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Wolfram Zillig - One of the best experts on this subject based on the ideXlab platform.

  • dna dependent rna polymerases of thermoacidophilic archaebacteria
    FEBS Journal, 2005
    Co-Authors: David Prangishvilli, Alfons Gierl, Wolfram Zillig, Lothar Biesert, Ingelore Holz
    Abstract:

    The component compositions of the DNA-dependent RNA polymerases of the extremely thermophilic. anaerobic sulfur-respiring archaebacteria Thermoproteus tenax and Desu/furococcus mucosus strongly resemble each other but also that of the RNA polymerase of Suljolobus acidocaldarius suggesting that both organisms belong to the same novel order Thermoproteales, which together with the order represented by Sulfolobus, forms the thermoacidophilic branch of archaebacteria. The component pattern of the RNA polymerase of T/iermoplasmu acidophilum, which does not belong to this branch, also appears homologuous. The archaebacterial type of the DNA-dependent RNA polymerase is thus characterized by 9–10 components yielding a characteristic pattern which resembles that of yeast RNA polymerase A(1). In contrast to the α subunit of eubacterial RNA polymerases, the third largest component of archaebacterial RNA polymerases, although similar in size, is present only once per enzyme monomer. The polymerases of T. tenax and D. mucosus, like those previously isolated from other archaebacteria, are completely resistant against 100 μ/ml rifampicin and streptolydigin. The RNA polymerases of both organisms are highly thermostable. The enzyme from D. inucosus transcribes selectively and almost completely the H strand of phage T7 DNA.

  • Novel RepA-MCM proteins encoded in plasmids pTAU4, pORA1 and pTIK4 from Sulfolobus neozealandicus
    Hindawi Limited, 2005
    Co-Authors: Bo Greve, Wolfram Zillig, Qunxin She, Susanne Jensen, Hoa Phan, Kim Brügger, Roger A Garrett
    Abstract:

    Three plasmids isolated from the crenarchaeal thermoacidophile Sulfolobus neozealandicus were characterized. Plasmids pTAU4 (7,192 bp), pORA1 (9,689 bp) and pTIK4 (13,638 bp) show unusual properties that distinguish them from previously characterized cryptic plasmids of the genus Sulfolobus. Plasmids pORA1 and pTIK4 encode RepA proteins, only the former of which carries the novel polymerase–primase domain of other known Sulfolobus plasmids. Plasmid pTAU4 encodes a mini-chromosome maintenance protein homolog and no RepA protein; the implications for DNA replication are considered. Plasmid pORA1 is the first Sulfolobus plasmid to be characterized that does not encode the otherwise highly conserved DNA-binding PlrA protein. Another encoded protein appears to be specific for the New Zealand plasmids. The three plasmids should provide useful model systems for functional studies of these important crenarchaeal proteins

  • relationships between fuselloviruses infecting the extremely thermophilic archaeon Sulfolobus ssv1 and ssv2
    Research in Microbiology, 2003
    Co-Authors: Kenneth M Stedman, Hans Peter Arnold, Ingelore Holz, Hien Phan, Roger A Garrett, Wolfram Zillig
    Abstract:

    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.

  • viruses of the extremely thermophilic archaeon Sulfolobus
    Trends in Microbiology, 2001
    Co-Authors: David Prangishvili, Kenneth M Stedman, Wolfram Zillig
    Abstract:

    Abstract 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.

  • ping family of conjugative plasmids from the extremely thermophilic archaeon Sulfolobus islandicus insights into recombination and conjugation in crenarchaeota
    Journal of Bacteriology, 2000
    Co-Authors: Kenneth M Stedman, Ingelore Holz, Hien Phan, David Prangishvili, Roger A Garrett, Harpreet Singh, Wolfram Zillig
    Abstract:

