A new ribosome structure.

Ribosomes derived from the sulfur-dependent archaebacteria are structurally distinct from those types found in ribosomes from eubacteria, eukaryotes, and other archaebacteria. All four ribosome types share a common structural core, but each type also has additional independent structural features. In the smaller subunit derived from sulfur-dependent archaebacteria ("eocytes"), lobes, similar to those found at the base of the eukaryotic small subunits, and an archaebacterial bill, similar to those found on the smaller subunit of archaebacteria and eukaryotes, are present. On the larger subunit from sulfur-dependent archaebacteria, an eocytic lobe, eocytic gap, and eocytic bulge are present. These features, with the exception of the eocytic gap, are found in a slightly modified form on eukaryotic large subunits. These novel ribosomal properties are in general consistent with other molecular biological properties peculiar to these organisms.

Comparative biochemistry of Archaea and Bacteria.

This review compares exemplary molecular and metabolic features of Archaea and Bacteria in terms of phylogenetic aspects. The results of the comparison confirm the coherence of the Archaea as postulated by Woese. Archaea and Bacteria share many basic features of their genetic machinery and their central metabolism. Similarities and distinctions allow projections regarding the nature of the common ancestor and the process of lineage diversification.

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and the orders Thermococcales and Thermoplasmales in the kingdom Euryarchaeota of the archael domain, including unpublished data from our laboratory.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.

Viruses of the extremely thermophilic archaeon Sulfolobus.

Viruses of Sulfolobus are highly unusual in their morphology, and genome structure and sequence. Certain characteristics of the replication strategies of these viruses and the virus-host interactions suggest relationships with eukaryal and bacterial viruses, and the primeval existence of common ancestors. Moreover, studying these viruses led to the discovery of archaeal promoters and has provided tools for the development of the molecular genetics of these organisms. The Sulfolobus viruses contain unique regulatory features and structures that undoubtedly hold surprises for researchers in the future.

Nucleotide sequence of the genes encoding proline tRNA(UGG) and threonine tRNA(GGU) and consensus promoter model of Thermococcus celer.

The nucleotide sequences of the genes encoding tRNA(Pro)(UGG) and tRNA(Thr)(GGU) from the extremely thermophilic archaeon (archaebacterium) Thermococcus celer have been determined. A consensus promoter model was deduced from the comparison of the upstream regions of several stable RNA genes with S1-mapped promoter regions of genes coding for ribosomal proteins and DNA-dependent RNA polymerase components.

Sequence, organization, transcription and evolution of RNA polymerase subunit genes from the archaebacterial extreme halophiles Halobacterium halobium and Halococcus morrhuae.

The genes for the four largest subunits, A, B', B" and C, of the DNA-dependent RNA polymerase were cloned from the extreme halophile Halobacterium halobium and sequenced and their transcription was analyzed. The downstream half of this gene cluster from another extreme halophile Halococcus morrhuae was also cloned, sequenced and its transcription products characterized. The H. halobium genes were transcribed into a common transcript from an upstream promoter in the order B", B', A and C. They are flanked by, and co-transcribed with, two smaller genes coding for 75 and 139 amino acid residues, respectively. Immediately downstream from these genes were two open reading frames that are homologous to ribosomal proteins S12 and S7 from Escherichia coli. In both extreme halophiles these genes were transcribed from their own promoter, but in Hc. morrhuae there was also considerable read-through from the RNA polymerase genes. Sequence alignment studies showed that the combined B" + B' subunits are equivalent to the B subunits of the eukaryotic polymerases I and II and to the eubacterial beta subunit, while the combined A + C subunits correspond to the A subunits of eukaryotic RNA polymerases I, II and III and to the eubacterial beta' subunit. The sequence similarity to the eukaryotic subunits was always much higher than to the eubacterial subunits. Conserved sequence regions within the individual subunits were located which are likely to constitute functionally important domains; they include sites associated with rifampicin and alpha-amanitin binding and two possible zinc binding fingers. Phylogenetic analyses based on sequence alignments confirmed that the extreme halophiles belong to the archaebacterial kingdom.

