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Sample records for archaeon sulfolobus solfataricus

  1. Sugar transport in the thermoacidophilic archaeon Sulfolobus solfataricus

    NARCIS (Netherlands)

    Albers, Sonja-Verena

    2001-01-01

    Summary and concluding remarks Introduction The archaeon Sulfolobus solfataricus is a thermoacidophile preferring growth at around 80oC and a pH of 2.5 to 3.5. As a thermoacidophile S. solfataricus faces two major problems: firstly, the proton permeability of membranes increases with temperature res

  2. "Hot standards" for the thermoacidophilic archaeon Sulfolobus solfataricus

    NARCIS (Netherlands)

    Zaparty, Melanie; Esser, Dominik; Gertig, Susanne; Haferkamp, Patrick; Kouril, Theresa; Manica, Andrea; Pham, Trong K.; Reimann, Julia; Schreiber, Kerstin; Sierocinski, Pawel; Teichmann, Daniela; van Wolferen, Marleen; von Jan, Mathias; Wieloch, Patricia; Albers, Sonja V.; Driessen, Arnold J. M.; Klenk, Hans-Peter; Schleper, Christa; Schomburg, Dietmar; van der Oost, John; Wright, Phillip C.; Siebers, Bettina

    2010-01-01

    Within the archaea, the thermoacidophilic crenarchaeote Sulfolobus solfataricus has become an important model organism for physiology and biochemistry, comparative and functional genomics, as well as, more recently also for systems biology approaches. Within the Sulfolobus Systems Biology ("SulfoSYS

  3. UV-inducible cellular aggregation of the hyperthermophilic archaeon Sulfolobus solfataricus is mediated by pili formation

    NARCIS (Netherlands)

    Froels, Sabrina; Ajon, Malgorzata; Wagner, Michaela; Teichmann, Daniela; Zolghadr, Behnam; Folea, Mihaela; Boekema, Egbert J.; Driessen, Arnold J. M.; Schleper, Christa; Albers, Sonja-Verena

    2008-01-01

    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 trans

  4. Preliminary characterization of two different crystal forms of acylphosphatase from the hyperthermophile archaeon Sulfolobus solfataricus

    Energy Technology Data Exchange (ETDEWEB)

    Zuccotti, Simone [Department of Physics-INFM and Center of Excellence for Biomedical Research, University of Genova, Via Dodecaneso 33, 16132 Genova (Italy); Rosano, Camillo [National Institute for Cancer Research (IST), X-ray Structural Biology Unit, Largo R. Benzi 10, 16132 Genova (Italy); Bemporad, Francesco [Department of Biochemical Sciences, University of Firenze, Viale Morgagni 50, 50134 Florence (Italy); Stefani, Massimo [Department of Biochemical Sciences, University of Firenze, Viale Morgagni 50, 50134 Florence (Italy); Centro di Ricerca, Trasferimento e Alta Formazione MCIDNENT, University of Firenze, Viale Morgagni 50, 50134 Florence (Italy); Bolognesi, Martino, E-mail: bolognes@fisica.unige.it [Department of Physics-INFM and Center of Excellence for Biomedical Research, University of Genova, Via Dodecaneso 33, 16132 Genova (Italy)

    2005-01-01

    S. solfataricus acylphosphatase has been expressed, purified and crystallized in two different crystal forms. Preliminary characterization of a triclinic and a monoclinic crystal form is reported and data were collected to 1.27 and 1.90 Å, respectively. Acylphosphatase is a ubiquitous small enzyme that was first characterized in mammals. It is involved in the hydrolysis of carboxyl-phosphate bonds in several acylphosphate substrates, such as carbamoylphosphate and 1,3-biphosphoglycerate; however, a consensus on acylphosphatase action in vivo has not yet been reached. Recent investigations have focused on acylphosphatases from lower phyla, such as Drosophila melanogaster and Escherichia coli, in view of the application of these small proteins as models in the study of folding, misfolding and aggregation processes. An acylphosphatase from the hyperthermophilic archaeon Sulfolobus solfataricus has been cloned, expressed and purified. Here, the growth and characterization of a triclinic and a monoclinic crystal form of the hyperthermophilic enzyme are reported; X-ray diffraction data have been collected to 1.27 and 1.90 Å resolution, respectively.

  5. Identification of a novel alpha-galatosidase from the hyperthermophilic archaeon Sulfolobus solfataricus

    NARCIS (Netherlands)

    Brouns, S.J.J.; Smits, N.; Wu, H.; Wright, P.C.; Snijders, A.P.L.; Vos, de W.M.; Oost, van der J.

    2006-01-01

    Sulfolobus solfataricus is an aerobic crenarchaeon that thrives in acidic volcanic pools. In this study, we have purified and characterized a thermostable -galactosidase from cell extracts of S. solfataricus P2 grown on the trisaccharide raffinose. The enzyme, designated GalS, is highly specific for

  6. Identification and characterization of small RNAs in the hyperthermophilic archaeon Sulfolobus solfataricus.

    Directory of Open Access Journals (Sweden)

    Ning Xu

    Full Text Available The term RNA silencing (RNA interference, RNAi describes a set of mechanisms that regulate gene expression in eukaryotes. Small interfering RNAs (siRNA and microRNAs (miRNAs are two major types of RNAi-associated small RNAs (smRNAs found in most eukaryotic organisms. Despite the presence of a plethora of non-coding RNAs longer than 50-nucleotide (nt in length in various species of Archaea, little is known about smRNAs in archaea that resemble the 20-24-nt long smRNAs found in eukaryotes, which have been implicated in the post-transcriptional control of gene expression. Here, we report the finding of a large number of smRNAs approximatelly 20-nt in length, including phased smRNAs and potential miRNAs, from the hyperthermophilic archaeon Sulfolobus solfataricus p2 (Ssp2 based on deep sequencing. The expression of some of the miRNA candidates in Ssp2 was confirmed. Consistent with the Ssp2 hyperthermophilic properties, we found that higher temperatures more efficiently induced the production of the miRNA candidates in an in vitro system using the putative foldback precursor transcripts incubated with Ssp2 extract. Although we initially predicted putative target genes of some miRNA candidates, further analysis mapped the cleavage sites downstream of the miRNA candidate complementary regions, similar to those involved in plant miRNA-mediated TAS transcript cleavage. We also identified smRNAs from clustered, regularly interspaced, short palindromic repeat (CRISPR loci, which play important roles in prokaryotic microbial defense systems. Archaea represent a unique life form next to Bacteria and Eukarya, and our results may provide a useful resource for further in-depth study on the regulation and evolution of smRNAs in this special organism.

  7. Allosteric regulation of the GTP activated and CTP inhibited uracil phosphoribosyltransferase from the thermophilic archaeon Sulfolobus solfataricus

    DEFF Research Database (Denmark)

    Jensen, Kaj Frank; Arent, Susan; Larsen, Sine;

    2005-01-01

    The upp gene, encoding uracil phosphoribosyltransferase (UPRTase) from the thermoacidophilic archaeon Sulfolobus solfataricus, was cloned and expressed in Escherichia coli. The enzyme was purified to homogeneity. It behaved as a tetramer in solution and showed optimal activity at pH 5.5 when...

  8. Targeted Disruption of the α-Amylase Gene in the Hyperthermophilic Archaeon Sulfolobus solfataricus

    OpenAIRE

    Worthington, Penny; Hoang, Viet; Perez-Pomares, Francisco; Blum, Paul

    2003-01-01

    Sulfolobus solfataricus secretes an acid-resistant α-amylase (amyA) during growth on starch as the sole carbon and energy source. Synthesis of this activity is subject to catabolite repression. To better understand α-amylase function and regulation, the structural gene was identified and disrupted and the resulting mutant was characterized. Internal α-amylase peptide sequences obtained by tandem mass spectroscopy were used to identify the amyA coding sequence. Anti-α-amylase antibodies raised...

  9. Genome-scale reconstruction and analysis of the metabolic network in the hyperthermophilic archaeon Sulfolobus solfataricus.

    Directory of Open Access Journals (Sweden)

    Thomas Ulas

    Full Text Available We describe the reconstruction of a genome-scale metabolic model of the crenarchaeon Sulfolobus solfataricus, a hyperthermoacidophilic microorganism. It grows in terrestrial volcanic hot springs with growth occurring at pH 2-4 (optimum 3.5 and a temperature of 75-80°C (optimum 80°C. The genome of Sulfolobus solfataricus P2 contains 2,992,245 bp on a single circular chromosome and encodes 2,977 proteins and a number of RNAs. The network comprises 718 metabolic and 58 transport/exchange reactions and 705 unique metabolites, based on the annotated genome and available biochemical data. Using the model in conjunction with constraint-based methods, we simulated the metabolic fluxes induced by different environmental and genetic conditions. The predictions were compared to experimental measurements and phenotypes of S. solfataricus. Furthermore, the performance of the network for 35 different carbon sources known for S. solfataricus from the literature was simulated. Comparing the growth on different carbon sources revealed that glycerol is the carbon source with the highest biomass flux per imported carbon atom (75% higher than glucose. Experimental data was also used to fit the model to phenotypic observations. In addition to the commonly known heterotrophic growth of S. solfataricus, the crenarchaeon is also able to grow autotrophically using the hydroxypropionate-hydroxybutyrate cycle for bicarbonate fixation. We integrated this pathway into our model and compared bicarbonate fixation with growth on glucose as sole carbon source. Finally, we tested the robustness of the metabolism with respect to gene deletions using the method of Minimization of Metabolic Adjustment (MOMA, which predicted that 18% of all possible single gene deletions would be lethal for the organism.

  10. SulfoSYS (Sulfolobus Systems Biology) : towards a silicon cell model for the central carbohydrate metabolism of the archaeon Sulfolobus solfataricus under temperature variation

    NARCIS (Netherlands)

    Albers, Sonja-Verena; Birkeland, Nils-Kare; Driessen, Arnold J. M.; Gertig, Susanne; Haferkamp, Patrick; Klenk, Hans-Peter; Kouril, Theresa; Manica, Andrea; Pham, Trong K.; Ruoff, Peter; Schleper, Christa; Schomburg, Dietmar; Sharkey, Kieran J.; Siebers, Bettina; Sierocinski, Pawel; Steuer, Ralf; van der Oost, John; Westerhoff, Hans V.; Wieloch, Patricia; Wright, Phillip C.; Zaparty, Melanie; Birkeland, Nils-Kåre

    2009-01-01

    SulfoSYS (Sulfolobus Systems Biology) focuses on the study of the CCM (central carbohydrate metabolism) of Sulfolobus solfataricus and its regulation under temperature variation at the systems level. in Archaea, carbohydrates are metabolized by modifications of the classical pathways known from Bact

  11. SulfoSYS (Sulfolobus Systems Biology): towards a silicon cell model for the central carbohydrate metabolism of the archaeon Sulfolobus solfataricus under temperature variation.

    NARCIS (Netherlands)

    Albers, S.V.; Birkeland, N.K.; Driessen, A.J.; Gertig, S.; Haferkamp, P.; Klenk, H.P.; Kouril, T.; Manica, A.; Pham, T.K.; Ruoff, P.; Schleper, C.; Schomburg, D.; Sharkey, K.J.; Siebers, A.G.; Sierocinski, P.; Steuer, R.; Oost, J. van der; Westerhoff, H.V.; Wieloch, P.; Wright, P.C.; Zaparty, M.

    2009-01-01

    SulfoSYS (Sulfolobus Systems Biology) focuses on the study of the CCM (central carbohydrate metabolism) of Sulfolobus solfataricus and its regulation under temperature variation at the systems level. In Archaea, carbohydrates are metabolized by modifications of the classical pathways known from Bact

  12. SulfoSYS (Sulfolobus Systems Biology): towards a silicon cell model for the central carbohydrate metabolism of the archaeon Sulfolobus solfataricus under temperature variation

    NARCIS (Netherlands)

    Albers, S.V.; Birkeland, N.K.; Driessen, A.J.M.; Gertig, S.; Haferkamp, P.; Klenk, H.P.; Kouril, T.; Manica, A.; Pham, T.K.; Ruoff, P.; Schleper, C.; Schomburg, D.; Sharkey, K.; Siebers, B.; Sierocinski, P.; Steur, R.; Oost, van der J.; Westerhoff, H.V.; Wieloch, P.; Wright, P.C.; Zaparty, M.

    2009-01-01

    SulfoSYS (Sulfolobus Systems Biology) focuses on the study of the CCM (central carbohydrate metabolism) of Sulfolobus solfataricus and its regulation under temperature variation at the systems level. In Archaea, carbohydrates are metabolized by modifications of the classical pathways known from Bact

  13. Active-site residues in the type IV prepilin peptidase homologue PibD from the archaeon Sulfolobus solfataricus

    NARCIS (Netherlands)

    Szabo, Z; Albers, SV; Driessen, AJM

    2006-01-01

    Archaeal preflagellin peptidases and bacterial type IV prepilin peptidases belong to a family of aspartic acid proteases that cleave the leader peptides of precursor proteins with type W prepilin signal sequences. The substrate repertoire of PibD from the crenarchaeon Sulfolobus solfataricus is unus

  14. Active-Site Residues in the Type IV Prepilin Peptidase Homologue PibD from the Archaeon Sulfolobus solfataricus

    NARCIS (Netherlands)

    Szabo, Zalan; Albers, Sonja-Verena; Driessen, Arnold J.M.

    2006-01-01

    Archaeal preflagellin peptidases and bacterial type IV prepilin peptidases belong to a family of aspartic acid proteases that cleave the leader peptides of precursor proteins with type IV prepilin signal sequences. The substrate repertoire of PibD from the crenarchaeon Sulfolobus solfataricus is unu

  15. Dynamic metabolic adjustments and genome plasticity are implicated in the heat shock response of the extremely thermoacidophilic archaeon Sulfolobus solfataricus.

    Science.gov (United States)

    Tachdjian, Sabrina; Kelly, Robert M

    2006-06-01

    Approximately one-third of the open reading frames encoded in the Sulfolobus solfataricus genome were differentially expressed within 5 min following an 80 to 90 degrees C temperature shift at pH 4.0. This included many toxin-antitoxin loci and insertion elements, implicating a connection between genome plasticity and metabolic regulation in the early stages of stress response.

  16. Identification of novel non-coding RNAs as potential antisense regulators in the archaeon Sulfolobus solfataricus

    DEFF Research Database (Denmark)

    tang, T. H.; Polacek, N.; Zywicki, M.;

    2005-01-01

    to target the 3'-untranslated regions of certain mRNAs. Furthermore, one of the ncRNAs that does not show antisense elements is transcribed from a repeat unit of a cluster of small regularly spaced repeats in S. solfataricus which is potentially involved in replicon partitioning. In conclusion...

  17. SSoNΔ and SsoNΔlong: two thermostable esterases from the same ORF in the archaeon Sulfolobus solfataricus?

    Directory of Open Access Journals (Sweden)

    Luigi Mandrich

    2006-01-01

    Full Text Available Previously, we reported from the Sulfolobus solfataricus open reading frame (ORF SSO2517 the cloning, overexpression and characterization of an esterase belonging to the hormone-sensitive lipase (HSL family and apparently having a deletion at the N-terminus, which we named SsoNΔ. Searching the recently reported Sulfolobus acidocaldarius genome by sequence alignment, using SSO2517 as a query, allowed identity of a putative esterase (ORF SAC1105 sharing high sequence similarity (82% with SSO2517. This esterase displays an N-terminus and total length similar to other known esterases of the HSL family. Analysis of the upstream DNA sequence of SS02517 revealed the possibility of expressing a longer version of the protein with an extended N-terminus; however, no clear translation signal consistent with a longer protein version was detected. This new version of SSO2517 was cloned, over-expressed, purified and characterized. The resulting protein, named SsoNΔlong, was 15-fold more active with the substrate p-nitrophenyl hexanoate than SsoNΔ. Furthermore, SsoNΔlong and SsoNΔ displayed different substrate specificities for triacylglycerols. These results and the phylogenetic relationship between S. solfataricus and S. acidocaldarius suggest a common origin of SSO2517 and SAC1105 from an ancestral gene, followed by divergent evolution. Alternatively, a yet-to-be discovered mechanism of translation that directs the expression of SsoNΔlong under specific metabolic conditions could be hypothesized.

  18. Specificities and pH profiles of adenine and hypoxanthine-guanine-xanthine phosphoribosyltransferases (nucleotide synthases) of the thermoacidophile archaeon Sulfolobus solfataricus

    DEFF Research Database (Denmark)

    Hansen, Michael Riis; Jensen, Kristine Steen; Rasmussen, Mads Skytte;

    2014-01-01

    Two open reading frames in the genome of Sulfolobus solfataricus (SSO2341 and SSO2424) were cloned and expressed in E. coli. The protein products were purified and their enzymatic activity characterized. Although SSO2341 was annotated as a gene (gpT-1) encoding a 6-oxopurine phosphoribosyltransfe......Two open reading frames in the genome of Sulfolobus solfataricus (SSO2341 and SSO2424) were cloned and expressed in E. coli. The protein products were purified and their enzymatic activity characterized. Although SSO2341 was annotated as a gene (gpT-1) encoding a 6-oxopurine...... phosphoribosyltransferase (PRTase), the protein product turned out to be a PRTase highly specific for adenine and we suggest that the reading frame should be renamed apT. The other reading frame SSO2424 (gpT-2) proved to be a true 6-oxopurine PRTase active with hypoxanthine, xanthine and guanine as substrates, and we...... suggest that the gene should be renamed gpT. Both enzymes exhibited unusual profiles of activity versus pH. The adenine PRTase showed the highest activity at pH 7.5-8.5, but had a distinct peak of activity also at pH 4.5. The 6-oxo PRTase showed maximal activity with hypoxanthine and guanine around pH 4...

  19. The magic spot ppGpp influences in vitro the molecular and functional properties of the elongation factor 1α from the archaeon Sulfolobus solfataricus.

    Science.gov (United States)

    Martucci, Nicola M; Lamberti, Anna; Vitagliano, Luigi; Cantiello, Piergiuseppe; Ruggiero, Immacolata; Arcari, Paolo; Masullo, Mariorosario

    2012-09-01

    Guanosine tetra-phosphate (ppGpp), also known as "magic spot I", is a key molecule in the stringent control of most eubacteria and some eukarya. Here, we show that ppGpp affects the functional and molecular properties of the archaeal elongation factor 1α from Sulfolobus solfataricus (SsEF-1α). Indeed, ppGpp inhibited archaeal protein synthesis in vitro, even though the concentration required to get inhibition was higher than that required for the eubacterial and eukaryal systems. Regarding the partial reactions catalysed by SsEF-1α the effect produced by ppGpp on the affinity for aa-tRNA was lower than that measured in the presence of GTP but higher than that for GDP. Magic spot I was also able to bind SsEF-1α with an intermediate affinity in comparison to that displayed by GDP and GTP. Furthermore, ppGpp inhibited the intrinsic GTPase of SsEF-1α with a competitive behaviour. Finally, the binding of ppGpp to SsEF-1α rendered the elongation factor more resistant to heat treatment and the analysis of the molecular model of the complex between SsEF-1α and ppGpp suggests that this stabilisation arises from the charge optimisation on the surface of the protein.

  20. Crystallization and preliminary X-ray diffraction analysis of the hyperthermophilic Sulfolobus solfataricus phosphotriesterase

    Energy Technology Data Exchange (ETDEWEB)

    Elias, Mikael [Laboratoire de Cristallographie et Modélisation des Matériaux Minéraux et Biologiques, CNRS-Université Henri Poincaré, 54506 Vandoeuvre-lès-Nancy (France); Dupuy, Jérôme [Laboratoire de Cristallogenèse et Cristallographie des Protéines, Institut de Biologie Structurale J.-P. Ebel, 38027 Grenoble (France); Merone, Luigia [Istituto di Biochimica delle Proteine, Consiglio Nazionale delle Ricerche, Via P. Castellino 111, 80131 Napoli (Italy); Lecomte, Claude [Laboratoire de Cristallographie et Modélisation des Matériaux Minéraux et Biologiques, CNRS-Université Henri Poincaré, 54506 Vandoeuvre-lès-Nancy (France); Rossi, Mosè [Istituto di Biochimica delle Proteine, Consiglio Nazionale delle Ricerche, Via P. Castellino 111, 80131 Napoli (Italy); Masson, Patrick [Unité d’Enzymologie, Département de Toxicologie, Centre de Recherches du Service de Santé des Armées, 38702 La Tronche (France); Manco, Giuseppe [Istituto di Biochimica delle Proteine, Consiglio Nazionale delle Ricerche, Via P. Castellino 111, 80131 Napoli (Italy); Chabriere, Eric, E-mail: eric.chabriere@lcm3b.uhp-nancy.fr [Laboratoire de Cristallographie et Modélisation des Matériaux Minéraux et Biologiques, CNRS-Université Henri Poincaré, 54506 Vandoeuvre-lès-Nancy (France); Unité d’Enzymologie, Département de Toxicologie, Centre de Recherches du Service de Santé des Armées, 38702 La Tronche (France)

    2007-07-01

    A phosphotriesterase (PTE) from the hyperthermophilic archaeon S. solfataricus has been crystallized. Combined with biochemical and bioengineering studies, it is expected that the structure of this protein will provide insight into the natural function of the PTE family and provide important data for achieving an efficient organophosphate biodecontaminant. Organophosphates constitute the largest class of insecticides used worldwide and some of them are potent nerve agents. Consequently, organophosphate-degrading enzymes are of paramount interest as they could be used as bioscavengers and biodecontaminants. Phosphotriesterases (PTEs) are capable of hydrolyzing these toxic compounds with high efficiency. A distant and hyperthermophilic representative of the PTE family was cloned from the archeon Sulfolobus solfataricus MT4, overexpressed in Escherichia coli and crystallized; the crystals diffracted to 2.54 Å resolution. Owing to its exceptional thermostability, this PTE may be an excellent candidate for obtaining an efficient organophosphate biodecontaminant. Here, the crystallization conditions and data collection for the hyperthermophilic S. solfataricus PTE are reported.