    A novel family of conjugative plasmids from Sulfolobus comprising the active variants pING1, -4, and -6 and the functionally defective variants pING2 and -3, which require the help of an active variant for spreading, has been extensively characterized both functionally and molecularly. In view of the sparse similarity between bacterial and archaeal conjugation and the lack of a practical genetic system for Sulfolobus, we compared the functions and sequences of these variants and the previously described archaeal conjugative plasmid pNOB8 in order to identify open reading frames (ORFs) and DNA sequences that are involved in conjugative transfer and maintenance of these plasmids in Sulfolobus. The variants pING4 and -6 are reproducibly derived from pING1 in vivo by successive transpositions of an element from the Sulfolobus genome. The small defective but mobile variants pING2 and -3, which both lack a cluster of highly conserved ORFs probably involved in plasmid transfer, were shown to be formed in vivo by recombinative deletion of the larger part of the genomes of pING4 and pING6, respectively. The efficient occurrence of these recombination processes is further evidence for the striking plasticity of the Sulfolobus genome.

Takayoshi Wakagi - One of the best experts on this subject based on the ideXlab platform.

  • Comparative analysis of two glyceraldehyde-3-phosphate dehydrogenases from a thermoacidophilic archaeon, Sulfolobus tokodaii
    FEBS letters, 2012
    Co-Authors: Fumiaki Ito, Hidehiro Chishiki, Shinya Fushinobu, Takayoshi Wakagi
    Abstract:

    Abstract Sulfolobus tokodaii, a thermoacidophilic archaeon, possesses two structurally and functionally different enzymes that catalyze the oxidation of glyceraldehyde-3-phosphate (GAP): non-phosphorylating GAP dehydrogenase (St-GAPN) and phosphorylating GAP dehydrogenase (St-GAPDH). In contrast to previously characterized GAPN from Sulfolobus solfataricus, which exhibits V-type allosterism, St-GAPN showed K-type allosterism in which the positive cooperativity was abolished with concomitant activation by glucose 1-phosphate (G1P). St-GAPDH catalyzed the reversible oxidation of GAP to 1,3-bisphosphoglycerate (1,3-BPG) with high gluconeogenic activity, which was specific for NADPH, while both NAD+ and NADP+ were utilized in the glycolytic direction. Structured summary of protein interactions GAPDH and GAPDH bind by molecular sieving ( View interaction ) GAPN and GAPN bind by 2.2 molecular sieving ( View interaction ).

  • Membrane-bound acid pyrophosphatase from Sulfolobus tokodaii, a thermoacidophilic archaeon: heterologous expression of the gene and characterization of the product
    Extremophiles, 2009
    Co-Authors: Fumitoshi Manabe, Yuko H. Itoh, Hirofumi Shoun, Takayoshi Wakagi
    Abstract:

    Membranes of Sulfolobus tokodaii , a thermoacidophilic archaeon that grows optimally at pH 2–3, 75–80°C, show the ability to hydrolyze PPi with an optimum pH of 2–3. This acid PPase is proposed to be a dolicholpyrophosphatase that participates in glycoprotein biosynthesis. In the present study, the archaeal membranes hydrolyzed isopentenylpyrophosphate and geranylpyrophosphate, compounds related to dolicholpyrophosphate, at pH 3. However, the dolicholpyrophosphate-binding antibiotic bacitracin failed to inhibit the acid PPase. To investigate further the function and structure of the acid PPase, the gene was cloned and heterologously expressed in Escherichia coli. The membranes from recombinant E. coli showed PPase activity with similar pH and temperature dependence, substrate specificity, and kinetic parameters to those reported for Sulfolobus membranes. The acid PPase was solubilized and purified to electrophoretic homogeneity from the recombinant E. coli . The purified enzyme showed similar K _m values for PPi, ATP, and ADP to the membrane-bound enzyme. Lipids from the Sulfolobus membranes enhanced the activity to about threefold. Studies involving deletion mutants indicated that basic amino acids in the N-terminal (Arg2 and Lys3), as well as the residues (4th–69th) possibly twice-spanning the membrane, are essential for integration of the enzyme into membranes.

