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W Zillig

Publications and source records attributed to W Zillig.

At least 19 recordsLinked to original sources
PubMed1998

Biochemical and phylogenetic characterization of the dUTPase from the archaeal virus SIRV.

The derived amino acid sequence from a 474-base pair open reading frame in the genome of the Sulfolobus islandicus rod-shaped virus SIRV shows striking similarity to bacterial dCTP deaminases and to dUTPases from eukaryotes, bacteria, Poxviridae, and Retroviridae. The putative gene was expressed in Escherichia coli, and dUTPase activity of the recombinant enzyme was demonstrated by hydrolysis of dUTP to dUMP. Deamination of dCTP by the enzyme was not detected. Phylogenetic analysis based on amino acid sequences of the characterized enzyme and its homologues showed that the dUTPase-encoding dut genes and the dCTP deaminase-encoding dcd genes constitute a paralogous gene family. This report is the first identification and functional characterization of an archaeal dUTPase and the first phylogeny derived for the dcd-dut gene family.

Amino Acid Sequence↗
PubMed1998

Conjugation in archaea: frequent occurrence of conjugative plasmids in Sulfolobus.

We describe five novel conjugative plasmids (CPs) and two subfamilies, each comprising several closely related variants of CPs isolated from colony-cloned strains of the extremely thermophilic, heterotrophic archaeon Sulfolobus islandicus, which were obtained by plating of samples from Icelandic solfataras after liquid enrichment. They are related to each other and to the previously described CP pNOB8 from a Japanese Sulfolobus strain in that they share essential functions and limited similarity of genomes as demonstrated by DNA cross-hybridization and sequences. All these plasmids thus form a family of highly efficient self-spreading elements directly transferred from donor into recipient cells. Conjugation is initiated by pair formation, followed by selective transfer of the plasmids into the recipient and expression of transfer functions. Some of these CPs exclude superconjugation of the transcipients with closely related CPs. The novel CPs are stable upon conjugative transfer, but vary upon growth of transcipients. The stability of the CPs is higher in their original hosts or in related S. islandicus strains, than in Sulfolobus solfataricus strain PH1 as recipient. The deletion variant pING3 has lost the ability to transfer itself but is still subject to being transferred by the transfer apparatus of its complete relative, pING6. The dissection of genes and functions has been initiated by characterizing this incomplete variant.

Bacteriological Techniques↗
PubMed1998

Bioenergetics and cytoplasmic membrane stability of the extremely acidophilic, thermophilic archaeon Picrophilus oshimae.

Picrophilus oshimae is an extremely acidophilic, thermophilic archaeon that grows optimally at 60 degrees C and at pH 0.7. It is an obligatory acidophile that does not grow at pH values above 4.0. The proton motive force in respiring cells is composed of a large transmembrane pH gradient, inside less acid, and a reversed transmembrane electrical potential, inside positive. Cells maintain an intracellular pH at around 4.6 at extracellular pH values ranging from 0.8 to 4.0. Above pH 4.0 cells lyse rapidly and lose their viability. Liposomes prepared from lipids derived from P. oshimae have an extremely low proton permeability at acidic pH. However, at neutral pH, the lipids are unable to assemble into regular liposomal structures. These observations suggest that the loss of viability and cell integrity above pH 4.0 is due to an impairment of the barrier function of the cytoplasmic membrane.

Cell Membrane↗
PubMed1998

Genetic elements in the extremely thermophilic archaeon Sulfolobus.

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.

Chromosome Mapping↗
PubMed1998

Sulfolobus islandicus plasmids pRN1 and pRN2 share distant but common evolutionary ancestry.