  1. Mutations and Rearrangements in the Genome of Sulfolobus solfataricus P2

    DEFF Research Database (Denmark)

    Redder, P.; Garrett, R. A.

    2006-01-01

    The genome of Sulfolobus solfataricus P2 carries a larger number of transposable elements than any other sequenced genome from an archaeon or bacterium and, as a consequence, may be particularly susceptible to rearrangement and change. In order to gain more insight into the natures and frequencies...... were defined using a specially developed "in vitro library" strategy. Moreover, while searching for the donor mobile elements, evidence was found for two major changes that had occurred in the genome of strain P2, one constituting a single deletion of about 4% of the total genome (124 kb), while...

  2. Metabolism of pentose sugars in the hyperthermophilic archaea Sulfolobus solfataricus and Sulfolobus acidocaldarius.

    Science.gov (United States)

    Nunn, Charlotte E M; Johnsen, Ulrike; Schönheit, Peter; Fuhrer, Tobias; Sauer, Uwe; Hough, David W; Danson, Michael J

    2010-10-29

    We have previously shown that the hyperthermophilic archaeon, Sulfolobus solfataricus, catabolizes d-glucose and d-galactose to pyruvate and glyceraldehyde via a non-phosphorylative version of the Entner-Doudoroff pathway. At each step, one enzyme is active with both C6 epimers, leading to a metabolically promiscuous pathway. On further investigation, the catalytic promiscuity of the first enzyme in this pathway, glucose dehydrogenase, has been shown to extend to the C5 sugars, D-xylose and L-arabinose. In the current paper we establish that this promiscuity for C6 and C5 metabolites is also exhibited by the third enzyme in the pathway, 2-keto-3-deoxygluconate aldolase, but that the second step requires a specific C5-dehydratase, the gluconate dehydratase being active only with C6 metabolites. The products of this pathway for the catabolism of D-xylose and L-arabinose are pyruvate and glycolaldehyde, pyruvate entering the citric acid cycle after oxidative decarboxylation to acetyl-coenzyme A. We have identified and characterized the enzymes, both native and recombinant, that catalyze the conversion of glycolaldehyde to glycolate and then to glyoxylate, which can enter the citric acid cycle via the action of malate synthase. Evidence is also presented that similar enzymes for this pentose sugar pathway are present in Sulfolobus acidocaldarius, and metabolic tracer studies in this archaeon demonstrate its in vivo operation in parallel with a route involving no aldol cleavage of the 2-keto-3-deoxy-pentanoates but direct conversion to the citric acid cycle C5-metabolite, 2-oxoglutarate.

  3. An Autonomously Replicating Transforming Vector for Sulfolobus solfataricus

    Science.gov (United States)

    Cannio, Raffaele; Contursi, Patrizia; Rossi, Mosè; Bartolucci, Simonetta

    1998-01-01

    A plasmid able to transform and to be stably maintained both in Sulfolobus solfataricus and in Escherichia coli was constructed by insertion into an E. coli plasmid of the autonomously replicating sequence of the virus particle SSV1 and a suitable mutant of the hph (hygromycin phosphotransferase) gene as the transformation marker. The vector suffered no rearrangement and/or chromosome integration, and its copy number in Sulfolobus was increased by exposure of the cells to mitomycin C. PMID:9620978

  4. Identification of a system required for the functional surface localization of sugar binding proteins with class III signal peptides in Sulfolobus solfataricus

    NARCIS (Netherlands)

    Zolghadr, Behnam; Weber, Stefan; Szabo, Zalan; Driessen, Arnold J. M.; Albers, Sonja-Verena

    2007-01-01

    The hyperthermophilic archaeon Sulfolobus solfataricus contains an unusual large number of sugar binding proteins that are synthesized as precursors with a class III signal peptide. Such signal peptides are commonly used to direct archaeal flagellin subunits or bacterial (pseudo)pilins into extracel

  5. Post-genomic characterization of metabolic pathways in Sulfolobus solfataricus

    NARCIS (Netherlands)

    Walther, J.

    2012-01-01

    The physiological functions and mode of actions of different biomolecules are of continuous interest and a prerequisite to fully understand and appreciate the potential of Archaea and their molecules. We chose to study Sulfolobus solfataricus for its stable (heat-resistant) enzymes and specific meta

  6. Modeling DNA Repair: Approaching In Vivo Techniques in the Hyperthermophile Sulfolobus Solfataricus

    Energy Technology Data Exchange (ETDEWEB)

    Blanton, J.; Fuss, J.; Yannone, S.M.; Tainer, J.A.; Cooper, P.K.

    2005-01-01

    Archaea are found in some of the most extreme environments on earth and represent a third domain of life distinct from Eukarya and Eubacteria. The hyperthermophilic archaeon Sulfolobus solfataricus, isolated from acidic hot springs (80oC, pH 3) in Yellowstone National Park, has emerged as a potential model system for studying human DNA repair processes. Archaea are more closely related to Eukarya than to Eubacteria, suggesting that archaeal DNA repair machinery may model the complex human system much more closely than that of other prokaryotes. DNA repair requires coordinated protein-protein interactions that are frequently transient. Protein complexes that are transient at extreme temperatures where archaea thrive may be more stable at room temperature, allowing for the characterization of otherwise short-lived complexes. However, characterization of these systems in archaea has been limited by the absence of a stable in vivo transformation and expression system. The work presented here is a pilot study in gene cloning and recombinant protein expression in S. solfataricus. Three genes associated with DNA repair were selected for expression: MRE11, PCNA1, and a putative CSB homologue. Though preparation of these recombinant genes followed standard methods, preparation of a suitable vector proved more challenging. The shuttle vector pSSV64, derived from the SSV1 virus and the E. coli vector pBSSK+, was most successfully isolated from the DH5α E. coli strain. Currently, alternative vectors are being designed for more efficient genetic manipulations in S. solfataricus.

  7. Structure of dimeric, recombinant Sulfolobus solfataricus phosphoribosyl diphosphate synthase

    DEFF Research Database (Denmark)

    Andersen, Rune W.; Lo Leggio, Leila; Hove-Jensen, Bjarne

    2015-01-01

    PRPP synthase as a search model. The two amino acid sequences share 35 % identity. The resulting asymmetric unit consists of three separated dimers. The protein was co-crystallised in the presence of AMP and ribose 5-phosphate, but in the electron density map of the active site only AMP and a sulphate......The enzyme 5-phosphoribosyl-1-α-diphosphate (PRPP) synthase (EC 2.7.6.1) catalyses the Mg2+-dependent transfer of a diphosphoryl group from ATP to the C1 hydroxyl group of ribose 5-phosphate resulting in the production of PRPP and AMP. A nucleotide sequence specifying Sulfolobus solfataricus PRPP....... A bent dimer oligomerisation was revealed, which seems to be an abundant feature among PRPP synthases for defining the adenine specificity of the substrate ATP. Molecular replacement was used to determine the S. solfataricus PRPP synthase structure with a monomer subunit of Methanocaldococcus jannaschii...

  8. Sulfolobus hakonensis sp. nov., a novel species of acidothermophilic archaeon.

    Science.gov (United States)

    Takayanagi, S; Kawasaki, H; Sugimori, K; Yamada, T; Sugai, A; Ito, T; Yamasato, K; Shioda, M

    1996-04-01

    We characterized a microbial strain that was isolated from a hot spring at a geothermal area in Hakone, Japan. This isolate, whose lobed-shaped cells were about 1.0 micron in diameter, was a facultative chemolitho-autotroph that required aerobic conditions for growth. The optimum pH was 3.0 (pH range, 1.0 to 4.0), and the optimum temperature was 70 degrees C (temperature range, 50 to 80 degrees C). Lithotrophically, this strain grew on elemental sulfur and reduced sulfur compounds. The G+C content of the genomic DNA was 38.4 mol%. This organism contained calditoglycerocaldarchaeol, which is characteristic of members of the Sulfolobaceae. The levels of 16S rRNA sequence similarity between the new isolate and Sulfolobus acidocaldarius, Sulfolobus solfataricus, and Sulfolobus shibatae were less than 89.8%. Unlike S. acidocaldarius, S. solfataricus, and S. shibatae, the new isolate utilized sugars and amino acids poorly as sole carbon sources, and the levels of DNA-DNA hybridization between the new isolate and these Sulfolobus species were very low. Phenotypically, the new isolate was also distinct from the obligately lithotrophic organism Sulfolobus metallicus. We concluded that the new organism belongs to a new Sulfolobus species, for which we propose the name Sulfolobus hakonensis.

  9. Purification and Properties of an ATPase from Sulfolobus solfataricus

    Science.gov (United States)

    Hochstein, Lawrence I.; Stan-Lotter, Helga

    1992-01-01

    A sulfite-activated ATPase isolated from Sulfolobus solfataricus had a relative molecular mass of 370,000. It was composed of three subunits whose relative molecular masses were 63,000, 48,000, and 24,000. The enzyme was inhibited by the vacuolar ATPase inhibitors nitrate and N-ethylmaleimide; 4-chloro-7-nitrobenzo-furazan (NBD-Cl) was also inhibitory. N-Ethylmaleimide was predominately bound to the largest subunit while NBD-CL was bound to both subunits. ATPase activity was inhibited by low concentrations of p-chloromercuri-phenyl sulfonate and the inhibition was reversed by cysteine which suggested that thiol groups were essential for activity. While the ATPase from S. solfataricus shared several properties with the ATPase from S. acidocaldarius there were significant differences. The latter enzyme was activated by sulfate and chloride and was unaffected by N-ethylmaleimide, whereas the S. solfataricus ATPase was inhibited by these anions as well as N-ethyimaleimide. These differences as well as differences that occur in other vacuolar-like ATPases isolated from the methanogenic and the extremely halophilic bacteria suggest the existence of several types of archaeal ATPases, none of which have been demonstrated to synthesize ATP.

  10. Allosteric regulation and communication between subunits in uracil phosphoribosyltransferase from Sulfolobus solfataricus

    DEFF Research Database (Denmark)

    Arent, Susan; Harris, Pernille; Jensen, Kaj Frank

    2005-01-01

    Uracil phosphoribosyltransferase (UPRTase) catalyzes the conversion of 5-phosphate-alpha-1-diphosphate (PRPP) and uracil to uridine 5'-monophosphate (UMP) and diphosphate. The UPRTase from Sulfolobus solfataricus has a unique regulation by nucleoside triphosphates compared to UPRTases from other...

  11. A new phosphotriesterase from Sulfolobus acidocaldarius and its comparison with the homologue from Sulfolobus solfataricus.

    Science.gov (United States)

    Porzio, Elena; Merone, Luigia; Mandrich, Luigi; Rossi, Mosè; Manco, Giuseppe

    2007-05-01

    The phosphotriesterase PTE, identified in the soil bacterium Pseudomonas diminuta, is thought to have evolved in the last several decades to degrade the pesticide paraoxon with proficiency approaching the limit of substrate diffusion (k(cat)/K(M) of 4 x 10(7)M(-1)s(-1)). It belongs to the amidohydrolase superfamily, but its evolutionary origin remains obscure. The enzyme has important potentiality in the field of the organophosphate decontamination. Recently we reported on the characterization of an archaeal member of the amidohydrolase superfamily, namely Sulfolobus solfataricus, showing low but significant and extremely thermostable paraoxonase activity (k(cat)/K(M) of 4 x 10(3)M(-1)s(-1)). Looking for other thermostable phosphotriesterases we assayed, among others, crude extracts of Sulfolobus acidocaldarius and detected activity. Since the genome of S. acidocaldarius has been recently reported, we identified there an open reading frame highly related to the S. solfataricus enzyme. The gene was cloned, the protein overexpressed in Escherichia coli, purified, and proven to have paraoxonase activity. A comparative analysis detected some significant differences between the two archaeal enzymes.

  12. Cloning, expression and evolution of the gene encoding the elongation factor 1alpha from a low thermophilic Sulfolobus solfataricus strain.

    Science.gov (United States)

    Masullo, Mariorosario; Cantiello, Piergiuseppe; Lamberti, Annalisa; Longo, Olimpia; Fiengo, Antonio; Arcari, Paolo

    2003-01-28

    The gene encoding the elongation factor 1alpha (EF-1alpha) from the archaeon Sulfolobus solfataricus strain MT3 (optimum growth temperature 75 degrees C) was cloned, sequenced and expressed in Escherichia coli. The structural and biochemical properties of the purified enzyme were compared to those of EF-1alpha isolated from S. solfataricus strain MT4 (optimum growth temperature 87 degrees C). Only one amino acid change (Val15-->Ile) was found. Interestingly, the difference was in the first guanine nucleotide binding consensus sequence G(13)HIDHGK and was responsible for a reduced efficiency in protein synthesis, which was accompanied by an increased affinity for both guanosine diphosphate (GDP) and guanosine triphosphate (GTP), and an increased efficiency in the intrinsic GTPase activity. Despite the different thermophilicities of the two microorganisms, only very marginal effects on the thermal properties of the enzyme were observed. Molecular evolution among EF-1alpha genes from Sulfolobus species showed that the average rate of nucleotide substitution per site per year (0.0312x10(-9)) is lower than that reported for other functional genes.

  13. The Sso7d DNA-binding protein from Sulfolobus solfataricus has ribonuclease activity.

    Science.gov (United States)

    Shehi, E; Serina, S; Fumagalli, G; Vanoni, M; Consonni, R; Zetta, L; Dehò, G; Tortora, P; Fusi, P

    2001-05-25

    Sso7d is a small, basic, abundant protein from the thermoacidophilic archaeon Sulfolobus solfataricus. Previous research has shown that Sso7d can bind double-stranded DNA without sequence specificity by placing its triple-stranded beta-sheet across the minor groove. We previously found RNase activity both in preparations of Sso7d purified from its natural source and in recombinant, purified protein expressed in Escherichia coli. This paper provides conclusive evidence that supports the assignment of RNase activity to Sso7d, shown by the total absence of activity in the single-point mutants E35L and K12L, despite the preservation of their overall structure under the assay conditions. In keeping with our observation that the residues putatively involved in RNase activity and those playing a role in DNA binding are located on different surfaces of the molecule, the activity was not impaired in the presence of DNA. If a small synthetic RNA was used as a substrate, Sso7d attacked both predicted double- and single-stranded RNA stretches, with no evident preference for specific sequences or individual bases. Apparently, the more readily attacked bonds were those intrinsically more unstable.

  14. Metal-ion dependent catalytic properties of Sulfolobus solfataricus class II α-mannosidase

    DEFF Research Database (Denmark)

    Nielsen, Jonas Willum; Poulsen, Nina Rødtness; Johnsson, Anna Margit Susanne;

    2012-01-01

    The active site for the family GH38 class II α-mannosidase is constituted in part by a divalent metal ion, mostly Zn(2+), as revealed in the crystal structures of enzymes from both animal and bacterial sources. The metal ion coordinates to the bound substrate and side chains of conserved amino acid...... residues. Recently, evidence has accumulated that class II α-mannosidase is active in complex with a range of divalent metal ions. In the present work, with employment of the class II α-mannosidase, ManA, from the hyperthermophilic archaeon Sulfolobus solfataricus, we explored the influence of the divalent...... metal ion on the associated steady-state kinetic parameters, K(M) and k(cat), for various substrates. With p-nitrophenyl-α-d-mannoside as a substrate, the enzyme showed activity in the presence of Co(2+), Cd(2+), Mn(2+), and Zn(2+), whereas Ni(2+) and Cu(2+) were inhibitory and nonactivating. Co(2...

  15. Production of steviol from steviol glucosides using β-glycosidase from Sulfolobus solfataricus.

    Science.gov (United States)

    Nguyen, Thi Thanh Hanh; Kim, Seong-Bo; Kim, Nahyun M; Kang, Choongil; Chung, Byoungsang; Park, Jun-Seong; Kim, Doman

    2016-11-01

    Steviol is a diterpene isolated from the plant Stevia rebaudiana that has a potential role as an antihyperglycemic agent by stimulating insulin secretion from pancreatic beta cells and also has significant potential to diminish the renal clearance of anionic drugs and their metabolites. In this study, the lacS gene, which encodes a thermostable β-glycosidase (SSbgly) enzyme from the extremely thermoacidophillic archaeon Sulfolobus solfataricus, was cloned and expressed in E. coli Rossetta BL21(DE3)pLyS using lactose as an inducer. Through fermentation, SSbgly was expressed as a 61kDa protein with activity of 24.3U/mg and the OD600 of 23 was reached after 18h induction with 10mM lactose. Purified protein was obtained by Ni-Sepharose chromatography with a yield of 92.3%. SSbgly hydrolyzed steviol glycosides to produce steviol with a yield of 99.2%. The optimum conditions for steviol production were 50U/ml SSbgly and 90mg/ml Ste at 75°C as determined by the response surface method.

  16. Conditions for gene disruption by homologous recombination of exogenous DNA into the Sulfolobus solfataricus genome

    Directory of Open Access Journals (Sweden)

    Sonja-Verena Albers

    2008-01-01

    Full Text Available The construction of directed gene deletion mutants is an essential tool in molecular biology that allows functional studies on the role of genes in their natural environment. For hyperthermophilic archaea, it has been difficult to obtain a reliable system to construct such mutants. However, during the past years, systems have been developed for Thermococcus kodakarensis and two Sulfolobus species, S. acidocaldarius and derivatives of S. solfataricus 98/2. Here we describe an optimization of the method for integration of exogenous DNA into S. solfataricus PBL 2025, an S. solfataricus 98/2 derivative, based on lactose auxotrophy that now allows for routine gene inactivation.

  17. The bindosome is a structural component of the Sulfolobus solfataricus cell envelope

    NARCIS (Netherlands)

    Zolghadr, Benham; Klingl, Andreas; Rachel, Reinard; Driessen, Arnold J. M.; Albers, Sonja-Verena

    2011-01-01

    Sugar binding proteins of the thermoacidophile Sulfolobus solfataricus function together with ABC transporters in the uptake of sugars. They are synthesized as precursors with a class III signal peptide that are normally found in archaeal flagellins and bacterial type IV pilins. The functional expre

  18. Functional genomics of the thermo-acidophilic archaeon Sulfolobus solfataricus

    NARCIS (Netherlands)

    Oost, van der J.; Walther, J.; Brouns, S.J.J.; Werken, van de H.J.G.; Snijders, A.P.L.; Wright, P.C.; Andersson, A.; Bernander, R.; Vos, de W.M.

    2006-01-01

    Archaea and bacteria that optimally grow at temperatures above 60C and 80C are referred to as thermophiles and hyperthermophiles, respectively (Stetter, 1996). Since their discovery in the late 1960s (Brock and Freeze, 1969), attempts were made to reveal the secrets of the thermal resistance of thes

  19. Flagellar motility and structure in the hyperthermoacidophilic archaeon Sulfolobus solfataricus

    NARCIS (Netherlands)

    Szabo, Zalan; Sani, Musa; Groeneveld, Maarten; Zolghadr, Benham; Schelert, James; Albers, Sonja-Verena; Blum, Paul; Boekema, Egbert J.; Driessen, Arnold J. M.

    2007-01-01

    Flagellation in archaea is widespread and is involved in swimming motility. Here, we demonstrate that the structural flagellin gene from the crenarchaeaon Suffolobus soffiataricus is highly expressed in stationary-phase-grown cells and under unfavorable nutritional conditions. A mutant in a flagella

  20. Characterization of a β-glucosidase from Sulfolobus solfataricus for isoflavone glycosides.

    Science.gov (United States)

    Kim, Bi-Na; Yeom, Soo-Jin; Kim, Yeong-Su; Oh, Deok-Kun

    2012-01-01

    The specific activity of a recombinant β-glucosidase from Sulfolobus solfataricus for isoflavones was: daidzin > glycitin > genistin > malonyl genistin > malonyl daidzin > malonyl glycitin. The hydrolytic activity of this enzyme for daidzin was highest at pH 5.5 and 90°C with a half-life of 18 h, a K (m) of 0.5 mM, and a k (cat) of 2532 s(-1). The enzyme converted 1 mM daidzin to 1 mM daidzein after 1 h with a molar yield of 100% and a productivity of 1 mM h(-1). Among β-glucosidases, that from S. solfataricus β had the highest thermostability, k (cat), k (cat)/K (m), conversion yield, and productivity in the hydrolysis of daidzin.

  1. Functional curation of the Sulfolobus solfataricus P2 and S. acidocaldarius 98-3 complete genome sequences.

    Science.gov (United States)

    Esser, Domink; Kouril, Theresa; Zaparty, Melanie; Sierocinski, Pawel; Chan, Patricia P; Lowe, Todd; Van der Oost, John; Albers, Sonja-Verena; Schomburg, Dietmar; Makarova, Kira S; Siebers, Bettina

    2011-11-01

    The thermoacidophiles Sulfolobus solfataricus P2 and S. acidocaldarius 98-3 are considered key model organisms representing a major phylum of the Crenarchaeota. Because maintaining current, accurate genome information is indispensable for modern biology, we have updated gene function annotation using the arCOGs database, plus other available functional, structural and phylogenetic information. The goal of this initiative is continuous improvement of genome annotation with the support of the Sulfolobus research community.

  2. Functional curation of the Sulfolobus solfataricus P2 and S. acidocaldarius 98-3 complete genome sequences

    NARCIS (Netherlands)

    Esser, D.; Kouril, T.; Zaparty, M.; Sierocinski, P.; Oost, van der J.; Makarova, K.S.; Siebers, B.