  • molecular and phylogenetic characterization of isopropylmalate dehydrogenase of a thermoacidophilic archaeon Sulfolobus sp strain 7
    Journal of Bacteriology, 1997
    Co-Authors: Toshiharu Suzuki, Toshio Iwasaki, Y Inoki, Akihiko Yamagishi, Takayoshi Wakagi
    Abstract:

    The archaeal leuB gene encoding isopropylmalate dehydrogenase of Sulfolobus sp. strain 7 was cloned, sequenced, and expressed in Escherichia coli. The recombinant Sulfolobus sp. enzyme was extremely stable to heat. The substrate and coenzyme specificities of the archaeal enzyme resembled those of the bacterial counterparts. Sedimentation equilibrium analysis supported an earlier proposal that the archaeal enzyme is homotetrameric, although the corresponding enzymes studied so far have been reported to be dimeric. Phylogenetic analyses suggested that the archaeal enzyme is homologous to mitochondrial NAD-dependent isocitrate dehydrogenases (which are tetrameric or octameric) as well as to isopropylmalate dehydrogenases from other sources. These results suggested that the present enzyme is the most primitive among isopropylmalate dehydrogenases belonging in the decarboxylating dehydrogenase family.

  • novel zinc binding centre in thermoacidophilic archaeal ferredoxins
    Nature Structural & Molecular Biology, 1996
    Co-Authors: Tomomi Fujii, Takayoshi Wakagi, Yasuo Hata, Nobuo Tanaka
    Abstract:

    The crystal structure of ferredoxin from Sulfolobus species strain 7 reveals a novel zinc-binding centre that may play an important role in stabilizing the protein and may be common to thermoacidophilic archaeal ferredoxins.

  • Alternative Form of the Dicluster Ferredoxin from the Thermoacidophilic Archaeon, Sulfolobus Sp. Strain 7
    Biochemical and biophysical research communications, 1995
    Co-Authors: Toshio Iwasaki, Tomoyuki Fujii, Takayoshi Wakagi
    Abstract:

    Abstract An alternative form of a 7Fe dicluster-type ferredoxin (Fd-B) with a different charge density was purified as a minor component from the aerobic and thermoacidophilic archaeon, Sulfolobus sp. strain 7. Comparison of its properties with those of native 7Fe ferredoxin (Fd-A), a major ferredoxin, was made in terms of the molecular properties, the absorption, circular dichroism and electron paramagnetic resonance spectral properties, and the reactivity coupled with the cognate 2-oxoacid:ferredoxin oxidoreductase at 50°C. Our results suggest that the Sulfolobus 7Fe ferredoxin has two spectroscopically distinct forms with slightly different conformations.

Christa Schleper - One of the best experts on this subject based on the ideXlab platform.

  • uv inducible cellular aggregation of the hyperthermophilic archaeon Sulfolobus solfataricus is mediated by pili formation
    Molecular Microbiology, 2008
    Co-Authors: Sabrina Frols, Michaela Wagner, Christa Schleper, Arnold J. M. Driessen, Mihaela Folea, Egbert J Boekema, Malgorzata Ajon, Daniela Teichmann, Behnam Zolghadr
    Abstract:

    The hyperthermophilic archaeon Sulfolobus solfataricus has been shown to exhibit a complex transcriptional response to UV irradiation involving 55 genes. Among the strongest UV-induced genes was a putative pili biogenesis operon encoding a potential secretion ATPase, two pre-pilins, a putative transmembrane protein and a protein of unknown function. Electron microscopy and image reconstruction of UV-treated cells showed straight pili with 10 nm in diameter, variable in length, not bundled or polarized and composed of three evenly spaced helices, thereby clearly being distinguishable from archaeal flagella. A deletion mutant of SSO0120, the central type II/IV secretion ATPase, did not produce pili. It could be complemented by reintroducing the gene on a plasmid vector. We have named the operon ups operon for UV-inducible pili operon of Sulfolobus. Overexpression of the pre-pilins, Ups-A/B (SSO0117/0118) in Sulfolobus resulted in production of extremely long filaments. Pronounced cellular aggregation was observed and quantified upon UV treatment. This aggregation was a UV-dose-dependent, dynamic process, not inducible by other physical stressors (such as pH or temperature shift) but stimulated by chemically induced double-strand breaks in DNA. We hypothesize that pili formation and subsequent cellular aggregation enhance DNA transfer among Sulfolobus cells to provide increased repair of damaged DNA via homologous recombination.