The complete sequence of the plasmid pRN2 from the thermoacidophile Sulfolobus islandicus has been determined. The plasmid was found to be circular and 6959 bp in length. S. islandicus harbors another endogenous plasmid, pRN1, and comparison of pRN1 and pRN2 revealed that these two plasmids are essentially homologous, although very distantly related. pRN1 and pRN2 share several stretches of highly conserved noncoding DNA and three common open reading frames. Two of these reading frames are likely related to replication, one encoding a large protein with a helicase domain similar to viral helicases, and the other a copy number control protein, CopG.

Base Sequence↗
PubMed1998

Genetic profile of pNOB8 from Sulfolobus: the first conjugative plasmid from an archaeon.

The complete nucleotide sequence of the archaeal conjugative plasmid, pNOB8, from the Sulfolobus isolate NOB8-H2, was determined. The plasmid is 41,229 bp in size and contains about 50 ORFs. Several direct sequence repeats are present, the largest of which is a perfect 85-bp repeat and a site of intraplasmid recombination in foreign Sulfolobus hosts. This recombination event produces a major deletion variant, pNOB8-33, which is not stably maintained. Less than 20% of the ORFs could be assigned putative functions after extensive database searches. Tandem ORFs 315 and 470, within the deleted 8-kb region, show significant sequence similarity to the protein superfamilies of ParA (whole protein) and ParB (N-terminal half), respectively, that are important for plasmid and chromosome partitioning in bacteria. A putative cis-acting element is also present that exhibits six 24-mer repeats containing palindromic sequences which are separated by 39 or 42 bp. By analogy with bacterial systems, this element may confer plasmid incompatibility and define a group of incompatible plasmids in Archaea. Although several ORFs can form putative trans-membrane or membrane-binding segments, only two ORFs show significant sequence similarity to bacterial conjugative proteins. ORF630b aligns with the TrbE protein superfamily, which contributes to mating pair formation in Bacteria, while ORF1025 aligns with the TraG protein superfamily. We infer that the conjugative mechanism for Sulfolobus differs considerably from known bacterial mechanisms. Finally, two transposases were detected; ORF413 is flanked by an imperfect 32-bp inverted repeat with a 5-bp direct repeat at the ends, and ORF406 is very similar in sequence to an insertion element identified in the Sulfolobus solfataricus P2 genome.

Amino Acid Sequence↗
PubMed1998

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.

Automation↗
PubMed1996

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.

Bacterial Proteins↗
PubMed1996

Complete nucleotide sequence of the Sulfolobus islandicus multicopy plasmid pRN1.

The complete sequence of the 5350-bp plasmid pRN1 from the crenarchaeote Sulfolobus islandicus has been determined. This plasmid is the first to be sequenced from this group of thermoacidophilic archaebacteria (Archaea) and its high copy number and wide host range make it a good candidate for a cloning vector. pRN1 contains several open reading frames, including one that spans over half the plasmid and has significant similarity to the helicase domain of viral primase proteins. Directly upstream of this putative primase is a homologue of Cop, a family of small proteins from promiscuous eubacterial plasmids which control copy number by repressing the expression of the replication initiation protein. In eubacterial plasmids cop is found upstream of the replication initiator protein. The location of a cop homologue upstream of a primase-like gene in pRN1 suggests that it controls DNA replication in a manner similar to these eubacterial plasmids, but does so using a mixture of components from plasmids and viruses.

Amino Acid Sequence↗
PubMed1996

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.

Archaea↗
PubMed1995

Transcription in archaea: similarity to that in eucarya.