    2011-01-01

    The thermoacidophiles Sulfolobus solfataricus P2 and S. acidocaldarius 98-3 are considered key model organisms representing a major phylum of the Crenarchaeota. Because maintaining current, accurate genome information is indispensable for modern biology, we have updated gene function annotation usin

  3. Identification and sequence analysis of Sulfolobus solfataricus purE and purK genes

    DEFF Research Database (Denmark)

    Sørensen, Iben Schildt; Dandanell, Gert

    1997-01-01

    From a genomic library of Sulfolobus solfataricus DSM1617 we have isolated and identified the purEK locus. Two open reading frames are identified as homologs of the purE and purK purine biosynthetic genes in Escherichia coli. The C-terminus of purE overlaps with the N-terminus of purK. When either...

  4. Reconstruction of central carbon metabolism in Sulfolobus solfataricus using a two-dimensional gel electrophoresis map, stable isotope labelling and DNA microarray analysis

    NARCIS (Netherlands)

    Snijders, B.P.L.; Walther, J.; Peter, S.; Kinnman, I.; Vos, de M.J.G.; Werken, van de H.J.G.; Brouns, S.J.J.; Oost, van der J.; Wright, P.C.

    2006-01-01

    In the last decade, an increasing number of sequenced archaeal genomes have become available, opening up the possibility for functional genomic analyses. Here, we reconstructed the central carbon metabolism in the hyperthermophilic crenarchaeon Sulfolobus solfataricus (glycolysis, gluconeogenesis an

  5. Overexpression, purification, crystallization and data collection of Sulfolobus solfataricus Sso6206, a novel highly conserved protein

    Energy Technology Data Exchange (ETDEWEB)

    McEwan, Andrew R.; Liu, Huanting; Oke, Muse; Carter, Lester; Powers, Helen; Dorward, Mark; McMahon, Stephen A.; White, Malcolm F.; Naismith, James H., E-mail: naismith@st-and.ac.uk [The Centre for Biomolecular Sciences, The University, St Andrews KY16 9ST,Scotland (United Kingdom)

    2006-03-01

    The novel protein Sso6206 has been crystallized; interestingly, the protein may form large multi-subunit oligomers. Sso6206, a 10.5 kDa protein from Sulfolobus solfataricus, has been overexpressed, purified and crystallized. The protein crystallizes in space group P6{sub 1/5}22, with unit-cell parameters a = b = 157.8, c = 307.3 Å. The crystals are hexagonal bipyramids and a data set has been collected to 2.4 Å resolution. Molecular replacement cannot be attempted as no convincing model can be identified. Crystals of selenomethionine-variant protein have not yet been obtained. Interestingly, crystal packing, gel filtration and mass spectrometry all suggest the native protein forms a multi-subunit oligomer consisting of >9 subunits.

  6. Small multicopy, non-integrative shuttle vectors based on the plasmid pRN1 for Sulfolobus acidocaldarius and Sulfolobus solfataricus, model organisms of the (cren-)archaea.

    Science.gov (United States)

    Berkner, Silvia; Grogan, Dennis; Albers, Sonja-Verena; Lipps, Georg

    2007-01-01

    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.

  7. VapC6, a ribonucleolytic toxin regulates thermophilicity in the crenarchaeote Sulfolobus solfataricus.

    Science.gov (United States)

    Maezato, Yukari; Daugherty, Amanda; Dana, Karl; Soo, Edith; Cooper, Charlotte; Tachdjian, Sabrina; Kelly, Robert M; Blum, Paul

    2011-07-01

    The phylum Crenarchaeota includes hyperthermophilic micro-organisms subjected to dynamic thermal conditions. Previous transcriptomic studies of Sulfolobus solfataricus identified vapBC6 as a heat-shock (HS)-inducible member of the Vap toxin-antitoxin gene family. In this study, the inactivation of the vapBC6 operon by targeted gene disruption produced two recessive phenotypes related to fitness, HS sensitivity and a heat-dependent reduction in the rate of growth. In-frame vapBC6 deletion mutants were analyzed to examine the respective roles of each protein. Since vapB6 transcript abundance was elevated in the vapC6 deletion, the VapC6 toxin appears to regulate abundance of its cognate antitoxin. In contrast, vapC6 transcript abundance was reduced in the vapB6 deletion. A putative intergenic terminator may underlie these observations by coordinating vapBC6 expression. As predicted by structural modeling, recombinant VapC6 produced using chaperone cosynthesis exhibited heat-dependent ribonucleolytic activity toward S. solfataricus total RNA. This activity could be blocked by addition of preheated recombinant VapB6. In vivo transcript targets were identified by assessing the relative expression of genes that naturally respond to thermal stress in VapBC6-deficient cells. Preferential increases were observed for dppB-1 and tetR, and preferential decreases were observed for rpoD and eIF2 gamma. Specific VapC6 ribonucleolytic action could also be demonstrated in vitro toward RNAs whose expression increased in the VapBC6-deficient strain during heat shock. These findings provide a biochemical mechanism and identify cellular targets underlying VapBC6-mediated control over microbial growth and survival at temperature extremes.

  8. Identification of amino acids related to catalytic function of Sulfolobus solfataricus P1 carboxylesterase by site-directed mutagenesis and molecular modeling

    Science.gov (United States)

    Choi, Yun-Ho; Lee, Ye-Na; Park, Young-Jun; Yoon, Sung-Jin; Lee, Hee-Bong

    2016-01-01

    The archaeon Sulfolobus solfataricus P1 carboxylesterase is a thermostable enzyme with a molecular mass of 33.5 kDa belonging to the mammalian hormone-sensitive lipase (HSL) family. In our previous study, we purified the enzyme and suggested the expected amino acids related to its catalysis by chemical modification and a sequence homology search. For further validating these amino acids in this study, we modified them using site-directed mutagenesis and examined the activity of the mutant enzymes using spectrophotometric analysis and then estimated by homology modeling and fluorescence analysis. As a result, it was identified that Ser151, Asp244, and His274 consist of a catalytic triad, and Gly80, Gly81, and Ala152 compose an oxyanion hole of the enzyme. In addition, it was also determined that the cysteine residues are located near the active site or at the positions inducing any conformational changes of the enzyme by their replacement with serine residues. [BMB Reports 2016; 49(6): 349-354] PMID:27222124

  9. Discovering Antioxidant Molecules in the Archaea Domain: Peroxiredoxin Bcp1 from Sulfolobus solfataricus Protects H9c2 Cardiomyoblasts from Oxidative Stress

    Science.gov (United States)

    Sarcinelli, Carmen; Pizzo, Elio

    2016-01-01

    Peroxiredoxins (Prxs) are ubiquitous thiol peroxidases that are involved in the reduction of peroxides. It has been reported that prokaryotic Prxs generally show greater structural robustness than their eukaryotic counterparts, making them less prone to inactivation by overoxidation. This difference has inspired the search for new antioxidants from prokaryotic sources that can be used as possible therapeutic biodrugs. Bacterioferritin comigratory proteins (Bcps) of the hyperthermophilic archaeon Sulfolobus solfataricus that belong to the Prx family have recently been characterized. One of these proteins, Bcp1, was chosen to determine its antioxidant effects in H9c2 rat cardiomyoblast cells. Bcp1 activity was measured in vitro under physiological temperature and pH conditions that are typical of mammalian cells; the yeast thioredoxin reductase (yTrxR)/thioredoxin (yTrx) reducing system was used to evaluate enzyme activity. A TAT-Bcp1 fusion protein was constructed to allow its internalization and verify the effect of Bcp1 on H9c2 rat cardiomyoblasts subjected to oxidative stress. The results reveal that TAT-Bcp1 is not cytotoxic and inhibits H2O2-induced apoptosis in H9c2 cells by reducing the H2O2 content inside these cells. PMID:27752237

  10. Discovering Antioxidant Molecules in the Archaea Domain: Peroxiredoxin Bcp1 from Sulfolobus solfataricus Protects H9c2 Cardiomyoblasts from Oxidative Stress

    Directory of Open Access Journals (Sweden)

    Carmen Sarcinelli

    2016-01-01

    Full Text Available Peroxiredoxins (Prxs are ubiquitous thiol peroxidases that are involved in the reduction of peroxides. It has been reported that prokaryotic Prxs generally show greater structural robustness than their eukaryotic counterparts, making them less prone to inactivation by overoxidation. This difference has inspired the search for new antioxidants from prokaryotic sources that can be used as possible therapeutic biodrugs. Bacterioferritin comigratory proteins (Bcps of the hyperthermophilic archaeon Sulfolobus solfataricus that belong to the Prx family have recently been characterized. One of these proteins, Bcp1, was chosen to determine its antioxidant effects in H9c2 rat cardiomyoblast cells. Bcp1 activity was measured in vitro under physiological temperature and pH conditions that are typical of mammalian cells; the yeast thioredoxin reductase (yTrxR/thioredoxin (yTrx reducing system was used to evaluate enzyme activity. A TAT-Bcp1 fusion protein was constructed to allow its internalization and verify the effect of Bcp1 on H9c2 rat cardiomyoblasts subjected to oxidative stress. The results reveal that TAT-Bcp1 is not cytotoxic and inhibits H2O2-induced apoptosis in H9c2 cells by reducing the H2O2 content inside these cells.

  11. Understanding Molecular Recognition of Promiscuity of Thermophilic Methionine Adenosyltransferase, sMAT from Sulfolobus solfataricus

    Science.gov (United States)

    Wang, Fengbin; Singh, Shanteri; Zhang, Jianjun; Huber, Tyler D.; Helmich, Kate E.; Sunkara, Manjula; Hurley, Katherine A.; Goff, Randal D.; Bingman, Craig A.; Morris, Andrew J.; Thorson, Jon S.; Phillips, George N.

    2014-01-01

    Methionine adenosyltransferase (MAT) is a family of enzymes that utilizes ATP and methionine to produce S-adenosylmethionine (AdoMet), the most crucial methyl donor in the biological methylation of biomolecules and bioactive natural products. Here, we report that the MAT from Sulfolobus solfataricus (sMAT), an enzyme from a poorly explored class of the MAT family, has the ability to produce a range of differentially alkylated AdoMet analogs in the presence of non-native methionine analogs and ATP. To investigate the molecular basis for AdoMet analog production, we have crystallized the sMAT in the AdoMet bound, S-adenosylethionine (AdoMet) bound, and unbound forms. Notably, among these structures, the AdoEth-bound form offers the first MAT structure containing a non-native product and cumulatively, these structures add new structural insight into the MAT family and allow for detailed active site comparison with its homologs in E. coli and human. As a thermostable MAT structure from archaea, the structures herein also provide as a basis for future engineering to potentially broaden AdoMet analog production as reagents for methyltransferase-catalyzed ‘alkylrandomization’ and/or the study of methylation in the context of biological processes. PMID:24649856

  12. The Sso7d protein of Sulfolobus solfataricus: in vitro relationship among different activities

    Science.gov (United States)

    Guagliardi, Annamaria; Cerchia, Laura; Rossi, Mosè

    2002-01-01

    The physiological role of the nonspecific DNA-binding protein Sso7d from the crenarchaeon Sulfolobus solfataricus is unknown. In vitro studies have shown that Sso7d promotes annealing of complementary DNA strands (Guagliardi et al. 1997), induces negative supercoiling (Lopez-Garcia et al. 1998), and chaperones the disassembly and renaturation of protein aggregates in an ATP hydrolysis-dependent manner (Guagliardi et al. 2000). In this study, we examined the relationships among the binding of Sso7d to double-stranded DNA, its interaction with protein aggregates, and its ATPase activity. Experiments with 1-anilinonaphthalene-8-sulfonic acid as probe demonstrated that exposed hydrophobic surfaces in Sso7d are responsible for interactions with protein aggregates and double-stranded DNA, whereas the site of ATPase activity has a non-hydrophobic character. The interactions of Sso7d with double-stranded DNA and with protein aggregates are mutually exclusive events, suggesting that the disassembly activity and the DNA-related activities of Sso7d may be competitive in vivo. In contrast, the hydrolysis of ATP by Sso7d is independent of the binding of Sso7d to double-stranded DNA or protein aggregates. PMID:15803646

  13. Processing of DNA lesions by archaeal DNA polymerases from Sulfolobus solfataricus

    Science.gov (United States)

    Grúz, Petr; Shimizu, Masatomi; Pisani, Francesca M.; Felice, Mariarita De; Kanke, Yusuke; Nohmi, Takehiko

    2003-01-01

    Spontaneous damage to DNA as a result of deamination, oxidation and depurination is greatly accelerated at high temperatures. Hyperthermophilic microorganisms constantly exposed to temperatures exceeding 80°C are endowed with powerful DNA repair mechanisms to maintain genome stability. Of particular interest is the processing of DNA lesions during replication, which can result in fixed mutations. The hyperthermophilic crenarchaeon Sulfolobus solfataricus has two functional DNA polymerases, PolB1 and PolY1. We have found that the replicative DNA polymerase PolB1 specifically recognizes the presence of the deaminated bases hypoxanthine and uracil in the template by stalling DNA polymerization 3–4 bases upstream of these lesions and strongly associates with oligonucleotides containing them. PolB1 also stops at 8-oxoguanine and is unable to bypass an abasic site in the template. PolY1 belongs to the family of lesion bypass DNA polymerases and readily bypasses hypoxanthine, uracil and 8-oxoguanine, but not an abasic site, in the template. The specific recognition of deaminated bases by PolB1 may represent an initial step in their repair while PolY1 may be involved in damage tolerance at the replication fork. Additionally, we reveal that the deaminated bases can be introduced into DNA enzymatically, since both PolB1 and PolY1 are able to incorporate the aberrant DNA precursors dUTP and dITP. PMID:12853619

  14. Structure of dimeric, recombinant Sulfolobus solfataricus phosphoribosyl diphosphate synthase: a bent dimer defining the adenine specificity of the substrate ATP.

    Science.gov (United States)

    Andersen, Rune W; Leggio, Leila Lo; Hove-Jensen, Bjarne; Kadziola, Anders

    2015-03-01

    The enzyme 5-phosphoribosyl-1-α-diphosphate (PRPP) synthase (EC 2.7.6.1) catalyses the Mg(2+)-dependent transfer of a diphosphoryl group from ATP to the C1 hydroxyl group of ribose 5-phosphate resulting in the production of PRPP and AMP. A nucleotide sequence specifying Sulfolobus solfataricus PRPP synthase was synthesised in vitro with optimised codon usage for expression in Escherichia coli. Following expression of the gene in E. coli PRPP synthase was purified by heat treatment and ammonium sulphate precipitation and the structure of S. solfataricus PRPP synthase was determined at 2.8 Å resolution. A bent dimer oligomerisation was revealed, which seems to be an abundant feature among PRPP synthases for defining the adenine specificity of the substrate ATP. Molecular replacement was used to determine the S. solfataricus PRPP synthase structure with a monomer subunit of Methanocaldococcus jannaschii PRPP synthase as a search model. The two amino acid sequences share 35 % identity. The resulting asymmetric unit consists of three separated dimers. The protein was co-crystallised in the presence of AMP and ribose 5-phosphate, but in the electron density map of the active site only AMP and a sulphate ion were observed. Sulphate ion, reminiscent of the ammonium sulphate precipitation step of the purification, seems to bind tightly and, therefore, presumably occupies and blocks the ribose 5-phosphate binding site. The activity of S. solfataricus PRPP synthase is independent of phosphate ion.

  15. Role of vapBC toxin-antitoxin loci in the thermal stress response of Sulfolobus solfataricus.

    Science.gov (United States)

    Cooper, Charlotte R; Daugherty, Amanda J; Tachdjian, Sabrina; Blum, Paul H; Kelly, Robert M

    2009-02-01

    TA (toxin-antitoxin) loci are ubiquitous in prokaryotic micro-organisms, including archaea, yet their physiological function is largely unknown. For example, preliminary reports have suggested that TA loci are microbial stress-response elements, although it was recently shown that knocking out all known chromosomally located TA loci in Escherichia coli did not have an impact on survival under certain types of stress. The hyperthermophilic crenarchaeon Sulfolobus solfataricus encodes at least 26 vapBC (where vap is virulence-associated protein) family TA loci in its genome. VapCs are PIN (PilT N-terminus) domain proteins with putative ribonuclease activity, while VapBs are proteolytically labile proteins, which purportedly function to silence VapCs when associated as a cognate pair. Global transcriptional analysis of S. solfataricus heat-shock-response dynamics (temperature shift from 80 to 90 degrees C) revealed that several vapBC genes were triggered by the thermal shift, suggesting a role in heat-shock-response. Indeed, knocking out a specific vapBC locus in S. solfataricus substantially changed the transcriptome and, in one case, rendered the crenarchaeon heat-shock-labile. These findings indicate that more work needs to be done to determine the role of VapBCs in S. solfataricus and other thermophilic archaea, especially with respect to post-transcriptional regulation.

  16. Role of vapBC toxin–antitoxin loci in the thermal stress response of Sulfolobus solfataricus

    Science.gov (United States)

    Cooper, Charlotte R.; Daugherty, Amanda J.; Tachdjian, Sabrina; Blum, Paul H.; Kelly, Robert M.

    2010-01-01

    TA (toxin–antitoxin) loci are ubiquitous in prokaryotic microorganisms, including archaea, yet their physiological function is largely unknown. For example, preliminary reports have suggested that TA loci are microbial stress-response elements, although it was recently shown that knocking out all known chromosomally located TA loci in Escherichia coli did not have an impact on survival under certain types of stress. The hyperthermophilic crenarchaeon Sulfolobus solfataricus encodes at least 26 vapBC (where vap is virulence-associated protein) family TA loci in its genome. VapCs are PIN (PilT N-terminus) domain proteins with putative ribonuclease activity, while VapBs are proteolytically labile proteins, which purportedly function to silence VapCs when associated as a cognate pair. Global transcriptional analysis of S. solfataricus heat-shock-response dynamics (temperature shift from 80 to 90°C) revealed that several vapBC genes were triggered by the thermal shift, suggesting a role in heat-shock-response. Indeed, knocking out a specific vapBC locus in S. solfataricus substantially changed the transcriptome and, in one case, rendered the crenarchaeon heat-shock-labile. These findings indicate that more work needs to be done to determine the role of VapBCs in S. solfataricus and other thermophilic archaea, especially with respect to post-transcriptional regulation. PMID:19143615

  17. Interfacial air/water proton conduction from long distances by sulfolobus solfataricus archaeal bolaform lipids.

    Science.gov (United States)

    Vilalta, I; Gliozzi, A; Prats, M

    1996-08-15

    The stability, structural organization, and the ability to transfer protons long distances have been investigated in monolayers formed from archael bolaform lipids at the air/water interface. The lipids employed were the fractions GroR2Gro (R represents an acyl group with variable chain length typically consisting of 0-4 cyclopentane rings and 40 isoprenoid residues) and GroR2GroNon-Ol (Non-ol represents nonitol) extracted from Sulfolobus solfataricus by hydrolysis of the cytoplasmic membrane. GroR2-GroNon-ol films exhibit a very peculiar behaviour: the monolayer surface pressure increases with time, regardless of its low or high initial value. This finding is related to the possibility of GroR2GroNon-ol molecules to assume an upright (a metastable) or a U-shaped (stable) configuration. In the gaseous state and in the collapsed state of the film, no lateral proton conduction was observed. However, in the pressure range 0 < pi < 25 mN/m for GroR2Gro and 0 < pi < 30 mN/m for GroR2GroNon-ol monolayers, a lateral proton conduction at the air/water interface was observed. The structural organization of these bipolar lipids at the air/water interface can be related to the lateral proton conduction; it is possible to conclude that whatever configuration these lipids may adopt, they are able to structure the air/water interface in a hydrogen bond network that supports lateral proton conduction. This process may be ascribed to a percolation phenomenon occurring when the polar lipid head groups form a structured lattice of hydrogen bonds.

  18. Crystal structure of a novel archaeal AAA+ ATPase SSO1545 from Sulfolobus solfataricus

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Qingping; Rife, Christopher L.; Carlton, Dennis; Miller, Mitchell D.; Krishna, S. Sri; Elsliger, Marc-André; Abdubek, Polat; Astakhova, Tamara; Chiu, Hsiu-Ju; Clayton, Thomas; Duan, Lian; Feuerhelm, Julie; Grzechnik, Slawomir K.; Hale, Joanna; Han, Gye Won; Jaroszewski, Lukasz; Jin, Kevin K.; Klock, Heath E.; Knuth, Mark W.; Kumar, Abhinav; McMullan, Daniel; Morse, Andrew T.; Nigoghossian, Edward; Okach, Linda; Oommachen, Silvya; Paulsen, Jessica; Reyes, Ron; van den Bedem, Henry; Hodgson, Keith O.; Wooley, John; Deacon, Ashley M.; Godzik, Adam; Lesley, Scott A.; Wilson, Ian A.; (Scripps); (SSR); (SSRL); (JCSG); (UCSD)

    2009-08-28

    Signal transduction ATPases with numerous domains (STAND), a large class of P-loop NTPases, belong to AAA+ ATPases. They include AP(apoptotic)-ATPases (e.g., animal apoptosis regulators CED4/Apaf-1, plant disease resistance proteins, and bacterial AfsR-like transcription regulators), NACHT NTPases (e.g. CARD4, NAIP, Het-E-1, TLP1), and several other less well-characterized families. STAND differ from other P-loop NTPases by their unique sequence motifs, which include an hhGRExE (h, hydrophobic; x, any residue) motif at the N-terminal region, a GxP/GxxP motif at the C-terminal region of the NTPase domain, in addition to a C-terminal helical domain and additional domains such as WD40, TPR, LRR or catalytic modules. Despite significant biological interests, structural coverage of STAND proteins is very limited and only two other structures are currently known: the cell death regulators Apaf-1 and CED-4. Here, we report the crystal structure of SSO1545 from Sulfolobus solfataricus, which was determined using the semi-automated, high-throughput pipeline of the Joint Center for Structural Genomics (JCSG; http://www.jcsg.org), as part of the National Institute of General Medical Sciences' Protein Structure Initiative (PSI). SSO1545 (NP-342973.1), a representative of the archaeal STANDs, is a member of Pfam PF01637 and encodes a protein of 356 residues with calculated molecular weight and isoelectric point of 41.7 kD and 8.2, respectively.