  • a reporter gene system for the hyperthermophilic archaeon Sulfolobus solfataricus based on a selectable and integrative shuttle vector
    Molecular Microbiology, 2003
    Co-Authors: Melanie Jonuscheit, Kenneth M Stedman, Erika Martusewitsch, Christa Schleper
    Abstract:

    Summary Sulfolobus solfataricus has developed into an important model organism for molecular and biochemical studies of hyperthermophilic archaea. Although a number of in vitro systems have been established for the organism, efficient tools for genetic manipulations have not yet been available for any hyperthermophile. In this work, we have developed a stable and selectable shuttle vector based on the virus SSV1 of Sulfolobus shibatae. We have introduced pUC18 for propagation in Escherichia coli and the genes pyrEF coding for orotidine-5′-monophosphate pyrophosphorylase and orotidine-5′-monophosphate decarboxylase of Sulfolobus solfataricus as selectable marker to complement pyrimidine auxotrophic mutants. Furthermore, the beta-galactosidase gene (lacS) was introduced into this vector as a reporter under the control of the strong and heat-inducible promoter of the Sulfolobus chaperonin (thermosome). After transformation of a S. solfataricus pyrEF/lacS double mutant, the vector was found to reside as a single-copy vector, stably integrated into the host chromosome via the site-specific recombination system of SSV1. Specific beta-galactosidase activities in transformants were found to be fourfold higher than in wild-type S. solfataricus cells, and increased to more than 10-fold after heat shock. Greatly increased levels of lacS mRNA were detected in Northern analyses, demonstrating that this reporter gene system is suitable for the study of regulated promoters in Sulfolobus and that the vector can also be used for the high-level expression of genes from hyperthermophilic archaea.

  • Transformation of the extremely thermoacidophilic archaeon Sulfolobus solfataricus via a self-spreading vector
    FEMS microbiology letters, 1996
    Co-Authors: Marieke G.l. Elferink, Christa Schleper, Wolfram Zillig
    Abstract:

    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 β-galactosidase gene downstream of a strong promotor. Transformation of a β-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 β-galactosidase activity

  • the particle ssv1 from the extremely thermophilic archaeon Sulfolobus is a virus demonstration of infectivity and of transfection with viral dna
    Proceedings of the National Academy of Sciences of the United States of America, 1992
    Co-Authors: Christa Schleper, K. Kubo, Wolfram Zillig
    Abstract:

    Abstract The lemon-shaped "virus-like" particle SSV1 produced by the thermophilic archaeon Sulfolobus shibatae has not previously been observed to infect any host. Using a plaque assay suitable for the extreme growth conditions of this archaeon, we have shown infection of Sulfolobus solfataricus by SSV1. Upon infection, the viral genome was always found integrated into a tRNA gene of the host chromosome, a situation similar to that in S. shibatae, proving that site-specific integration is involved in establishing the lysogenic state. As in S. shibatae, UV-irradiation of lysogenized S. solfataricus led to virus production apparently not accompanied by cell lysis. We have also demonstrated the efficient uptake of exogenous DNA and its expression in Sulfolobus by transfecting S. solfataricus with SSV1 DNA by electroporation. Transfection efficiencies of up to 10(6) transfectants per microgram of DNA were obtained.

Toshio Iwasaki - One of the best experts on this subject based on the ideXlab platform.