We present homologies between archaeal and eucaryal DNA-dependent RNA polymerase (RNAP) subunits and transcription factors. The sequences of the Sulfolobus acidocaldarius subunits D, E, and N and alignments with eucaryal homologs are presented here. The similarities between archaeal transcription factors and their eucaryal homologs TFIIB and TBP have been established in other laboratories. The archaeal RNAP subunits H, K, and N, respectively, show high sequence similarity to ABC27, ABC23, and ABC10 beta (found in all three eucaryal RNAPs); subunit D, to AC40 (common to polymerase II and polymerase III) and B44 (polymerase II); and subunit L, to AC19 and B12.5. The similarity of subunit D and its eucaryal homologs to bacterial alpha is limited to the "alpha-motif," which is also present in subunit L and its eucaryal homologs. Genes encoding homologs of the related eucaryal RNAP subunits A12.2/B12.6 and also homologs of eucaryal transcription elongation factors of the TFIIS family have been detected in Sulfolobus acidocaldarius and Thermococcus celer. In archaea, the protein is not an RNAP subunit. Together with the sequence similarities between archaeal box A-containing and eucaryal TATA box-containing promoters, this shows that the archaeal and eucaryal transcription systems are truly homologous and that they differ structurally and functionally from the bacterial transcription machinery. In contrast, however, a number of genes for the archaeal transcription apparatus are organized in clusters resembling the clusters of transcription-associated genes in Bacteria.

Amino Acid Sequence↗
PubMed1995

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.

Cell Aggregation↗
PubMed1995

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.

Archaea↗
PubMed1994

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.

Bacteriophages↗
PubMed1994

DNA-dependent RNA polymerase subunit B as a tool for phylogenetic reconstructions: branching topology of the archaeal domain.

The branching topology of the archaeal (archaebacterial) domain was inferred from sequence comparisons of the largest subunit (B) of DNA-dependent RNA polymerases (RNAP). Both the nucleic acid sequences of the genes coding for RNAP subunit B and the amino acid sequences of the derived gene products were used for phylogenetic reconstructions. Individual analysis of the three nucleotide positions of codons revealed significant inequalities with respect to guanosine and cytosine (GC) content and evolutionary rates. Only the nucleotides at the second codon positions were found to be unbiased by varied GC contents and sufficiently conserved for reliable phylogenetic reconstructions. A decision matrix was used for the combination of the results of distance matrix, maximum parsimony, and maximum likelihood methods. For this purpose the original results (sums of squares, steps, and logarithms of likelihoods) were transformed into comparable effective values and analyzed with methods known from the theory of statistical decisions. Phylogenetic invariants and statistical analysis with resampling techniques (bootstrap and jackknife) confirmed the preferred branching topology, which is significantly different from the topology known from phylogenetic trees based on 16S rRNA sequences. The preferred topology reconstructed by this analysis shows a common stem for the Methanococcales and Methanobacteriales and a separation of the thermophilic sulfur archaea from the methanogens and halophiles. The latter coincides with a unique phylogenetic location of a characteristic splitting event replacing the largest RNAP subunit of thermophilic sulfur archaea by two fragments in methanogens and halophiles. This topology is in good agreement with physiological and structural differences between the various archaea and demonstrates RNAP to be a suitable phylogenetic marker molecule.

Amino Acid Sequence↗
PubMed1994

An insertion element of the extremely thermophilic archaeon Sulfolobus solfataricus transposes into the endogenous beta-galactosidase gene.

Three phenotypically stable mutants of the extremely thermophilic archaeon Sulfolobus solfataricus have been isolated by screening for beta-galactosidase negative colonies on plates with X-Gal (5-bromo-4-chloro-3-indolyl-beta-D- galactopyranoside). From one of these mutants an insertion element, designated ISC1217, was isolated and characterized. Sequence analysis of ISC1217 and of the regions adjacent to the insertion site in the beta-galactosidase gene revealed features typical of a transposable element: ISC1217 contained terminal inverted repeats and was flanked by a direct repeat of 6 bp. The 1147 bp sequence contained an open reading frame encoding a putative protein of 354 amino acid residues and, overlapping this, two smaller open reading frames on the opposite strand. There were approximately 8 copies of the insertion element in the S. solfataricus genome. ISC1217 did not cross-hybridize with DNA of other Sulfolobus species. All three independently isolated beta-galactosidase mutants of S. solfataricus arose by transposition of ISC1217 or a related element.

Amino Acid Sequence↗