  19. Conformational changes during nucleotide selection by Sulfolobus solfataricus DNA polymerase Dpo4.

    Science.gov (United States)

    Eoff, Robert L; Sanchez-Ponce, Raymundo; Guengerich, F Peter

    2009-07-31

    The mechanism of nucleotide selection by Y-family DNA polymerases has been the subject of intense study, but significant structural contacts and/or conformational changes that relate to polymerase fidelity have been difficult to identify. Here we report on the conformational dynamics of a model Y-family polymerase Dpo4 from Sulfolobus solfataricus. Hydrogen-deuterium exchange in tandem with mass spectrometry was used to monitor changes in Dpo4 structure as a function of time and the presence or absence of specific substrates and ligands. Analysis of the data revealed previously unrecognized structural changes that accompany steps in the catalytic cycle leading up to phosphoryl transfer. For example, the solvent accessibility of the alphaB-loop-alphaC region in the finger domain decreased in the presence of all four dNTP insertion events, but the rate of deuterium exchange, an indicator of conformational flexibility, only decreased during an accurate insertion event. Of particular note is a change in the region surrounding the H-helix of the thumb domain. Upon binding DNA and Mg2+, the H-helix showed a decrease in solvent accessibility and flexibility that was relaxed only upon addition of dCTP, which forms a Watson-Crick base pair with template dG and not during mispairing events. The current study expands upon a previous report from our group that used a fluorescent probe located near the thumb domain to measure the kinetic properties of Dpo4 conformational changes. We now present a model for nucleotide selection by Dpo4 that arises from a synthesis of both structural and kinetic data.

  20. Chromosome replication dynamics in the archaeon Sulfolobus acidocaldarius.

    Science.gov (United States)

    Duggin, Iain G; McCallum, Simon A; Bell, Stephen D

    2008-10-28

    The "baby machine" provides a means of generating synchronized cultures of minimally perturbed cells. We describe the use of this technique to establish the key cell-cycle parameters of hyperthermophilic archaea of the genus Sulfolobus. The 3 DNA replication origins of Sulfolobus acidocaldarius were mapped by 2D gel analysis to near 0 (oriC2), 579 (oriC1), and 1,197 kb (oriC3) on the 2,226-kb circular genome, and we present a direct demonstration of their activity within the first few minutes of a synchronous cell cycle. We also detected X-shaped DNA molecules at the origins in log-phase cells, but these were not directly associated with replication initiation or ongoing chromosome replication in synchronized cells. Whole-genome marker frequency analyses of both synchronous and log-phase cultures showed that origin utilization was close to 100% for all 3 origins per round of replication. However, oriC2 was activated slightly later on average compared with oriC1 and oriC3. The DNA replication forks moved bidirectionally away from each origin at approximately 88 bp per second in synchronous culture. Analysis of the 3 Orc1/Cdc6 initiator proteins showed a uniformity of cellular abundance and origin binding throughout the cell cycle. In contrast, although levels of the MCM helicase were constant across the cell cycle, its origin localization was regulated, because it was strongly enriched at all 3 origins in early S phase.

  1. Formation of the productive ATP-Mg2+-bound dimer of GlcV, an ABC-ATPase from Sulfolobus solfataricus

    NARCIS (Netherlands)

    Verdon, G; Albers, SV; van Oosterwijk, N; Dijkstra, BW; Driessen, AJM; Thunnissen, AMWH; Dijkstra, Bauke W.

    2003-01-01

    The ABC-ATPase GlcV from Sulfolobus solfataricus energizes an ABC transporter mediating glucose uptake. In ABC transporters, two ABC-ATPases are believed to form a head-to-tail dimer, with both monomers contributing conserved residues to each of the two productive active sites. In contrast, isolated

  2. SMV1 virus-induced CRISPR spacer acquisition from the conjugative plasmid pMGB1 in Sulfolobus solfataricus P2

    DEFF Research Database (Denmark)

    Erdmann, Susanne; Shah, Shiraz Ali; Garrett, Roger Antony

    2013-01-01

    complexity and diversity of the CRISPR immune systems that are found among the Sulfolobales. In the present article, we re-examine the first successful induction of archaeal spacer acquisition in our laboratory that occurred exclusively for the conjugative plasmid pMGB1 in Sulfolobus solfataricus P2...

  3. Biochemical, transcriptional and translational evidences of the phenol-meta-degradation pathway by the hyperthermophilic Sulfolobus solfataricus 98/2.

    Directory of Open Access Journals (Sweden)

    Alexia Comte

    Full Text Available Phenol is a widespread pollutant and a model molecule to study the biodegradation of monoaromatic compounds. After a first oxidation step leading to catechol in mesophilic and thermophilic microorganisms, two main routes have been identified depending on the cleavage of the aromatic ring: ortho involving a catechol 1,2 dioxygenase (C12D and meta involving a catechol 2,3 dioxygenase (C23D. Our work aimed at elucidating the phenol-degradation pathway in the hyperthermophilic archaea Sulfolobus solfataricus 98/2. For this purpose, the strain was cultivated in a fermentor under different substrate and oxygenation conditions. Indeed, reducing dissolved-oxygen concentration allowed slowing down phenol catabolism (specific growth and phenol-consumption rates dropped 55% and 39%, respectively and thus, evidencing intermediate accumulations in the broth. HPLC/Diode Array Detector and LC-MS analyses on culture samples at low dissolved-oxygen concentration (DOC  =  0.06 mg x L(-1 suggested, apart for catechol, the presence of 2-hydroxymuconic acid, 4-oxalocrotonate and 4-hydroxy-2-oxovalerate, three intermediates of the meta route. RT-PCR analysis on oxygenase-coding genes of S. solfataricus 98/2 showed that the gene coding for the C23D was expressed only on phenol. In 2D-DIGE/MALDI-TOF analysis, the C23D was found and identified only on phenol. This set of results allowed us concluding that S. solfataricus 98/2 degrade phenol through the meta route.

  4. Surface-exposed glycoproteins of hyperthermophilic Sulfolobus solfataricus P2 show a common N-glycosylation profile.

    Science.gov (United States)

    Palmieri, Gianna; Balestrieri, Marco; Peter-Katalinić, Jasna; Pohlentz, Gottfried; Rossi, Mosè; Fiume, Immacolata; Pocsfalvi, Gabriella

    2013-06-07

    Cell surface proteins of hyperthermophilic Archaea actively participate in intercellular communication, cellular uptake, and energy conversion to sustain survival strategies in extreme habitats. Surface (S)-layer glycoproteins, the major component of the S-layers in many archaeal species and the best-characterized prokaryotic glycoproteins, were shown to have a large structural diversity in their glycan compositions. In spite of this, knowledge on glycosylation of proteins other than S-layer proteins in Archaea is quite limited. Here, the N-glycosylation pattern of cell-surface-exposed proteins of Sulfolobus solfataricus P2 were analyzed by lectin affinity purification, HPAEC-PAD, and multiple mass spectrometry-based techniques. Detailed analysis of SSO1273, one of the most abundant ABC transporters present in the cell surface fraction of S. solfataricus, revealed a novel glycan structure composed of a branched sulfated heptasaccharide, Hex4(GlcNAc)2 plus sulfoquinovose where Hex is d-mannose and d-glucose. Having one monosaccharide unit more than the glycan of the S-layer glycoprotein of S. acidocaldarius, this is the most complex archaeal glycan structure known today. SSO1273 protein is heavily glycosylated and all 20 theoretical N-X-S/T (where X is any amino acid except proline) consensus sequence sites were confirmed. Remarkably, we show that several other proteins in the surface fraction of S. solfataricus are N-glycosylated by the same sulfated oligosaccharide and we identified 56 N-glycosylation sites in this subproteome.

  5. Dynamic Metabolic Adjustments and Genome Plasticity Are Implicated in the Heat Shock Response of the Extremely Thermoacidophilic Archaeon Sulfolobus solfataricus†

    Science.gov (United States)

    Tachdjian, Sabrina; Kelly, Robert M.

    2006-01-01

    Approximately one-third of the open reading frames encoded in the Sulfolobus solfataricus genome were differentially expressed within 5 min following an 80 to 90°C temperature shift at pH 4.0. This included many toxin-antitoxin loci and insertion elements, implicating a connection between genome plasticity and metabolic regulation in the early stages of stress response. PMID:16740961

  6. Effect of DNA binding protein Ssh12 from hyperthermophilic archaeon Sulfolobus shibatae on DNA supercoiling

    Institute of Scientific and Technical Information of China (English)

    楼慧强; 黄力; VietQ.Mai

    1999-01-01

    An 11.5-ku DNA binding protein, designated as Sshl2, was purified from the hyperthermophilic archaeon Sulfolobus shibatae by column chromatography in SP Sepharose, DNA cellulose and phosphocellulose. Sshl2 accounts for about 4 % of the total cellular protein. The protein is capable of binding to both negatively supercoiled and relaxed DNAs. Nick closure analysis revealed that Sshl2 constrains negative supercoils upon binding to DNA. While the ability of the protein to constrain supercoils is weak at 22℃ , it is enhanced substantially at temperatures higher than 37℃ . Both the cellular content and supercoil-constraining ability of Sshl2 suggest that the protein may play an important role in the organization and stabilization of the chromosome of S. shibatae.

  7. Biochemical characterization and helix stabilizing properties of HSNP-C' from the thermoacidophilic archaeon Sulfolobus acidocaldarius.

    Science.gov (United States)

    Celestina, F; Suryanarayana, T

    2000-01-19

    Helix stabilizing nucleoid protein HSNP-C' from the thermophilic archaeon Sulfolobus acidocaldarius has been characterized with respect to its interactions with nucleic acids by gel retardation assay, affinities to immobilized matrices, electron microscopy, and fluorescence titration. The amino acids implicated in the DNA binding site of the protein have been shown by selectively modifying specific amino acyl functional groups and looking at their effects on the DNA binding properties of the protein. Lysine, arginine, tryptophan, and tyrosine residues of the protein HSNP-C' were modified with pyridoxal-5-phosphate; 2,3-butanedione; BNPS-skatole; and tetranitromethane, respectively. The modification of residues was assessed according to standard procedures. The effect of the chemical modification on the function of the protein HSNP-C' with respect to DNA protein interactions was studied and the results indicate the definite involvement of tyrosines and also the significant involvement of the flanking tryptophan residues in the DNA binding domain on the protein.

  8. A New Pepstatin-Insensitive Thermopsin-Like Protease Overproduced in Peptide-Rich Cultures of Sulfolobus solfataricus

    Directory of Open Access Journals (Sweden)

    Marta Gogliettino

    2014-02-01

    Full Text Available In this study, we gain insight into the extracellular proteolytic system of Sulfolobus solfataricus grown on proteinaceous substrates, providing further evidence that acidic proteases were specifically produced in response to peptide-rich media. The main proteolytic component was the previously isolated SsMTP (Sulfolobus solfataricus multi-domain thermopsin-like protease, while the less abundant (named SsMTP-1 one was purified, characterized and identified as the sso1175 gene-product. The protein revealed a multi-domain organization shared with the cognate SsMTP with a catalytic domain followed by several tandemly-repeated motifs. Moreover, both enzymes were found spread across the Crenarchaeota phylum and belonging to the thermopsin family, although segregated into diverse phylogenetic clusters. SsMTP-1 showed a 75-kDa molecular mass and was stable in the temperature range 50–90 °C, with optimal activity at 70 °C and pH 2.0. Serine, metallo and aspartic protease inhibitors did not affect the enzyme activity, designating SsMTP-1 as a new member of the pepstatin-insensitive aspartic protease family. The peptide-bond-specificity of SsMTP-1 in the cleavage of the oxidized insulin B chain was uncommon amongst thermopsins, suggesting that it could play a distinct, but cooperative role in the protein degradation machinery. Interestingly, predictions of the transmembrane protein topology of SsMTP and SsMTP-1 strongly suggest a possible contribution in signal-transduction pathways.

  9. Bypass of Aflatoxin B[subscript 1] Adducts by the Sulfolobus solfataricus DNA Polymerase IV

    Energy Technology Data Exchange (ETDEWEB)

    Banerjee, Surajit; Brown, Kyle L.; Egli, Martin; Stone, Michael P. (Vanderbilt)

    2012-07-18

    Aflatoxin B{sub 1} (AFB{sub 1}) is oxidized to an epoxide in vivo, which forms an N7-dG DNA adduct (AFB{sub 1}-N7-dG). The AFB{sub 1}-N7-dG can rearrange to a formamidopyrimidine (AFB{sub 1}-FAPY) derivative. Both AFB{sub 1}-N7-dG and the {beta}-anomer of the AFB{sub 1}-FAPY adduct yield G {yields} T transversions in Escherichia coli, but the latter is more mutagenic. We show that the Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4) bypasses AFB{sub 1}-N7-dG in an error-free manner but conducts error-prone replication past the AFB{sub 1}-FAPY adduct, including misinsertion of dATP, consistent with the G {yields} T mutations observed in E. coli. Three ternary (Dpo4-DNA-dNTP) structures with AFB{sub 1}-N7-dG adducted template:primers have been solved. These demonstrate insertion of dCTP opposite the AFB{sub 1}-N7-dG adduct, and correct vs incorrect insertion of dATP vs dTTP opposite the 5'-template neighbor dT from a primed AFB{sub 1}-N7-dG:dC pair. The insertion of dTTP reveals hydrogen bonding between the template N3 imino proton and the O{sup 2} oxygen of dTTP, and between the template T O{sup 4} oxygen and the N3 imino proton of dTTP, perhaps explaining why this polymerase does not efficiently catalyze phosphodiester bond formation from this mispair. The AFB{sub 1}-N7-dG maintains the 5'-intercalation of the AFB{sub 1} moiety observed in DNA. The bond between N7-dG and C8 of the AFB{sub 1} moiety remains in plane with the alkylated guanine, creating a 16{sup o} inclination of the AFB{sub 1} moiety with respect to the guanine. A binary (Dpo4-DNA) structure with an AFB{sub 1}-FAPY adducted template:primer also maintains 5'-intercalation of the AFB{sub 1} moiety. The {beta}-deoxyribose anomer is observed. Rotation about the FAPY C5-N{sup 5} bond orients the bond between N{sup 5} and C8 of the AFB{sub 1} moiety out of plane in the 5'-direction, with respect to the FAPY base. The formamide group extends in the 3'-direction. This improves

  10. EXPRESSION, PURIFICATION, AND SMALL ANGLE X-RAY SCATTERING OF DNA REPLICATION AND REPAIR PROTEINS FROM THE HYPERTHERMOPHILE SULFOLOBUS SOLFATARICUS

    Energy Technology Data Exchange (ETDEWEB)

    Patterson, S.M.; Hatherill, J.R.; Hammel, M.; Hura, G.L.; Tainer, J.A.; Yannone, S.M.

    2008-01-01

    Vital molecular processes such as DNA replication, transcription, translation, and maintenance occur through transient protein interactions. Elucidating the mechanisms by which these protein complexes and interactions function could lead to treatments for diseases related to DNA damage and cell division control. In the recent decades since its introduction as a third domain, Archaea have shown to be simpler models for complicated eukaryotic processes such as DNA replication, repair, transcription, and translation. Sulfolobus solfataricus is one such model organism. A hyperthermophile with an optimal growth temperature of 80°C, Sulfolobus protein-protein complexes and transient protein interactions should be more stable at moderate temperatures, providing a means to isolate and study their structure and function. Here we provide the initial steps towards characterizing three DNA-related Sulfolobus proteins with small angle X-ray scattering (SAXS): Sso0257, a cell division control and origin recognition complex homolog, Sso0768, the small subunit of the replication factor C, and Sso3167, a Mut-T like protein. SAXS analysis was performed at multiple concentrations for both short and long exposure times. The Sso0257 sample was determined to be either a mixture of monomeric and dimeric states or a population of dynamic monomers in various conformational states in solution, consistent with a fl exible winged helix domain. Sso0768 was found to be a complex mixture of multimeric states in solution. Finally, molecular envelope reconstruction from SAXS data for Sso3167 revealed a novel structural component which may function as a disordered to ordered region in the presence of its substrates and/or protein partners.

  11. Characterization of a trehalose-degrading enzyme from the hyperthermophilic archaeon Sulfolobus acidocaldarius.

    Science.gov (United States)

    Moon, Jeong Hyun; Lee, Whiso; Park, Jihee; Choi, Kyoung-Hwa; Cha, Jaeho

    2016-07-01

    We purified a cytosolic trehalase (TreH) from a thermoacidophilic archaeon Sulfolobus acidocaldarius. Enzyme activity in cell-free extracts indicated that trehalose degradation in the cell occurred via the hydrolytic activity of TreH, and not via TreP (phosphorolytic activity) or TreT (transfer activity). TreH was purified to near-homogeneity by DEAE anion-exchange chromatography, followed by size exclusion and HiTrap Q anion-exchange chromatography, and its molecular mass was estimated as 40 kDa. Maximum activity was observed at 85°C and pH 4.5. The half-life of TreH was 53 and 41 min at 90°C and 95°C, respectively. TreH was highly specific for trehalose and was inhibited by glucose with a Ki of 0.05 mM. Compared with TreH from other trehalases, TreH from S. acidocaldarius is the most thermostable trehalase reported so far. Furthermore, this is the first trehalase characterized in the Archaea domain.

  12. Molecular characteristics of spontaneous deletions in the hyperthermophilic archaeon Sulfolobus acidocaldarius.

    Science.gov (United States)

    Grogan, Dennis W; Hansen, Josh E

    2003-02-01

    Prokaryotic genomes acquire and eliminate blocks of DNA sequence by lateral gene transfer and spontaneous deletion, respectively. The basic parameters of spontaneous deletion, which are expected to influence the course of genome evolution, have not been determined for any hyperthermophilic archaeon. We therefore screened a number of independent pyrimidine auxotrophs of Sulfolobus acidocaldarius for deletions and sequenced those detected. Deletions accounted for only 0.4% of spontaneous pyrE mutations, corresponding to a frequency of about 10(-8) per cell. Nucleotide sequence analysis of five independent deletions showed no significant association of the endpoints with short direct repeats, despite the fact that several such repeats occur within the pyrE gene and that duplication mutations in pyrE reverted at high frequencies. Endpoints of the spontaneous deletions did not coincide with short inverted repeats or potential stem-loop structures. No consensus sequence common to all the deletions could be identified, although two deletions showed the potential of being stabilized by octanucleotide sequences elsewhere in pyrE, and another pair of deletions shared an octanucleotide at their 3' ends. The unusually low frequency and low sequence dependence of spontaneous deletions in the S. acidocaldarius pyrE gene compared to other genetic systems could not be explained in terms of possible constraints imposed by the 5-fluoroorotate selection.

  13. The thermoacidophilic archaeon Sulfolobus acidocaldarius contains an unusually short, highly reduced dolichyl phosphate.

    Science.gov (United States)

    Guan, Ziqiang; Meyer, Benjamin H; Albers, Sonja-Verena; Eichler, Jerry

    2011-10-01

    Polyprenoids, polymers containing varied numbers of isoprene subunits, serve numerous roles in biology. In Eukarya, dolichyl phosphate, a phosphorylated polyprenol bearing a saturated α-end isoprene subunit, serves as the glycan carrier during N-glycosylation, namely that post-translational modification whereby glycans are covalently linked to select asparagine residues of a target protein. As in Eukarya, N-glycosylation in Archaea also relies on phosphorylated dolichol. In this report, LC-ESI/MS/MS was employed to identify a novel dolichyl phosphate (DolP) in the thermoacidophilic archaeon, Sulfolobus acidocaldarius. The unusually short S. acidocaldarius DolP presents a degree of saturation not previously reported. S. acidocaldarius DolP contains not only the saturated α- and ω-end isoprene subunits observed in other archaeal DolPs, but also up to five saturated intra-chain isoprene subunits. The corresponding dolichol and hexose-charged DolP species were also detected. The results of the present study offer valuable information on the biogenesis and potential properties of this unique DolP. Furthermore, elucidation of the mechanism of α-isoprene unit reduction in S. acidocaldarius dolichol may facilitate the identification of the alternative, as yet unknown polyprenol reductase in Eukarya.

  14. Growth phase-dependent gene regulation in vivo in Sulfolobus solfataricus

    NARCIS (Netherlands)

    DeYoung, M.; Oost, van der J.

    2011-01-01

    Ribosomal genes are strongly regulated dependent on growth phase in all organisms, but this regulation is poorly understood in Archaea. Moreover, very little is known about growth phase-dependent gene regulation in Archaea. SSV1-based lacS reporter gene constructs containing the Sulfolobus 16S/23S r

  15. Mutational analysis of divalent metal ion binding in the active site of class II α-mannosidase from sulfolobus solfataricus

    DEFF Research Database (Denmark)

    Hansen, Dennis K.; Webb, Helen; Nielsen, Jonas Willum;

    2015-01-01

    Mutational analysis of Sulfolobus solfataricus class II α-mannosidase was focused on side chains that interact with the hydroxyls of the-1 mannosyl of the substrate (Asp-534) or form ligands to the active site divalent metal ion (His-228 and His-533) judged from crystal structures of homologous...... enzymes. D534A and D534N appeared to be completely inactive. When compared to the wild-type enzyme, the mutant enzymes in general showed only small changes in KM for the substrate, p-nitrophenyl-α-mannoside, but elevated activation constants, KA, for the divalent metal ion (Co2+, Zn2+, Mn2+, or Cd2......+). Some mutant enzyme forms displayed an altered preference for the metal ion compared to that of the wild type-enzyme. Furthermore, the H228Q, H533E, and H533Q enzymes were inhibited at increasing Zn2+ concentrations. The catalytic rate was reduced for all enzymes compared to that of the wild-type enzyme...