  • Sulfolobus tokodaii sp nov f Sulfolobus sp strain 7 a new member of the genus Sulfolobus isolated from beppu hot springs japan
    Extremophiles, 2002
    Co-Authors: Toshiharu Suzuki, Toshio Iwasaki, Akihiko Yamagishi, Taketoshi Uzawa, Kurt Hara, Naoki Nemoto, Takahide Kon, Toshiaki Ueki, Tairo Oshima
    Abstract:

    The taxonomic position of a thermoacidophilic crenarchaeote Sulfolobus sp. strain 7, previously isolated from the Beppu Hot Springs in the geothermal area of Kyushu Island, Japan, was investigated by cloning and sequencing, by phylogenetic analysis of the 16S rRNA gene sequence, by DNA-DNA homology with similar species, and by biochemical characterization of the isolate. This isolate is an obligate aerobe and grows optimally at 80 degrees C and pH2.5-3 under aerobic and chemoheterotrophic growth conditions by aerobic respiration rather than simple fermentation. In conjunction with the phenotypic properties, the present phylogenetic analysis based on the 16S rRNA gene sequence and DNA-DNA hybridization experiments indicate that this isolate is related to the described Sulfolobus taxon and should be considered a novel species of the genus. We propose that this isolate is a novel species of the genus Sulfolobus that we name Sulfolobus tokodaii sp. nov. The type strain is strain 7 (JCM 10545).

  • molecular and phylogenetic characterization of isopropylmalate dehydrogenase of a thermoacidophilic archaeon Sulfolobus sp strain 7
    Journal of Bacteriology, 1997
    Co-Authors: Toshiharu Suzuki, Toshio Iwasaki, Y Inoki, Akihiko Yamagishi, Takayoshi Wakagi
    Abstract:

    The archaeal leuB gene encoding isopropylmalate dehydrogenase of Sulfolobus sp. strain 7 was cloned, sequenced, and expressed in Escherichia coli. The recombinant Sulfolobus sp. enzyme was extremely stable to heat. The substrate and coenzyme specificities of the archaeal enzyme resembled those of the bacterial counterparts. Sedimentation equilibrium analysis supported an earlier proposal that the archaeal enzyme is homotetrameric, although the corresponding enzymes studied so far have been reported to be dimeric. Phylogenetic analyses suggested that the archaeal enzyme is homologous to mitochondrial NAD-dependent isocitrate dehydrogenases (which are tetrameric or octameric) as well as to isopropylmalate dehydrogenases from other sources. These results suggested that the present enzyme is the most primitive among isopropylmalate dehydrogenases belonging in the decarboxylating dehydrogenase family.

  • sulredoxin a novel iron sulfur protein of the thermoacidophilic archaeon Sulfolobus sp strain 7 with a rieske type 2fe 2s center
    Journal of Bacteriology, 1995
    Co-Authors: Toshio Iwasaki, Y Isogai, T Iizuka
    Abstract:

    A novel pink [2Fe-2S] protein has been purified from the cytosol fraction of the thermoacidophilic archaeon Sulfolobus sp. strain 7 (originally named Sulfolobus acidocaldarius 7) and called ‘‘sulredoxin.’’ Its absorption, circular dichroism, and electron paramagnetic resonance spectra suggest the presence of a Rieske-type [2Fe-2S] cluster (g-factors of 2.01, 1.91, and 1.79; averageg- factor [gav] 51.90) which is remarkably similar to that ofThermus thermophilusrespiratory Rieske FeS protein (J. A. Fee, K. L. Findling, T. Yoshida, R. Hille,

  • Alternative Form of the Dicluster Ferredoxin from the Thermoacidophilic Archaeon, Sulfolobus Sp. Strain 7
    Biochemical and biophysical research communications, 1995
    Co-Authors: Toshio Iwasaki, Tomoyuki Fujii, Takayoshi Wakagi
    Abstract:

    Abstract An alternative form of a 7Fe dicluster-type ferredoxin (Fd-B) with a different charge density was purified as a minor component from the aerobic and thermoacidophilic archaeon, Sulfolobus sp. strain 7. Comparison of its properties with those of native 7Fe ferredoxin (Fd-A), a major ferredoxin, was made in terms of the molecular properties, the absorption, circular dichroism and electron paramagnetic resonance spectral properties, and the reactivity coupled with the cognate 2-oxoacid:ferredoxin oxidoreductase at 50°C. Our results suggest that the Sulfolobus 7Fe ferredoxin has two spectroscopically distinct forms with slightly different conformations.