Evolution of the family of pRN plasmids and their integrase-mediated insertion into the chromosome of the crenarchaeon Sulfolobus solfataricus.

Plasmid pHEN7 from Sulfolobus islandicus was sequenced (7.83 kb) and shown to belong to the archaeal pRN family, which includes plasmids pRN1, pRN2, pSSVx and pDL10 that share a large conserved sequence region. pHEN7 is most closely related to pRN1 in this conserved region. It also shares a large variant region containing several homologous genes with pDL10, which is absent from the other plasmids. The variant region is flanked by the sequence motif TTAGAATGGGGATTC and similar duplicated motifs occur in plasmids pRN1 and pRN2, separated by a few bases. It is inferred that recombination at these sites produces the main genetic variability in the plasmid family. The conserved region of the plasmid, and duplicated copies of the motif, are also present in the genome of Sulfolobus solfataricus P2. Moreover, they are bordered by a partitioned integrase gene (int) and by a 45 bp perfect direct repeat corresponding to the downstream half of a tRNA(Val) gene. The integrase and the direct repeat are highly similar in sequence to the integrase and the chromosomal integration site (att), respectively, of the SSV1 virus, which integrates into the chromosome of Sulfolobus shibatae. Recombination at the att repeats in S. solfataricus would produce a novel plasmid, pXQ1, which carries both an intact integrase gene and a single integration site (att). This strongly suggests that the same mechanism of site-specific integration at a tRNA gene is used for both viruses and plasmids in Sulfolobus.

Genetic requirements for the function of the archaeal virus SSV1 in Sulfolobus solfataricus: construction and testing of viral shuttle vectors.

Directed open reading frame (ORF) disruption and a serial selection technique in Escherichia coli and the extremely thermophilic archaeon Sulfolobus solfataricus allowed the identification of otherwise cryptic crucial and noncrucial viral open reading frames in the genome of the archaeal virus SSV1. It showed that the 15. 5-kbp viral genome can incorporate a 2.96-kbp insertion without loss of viral function and package this DNA properly into infectious virus particles. The selection technique, based on the preferential binding of ethidium bromide to relaxed DNA and the resulting inhibition of endonuclease cleavage to generate a pool of mostly singly cut molecules, should be generally applicable. A fully functional viral shuttle vector for S. solfataricus and E. coli was made. This vector spreads efficiently through infected cultures of S. solfataricus, its replication is induced by UV irradiation, it forms infectious virus particles, and it is stable at high copy number in both S. solfataricus and E. coli. The classification of otherwise unidentifiable ORFs in SSV1 facilitates genetic analysis of this virus, and the shuttle vector should be useful for the development of genetic systems for Crenarchaeota.

A novel virus family, the Rudiviridae: Structure, virus-host interactions and genome variability of the sulfolobus viruses SIRV1 and SIRV2.

The unenveloped, stiff-rod-shaped, linear double-stranded DNA viruses SIRV1 and SIRV2 from Icelandic Sulfolobus isolates form a novel virus family, the Rudiviridae. The sizes of the genomes are 32. 3 kbp for SIRV1 and 35.8 kbp for SIRV2. The virions consist of a tube-like superhelix formed by the DNA and a single basic 15.8-kD DNA-binding protein. The tube carries a plug and three tail fibers at each end. One turn of the DNA-protein superhelix measures 4.3 nm and comprises 16.5 turns of B DNA. The linear DNA molecules appear to have covalently closed hairpin ends. The viruses are not lytic and are present in their original hosts in carrier states. Both viruses are quite stable in these carrier states. In several laboratory hosts SIRV2 was invariant, but SIRV1 formed many different variants that completely replaced the wild-type virus. Some of these variants were still variable, whereas others were stable. Up to 10% nucleotide substitution was found between corresponding genome fragments of three variants. Some variants showed deletions. Wild-type SIRV1, but not SIRV2, induces an SOS-like response in Sulfolobus. We propose that wild-type SIRV1 is unable to propagate in some hosts but surmounts this host range barrier by inducing a host response effecting extensive variation of the viral genome.