  16. Analysis of ATPases of putative secretion operons in the thermoacidophilic archaeon Sulfolobus solfataricus

    NARCIS (Netherlands)

    Albers, SV; Driessen, AJM

    2005-01-01

    Gram-negative bacteria use a wide variety of complex mechanisms to secrete proteins across their membranes or to assemble secreted proteins into surface structures. As most archaea only possess a cytoplasmic membrane surrounded by a membrane-anchored S-layer, the organization of such complexes might

  17. Production of Recombinant and Tagged Proteins in the Hyperthermophilic Archaeon Sulfolobus solfataricus

    NARCIS (Netherlands)

    Albers, S.-V.; Jonuscheit, M.; Dinkelaker, S.; Urich, T.; Kletzin, A.; Tampé, R.; Driessen, A.J.M.; Schleper, C.

    2006-01-01

    Many systems are available for the production of recombinant proteins in bacterial and eukaryotic model organisms, which allow us to study proteins in their native hosts and to identify protein-protein interaction partners. In contrast, only a few transformation systems have been developed for archa

  18. Structural and kinetic studies of the allosteric transition in Sulfolobus solfataricus uracil phosphoribosyltransferase: Permanent activation by engineering of the C-terminus

    DEFF Research Database (Denmark)

    Christoffersen, Stig; Kadziola, Anders; Johansson, Eva

    2009-01-01

    Uracil phosphoribosyltransferase catalyzes the conversion of 5-phosphoribosyl- a-1-diphosphate (PRPP) and uracil to uridine monophosphate (UMP) and diphosphate (PPi). The tetrameric enzyme from Sulfolobus solfataricus has a unique type of allosteric regulation by cytidine triphosphate (CTP......) and guanosine triphosphate (GTP). Here we report two structures of the activated state in complex with GTP. One structure (refined at 2.8-Å resolution) contains PRPP in all active sites, while the other structure (refined at 2.9-Å resolution) has PRPP in two sites and the hydrolysis products, ribose-5-phosphate...

  19. Crystal structure of T state aspartate carbamoyltransferase of the hyperthermophilic archaeon Sulfolobus acidocaldarius.

    Science.gov (United States)

    De Vos, Dirk; Van Petegem, Filip; Remaut, Han; Legrain, Christianne; Glansdorff, Nicolas; Van Beeumen, Jozef J

    2004-06-11

    Aspartate carbamoyltransferase (ATCase) is a model enzyme for understanding allosteric effects. The dodecameric complex exists in two main states (T and R) that differ substantially in their quaternary structure and their affinity for various ligands. Many hypotheses have resulted from the structure of the Escherichia coli ATCase, but so far other crystal structures to test these have been lacking. Here, we present the tertiary and quaternary structure of the T state ATCase of the hyperthermophilic archaeon Sulfolobus acidocaldarius (SaATC(T)), determined by X-ray crystallography to 2.6A resolution. The quaternary structure differs from the E.coli ATCase, by having altered interfaces between the catalytic (C) and regulatory (R) subunits, and the presence of a novel C1-R2 type interface. Conformational differences in the 240 s loop region of the C chain and the C-terminal region of the R chain affect intersubunit and interdomain interfaces implicated previously in the allosteric behavior of E.coli ATCase. The allosteric-zinc binding domain interface is strengthened at the expense of a weakened R1-C4 type interface. The increased hydrophobicity of the C1-R1 type interface may stabilize the quaternary structure. Catalytic trimers of the S.acidocaldarius ATCase are unstable due to a drastic weakening of the C1-C2 interface. The hyperthermophilic ATCase presents an interesting example of how an allosteric enzyme can adapt to higher temperatures. The structural rearrangement of this thermophilic ATCase may well promote its thermal stability at the expense of changes in the allosteric behavior.

  20. Calcium-induced aggregation of archaeal bipolar tetraether liposomes derived from the thermoacidophilic archaeon Sulfolobus acidocaldarius

    Directory of Open Access Journals (Sweden)

    Roby Kanichay

    2003-01-01

    Full Text Available Previously, we showed that the proton permeability of small unilamellar vesicles (SUVs composed of polar lipid fraction E (PLFE from the thermoacidophilic archaeon Sulfolobus acidocaldarius was remarkably low and insensitive to temperature (Komatsu and Chong 1998. In this study, we used photon correlation spectroscopy to investigate the time dependence of PLFE SUV size as a function of Ca2+ concentration. In the absence of Ca2+, vesicle diameter changed little over 6 months. Addition of Ca2+, however, immediately induced formation of vesicle aggregates with an irregular shape, as revealed by confocal fluorescence microscopy. Aggregation was reversible upon addition of EDTA; however, the reversibility varied with temperature as well as incubation time with Ca2+. Freeze-fracture electron microscopy showed that, after a long period of incubation (2 weeks with Ca2+, the PLFE vesicles had not just aggregated, but had fused or coalesced. The initial rate of vesicle aggregation varied sigmoidally with Ca2+ concentration. At pH 6.6, the threshold calcium concentration (Cr for vesicle aggregation at 25 and 40 °C was 11 and 17 mM, respectively. At pH 3.0, the Cr at 25 °C increased to 25 mM. The temperature dependence of Cr may be attributable to changes in membrane surface potential, which was –22.0 and –13.2 mV at 25 and 40 °C, respectively, at pH 6.6, as determined by 2-(p-toluidinylnaphthalene-6-sulfonic acid fluorescence. The variation in surface potential with temperature is discussed in terms of changes in lipid conformation and membrane organization.

  1. Isolation of Extracellular Polymeric Substances from Biofilms of the Thermoacidophilic Archaeon Sulfolobus acidocaldarius.

    Science.gov (United States)

    Jachlewski, Silke; Jachlewski, Witold D; Linne, Uwe; Bräsen, Christopher; Wingender, Jost; Siebers, Bettina

    2015-01-01

    Extracellular polymeric substances (EPS) are the major structural and functional components of microbial biofilms. The aim of this study was to establish a method for EPS isolation from biofilms of the thermoacidophilic archaeon, Sulfolobus acidocaldarius, as a basis for EPS analysis. Biofilms of S. acidocaldarius were cultivated on the surface of gellan gum-solidified Brock medium at 78°C for 4 days. Five EPS extraction methods were compared, including shaking of biofilm suspensions in phosphate buffer, cation-exchange resin (CER) extraction, and stirring with addition of EDTA, crown ether, or NaOH. With respect to EPS yield, impact on cell viability, and compatibility with subsequent biochemical analysis, the CER extraction method was found to be the best suited isolation procedure resulting in the detection of carbohydrates and proteins as the major constituents and DNA as a minor component of the EPS. Culturability of CER-treated cells was not impaired. Analysis of the extracellular proteome using two-dimensional gel electrophoresis resulted in the detection of several hundreds of protein spots, mainly with molecular masses of 25-116 kDa and pI values of 5-8. Identification of proteins suggested a cytoplasmic origin for many of these proteins, possibly released via membrane vesicles or biofilm-inherent cell lysis during biofilm maturation. Functional analysis of EPS proteins, using fluorogenic substrates as well as zymography, demonstrated the activity of diverse enzyme classes, such as proteases, lipases, esterases, phosphatases, and glucosidases. In conclusion, the CER extraction method, as previously applied to bacterial biofilms, also represents a suitable method for isolation of water soluble EPS from the archaeal biofilms of S. acidocaldarius, allowing the investigation of composition and function of EPS components in these types of biofilms.

  2. Calcium-induced aggregation of archaeal bipolar tetraether liposomes derived from the thermoacidophilic archaeon Sulfolobus acidocaldarius.

    Science.gov (United States)

    Kanichay, Roby; Boni, Lawrence T; Cooke, Peter H; Khan, Tapan K; Chong, Parkson Lee-Gau

    2003-10-01

    Previously, we showed that the proton permeability of small unilamellar vesicles (SUVs) composed of polar lipid fraction E (PLFE) from the thermoacidophilic archaeon Sulfolobus acidocaldarius was remarkably low and insensitive to temperature (Komatsu and Chong 1998). In this study, we used photon correlation spectroscopy to investigate the time dependence of PLFE SUV size as a function of Ca2+ concentration. In the absence of Ca2+, vesicle diameter changed little over 6 months. Addition of Ca2+, however, immediately induced formation of vesicle aggregates with an irregular shape, as revealed by confocal fluorescence microscopy. Aggregation was reversible upon addition of EDTA; however, the reversibility varied with temperature as well as incubation time with Ca2+. Freeze-fracture electron microscopy showed that, after a long period of incubation (2 weeks) with Ca2+, the PLFE vesicles had not just aggregated, but had fused or coalesced. The initial rate of vesicle aggregation varied sigmoidally with Ca2+ concentration. At pH 6.6, the threshold calcium concentration (Cr) for vesicle aggregation at 25 and 40 degrees C was 11 and 17 mM, respectively. At pH 3.0, the Cr at 25 degrees C increased to 25 mM. The temperature dependence of Cr may be attributable to changes in membrane surface potential, which was -22.0 and -13.2 mV at 25 and 40 degrees C, respectively, at pH 6.6, as determined by 2-(p-toluidinyl)naphthalene-6-sulfonic acid fluorescence. The variation in surface potential with temperature is discussed in terms of changes in lipid conformation and membrane organization.

  3. Isolation of extracellular polymeric substances from biofilms of the thermoacidophilic archaeon Sulfolobus acidocaldarius

    Directory of Open Access Journals (Sweden)

    Silke eJachlewski

    2015-08-01

    Full Text Available Extracellular polymeric substances (EPS are the major structural and functional components of microbial biofilms. The aim of this study was to establish a method for EPS isolation from biofilms of the thermoacidophilic archaeon Sulfolobus acidocaldarius as a basis for EPS analysis. Biofilms of S. acidocaldarius were cultivated on the surface of gellan gum-solidified Brock medium at 78 °C for 4 days. Five EPS extraction methods were compared, including shaking of biofilm suspensions in phosphate buffer, cation-exchange resin (CER extraction and stirring with addition of EDTA, crown ether or NaOH. With respect to EPS yield, impact on cell viability and compatibility with subsequent biochemical analysis, the CER extraction method was found to be the best suited isolation procedure resulting in the detection of carbohydrates and proteins as the major constituents and DNA as a minor component of the EPS. Culturability of CER-treated cells was not impaired. Analysis of the extracellular proteome using two-dimensional gel electrophoresis resulted in the detection of several hundredshundred of protein spots, mainly with molecular masses of 25 kDa to 116 kDa and pI values of 5 to 8. Identification of proteins suggested a cytoplasmic origin for many of these proteins, possibly released via membrane vesicles or biofilm-inherent cell lysis during biofilm maturation. Functional analysis of EPS proteins, using fluorogenic substrates as well as zymography, demonstrated the activity of diverse groups of enzymes such as proteases, lipases, esterases, phosphatases and glucosidases. In conclusion, the CER extraction method, as previously applied to bacterial biofilms, also represents a suitable method for isolation of water soluble EPS from the archaeal biofilms of S. acidocaldarius, allowing the investigation of composition and function of EPS components in these types of biofilms.

  4. Involvement of a eukaryotic-like ubiquitin-related modifier in the proteasome pathway of the archaeon Sulfolobus acidocaldarius

    Science.gov (United States)

    Anjum, Rana S.; Bray, Sian M.; Blackwood, John K.; Kilkenny, Mairi L.; Coelho, Matthew A.; Foster, Benjamin M.; Li, Shurong; Howard, Julie A.; Pellegrini, Luca; Albers, Sonja-Verena; Deery, Michael J.; Robinson, Nicholas P.

    2015-09-01

    In eukaryotes, the covalent attachment of ubiquitin chains directs substrates to the proteasome for degradation. Recently, ubiquitin-like modifications have also been described in the archaeal domain of life. It has subsequently been hypothesized that ubiquitin-like proteasomal degradation might also operate in these microbes, since all archaeal species utilize homologues of the eukaryotic proteasome. Here we perform a structural and biochemical analysis of a ubiquitin-like modification pathway in the archaeon Sulfolobus acidocaldarius. We reveal that this modifier is homologous to the eukaryotic ubiquitin-related modifier Urm1, considered to be a close evolutionary relative of the progenitor of all ubiquitin-like proteins. Furthermore we demonstrate that urmylated substrates are recognized and processed by the archaeal proteasome, by virtue of a direct interaction with the modifier. Thus, the regulation of protein stability by Urm1 and the proteasome in archaea is likely representative of an ancient pathway from which eukaryotic ubiquitin-mediated proteolysis has evolved.

  5. SMV1 virus-induced CRISPR spacer acquisition from the conjugative plasmid pMGB1 in Sulfolobus solfataricus P2

    DEFF Research Database (Denmark)

    Erdmann, Susanne; Shah, Shiraz Ali; Garrett, Roger Antony

    2013-01-01

    Organisms of the crenarchaeal order Sulfolobales carry complex CRISPR (clustered regularly interspaced short palindromic repeats) adaptive immune systems. These systems are modular and show extensive structural and functional diversity, especially in their interference complexes. The primary...... targets are an exceptional range of diverse viruses, many of which propagate stably within cells and follow lytic life cycles without producing cell lysis. These properties are consistent with the difficulty of activating CRISPR spacer uptake in the laboratory, but appear to conflict with the high...... complexity and diversity of the CRISPR immune systems that are found among the Sulfolobales. In the present article, we re-examine the first successful induction of archaeal spacer acquisition in our laboratory that occurred exclusively for the conjugative plasmid pMGB1 in Sulfolobus solfataricus P2...

  6. Evidence that the xylanase activity from Sulfolobus solfataricus Oalpha is encoded by the endoglucanase precursor gene (sso1354) and characterization of the associated cellulase activity.

    Science.gov (United States)

    Maurelli, Luisa; Giovane, Alfonso; Esposito, Alessandra; Moracci, Marco; Fiume, Immacolata; Rossi, Mosè; Morana, Alessandra

    2008-09-01

    Sulfolobus solfataricus strain Oalpha was previously isolated for its ability to grow on minimal medium supplemented with xylan as a carbon source. The strain exhibited thermostable xylanase activity but several attempts to identify the gene encoding for the activity failed. Further studies showed that the xylanase displayed activity on carboxymethylcellulose (CMC) and the new activity was characterized. It exhibited an optimal temperature and pH of 95 degrees C and 3.5, respectively, and a half-life of 53 min at 95 degrees C. The enzyme, which was demonstrated to be glycosylated, hydrolyzed CMC in an endo-manner releasing cellobiose and other cello-oligomers. Analysis of the tryptic fragments by tandem mass spectrometry led to identification of the endoglucanase precursor, encoded by the sso1354 gene, as the protein possessing dual activity. The efficiency of the SSO1354 protein in degrading cellulosic and hemicellulosic fractions contained in agronomic residues was tested at low pH and high temperature. Cellulose and xylan were degraded to glucose and xylose at 90 degrees C, pH 4 by an enzyme mix consisting of SSO1354 and additional glycosyl hydrolases from S. solfataricus Oalpha. Given its role in saccharification processes requiring high temperatures and acidic environments, SSO1354 represents an interesting candidate for the utilization of agro-industrial waste for fuel production.

  7. The structure and evolution of the ribosomal proteins encoded in the spc operon of the archaeon (Crenarchaeota) Sulfolobus acidocaldarius.

    Science.gov (United States)

    Yang, D; Kusser, I; Köpke, A K; Koop, B F; Matheson, A T

    1999-07-01

    The genes for nine ribosomal proteins, L24, L5, S14, S8, L6, L18, S5, L30, and L15, have been isolated and sequenced from the spc operon in the archaeon (Crenarchaeota) Sulfolobus acidocaldarius, and the putative amino acid sequence of the proteins coded by these genes has been determined. In addition, three other genes in the spc operon, coding for ribosomal proteins S4E, L32E, and L19E (equivalent to rat ribosomal proteins S4, L32, and L19), were sequenced and the structure of the putative proteins was determined. The order of the ribosomal protein genes in the spc operon of the Crenarchaeota kingdom of Archaea is identical to that present in the Euryarchaeota kingdom of Archaea and also identical to that found in bacteria, except for the genes for r-proteins S4E, L32E, and L19E, which are absent in bacteria. Although AUG is the initiation codon in most of the spc genes, GUG (val) and UUG (leu) are also used as initiation codons in S. acidocaldarius. Over 70% of the codons in the Sulfolobus spc operon have A or U in the third position, reflecting the low GC content of Sulfolobus DNA. Phylogenetic analysis indicated that the archaeal r-proteins are a sister group of their eucaryotic counterparts but did not resolve the question of whether the Archaea is monophyletic, as suggested by the L6P, L15P, and L18P trees, or the question of whether the Crenarchaeota is separate from the Euryarchaeota and closer to the Eucarya, as suggested by the S8P, S5P, and L24P trees. In the case of the three Sulfolobus r-proteins that do not have a counterpart in the bacterial ribosome (S4E, L32E, and L19E), the archaeal r-proteins showed substantial identity to their eucaryotic equivalents, but in all cases the archaeal proteins formed a separate group from the eucaryotic proteins.

  8. Biological effects of DNA damage in the hyperthermophilic archaeon Sulfolobus acidocaldarius.

    Science.gov (United States)

    Reilly, Michelle S; Grogan, Dennis W

    2002-02-19

    To investigate the generality of efficient double-strand break repair and damage-induced mutagenesis in hyperthermophilic archaea, we systematically measured the effects of five DNA-damaging agents on Sulfolobus acidocaldarius and compared the results to those obtained for Escherichia coli under corresponding conditions. The observed lethality of gamma-radiation was very similar for S. acidocaldarius and E. coli, arguing against unusually efficient double-strand break repair in S. acidocaldarius. In addition, DNA-strand-breaking agents (gamma-radiation or bleomycin), as well as DNA-cross-linking agents (mechlorethamine, butadiene diepoxide or cisplatin) stimulated forward mutation, reverse mutation, and formation of recombinants via conjugation in Sulfolobus cells. Although two of the five DNA-damaging agents failed to revert the E. coli auxotrophs under these conditions, all five reverted S. acidocaldarius auxotrophs.

  9. Small multicopy, non-integrative shuttle vectors based on the plasmid pRN1 for Sulfolobus acidocaldarius and Sulfolobus solfataricus, model organisms of the (cren-)archaea

    NARCIS (Netherlands)

    Berkner, Silvia; Grogan, Dennis; Albers, Sonja-Verena; Lipps, Georg

    2007-01-01

    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 Sulfolo

  10. Lesion-Induced Mutation in the Hyperthermophilic Archaeon Sulfolobus acidocaldarius and Its Avoidance by the Y-Family DNA Polymerase Dbh.

    Science.gov (United States)

    Sakofsky, Cynthia J; Grogan, Dennis W

    2015-10-01

    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.

  11. SMV1 virus-induced CRISPR spacer acquisition from the conjugative plasmid pMGB1 in Sulfolobus solfataricus P2.

    Science.gov (United States)

    Erdmann, Susanne; Shah, Shiraz A; Garrett, Roger A

    2013-12-01

    Organisms of the crenarchaeal order Sulfolobales carry complex CRISPR (clustered regularly interspaced short palindromic repeats) adaptive immune systems. These systems are modular and show extensive structural and functional diversity, especially in their interference complexes. The primary targets are an exceptional range of diverse viruses, many of which propagate stably within cells and follow lytic life cycles without producing cell lysis. These properties are consistent with the difficulty of activating CRISPR spacer uptake in the laboratory, but appear to conflict with the high complexity and diversity of the CRISPR immune systems that are found among the Sulfolobales. In the present article, we re-examine the first successful induction of archaeal spacer acquisition in our laboratory that occurred exclusively for the conjugative plasmid pMGB1 in Sulfolobus solfataricus P2 that was co-infected with the virus SMV1 (Sulfolobus monocaudavirus 1). Although we reaffirm that protospacer selection is essentially a random process with respect to the pMGB1 genome, we identified single spacer sequences specific for each of CRISPR loci C, D and E that, exceptionally, occurred in many sequenced clones. Moreover, the same sequence was reproducibly acquired for a given locus in independent experiments, consistent with it being the first protospacer to be selected. There was also a small protospacer bias (1.6:1) to the antisense strand of protein genes. In addition, new experiments demonstrated that spacer acquisition in the previously inactive CRISPR locus A could be induced on freeze-thawing of the infected cells, suggesting that environmental stress can facilitate activation. Coincidentally with spacer acquisition, a mobile OrfB element was deleted from pMGB1, suggesting that interplay can occur between spacer acquisition and transposition.

  12. N-glycosylation in the thermoacidophilic archaeon Sulfolobus acidocaldarius involves a short dolichol pyrophosphate carrier.

    Science.gov (United States)

    Guan, Ziqiang; Delago, Antonia; Nußbaum, Phillip; Meyer, Benjamin; Albers, Sonja-Verena; Eichler, Jerry

    2016-09-01

    N-glycosylation is a post-translational modification that occurs across evolution. In the thermoacidophilic archaea Sulfolobus acidocaldarius, glycoproteins are modified by an N-linked tribranched hexasaccharide reminiscent of the N-glycans assembled in Eukarya. Previously, hexose-bearing dolichol phosphate was detected in a S. acidocaldarius Bligh-Dyer lipid extract. Here, we used a specialized protocol for extracting lipid-linked oligosaccharides to detect a dolichol pyrophosphate bearing the intact hexasaccharide, as well as its biosynthetic intermediates. Furthermore, evidence for N-glycosylation of two S. acidocaldarius proteins by the same hexasaccharide and its derivatives was collected. These findings thus provide novel insight into archaeal N-glycosylation.

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

    DEFF Research Database (Denmark)

    Stedman, Kenneth M; She, Qunxin; Phan, Hien

    2003-01-01

    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...... 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...

  14. Nucleic acid binding properties of a helix stabilising nucleoid protein from the thermoacidophilic archaeon Sulfolobus acidocaldarius that condenses DNA into compact structures.