Dennis W. Grogan - One of the best experts on this subject based on the ideXlab platform.

  • how a genetically stable extremophile evolves modes of genome diversification in the archaeon Sulfolobus acidocaldarius
    Journal of Bacteriology, 2017
    Co-Authors: Dominic Mao, Dennis W. Grogan
    Abstract:

    In order to analyze in molecular terms how Sulfolobus genomes diverge, damage-induced mutations and natural polymorphisms (PMs) were identified in laboratory constructs and wild-type isolates, respectively, of Sulfolobus acidocaldarius Among wild-type isolates drawn from one local population, pairwise nucleotide divergence averaged 4 × 10-6, which is about 0.15% of the corresponding divergence reported for Sulfolobus islandicus The most variable features of wild-type S. acidocaldarius genomes were homopolymer (mononucleotide) tracts and longer tandem repeats, consistent with the spontaneous mutations that occur under laboratory conditions. Natural isolates, however, also revealed large insertions/deletions and inversions, which did not occur in any of the laboratory-manipulated strains. Several of the large insertions/deletions could be attributed to the integration or excision of mobile genetic elements (MGEs), and each MGE represented a distinct system of site-specific recombination. The mode of recombination associated with one MGE, a provirus related to Sulfolobus turreted icosahedral virus, was also seen in certain chromosomal inversions. Artificially induced mutations, non-MGE insertions/deletions, and small PMs exhibited different distributions over the genome, suggesting that large-scale patterning of Sulfolobus genomes begins early in the divergence process. Unlike induced mutations, natural base pair substitutions occurred in clusters, and one cluster exhibited properties expected of nonreciprocal recombination (gene conversion) between dispersed imperfect repeats. Taken together, the results identify simple replication errors, slipped-strand events promoted by tandem repeats, homologous recombination, and rearrangements promoted by MGEs as the primary sources of genetic variation for this extremely acidophilic archaeon in its geothermal environment.IMPORTANCE The optimal growth temperatures of hyperthermophilic archaea accelerate DNA decomposition, which is expected to make DNA repair especially important for their genetic stability, yet these archaea lack certain broadly conserved types of DNA repair proteins. In this study, the genome of the extreme thermoacidophile Sulfolobus acidocaldarius was found to be remarkably stable, accumulating few mutations in many (though not all) laboratory manipulations and in natural populations. Furthermore, all the genetic processes that were inferred to diversify these genomes also operate in mesophilic bacteria and eukaryotes. This suggests that a common set of mechanisms produces most of the genetic variation in all microorganisms, despite the fundamental differences in physiology, DNA repair systems, and genome structure represented in the three domains of life.

  • lesion induced mutation in the hyperthermophilic archaeon Sulfolobus acidocaldarius and its avoidance by the y family dna polymerase dbh
    Genetics, 2015
    Co-Authors: Cynthia J Sakofsky, Dennis W. Grogan
    Abstract:

