kingdom is deeply divided into three major branches (Woese and Olsen, 1986; Klenk et al., 1986): (1) the methanogens (Methanococcales, Methanobacteriales, and Methanomicrobiales) (for review see Whitman, 1985) plus extreme halophiles (Halobacteriales and Thermoplasmales) (for review see Kushner, 1985); (2) the sulfur-dependent extremely thermophilic Thermococcales (Woese and Olsen, 1986; Zillig et al., 1987); and (3) the sulfur-dependent, extremely thermophilic Thermoproteales plus Sulfolobales (for review see Stetter and Zillig, 1985) (Fig. 1).

Homologies of components of DNA-dependent RNA polymerases of archaebacteria, eukaryotes and eubacteria

Summary Using an immunochemical approach homologies between single components of DNA-dependent RNA polymerases from eubacteria, archaebacteria and eukaryotes were investigated. The largest components of all RNA polymerases included in this study are homologous to one another indicating a monophyletic origin of these proteins. Immunological crossreactions show that one of the large subunits present in the enzymes of sulfur-dependent archaebacteria is split into two smaller components in methanogens and halophiles. One of these smaller components roughly corresponds to the second largest subunit of the three eukaryotic enzymes whereas the other one shares antigenic determinants with subunit s of eubacterial RNA polymerases. Semi-quantitative evaluation of the data suggests that the three nuclear RNA polymerases of eukaryotes have evolved from an ancestral enzyme of the type that is found in sulfur-dependent archaebacteria.

Identification and characterization of a defective SSV1 genome integrated into a tRNA gene in the archaebacterium Sulfolobus sp. B12

SummaryWithin the chromosome of the archaebacterium Sulfolobus sp. B12, a 7.4 kb region was identified which displayed extensive sequence similarities to the 15.5 kb genetic element SSV1 carried by the same strain both as a circular form and as a site-specifically integrated copy. DNA sequence analysis indicated that this 7.4 kb region (designated SSV1intB) represented an SSV1-like element distinguishable from the full-length integrated copy (designated SSV1intA) by extensive deletions and point mutations. The physical organization of DNA sequences of SSV1intB indicated that this element was integrated at the same attP site as previously identified for SSV1intA. A comparison of the DNA sequences at the left attachment sites of SSV1intA and SSV1intB revealed that they both represented very similar putative arginine tRNA genes followed by a 10 by inverted repeat sequence. S1 nuclease mapping experiments indicated that these tRNA genes are transcribed.

Archaebacterial virus host systems

Summary Within a review on archaebacterial virus host systems particular emphasis is laid on the description of the novel virus-like particle SSV1 (formerly SAV1) of Sulfolobus solfataricus strain B12, and of the novel viruses TTV1, 2, 3 and 4 of Thermoproteus tenax . Structure, virus host relationships, gene sequence and expression, genome organization and phylogenetic aspects are discussed.