    Science.gov (United States)

    Celestina, F; Suryanarayana, T

    1995-12-01

    Helix stabilising nucleoid protein (HSNP-C') from an acidothermophilic archaeon Sulfolobus acidocaldarius has been characterised with respect to interaction with nucleic acids by gel retardation assay, binding to nucleic acid columns, fluorescence titrations and electron microscopy. The protein exists in solution as very large multimeric aggregates as indicated by cross-linking studies. The protein binds strongly and co-operatively to double stranded DNA. Electron microscopy of the complexes of the protein with DNA shows compact structures suggesting that the protein condenses DNA.

  15. Homologous recombination in the archaeon Sulfolobus acidocaldarius: effects of DNA substrates and mechanistic implications.

    Science.gov (United States)

    Rockwood, Jananie; Mao, Dominic; Grogan, Dennis W

    2013-09-01

    Although homologous recombination (HR) is known to influence the structure, stability, and evolution of microbial genomes, few of its functional properties have been measured in cells of hyperthermophilic archaea. The present study manipulated various properties of the parental DNAs in high-resolution assays of Sulfolobus acidocaldarius transformation, and measured the impact on the efficiency and pattern of marker transfer to the recipient chromosome. The relative orientation of homologous sequences, the type and position of chromosomal mutation being replaced, and the length of DNA flanking the marked region all affected the efficiency, linkage, tract continuity, and other parameters of marker transfer. Effects predicted specifically by the classical reciprocal-exchange model of HR were not observed. One analysis observed only 90 % linkage between markers defined by adjacent bases; in another series of experiments, sequence divergence up to 4 % had no detectable impact on overall efficiency of HR or on the co-transfer of a distal non-selected marker. The effects of introducing DNA via conjugation, rather than transformation, were more difficult to assess, but appeared to increase co-transfer (i.e. linkage) of relatively distant non-selected markers. The results indicate that HR events between gene-sized duplex DNAs and the S. acidocaldarius chromosome typically involve neither crossing over nor interference from a mismatch-activated anti-recombination system. Instead, the donor DNA may anneal to a transient chromosomal gap, as in the mechanism proposed for oligonucleotide-mediated transformation of Sulfolobus and other micro-organisms.

  16. Pressure and temperature as tools for investigating the role of individual non-covalent interactions in enzymatic reactions Sulfolobus solfataricus carboxypeptidase as a model enzyme.

    Science.gov (United States)

    Occhipinti, Emanuela; Bec, Nicole; Gambirasio, Benedetta; Baietta, Gabriella; Martelli, Pier Luigi; Casadio, Rita; Balny, Claude; Lange, Reinhard; Tortora, Paolo

    2006-03-01

    Sulfolobus solfataricus carboxypeptidase, (CPSso), is a heat- and pressure-resistant zinc-metalloprotease. Thanks to its properties, it is an ideal tool for investigating the role of non-covalent interactions in substrate binding. It has a broad substrate specificity as it can cleave any N-blocked amino acid (except for N-blocked proline). Its catalytic and kinetic mechanisms are well understood, and the hydrolytic reaction is easily detectable spectrophotometrically. Here, we report investigations on the pressure- and temperature-dependence of the kinetic parameters (turnover number and Michaelis constant) of CPSso using several benzoyl- and 3-(2-furyl)acryloyl-amino acids as substrates. This approach enabled us to study these parameters in terms of individual rate constants and establish that the release of the free amino acid is the rate-limiting step, making it possible to dissect the individual non-covalent interactions participating in substrate binding. In keeping with molecular docking experiments performed on the 3D model of CPSso available to date, our results show that both hydrophobic and energetic interactions (i.e., stacking and van der Waals) are mainly involved, but their contribution varies strongly, probably due to changes in the conformational state of the enzyme.

  17. Heteroduplex formation, mismatch resolution, and genetic sectoring during homologous recombination in the hyperthermophilic archaeon Sulfolobus acidocaldarius

    Directory of Open Access Journals (Sweden)

    Dennis W. Grogan

    2012-06-01

    Full Text Available Hyperthermophilic archaea exhibit certain molecular-genetic features not seen in bacteria or eukaryotes, and their systems of homologous recombination (HR remain largely unexplored in vivo. We transformed a Sulfolobus acidocaldarius pyrE mutant with short DNAs that contained multiple non-selected genetic markers within the pyrE gene. From 20 to 40% of the resulting colonies were found to contain two Pyr+ clones with distinct sets of the non-selected markers. The dual-genotype colonies could not be attributed to multiple DNAs entering the cells or conjugation between transformed and non-transformed cells. These colonies thus appear to represent genetic sectoring in which stretches of heteroduplex DNA formed during HR and segregated without complete resolution of inter-strand differences. Surprisingly, sectoring was also frequent in transformation with single-stranded DNAs. Oligonucleotides, for example, produced somewhat more sectored transformants when electroporated as single strands than as a duplex, although all forms (positive-strand, negative-strand, and duplex produced a diversity of genotypes from the limited number of markers. The marker patterns in the recombinants indicate that S. acidocaldarius resolves individual mismatches through un-coordinated short-patch excision followed by re-filling of the resulting gap. These gene-conversion events exhibit little strand bias, and can occur in pre-formed heteroduplex. These properties suggest that this process does not play a central role in the fidelity of genome replication, but may generate 3’ single-strand tails, and thereby initiate the incorporation of duplex DNA into the recipient chromosome. Regardless of the molecular details of its mechanism, HR between the S. acidocaldarius chromosome and a multiply-marked DNA produces a strikingly high level of genetic diversity in a very short chromosomal interval, and suggests that HR in Sulfolobus has significant mutagenic potential if not

  18. Heteroduplex formation, mismatch resolution, and genetic sectoring during homologous recombination in the hyperthermophilic archaeon sulfolobus acidocaldarius.

    Science.gov (United States)

    Mao, Dominic; Grogan, Dennis W

    2012-01-01

    Hyperthermophilic archaea exhibit certain molecular-genetic features not seen in bacteria or eukaryotes, and their systems of homologous recombination (HR) remain largely unexplored in vivo. We transformed a Sulfolobus acidocaldariuspyrE mutant with short DNAs that contained multiple non-selected genetic markers within the pyrE gene. From 20 to 40% of the resulting colonies were found to contain two Pyr(+) clones with distinct sets of the non-selected markers. The dual-genotype colonies could not be attributed to multiple DNAs entering the cells, or to conjugation between transformed and non-transformed cells. These colonies thus appear to represent genetic sectoring in which regions of heteroduplex DNA formed and then segregated after partial resolution of inter-strand differences. Surprisingly, sectoring was also frequent in cells transformed with single-stranded DNAs. Oligonucleotides produced more sectored transformants when electroporated as single strands than as a duplex, although all forms of donor DNA (positive-strand, negative-strand, and duplex) produced a diversity of genotypes, despite the limited number of markers. The marker patterns in the recombinants indicate that S. acidocaldarius resolves individual mismatches through un-coordinated short-patch excision followed by re-filling of the resulting gap. The conversion events that occur during transformation by single-stranded DNA do not show the strand bias necessary for a system that corrects replication errors effectively; similar events also occur in pre-formed heteroduplex electroporated into the cells. Although numerous mechanistic details remain obscure, the results demonstrate that the HR system of S. acidocaldarius can generate remarkable genetic diversity from short intervals of moderately diverged DNAs.

  19. The catalytic mechanism of indole-3-glycerol phosphate synthase: crystal structures of complexes of the enzyme from Sulfolobus solfataricus with substrate analogue, substrate, and product.

    Science.gov (United States)

    Hennig, Michael; Darimont, B D; Jansonius, J N; Kirschner, K

    2002-06-07

    Indoleglycerol phosphate synthase catalyzes the ring closure of an N-alkylated anthranilate to a 3-alkyl indole derivative, a reaction requiring Lewis acid catalysis in vitro. Here, we investigated the enzymatic reaction mechanism through X-ray crystallography of complexes of the hyperthermostable enzyme from Sulfolobus solfataricus with the substrate 1-(o-carboxyphenylamino) 1-deoxyribulose 5-phosphate, a substrate analogue and the product indole-3-glycerol phosphate. The substrate and the substrate analogue are bound to the active site in a similar, extended conformation between the previously identified phosphate binding site and a hydrophobic pocket for the anthranilate moiety. This binding mode is unproductive, because the carbon atoms that are to be joined are too far apart. The indole ring of the bound product resides in a second hydrophobic pocket adjacent to that of the anthranilate moiety of the substrate. Although the hydrophobic moiety of the substrate moves during catalysis from one hydrophobic pocket to the other, the triosephosphate moiety remains rigidly bound to the same set of hydrogen-bonding residues. Simultaneously, the catalytically important residues Lys53, Lys110 and Glu159 maintain favourable distances to the atoms of the ligand undergoing covalent changes. On the basis of these data, the structures of two putative catalytic intermediates were modelled into the active site. This new structural information and the modelling studies provide further insight into the mechanism of enzyme-catalyzed indole synthesis. The charged epsilon-amino group of Lys110 is the general acid, and the carboxylate group of Glu159 is the general base. Lys53 guides the substrate undergoing conformational transitions during catalysis, by forming a salt-bridge to the carboxylate group of its anthranilate moiety.

  20. The Arginine Pairs and C-Termini of the Sso7c4 from Sulfolobus solfataricus Participate in Binding and Bending DNA

    Science.gov (United States)

    Huang, Chun-Hsiang; Ko, Tzu-Ping; Chiang, Cheng-Hung; Lin, Kuan-Fu; Chang, Yuan-Chih; Lin, Po-Yen; Tsai, Hui-Hsu Gavin; Wang, Andrew H.-J.

    2017-01-01

    The Sso7c4 from Sulfolobus solfataricus forms a dimer, which is believed to function as a chromosomal protein involved in genomic DNA compaction and gene regulation. Here, we present the crystal structure of wild-type Sso7c4 at a high resolution of 1.63 Å, showing that the two basic C-termini are disordered. Based on the fluorescence polarization (FP) binding assay, two arginine pairs, R11/R22′ and R11′/R22, on the top surface participate in binding DNA. As shown in electron microscopy (EM) images, wild-type Sso7c4 compacts DNA through bridging and bending interactions, whereas the binding of C-terminally truncated proteins rigidifies and opens DNA molecules, and no compaction of the DNA occurs. Moreover, the FP, EM and fluorescence resonance energy transfer (FRET) data indicated that the two basic and flexible C-terminal arms of the Sso7c4 dimer play a crucial role in binding and bending DNA. Sso7c4 has been classified as a repressor-like protein because of its similarity to Escherichia coli Ecrep 6.8 and Ecrep 7.3 as well as Agrobacterium tumefaciens ACCR in amino acid sequence. Based on these data, we proposed a model of the Sso7c4-DNA complex using a curved DNA molecule in the catabolite activator protein-DNA complex. The DNA end-to-end distance measured with FRET upon wild-type Sso7c4 binding is almost equal to the distance measured in the model, which supports the fidelity of the proposed model. The FRET data also confirm the EM observation showing that the binding of wild-type Sso7c4 reduces the DNA length while the C-terminal truncation does not. A functional role for Sso7c4 in the organization of chromosomal DNA and/or the regulation of gene expression through bridging and bending interactions is suggested. PMID:28068385

  1. Structural and Functional Analysis of Sulfolobus solfataricus Y-Family DNA Polymerase Dpo4-Catalyzed Bypass of the Malondialdehyde−Deoxyguanosine Adduct

    Energy Technology Data Exchange (ETDEWEB)

    Eoff, Robert L.; Stafford, Jennifer B.; Szekely, Jozsef; Rizzo, Carmelo J.; Egli, Martin; Guengerich, F. Peter; Marnett, Lawrence J.; (Vanderbilt)

    2010-01-12

    Oxidative stress can induce the formation of reactive electrophiles, such as DNA peroxidation products, e.g., base propenals, and lipid peroxidation products, e.g., malondialdehyde. Base propenals and malondialdehyde react with DNA to form adducts, including 3-(2'-deoxy-{beta}-d-erythro-pentofuranosyl)pyrimido[1,2-{alpha}]purin-10(3H)-one (M{sub 1}dG). When paired opposite cytosine in duplex DNA at physiological pH, M{sub 1}dG undergoes ring opening to form N{sup 2}-(3-oxo-1-propenyl)-dG (N{sup 2}-OPdG). Previous work has shown that M{sub 1}dG is mutagenic in bacteria and mammalian cells and that its mutagenicity in Escherichia coli is dependent on induction of the SOS response, indicating a role for translesion DNA polymerases in the bypass of M{sub 1}dG. To probe the mechanism by which translesion polymerases bypass M{sub 1}dG, kinetic and structural studies were conducted with a model Y-family DNA polymerase, Dpo4 from Sulfolobus solfataricus. The level of steady-state incorporation of dNTPs opposite M{sub 1}dG was reduced 260-2900-fold and exhibited a preference for dATP incorporation. Liquid chromatography-tandem mass spectrometry analysis of the full-length extension products revealed a spectrum of products arising principally by incorporation of dC or dA opposite M{sub 1}dG followed by partial or full-length extension. A greater proportion of -1 deletions were observed when dT was positioned 5' of M{sub 1}dG. Two crystal structures were determined, including a 'type II' frameshift deletion complex and another complex with Dpo4 bound to a dC-M{sub 1}dG pair located in the postinsertion context. Importantly, M{sub 1}dG was in the ring-closed state in both structures, and in the structure with dC opposite M{sub 1}dG, the dC residue moved out of the Dpo4 active site, into the minor groove. The results are consistent with the reported mutagenicity of M{sub 1}dG and illustrate how the lesion may affect replication events.

  2. Insertion of dNTPs Opposite the 1,N[superscript 2]-Propanodeoxyguanosine Adduct by Sulfolobus solfataricus P2 DNA Polymerase IV

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Yazhen; Musser, Sarah K.; Saleh, Sam; Marnett, Lawrence J.; Egli, Martin; Stone, Michael P. (Vanderbilt)

    2008-08-04

    1,N{sup 2}-Propanodeoxyguanosine (PdG) is a stable structural analogue for the 3-(2'-deoxy-{beta}-d-erythro-pentofuranosyl)pyrimido[1,2-?]purin-10(3H)-one (M{sub 1}dG) adduct derived from exposure of DNA to base propenals and to malondialdehyde. The structures of ternary polymerase-DNA-dNTP complexes for three template-primer DNA sequences were determined, with the Y-family Sulfolobus solfataricus DNA polymerase IV (Dpo4), at resolutions between 2.4 and 2.7 {angstrom}. Three template 18-mer-primer 13-mer sequences, 5'-d(TCACXAAATCCTTCCCCC)-3'{center_dot}5'-d(GGGGGAAGGATTT)-3' (template I), 5'-d(TCACXGAATCCTTCCCCC)-3'{center_dot}5'-d(GGGGGAAGGATTC)-3' (template II), and 5'-d(TCATXGAATCCTTCCCCC)-3'{center_dot}5'-d(GGGGGAAGGATTC)-3' (template III), where X is PdG, were analyzed. With templates I and II, diffracting ternary complexes including dGTP were obtained. The dGTP did not pair with PdG, but instead with the 5'-neighboring template dC, utilizing Watson-Crick geometry. Replication bypass experiments with the template-primer 5?-TCACXAAATCCTTACGAGCATCGCCCCC-3'{center_dot}5'-GGGGGCGATGCTCGTAAGGATTT-3', where X is PdG, which includes PdG in the 5'-CXA-3' template sequence as in template I, showed that the Dpo4 polymerase inserted dGTP and dATP when challenged by the PdG adduct. For template III, in which the template sequence was 5'-TXG-3', a diffracting ternary complex including dATP was obtained. The dATP did not pair with PdG, but instead with the 5'-neighboring T, utilizing Watson-Crick geometry. Thus, all three ternary complexes were of the 'type II' structure described for ternary complexes with native DNA [Ling, H., Boudsocq, F., Woodgate, R., and Yang, W. (2001) Cell 107, 91--102]. The PdG adduct remained in the anti conformation about the glycosyl bond in each of these threee ternary complexes. These results provide insight into how -1

  3. Adenine phosphoribosyltransferase from Sulfolobus solfataricus is an enzyme with unusual kinetic properties and a crystal structure that suggests it evolved from a 6-oxopurine phosphoribosyltransferase

    DEFF Research Database (Denmark)

    Jensen, Kaj Frank; Hansen, Michael Riis; Jensen, Kristine Steen

    2015-01-01

    The adenine phosphoribosyltransferase (APRTase) encoded by the open reading frame SSO2342 of Sulfolobus solfataricus P2, was subjected to crystallographic, kinetic and ligand binding analyses. The enzyme forms dimers in solution and in the crystals, and binds one molecule of the reactants 5......-phosphoribosyl-α-1-pyrophosphate (PRPP) and adenine or the product AMP or the inhibitor ADP in each active site. The individual subunit adopts an overall structure that resembles a 6-oxopurine phosphoribosyltransferase (PRTase) more than known APRTases implying that APRT functionality in Crenarchaeotae has its...... undergoes a conformational change upon binding of adenine and phosphate resulting in a slight contraction of the active site. The inhibitor, ADP binds like the product AMP with both the α- and β-phosphates occupying the 5’-phosphoribosyl binding site. The enzyme shows activity over a wide p...

  4. Replication bypass of the trans-4-Hydroxynonenal-derived (6S,8R,11S)-1,N(2)-deoxyguanosine DNA adduct by the sulfolobus solfataricus DNA polymerase IV.

    Science.gov (United States)

    Banerjee, Surajit; Christov, Plamen P; Kozekova, Albena; Rizzo, Carmelo J; Egli, Martin; Stone, Michael P

    2012-02-20

    trans-4-Hydroxynonenal (HNE) is the major peroxidation product of ω-6 polyunsaturated fatty acids in vivo. Michael addition of the N(2)-amino group of dGuo to HNE followed by ring closure of N1 onto the aldehyde results in four diastereomeric 1,N(2)-dGuo (1,N(2)-HNE-dGuo) adducts. The (6S,8R,11S)-HNE-1,N(2)-dGuo adduct was incorporated into the 18-mer templates 5'-d(TCATXGAATCCTTCCCCC)-3' and d(TCACXGAATCCTTCCCCC)-3', where X = (6S,8R,11S)-HNE-1,N(2)-dGuo adduct. These differed in the identity of the template 5'-neighbor base, which was either Thy or Cyt, respectively. Each of these templates was annealed with either a 13-mer primer 5'-d(GGGGGAAGGATTC)-3' or a 14-mer primer 5'-d(GGGGGAAGGATTCC)-3'. The addition of dNTPs to the 13-mer primer allowed analysis of dNTP insertion opposite to the (6S,8R,11S)-HNE-1,N(2)-dGuo adduct, whereas the 14-mer primer allowed analysis of dNTP extension past a primed (6S,8R,11S)-HNE-1,N(2)-dGuo:dCyd pair. The Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4) belongs to the Y-family of error-prone polymerases. Replication bypass studies in vitro reveal that this polymerase inserted dNTPs opposite the (6S,8R,11S)-HNE-1,N(2)-dGuo adduct in a sequence-specific manner. If the template 5'-neighbor base was dCyt, the polymerase inserted primarily dGTP, whereas if the template 5'-neighbor base was dThy, the polymerase inserted primarily dATP. The latter event would predict low levels of Gua → Thy mutations during replication bypass when the template 5'-neighbor base is dThy. When presented with a primed (6S,8R,11S)-HNE-1,N(2)-dGuo:dCyd pair, the polymerase conducted full-length primer extension. Structures for ternary (Dpo4-DNA-dNTP) complexes with all four template-primers were obtained. For the 18-mer:13-mer template-primers in which the polymerase was confronted with the (6S,8R,11S)-HNE-1,N(2)-dGuo adduct, the (6S,8R,11S)-1,N(2)-dGuo lesion remained in the ring-closed conformation at the active site. The incoming dNTP, either d

  5. Conjugational genetic exchange in the hyperthermophilic archaeon Sulfolobus acidocaldarius: intragenic recombination with minimal dependence on marker separation.

    Science.gov (United States)

    Hansen, Josh E; Dill, Amy C; Grogan, Dennis W

    2005-01-01

    In Sulfolobus acidocaldarius conjugation assays, recombinant frequency was relatively constant for marker separations from 1,154 bp down to about 50 bp and readily detectable at 10 bp. Three-factor crosses revealed little, if any, genetic linkage over distances of 500 to 600 bp, and large deletion mutants were good donors but poor recipients in matings. The results indicate that most intragenic recombination events occur at one of the mutations, not in the interval between them.

  6. The apt/6-Methylpurine Counterselection System and Its Applications in Genetic Studies of the Hyperthermophilic Archaeon Sulfolobus islandicus

    Science.gov (United States)

    Bi, Hongkai; Whitaker, Rachel J.

    2016-01-01

    ABSTRACT Sulfolobus islandicus serves as a model for studying archaeal biology as well as linking novel biology to evolutionary ecology using functional population genomics. In the present study, we developed a new counterselectable genetic marker in S. islandicus to expand the genetic toolbox for this species. We show that resistance to the purine analog 6-methylpurine (6-MP) in S. islandicus M.16.4 is due to the inactivation of a putative adenine phosphoribosyltransferase encoded by M164_0158 (apt). The application of the apt gene as a novel counterselectable marker was first illustrated by constructing an unmarked α-amylase deletion mutant. Furthermore, the 6-MP counterselection feature was employed in a forward (loss-of-function) mutation assay to reveal the profile of spontaneous mutations in S. islandicus M.16.4 at the apt locus. Moreover, the general conservation of apt genes in the crenarchaea suggests that the same strategy can be broadly applied to other crenarchaeal model organisms. These results demonstrate that the apt locus represents a new tool for genetic manipulation and sequence analysis of the hyperthermophilic crenarchaeon S. islandicus. IMPORTANCE Currently, the pyrEF/5-fluoroorotic acid (5-FOA) counterselection system remains the sole counterselection marker in crenarchaeal genetics. Since most Sulfolobus mutants constructed by the research community were derived from genetic hosts lacking the pyrEF genes, the pyrEF/5-FOA system is no longer available for use in forward mutation assays. Demonstration of the apt/6-MP counterselection system for the Sulfolobus model renders it possible to again study the mutation profiles in mutants that have already been constructed by the use of strains with a pyrEF-deficient background. Furthermore, additional counterselectable markers will allow us to conduct more sophisticated genetic studies, i.e., investigate mechanisms of chromosomal DNA transfer and quantify recombination frequencies among S

  7. Cytochrome b558/566 from the archaeon Sulfolobus acidocaldarius has a unique Asn-linked highly branched hexasaccharide chain containing 6-sulfoquinovose.