    Hyperthermophilic archaea offer certain advantages as models of genome replication, and Sulfolobus Y-family polymerases Dpo4 (S. solfataricus) and Dbh (S. acidocaldarius) have been studied intensively in vitro as biochemical and structural models of trans-lesion DNA synthesis (TLS). However, the genetic functions of these enzymes have not been determined in the native context of living cells. We developed the first quantitative genetic assays of replication past defined DNA lesions and error-prone motifs in Sulfolobus chromosomes and used them to measure the efficiency and accuracy of bypass in normal and dbh− strains of Sulfolobus acidocaldarius. Oligonucleotide-mediated transformation allowed low levels of abasic-site bypass to be observed in S. acidocaldarius and demonstrated that the local sequence context affected bypass specificity; in addition, most erroneous TLS did not require Dbh function. Applying the technique to another common lesion, 7,8-dihydro-8-oxo-deoxyguanosine (8-oxo-dG), revealed an antimutagenic role of Dbh. The efficiency and accuracy of replication past 8-oxo-dG was higher in the presence of Dbh, and up to 90% of the Dbh-dependent events inserted dC. A third set of assays, based on phenotypic reversion, showed no effect of Dbh function on spontaneous −1 frameshifts in mononucleotide tracts in vivo, despite the extremely frequent slippage at these motifs documented in vitro. Taken together, the results indicate that a primary genetic role of Dbh is to avoid mutations at 8-oxo-dG that occur when other Sulfolobus enzymes replicate past this lesion. The genetic evidence that Dbh is recruited to 8-oxo-dG raises questions regarding the mechanism of recruitment, since Sulfolobus spp. have eukaryotic-like replisomes but no ubiquitin.

  • small multicopy non integrative shuttle vectors based on the plasmid prn1 for Sulfolobus acidocaldarius and Sulfolobus solfataricus model organisms of the cren archaea
    Nucleic Acids Research, 2007
    Co-Authors: Silvia Berkner, Sonja-verena Albers, Dennis W. Grogan, Georg Lipps
    Abstract:

    The extreme thermoacidophiles of the genus Sulfolobus are among the best-studied archaea but have lacked small, reliable plasmid vectors, which have proven extremely useful for manipulating and analyzing genes in other microorganisms. Here we report the successful construction of a series of Sulfolobus-Escherichia coli shuttle vectors based on the small multicopy plasmid pRN1 from Sulfolobus islandicus. Selection in suitable uracil auxotrophs is provided through inclusion of pyrEF genes in the plasmid. The shuttle vectors do not integrate into the genome and do not rearrange. The plasmids allow functional overexpression of genes, as could be demonstrated for the beta-glycosidase (lacS) gene of S. solfataricus. In addition, we demonstrate that this beta-glycosidase gene could function as selectable marker in S. solfataricus. The shuttle plasmids differ in their interruption sites within pRN1 and allowed us to delineate functionally important regions of pRN1. The orf56/orf904 operon appears to be essential for pRN1 replication, in contrast interruption of the highly conserved orf80/plrA gene is tolerated. The new vector system promises to facilitate genetic studies of Sulfolobus and to have biotechnological uses, such as the overexpression or optimization of thermophilic enzymes that are not readily performed in mesophilic hosts.

  • organization and interactions of cell envelope proteins of the extreme thermoacidophile Sulfolobus acidocaldarius
    Canadian Journal of Microbiology, 1996
    Co-Authors: Dennis W. Grogan
    Abstract:

    To address basic questions concerning proteins of the Sulfolobus acidocaldarius cell envelope, cell ghosts (empty cells consisting of cytoplasmic membrane complexed with the glycoprotein S-layer) w...

  • isolation and fractionation of cell envelope from the extreme thermo acidophile Sulfolobus acidocaldarius
    Journal of Microbiological Methods, 1996
    Co-Authors: Dennis W. Grogan
    Abstract:

    Abstract Methods to produce membrane fragment of defined composition were evaluated for their applicability to the extremely thermophilic, acidophilic archaeon Sulfolobus acidocaldarius . Sonication was found to effectively disrupt Sulfolobus cells and to produce small vesicles of varying size. Alternatively, grinding cells with fine alumina yielded large, uniform vesicles retaining the characteristic cell envelope ultrastructure of Sulfolobus spp. Biochemical and electron-microscopic examination indicated that the ‘cell ghosts’ prepared from S. acidocaldarius by the latter method have a relatively simple protein composition dominated by the glycoprotein subunits of the organism's quasicrystalline surface (S) layer cell wall. By suitable treatments, the cell ghosts could, in turn, be used to prepare S layer without the associated lipid membrane, or conversely, to prepare cytoplasmic membrane lacking the apposed S layer. These results offer a basis for compositional and functional analyses of a cell surface that is normally exposed to extremely severe environmental conditions.