Transcription of the halophage phi H repressor gene is abolished by transcription from an inversely oriented lytic promoter.

The temperate phage phi H of the extremely halophilic archaebacterium Halobacterium salinarium encodes a repressor, Rep, which in the immune state represses the production of an early lytic transcript, denoted T4. Rep acts at the transcriptional level by blocking the promoter for T4. The promoter for the rep gene itself is positioned back to back to the promoter for T4, in a manner analogous to that of the cI/cro genes in bacteriophage lambda. Transcription of the rep gene does not occur when the phase is growing lytically. We show that this repressor of rep transcription during lytic growth is due to the transcription per se from the stronger, oppositely oriented promoter for T4, without the need of a phage gene product.

Heterologous in vitro transcription from two archaebacterial promoters.

A cell-free extract of Sulfolobus shibatae is able to specifically initiate transcription in vitro at the promoter of the plasmid-encoded gene for the major gas vesicle protein of Halobacterium halobium and at the promoter for the transcript T4 of the temperate H. halobium phage phi H. The corresponding promoter from the virulent phage mutant phi HL1 yields enhanced transcription in the heterologous system, in agreement with strongly increased in vivo expression.

Transformation of the extremely thermoacidophilic archaeon Sulfolobus solfataricus via a self-spreading vector.

We describe a transformation system for extremely thermophilic archaea of the genus Sulfolobus in the kingdom Crenarchaeota. We have constructed in vitro a recombinant derivative of the recently described conjugative plasmid pNOB8, containing a beta-galactosidase gene downstream of a strong promotor. Transformation of a beta-galactosidase negative mutant of Sulfolobus solfataricus with this construct resulted in its spreading through the culture containing the primary transformants and in efficient restoration of beta-galactosidase activity.

Rooting the archaebacterial tree: the pivotal role of Thermococcus celer in archaebacterial evolution.

The sequence of the 16S ribosomal RNA gene from the archaebacterium Thermococcus celer shows the organism to be related to the methanogenic archaebacteria rather than to its phenotypic counterparts, the extremely thermophilic archaebacteria. This conclusion turns on the position of the root of the archaebacterial phylogenetic tree, however. The problems encountered in rooting this tree are analyzed in detail. Under conditions that suppress evolutionary noise both the parsimony and evolutionary distance methods yield a root location (using a number of eubacterial or eukaryotic outgroup sequences) that is consistent with that determined by an "internal rooting" method, based upon an (approximate) determination of relative evolutionary rates.

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.

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 phylogenetic relationships of three sulfur dependent archaebacteria.

Oligonucleotide catalogs have been determined for the 16S ribosomal RNAs of three sulfur dependent (i.e. "thermoacidophilic") archaebacteria--Sulfolobus acidocaldarius, S. solfataricus, and Thermoproteus tenax. The three form a group specifically related to one another, but are only distantly related to the other archaebacteria--i.e. the group comprising the methanogens, extreme halophiles, and (peripherially) the genus Thermoplasma. The three catalogs exhibit two features unique among bacteria: (1) an unusually high number of long pyrimidine runs, and (2) a remarkably high number of (post-transcriptionally) modified nucleotides.

Genome organization and transcription in archaebacteria.

The genome organization of the archaebacteria is investigated in three model systems: a) rRNA genes of various archaebacteria, b) a plasmid of 15.6 kb from Sulfolobus acidocaldarius which exists in free or integrated form, c) the 59 kb genome of phage phi H of Halobacterium halobium as a model for the unusual structural variability of DNA in this organism. Several variants of this phage have been isolated, their genomes differ by several insertions, a deletion, and an inversion. The frequent inversion and circularization of a 12 kb segment of DNA appears to be linked to the presence of two copies of an IS element at its flanks. DNA-dependent RNA polymerases have been isolated from a large number of archaebacteria including representatives of 4 families of the novel order Thermoproteales . As shown by immunological methods, they are closely related to those of eukaryotes. Two different types of RNA polymerase exist in the two main branches of the archaebacteria. The role of one component of the enzyme of Thermoplasma acidophilum was elucidated using an in vitro transcription system.