    Science.gov (United States)

    Zähringer, U; Moll, H; Hettmann, T; Knirel, Y A; Schäfer, G

    2000-07-01

    Cytochrome b558/566 from the archaeon Sulfolobus acidocaldarius (DSM 639) has been described as a novel highly glycosylated membrane-bound b-type hemoprotein [Hettmann, T., Schmidt, C. L., Anemüller, S., Zähringer, U., Moll, H., Petersen, A. & Schäfer, G. (1998) J. Biol. Chem. 273, 12032-12040]. The purified cytochrome b558/566 was characterized by MALDI MS as a 64-kDa (glyco)protein expressing 17% glycosylation. Detailed chemical studies showed that it was exclusively O-mannosylated with monosaccharides and N-glycosylated with at least seven hexasaccharide units having the same unique structure. The hexasaccharide was released by cleavage with peptide:N-glycosidase (PNGase) F and found to consist of two residues each of Man and GlcNAc and one residue each of Glc and 6-deoxy-6-sulfoglucose (6-sulfoquinovose). The last sugar has been known as a component of glycolipids of plants and some prokaryotes, but has not been hitherto found in bacterial glycoproteins. Digestion with trypsin/pronase gave a mixture of glycopeptides with the same Asn-linked hexasaccharide chain, from which an N-glycosylated Tyr-Asn dipeptide was purified by gel chromatography and anion-exchange HPLC. Studies of the degradation products using methylation analysis, ESI MS, MALDI MS, and 1H and 13C NMR spectroscopy, including 1H,13C HMQC and NOESY experiments, established the structure of the unique Asn-linked hexasaccharide chain of cytochrome b558/566.

  8. Studying Extrachromosomal Genetic Elements in Sulfolobus

    DEFF Research Database (Denmark)

    Guannan, Liu

    facilitated the characterization of viruses, plasmids and membrane vesicles. Studying the interactions between Sulfolobus and extrachromosomal genetic elements has provided many new insights into basic molecular processes. Secreted membrane vesicle seems to be a common characteristic for Sulfolobus. In order...... of random chromosomal fragments, including IS elements. The results suggest that membrane vesicles could serve as vehicles for the inter-cellular transport of genetic material. A variant of ATV, ATV2, was isolated that infected a newly isolated Sulfolobus solfataricus P3 strain. Comparative genomics......, whereas the deactivation of pKEF9 in S. solfataricus was caused by mobile elements after it had integrated into the host genome....

  9. Doubling Power Output of Starch Biobattery Treated by the Most Thermostable Isoamylase from an Archaeon Sulfolobus tokodaii.

    Science.gov (United States)

    Cheng, Kun; Zhang, Fei; Sun, Fangfang; Chen, Hongge; Percival Zhang, Y-H

    2015-08-20

    Biobattery, a kind of enzymatic fuel cells, can convert organic compounds (e.g., glucose, starch) to electricity in a closed system without moving parts. Inspired by natural starch metabolism catalyzed by starch phosphorylase, isoamylase is essential to debranch alpha-1,6-glycosidic bonds of starch, yielding linear amylodextrin - the best fuel for sugar-powered biobattery. However, there is no thermostable isoamylase stable enough for simultaneous starch gelatinization and enzymatic hydrolysis, different from the case of thermostable alpha-amylase. A putative isoamylase gene was mined from megagenomic database. The open reading frame ST0928 from a hyperthermophilic archaeron Sulfolobus tokodaii was cloned and expressed in E. coli. The recombinant protein was easily purified by heat precipitation at 80 (o)C for 30 min. This enzyme was characterized and required Mg(2+) as an activator. This enzyme was the most stable isoamylase reported with a half lifetime of 200 min at 90 (o)C in the presence of 0.5 mM MgCl2, suitable for simultaneous starch gelatinization and isoamylase hydrolysis. The cuvett-based air-breathing biobattery powered by isoamylase-treated starch exhibited nearly doubled power outputs than that powered by the same concentration starch solution, suggesting more glucose 1-phosphate generated.

  10. Inducible and constitutive promoters for genetic systems in Sulfolobus acidocaldarius.

    Science.gov (United States)

    Berkner, Silvia; Wlodkowski, Alexander; Albers, Sonja-Verena; Lipps, Georg

    2010-05-01

    Central to genetic work in any organism are the availability of a range of inducible and constitutive promoters. In this work we studied several promoters for use in the hyperthermophilic archaeon Sulfolobus acidocaldarius. The promoters were tested with the aid of an E. coli-Sulfolobus shuttle vector in reporter gene experiments. As the most suitable inducible promoter a maltose inducible promoter was identified. It comprises 266 bp of the sequence upstream of the gene coding for the maltose/maltotriose binding protein (mbp, Saci_1165). Induction is feasible with either maltose or dextrin at concentrations of 0.2-0.4%. The highest increase in expression (up to 17-fold) was observed in late exponential and stationary phase around 30-50 h after addition of dextrin. Whereas in the presence of glucose and xylose higher basal activity and reduced inducibility with maltose is observed, sucrose can be used in the growth medium additionally without affecting the basal activity or the inducibility. The minimal promoter region necessary could be narrowed down to 169 bp of the upstream sequence. The ABCE1 protein from S. solfataricus was successfully expressed under control of the inducible promoter with the shuttle vector pC and purified from the S. acidocaldarius culture with a yield of about 1 mg L(-1) culture. In addition we also determined the promoter strength of several constitutive promoters.

  11. The Genome of Sulfolobus acidocaldarius, a Model Organism of the Crenarchaeota

    DEFF Research Database (Denmark)

    Chen, Lanming; Brügger, Kim; Skovgaard, M.

    2005-01-01

    to the Sulfolobus genus. Moreover, 82 genes for untranslated RNAs were identified and annotated. Owing to the probable absence of active autonomous and nonautonomous mobile elements, the genome stability and organization of S. acidocaldarius differ radically from those of Sulfolobus solfataricus and Sulfolobus...

  12. The genome of Sulfolobus acidocaldarius, a model organism of the Crenarchaeota

    DEFF Research Database (Denmark)

    Chen, L.M.; Brugger, K.; Skovgaard, Marie

    2005-01-01

    to the Sulfolobus genus. Moreover, 82 genes for untranslated RNAs were identified and annotated. Owing to the probable absence of active autonomous and nonautonomous mobile elements, the genome stability and organization of S. acidocaldarius differ radically from those of Sulfolobus solfataricus and Sulfolobus...

  13. Expanding and understanding the genetic toolbox of the hyperthermophilic genus Sulfolobus.

    Science.gov (United States)

    Wagner, Michaela; Berkner, Silvia; Ajon, Malgorzata; Driessen, Arnold J M; Lipps, Georg; Albers, Sonja-Verena

    2009-02-01

    Although Sulfolobus species are among the best studied archaeal micro-organisms, the development and availability of genetic tools has lagged behind. In the present paper, we discuss the latest progress in understanding recombination events of exogenous DNA into the chromosomes of Sulfolobus solfataricus and Sulfolobus acidocaldarius and their application in the construction of targeted-deletion mutant strains.

  14. Deletion of the topoisomerase III gene in the hyperthermophilic archaeon Sulfolobus islandicus results in slow growth and defects in cell cycle control

    DEFF Research Database (Denmark)

    Li, Xiyang; Guo, Li; Deng, Ling

    2011-01-01

    Topoisomerase III (topo III), a type IA topoisomerase, is widespread in hyperthermophilic archaea. In order to interrogate the in vivo role of archaeal topo III, we constructed and characterized a topo III gene deletion mutant of Sulfolobus islandicus. The mutant was viable but grew more slowly t...

  15. Specific partial reduction of geranylgeranyl diphosphate by an enzyme from the thermoacidophilic archaeon Sulfolobus acidocaldarius yields a reactive prenyl donor, not a dead-end product.

    Science.gov (United States)

    Sato, Sho; Murakami, Motomichi; Yoshimura, Tohru; Hemmi, Hisashi

    2008-06-01

    Geranylgeranyl reductase from Sulfolobus acidocaldarius was shown to catalyze the reduction of geranylgeranyl groups in the precursors of archaeal membrane lipids, generally reducing all four double bonds. However, when geranylgeranyl diphosphate was subjected to the reductase reaction, only three of the four double bonds were reduced. Mass spectrometry and acid hydrolysis indicated that the allylic double bond was preserved in the partially reduced product derived from geranylgeranyl diphosphate. Thus, the reaction product was shown to be phytyl diphosphate, which is a substrate for archaeal prenyltransferases, unlike the completely reduced compound phytanyl diphosphate.

  16. Efficient CRISPR-Mediated Post-Transcriptional Gene Silencing in a Hyperthermophilic Archaeon Using Multiplexed crRNA Expression

    Directory of Open Access Journals (Sweden)

    Ziga Zebec

    2016-10-01

    Full Text Available CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats-mediated RNA degradation is catalyzed by a type III system in the hyperthermophilic archaeon Sulfolobus solfataricus. Earlier work demonstrated that the system can be engineered to target specifically mRNA of an endogenous host reporter gene, namely the β-galactosidase in S. solfataricus. Here, we investigated the effect of single and multiple spacers targeting the mRNA of a second reporter gene, α-amylase, at the same, and at different, locations respectively, using a minimal CRISPR (miniCR locus supplied on a viral shuttle vector. The use of increasing numbers of spacers reduced mRNA levels at progressively higher levels, with three crRNAs (CRISPR RNAs leading to ∼ 70–80% reduction, and five spacers resulting in an α-amylase gene knockdown of > 90% measured on both mRNA and protein activity levels. Our results indicate that this technology can be used to increase or modulate gene knockdown for efficient post-transcriptional gene silencing in hyperthermophilic archaea, and potentially also in other organisms.

  17. The membrane-extrinsic domain of cytochrome b(558/566) from the archaeon Sulfolobus acidocaldarius performs pivoting movements with respect to the membrane surface.

    Science.gov (United States)

    Schoepp-Cothenet, B; Schütz, M; Baymann, F; Brugna, M; Nitschke, W; Myllykallio, H; Schmidt, C

    2001-01-05

    The orientation of the membrane-attached cytochrome b(558/566)-haem with respect to the membrane was determined by electron paramagnetic resonance spectroscopy on two-dimensionally ordered oxidised membrane fragments from Sulfolobus acidocaldarius. Unlike the other redox centres in the membrane, the cytochrome b(558/566)-haem was found to cover a range of orientations between 25 degrees and 90 degrees. The described results are reminiscent of those obtained on the Rieske cluster of bc complexes and indicate that the membrane-extrinsic domain of cytochrome b(558/566) can perform pivoting motion between two extreme positions. Such a conformational flexibility is likely to play a role in electron transfer with its redox partners.

  18. Evolutionary conservation and DNA binding properties of the Ssh7 proteins from Sulfolobus shibatae

    Institute of Scientific and Technical Information of China (English)

    CHEN; Xulin(陈绪林); GUO; Rong(郭荣); HUANG; Li(黄力); Ray; Hong

    2002-01-01

    The thermoacidophilic archaeon Sulfolobus shibatae synthesizes a large amount of the 7-ku DNA binding proteins known as Ssh7. Our hybridization experiments showed that two Ssh7-encoding genes existed in the genome of S. Shibatae. These two genes, designated ssh7a and ssh7b, have been cloned, sequenced and expressed in Escherichia coli. The two Ssh7 proteins differ only at three amino acid positions. In addition, the cis-regulatory sequences of the ssh7a and ssh7b genes are highly conserved. These results suggest the presence of a selective pressure to maintain not only the sequence but also the expression of the two genes. We have also found that there are two genes encoding the 7-ku protein in Sulfolobus solfataricus. Based on this and other studies, we suggest that the gene encoding the 7-ku protein underwent duplication before the separation of Sulfolobus species. Binding of native Ssh7 and recombinant (r)Ssh7 to short duplex DNA fragments was analyzed by electrophoretic mobility shift assays. Both native and recombinant forms of the protein behaved in a similar fashion in the assays, suggesting that the interaction of Ssh7 with DNA is not affected either by specific lysine methylation found in the native Ssh7 proteins or by the difference between the two Ssh7 isomers in amino acid sequence. Our data show that Ssh7 binds duplex DNA fragments with a binding size of ~ 6.6 base pairs and an apparent dissociation constant of (0.7-1.0)×10-7 mol/L under the assay conditions employed in the present study. In addition, Ssh7 binds more tightly to negatively supercoiled DNA than to linear or relaxed DNA.

  19. The saci_2123 gene of the hyperthermoacidophile Sulfolobus acidocaldarius encodes an ATP-binding cassette multidrug transporter.

    Science.gov (United States)

    Yang, Nuan; Driessen, Arnold J M

    2015-01-01

    Multidrug resistance (MDR) transporters are capable of secreting structurally and functionally unrelated toxic compounds from the cell. Among this group are ATP-binding cassette (ABC) transporters. These membrane proteins are typically arranged as either hetero- or homo-dimers of ABC half-transporters with each subunit consisting of a membrane domain fused at the C-terminus to an ATP-binding domain, or as full transporters in which the two subunits are fused into a single polypeptide. The saci_2123 gene of the thermoacidophilic archaeon Sulfolobus acidocaldarius is the only gene in the genome that encodes an ATP-binding cassette half-transporter, while a homologous gene is present in the genomes of S. solfataricus, S. tokodaii and S islandicus. Saci_2123 shares homology with well-characterized bacterial and mammalian MDR transporters. The saci_2132 gene is up-regulated when cells are exposed to drugs. A deletion mutant of saci_2132 was found to be more vulnerable to a set of toxic compounds, including detergents, antibiotics and uncouplers as compared to the wild-type strain, while the drug resistance could be restored through the plasmid-based expression of saci_2132. These data demonstrate that Saci_2132 is an archaeal ABC-MDR transporter and therefore it was termed Smr1 (Sulfolobus multidrug resistance transporter 1).

  20. Disruption of the gene encoding restriction endonuclease SuaI and development of a host-vector system for the thermoacidophilic archaeon Sulfolobus acidocaldarius.

    Science.gov (United States)

    Suzuki, Shoji; Kurosawa, Norio

    2016-03-01

    Sulfolobus acidocaldarius is a useful model organism for the genetic study of thermophilic archaea due to its ease of cultivation. Here we describe the development of a host-vector system for S. acidocaldarius consisting of SuaI restriction system-deficient strain SK-1 and shuttle vector pSAV2. The new host strain SK-1 was constructed by pop-out recombination based on the pyrE marker gene. Plasmid pSAV2 was constructed from the S. islandicus native plasmid pRN1, in which selectable markers and functional genes were inserted in suitable locations and orientations followed by the deletion of non-essential open reading frames. SK-1 allowed direct transformation without N(4)-methylation at SuaI restriction sites, so unmethylated vector pSAV2 could be introduced directly into SK-1 by electroporation. The transformants were selected by pyrEF complementation on xyrose-tryptone solid medium without prior liquid culturing. The transformation efficiency was approximately 1.0 × 10(3)/μg DNA. After replication in S. acidocaldarius, pSAV2 was successfully recovered from transformant cultures by the standard alkaline lysis method. Plasmid yield was approximately 40-50 ng/ml from late-log through stationary phase cultures. In addition, pSAV2 was maintained stably and at relatively high copy number in S. acidocaldarius.

  1. Bipolar tetraether lipids derived from thermoacidophilic archaeon Sulfolobus acidocaldarius for membrane stabilization of chlorin e6 based liposomes for photodynamic therapy.

    Science.gov (United States)

    Mahmoud, Gihan; Jedelská, Jarmila; Strehlow, Boris; Bakowsky, Udo

    2015-09-01

    The initial burst release of water-soluble photosensitizers is one of the major problems encountered the development of controlled release formulations. In this study, the freely water soluble chlorin e6 (Ce6) was assembled with cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) to improve its loading efficiency in the liposomal bilayer. Tetraether lipids (TELs) derived from Sulfolobus acidocaldarius were added to DOTAP:Ce6 assembly in a concentration range of 2.5×10(-4)-1.6×10(-3)M to stabilize the membrane rigidity of the liposomes and to provide controlled release system. From the comparative spectroscopic experiments, it has been shown that the assembled DOTAP:Ce6 along with addition of TELs have improved the loading efficiency of Ce6 in TELs-liposomes and obviously modified the release profile of Ce6. The in vitro cell viability of Ce6 in mouse neuro-blastoma (Neuro-2a) and ovarian cell carcinoma (SK-OV-3) confirmed neglected dark cytotoxicity and presented potential photo-induced cytotoxicity with the effect was being more pronounced in Neuro 2a than in SK-OV-3. In-situ IV-injection of chick chorioallantoic membrane (CAM) showed hemorrhage and necrosis 30 min post irradiation at 1.8 mol% TELs (19.9J/cm(2)). Higher TELs of 2.2 and 3.7 mol% in particular demonstrated localized vascular destruction within the irradiated area. Our results suggest that TELs favored slower release rates of Ce6. This, in turn, tetraether lipids can be considered as a versatile class of lipids for photodynamic modality for destruction of cancer cells and tumor vasculature while sparing the quiescent ones.

  2. Sulfolobicins, Specific Proteinaceous Toxins Produced by Strains of the Extremely Thermophilic Archaeal Genus Sulfolobus

    Science.gov (United States)

    Prangishvili, David; Holz, Ingelore; Stieger, Evelyn; Nickell, Stephan; Kristjansson, Jakob K.; Zillig, Wolfram

    2000-01-01

    Several novel strains of “Sulfolobus islandicus” produced proteinaceous toxins, termed sulfolobicins, which killed cells of other strains of the same species, as well as of Sulfolobus solfataricus P1 and Sulfolobus shibatae B12, but not of the producer strains and of Sulfolobus acidocaldarius DSM639. The sulfolobicin purified from the strain HEN2/2 had a molecular mass of about 20 kDa. It was found to be associated with the producer cells as well as with cell-derived S-layer-coated spherical membrane vesicles 90 to 180 nm in diameter and was not released from the cells in soluble form. PMID:10781574

  3. Improvement of a Sulfolobus-E. coli shuttle vector for heterologous gene expression in Sulfolobus acidocaldarius.

    Science.gov (United States)

    Hwang, Sungmin; Choi, Kyoung-Hwa; Yoon, Naeun; Cha, Jaeho

    2015-02-01

    A Sulfolobus-E. coli shuttle vector for an efficient expression of the target gene in S. acidocaldarius strain was constructed. The plasmid-based vector pSM21 and its derivative pSM21N were generated based on the pUC18 and Sulfolobus cryptic plasmid pRN1. They carried the S. solfataricus P2 pyrEF gene for the selection marker, a multiple cloning site (MCS) with C-terminal histidine tag, and a constitutive promoter of the S. acidocaldarius gdhA gene for strong expression of the target gene, as well as the pBR322 origin and ampicillin-resistant gene for E. coli propagation. The advantage of pSM21 over other Sulfolobus shuttle vectors is that it contains a MCS and a histidine tag for the simple and easy cloning of a target gene as well as one-step purification by histidine affinity chromatography. For successful expression of the foreign genes, two genes from archaeal origins (PH0193 and Ta0298) were cloned into pSM21N and the functional expression was examined by enzyme activity assay. The recombinant PH0193 was successfully expressed under the control of the gdhA promoter and purified from the cultures by His-tag affinity chromatography. The yield was approximately 1 mg of protein per liter of cultures. The enzyme activity measurements of PH0913 and Ta0298 revealed that both proteins were expressed as an active form in S. acidocaldarius. These results indicate that the pSM21N shuttle vector can be used for the functional expression of foreign archaeal genes that form insoluble aggregates in the E. coli system.

  4. Genome Sequencing of Sulfolobus sp. A20 from Costa Rica and Comparative Analyses of the Putative Pathways of Carbon, Nitrogen, and Sulfur Metabolism in Various Sulfolobus Strains.

    Science.gov (United States)

    Dai, Xin; Wang, Haina; Zhang, Zhenfeng; Li, Kuan; Zhang, Xiaoling; Mora-López, Marielos; Jiang, Chengying; Liu, Chang; Wang, Li; Zhu, Yaxin; Hernández-Ascencio, Walter; Dong, Zhiyang; Huang, Li

    2016-01-01

    The genome of Sulfolobus sp. A20 isolated from a hot spring in Costa Rica was sequenced. This circular genome of the strain is 2,688,317 bp in size and 34.8% in G+C content, and contains 2591 open reading frames (ORFs). Strain A20 shares ~95.6% identity at the 16S rRNA gene sequence level and Sulfolobus species (i.e., Sulfolobus islandicus and Sulfolobus solfataricus), suggesting that it represents a novel Sulfolobus species. Comparison of the genome of strain A20 with those of the type strains of S. solfataricus, Sulfolobus acidocaldarius, S. islandicus, and Sulfolobus tokodaii, which were isolated from geographically separated areas, identified 1801 genes conserved among all Sulfolobus species analyzed (core genes). Comparative genome analyses show that central carbon metabolism in Sulfolobus is highly conserved, and enzymes involved in the Entner-Doudoroff pathway, the tricarboxylic acid cycle and the CO2 fixation pathways are predominantly encoded by the core genes. All Sulfolobus species encode genes required for the conversion of ammonium into glutamate/glutamine. Some Sulfolobus strains have gained the ability to utilize additional nitrogen source such as nitrate (i.e., S. islandicus strain REY15A, LAL14/1, M14.25, and M16.27) or urea (i.e., S. islandicus HEV10/4, S. tokodaii strain7, and S. metallicus DSM 6482). The strategies for sulfur metabolism are most diverse and least understood. S. tokodaii encodes sulfur oxygenase/reductase (SOR), whereas both S. islandicus and S. solfataricus contain genes for sulfur reductase (SRE). However, neither SOR nor SRE genes exist in the genome of strain A20, raising the possibility that an unknown pathway for the utilization of elemental sulfur may be present in the strain. The ability of Sulfolobus to utilize nitrate or sulfur is encoded by a gene cluster flanked by IS elements or their remnants. These clusters appear to have become fixed at a specific genomic site in some strains and lost in other strains during the

  5. Strain identification and 5S rRNA gene characterization of the hyperthermophilic archaebacterium Sulfolobus acidocaldarius.

    OpenAIRE

    Durovic, P; Kutay, U.; Schleper, C; Dennis, P P

    1994-01-01

    A commonly used laboratory Sulfolobus strain has been unambiguously identified as Sulfolobus acidocaldarius DSM639. The 5S rRNA gene from this strain was cloned and sequenced. It differs at 17 of 124 positions from the identical 5S rRNA sequences from Sulfolobus solfataricus and a strain apparently misidentified as S. acidocaldarius. Analysis of the transcripts from the 5S rRNA gene failed to identify any precursor extending a significant distance beyond the 5' or 3' boundary of the 5S rRNA-c...

  6. Differential tolerance of Sulfolobus strains to transition metals

    Science.gov (United States)

    Miller, K.W.; Sass, Risanico S.; Risatti, J.B.

    1992-01-01

    Sulfolobus acidocaldarius strains 98-3 and B12, and S. solfataricus ATCC 35091 were evaluated for tolerance to Cd, Co, Cu, Ni, Zn and Mg. The tolerance of strains 98-3 and ATCC 35091 to these metals was Mg > Zn > Cd > Cu ??? Co > Ni. For B12, however, the order of tolerance was Mg > Cd > Zn = Co > Ni > Cu. Tolerance to these metals is also presented as a potentially useful taxonomic indicator.

  7. The sub-cellular localization of Sulfolobus DNA replication.

    Science.gov (United States)

    Gristwood, Tamzin; Duggin, Iain G; Wagner, Michaela; Albers, Sonja V; Bell, Stephen D

    2012-07-01

    Analyses of the DNA replication-associated proteins of hyperthermophilic archaea have yielded considerable insight into the structure and biochemical function of these evolutionarily conserved factors. However, little is known about the regulation and progression of DNA replication in the context of archaeal cells. In the current work, we describe the generation of strains of Sulfolobus solfataricus and Sulfolobus acidocaldarius that allow the incorporation of nucleoside analogues during DNA replication. We employ this technology, in conjunction with immunolocalization analyses of replisomes, to investigate the sub-cellular localization of nascent DNA and replisomes. Our data reveal a peripheral localization of replisomes in the cell. Furthermore, while the two replication forks emerging from any one of the three replication origins in the Sulfolobus chromosome remain in close proximity, the three origin loci are separated.

  8. Genetic tools for Sulfolobus spp.: vectors and first applications.

    Science.gov (United States)

    Berkner, Silvia; Lipps, Georg

    2008-09-01

    Sulfolobus species belong to the best-studied archaeal organisms but have lacked powerful genetic methods. Recently, there has been considerable progress in the field of Sulfolobus genetics. Urgently needed basic genetic tools, such as targeted gene knockout techniques and shuttle vectors are being developed at an increasing pace. For S. solfataricus knockout systems as well as different shuttle vectors are available. For the genetically more stable S. acidocaldarius shuttle vectors have been recently developed. In this review we summarize the currently available genetic tools and methods for the genus Sulfolobus. Different transformation protocols are discussed, as well as all so far developed knockout systems and Sulfolobus-Escherichia coli shuttle vectors are summarized. Special emphasis is put on the important vector components, i.e., selectable markers and Sulfolobus replicons. Additionally, the information gathered on different Sulfolobus strains with respect to their use as recipient strains is reviewed. The advantages and disadvantages of the different systems are discussed and aims for further improvement of genetic systems are identified.

  9. Genus-specific protein binding to the large clusters of DNA repeats (short regularly spaced repeats) present in Sulfolobus genomes

    DEFF Research Database (Denmark)

    Peng, Xu; Brügger, Kim; Shen, Biao;

    2003-01-01

    Short regularly spaced repeats (SRSRs) occur in multiple large clusters in archaeal chromosomes and as smaller clusters in some archaeal conjugative plasmids and bacterial chromosomes. The sequence, size, and spacing of the repeats are generally constant within a cluster but vary between clusters....... For the crenarchaeon Sulfolobus solfataricus P2, the repeats in the genome fall mainly into two closely related sequence families that are arranged in seven clusters containing a total of 441 repeats which constitute ca. 1% of the genome. The Sulfolobus conjugative plasmid pNOB8 contains a small cluster of six repeats...... that are identical in sequence to one of the repeat variants in the S. solfataricus chromosome. Repeats from the pNOB8 cluster were amplified and tested for protein binding with cell extracts from S. solfataricus. A 17.5-kDa SRSR-binding protein was purified from the cell extracts and sequenced. The protein is N...

  10. Generation of proton-motive force by an archaeal terminal quinol oxidase from Sulfolobus acidocaldarius

    NARCIS (Netherlands)

    Gleissner, Michael; Elferink, Maria; Driessen, Arnold J.M.; Konings, Wilhelmus; Anemüller, Stefan; Schäfer, Günter

    1994-01-01

    The terminal quinol oxidase of the cytochrome aa3 type was isolated from the extreme thermo-acidophilic archaeon Sulfolobus acidocaldarius. In micellar solution, the enzyme oxidized various quinols and exerted the highest activity with the physiological substrate caldariella quinol. The enzyme was f

  11. Genome sequencing of Sulfolobus sp. A20 from Costa Rica and comparative analyses of the putative pathways of carbon, nitrogen and sulfur metabolism in various Sulfolobus strains

    Directory of Open Access Journals (Sweden)

    Xin Dai

    2016-11-01

    Full Text Available The genome of Sulfolobus sp. A20 isolated from a hot spring in Costa Rica was sequenced. This circular genome of the strain is 2,688,317 bp in size and 34.8% in G+C content, and contains 2,591 open reading frames (ORFs. Strain A20 shares ~95.6% identity at the 16S rRNA gene sequence level and less than 30% DNA-DNA hybridization (DDH values with the most closely related known Sulfolobus species (i.e., S. islandicus and S. solfataricus, suggesting that it represents a novel Sulfolobus species. Comparison of the genome of strain A20 with those of the type strains of S. solfataricus, S. acidocaldarius, S. islandicus and S. tokodaii, which were isolated from geographically separated areas, identified 1,801 genes conserved among all Sulfolobus species analyzed (core genes. Comparative genome analyses show that central carbon metabolism in Sulfolobus is highly conserved, and enzymes involved in the Entner-Doudoroff pathway, the tricarboxylic acid cycle and the CO2 fixation pathways are predominantly encoded by the core genes. All Sulfolobus species encode genes required for the conversion of ammonium into glutamate/glutamine. Some Sulfolobus strains have gained the ability to utilize additional nitrogen source such as nitrate (i.e. S. islandicus strain REY15A, LAL14/1, M14.25 and M16.27 or urea (i.e. S. islandicus HEV10/4, S. tokodaii strain7 and S. metallicus DSM 6482. The strategies for sulfur metabolism are most diverse and least understood. S. tokodaii encodes sulfur oxygenase/reductase (SOR, whereas both S. islandicus and S. solfataricus contain genes for sulfur reductase (SRE. However, neither SOR nor SRE genes exist in the genome of strain A20, raising the possibility that an unknown pathway for the utilization of elemental sulfur may be present in the strain. The ability of Sulfolobus to utilize nitrate or sulfur is encoded by a gene cluster flanked by IS elements or their remnants. These clusters appear to have become fixed at a specific

  12. The Mre11 protein interacts with both Rad50 and the HerA bipolar helicase and is recruited to DNA following gamma irradiation in the archaeon Sulfolobus acidocaldarius

    Directory of Open Access Journals (Sweden)

    Forterre Patrick

    2008-02-01

    Full Text Available Abstract Background The ubiquitous Rad50 and Mre11 proteins play a key role in many processes involved in the maintenance of genome integrity in Bacteria and Eucarya, but their function in the Archaea is presently unknown. We showed previously that in most hyperthermophilic archaea, rad50-mre11 genes are linked to nurA encoding both a single-strand endonuclease and a 5' to 3' exonuclease, and herA, encoding a bipolar DNA helicase which suggests the involvement of the four proteins in common molecular pathway(s. Since genetic tools for hyperthermophilic archaea are just emerging, we utilized immuno-detection approaches to get the first in vivo data on the role(s of these proteins in the hyperthermophilic crenarchaeon Sulfolobus acidocaldarius. Results We first showed that S. acidocaldarius can repair DNA damage induced by high doses of gamma rays, and we performed a time course analysis of the total levels and sub-cellular partitioning of Rad50, Mre11, HerA and NurA along with the RadA recombinase in both control and irradiated cells. We found that during the exponential phase, all proteins are synthesized and display constant levels, but that all of them exhibit a different sub-cellular partitioning. Following gamma irradiation, both Mre11 and RadA are immediately recruited to DNA and remain DNA-bound in the course of DNA repair. Furthermore, we show by immuno-precipitation assays that Rad50, Mre11 and the HerA helicase interact altogether. Conclusion Our analyses strongly support that in Sulfolobus acidocaldarius, the Mre11 protein and the RadA recombinase might play an active role in the repair of DNA damage introduced by gamma rays and/or may act as DNA damage sensors. Moreover, our results demonstrate the functional interaction between Mre11, Rad50 and the HerA helicase and suggest that each protein play different roles when acting on its own or in association with its partners. This report provides the first in vivo evidence supporting the

  13. The protein ORF80 from the acidophilic and thermophilic archaeon Sulfolobus islandicus binds highly site-specifically to double-stranded DNA and represents a novel type of basic leucine zipper protein

    Science.gov (United States)

    Lipps, Georg; Ibanez, Pablo; Stroessenreuther, Thomas; Hekimian, Katya; Krauss, Gerhard

    2001-01-01

    The cryptic high copy number plasmid pRN1 from the thermophilic and acidophilic crenarchaeote Sulfolobus islandicus shares three conserved open reading frames with other S.islandicus plasmids. One of the open reading frames, namely orf80, encodes a 9.5 kDa protein that has no homology to any characterised protein. Recombinant ORF80 purified from Escherichia coli binds to double-stranded DNA in a sequence-specific manner as suggested by EMSA experiments and DNase I footprints. Two highly symmetrical binding sites separated by ∼60 bp were found upstream of the orf80 gene. Both binding sites contain two TTAA motifs as well as other conserved bases. Fluorescence measurements show that short duplex DNAs derived from a single binding site sequence are bound with submicromolar affinity and moderate cooperativity by ORF80. On DNA fragments carrying both binding sites, a rather large protein–DNA complex is formed in a highly cooperative manner. ORF80 contains an N-terminal leucine zipper motif and a highly basic domain at its C-terminus. Compared to all known basic leucine zipper proteins the order of the domains is reversed in ORF80. ORF80 may therefore constitute a new subclass of basic leucine zipper DNA-binding proteins. PMID:11812827

  14. A novel Sulfolobus non-conjugative extrachromosomal genetic element capable of integration into the host genome and spreading in the presence of a fusellovirus

    DEFF Research Database (Denmark)

    Wang, Ying; Duan, Zhenhong; Zhu, Haojun;

    2007-01-01

    An integrative non-conjugative extrachromosomal genetic element, denoted as pSSVi, has been isolated from a Sulfolobus solfataricus P2 strain and was characterized. This genetic element is a double-stranded DNA of 5740 bp in size and contains eight open reading frames (ORFs). It resembles members...

  15. The genome of Sulfolobus acidocaldarius, a model organism of the Crenarchaeota.

    Science.gov (United States)

    Chen, Lanming; Brügger, Kim; Skovgaard, Marie; Redder, Peter; She, Qunxin; Torarinsson, Elfar; Greve, Bo; Awayez, Mariana; Zibat, Arne; Klenk, Hans-Peter; Garrett, Roger A

    2005-07-01

    Sulfolobus acidocaldarius is an aerobic thermoacidophilic crenarchaeon which grows optimally at 80 degrees C and pH 2 in terrestrial solfataric springs. Here, we describe the genome sequence of strain DSM639, which has been used for many seminal studies on archaeal and crenarchaeal biology. The circular genome carries 2,225,959 bp (37% G+C) with 2,292 predicted protein-encoding genes. Many of the smaller genes were identified for the first time on the basis of comparison of three Sulfolobus genome sequences. Of the protein-coding genes, 305 are exclusive to S. acidocaldarius and 866 are specific to the Sulfolobus genus. Moreover, 82 genes for untranslated RNAs were identified and annotated. Owing to the probable absence of active autonomous and nonautonomous mobile elements, the genome stability and organization of S. acidocaldarius differ radically from those of Sulfolobus solfataricus and Sulfolobus tokodaii. The S. acidocaldarius genome contains an integrated, and probably encaptured, pARN-type conjugative plasmid which may facilitate intercellular chromosomal gene exchange in S. acidocaldarius. Moreover, it contains genes for a characteristic restriction modification system, a UV damage excision repair system, thermopsin, and an aromatic ring dioxygenase, all of which are absent from genomes of other Sulfolobus species. However, it lacks genes for some of their sugar transporters, consistent with it growing on a more limited range of carbon sources. These results, together with the many newly identified protein-coding genes for Sulfolobus, are incorporated into a public Sulfolobus database which can be accessed at http://dac.molbio.ku.dk/dbs/Sulfolobus.

  16. Sulfolobus Turreted Icosahedral Virus c92 Protein Responsible for the Formation of Pyramid-Like Cellular Lysis Structures

    DEFF Research Database (Denmark)

    Snyder, Jamie C; Brumfield, Susan K; Peng, Nan;

    2011-01-01

    Host cells infected by Sulfolobus turreted icosahedral virus (STIV) have been shown to produce unusual pyramid-like structures on the cell surface. These structures represent a virus-induced lysis mechanism that is present in Archaea and appears to be distinct from the holin/endolysin system...... described for DNA bacteriophages. This study investigated the STIV gene products required for pyramid formation in its host Sulfolobus solfataricus. Overexpression of STIV open reading frame (ORF) c92 in S. solfataricus alone is sufficient to produce the pyramid-like lysis structures in cells. Gene...... disruption of c92 within STIV demonstrates that c92 is an essential protein for virus replication. Immunolocalization of c92 shows that the protein is localized to the cellular membranes forming the pyramid-like structures....

  17. Sulfolobus turreted icosahedral virus c92 protein responsible for the formation of pyramid-like cellular lysis structures.

    Science.gov (United States)

    Snyder, Jamie C; Brumfield, Susan K; Peng, Nan; She, Qunxin; Young, Mark J

    2011-07-01

    Host cells infected by Sulfolobus turreted icosahedral virus (STIV) have been shown to produce unusual pyramid-like structures on the cell surface. These structures represent a virus-induced lysis mechanism that is present in Archaea and appears to be distinct from the holin/endolysin system described for DNA bacteriophages. This study investigated the STIV gene products required for pyramid formation in its host Sulfolobus solfataricus. Overexpression of STIV open reading frame (ORF) c92 in S. solfataricus alone is sufficient to produce the pyramid-like lysis structures in cells. Gene disruption of c92 within STIV demonstrates that c92 is an essential protein for virus replication. Immunolocalization of c92 shows that the protein is localized to the cellular membranes forming the pyramid-like structures.

  18. Macromolecular fingerprinting of sulfolobus species in biofilm: a transcriptomic and proteomic approach combined with spectroscopic analysis.

    Science.gov (United States)

    Koerdt, Andrea; Orell, Alvaro; Pham, Trong Khoa; Mukherjee, Joy; Wlodkowski, Alexander; Karunakaran, Esther; Biggs, Catherine A; Wright, Phillip C; Albers, Sonja-Verena

    2011-09-02

    Microorganisms in nature often live in surface-associated sessile communities, encased in a self-produced matrix, referred to as biofilms. Biofilms have been well studied in bacteria but in a limited way for archaea. We have recently characterized biofilm formation in three closely related hyperthermophilic crenarchaeotes: Sulfolobus acidocaldarius, S. solfataricus, and S. tokodaii. These strains form different communities ranging from simple carpet structures in S. solfataricus to high density tower-like structures in S. acidocaldarius under static condition. Here, we combine spectroscopic, proteomic, and transcriptomic analyses to describe physiological and regulatory features associated with biofilms. Spectroscopic analysis reveals that in comparison to planktonic life-style, biofilm life-style has distinctive influence on the physiology of each Sulfolobus spp. Proteomic and transcriptomic data show that biofilm-forming life-style is strain specific (eg ca. 15% of the S. acidocaldarius genes were differently expressed, S. solfataricus and S. tokodaii had ~3.4 and ~1%, respectively). The -omic data showed that regulated ORFs were widely distributed in basic cellular functions, including surface modifications. Several regulated genes are common to biofilm-forming cells in all three species. One of the most striking common response genes include putative Lrs14-like transcriptional regulators, indicating their possible roles as a key regulatory factor in biofilm development.

  19. Sulfolobus tengchongensis Spindle-Shaped Virus STSV1: Virus-Host Interactions and Genomic Features

    DEFF Research Database (Denmark)

    Xiang, X.; Chen, L.; Huang, X

    2005-01-01

    A virus infecting the hyperthermophilic archaeon Sulfolobus tengchongensis has been isolated from a field sample from Tengchong, China, and characterized. The virus, denoted STSV1 (Sulfolobus tengchongensis spindle-shaped virus 1), has the morphology of a spindle (230 by 107 nm) with a tail...... contains a total of 74 open reading frames (ORFs), among which 14 have a putative function. Five ORFs encode viral structural proteins, including a putative coat protein of high abundance. The products of the other nine ORFs are probably involved in polysaccharide biosynthesis, nucleotide metabolism...

  20. Comparative study of the extracellular proteome of Sulfolobus species reveals limited secretion.

    Science.gov (United States)

    Ellen, Albert F; Albers, Sonja-Verena; Driessen, Arnold J M

    2010-01-01

    Although a large number of potentially secreted proteins can be predicted on the basis of genomic distribution of signal sequence-bearing proteins, protein secretion in Archaea has barely been studied. A proteomic inventory and comparison of the growth medium proteins in three hyperthermoacidophiles, i.e., Sulfolobus solfataricus, S. acidocaldarius and S. tokodaii, indicates that only few proteins are freely secreted into the growth medium and that the majority originates from cell envelope bound forms. In S. acidocaldarius both cell-associated and secreted alpha-amylase activities are detected. Inactivation of the amyA gene resulted in a complete loss of activity, suggesting that the same protein is responsible for the a-amylase activity at both locations. It is concluded that protein secretion in Sulfolobus is a limited process, and it is suggested that the S-layer may act as a barrier for the free diffusion of folded proteins into the medium.

  1. Characterization of the peroxidase activity of CYP119, a thermostable P450 from Sulfolobus acidocaldarius.

    Science.gov (United States)

    Rabe, Kersten S; Kiko, Kathrin; Niemeyer, Christof M

    2008-02-15

    We report the cloning, expression, and purification of CYP119, a thermostable enzyme previously thought to derive from Sulfolobus solfataricus. Sequence analysis suggested that, in contrast to the conclusions of earlier studies, the enzyme stems from the closely related Sulfolobus acidocaldarius, and we were indeed able to clone the gene from the genomic DNA of this organism. For the first time, we report here on the peroxidase activity of this enzyme and the optimization of the associated reaction parameters. The optimized reaction conditions were then applied to the biocatalytic epoxidation of styrene. The values obtained for k(cat) (78.2+/-20.6 min(-1)) and K(M) (9.2+/-4.3 mM) indicated an approximately 100-fold increased catalytic activity over previously reported results.

  2. Lysine and arginine biosyntheses mediated by a common carrier protein in Sulfolobus.

    Science.gov (United States)

    Ouchi, Takuya; Tomita, Takeo; Horie, Akira; Yoshida, Ayako; Takahashi, Kento; Nishida, Hiromi; Lassak, Kerstin; Taka, Hikari; Mineki, Reiko; Fujimura, Tsutomu; Kosono, Saori; Nishiyama, Chiharu; Masui, Ryoji; Kuramitsu, Seiki; Albers, Sonja-Verena; Kuzuyama, Tomohisa; Nishiyama, Makoto

    2013-04-01

    LysW has been identified as a carrier protein in the lysine biosynthetic pathway that is active through the conversion of α-aminoadipate (AAA) to lysine. In this study, we found that the hyperthermophilic archaeon, Sulfolobus acidocaldarius, not only biosynthesizes lysine through LysW-mediated protection of AAA but also uses LysW to protect the amino group of glutamate in arginine biosynthesis. In this archaeon, after LysW modification, AAA and glutamate are converted to lysine and ornithine, respectively, by a single set of enzymes with dual functions. The crystal structure of ArgX, the enzyme responsible for modification and protection of the amino moiety of glutamate with LysW, was determined in complex with LysW. Structural comparison and enzymatic characterization using Sulfolobus LysX, Sulfolobus ArgX and Thermus LysX identify the amino acid motif responsible for substrate discrimination between AAA and glutamate. Phylogenetic analysis reveals that gene duplication events at different stages of evolution led to ArgX and LysX.

  3. Interactions of Sulfolobus acidocaldarius with uranium

    Energy Technology Data Exchange (ETDEWEB)

    Reitz, T.; Merroun, M.L.; Rossberg, A.; Selenska-Pobell, S. [Inst. of Radiochemistry, Forschungszentrum Rossendorf, Dresden (Germany)

    2010-07-01

    Interactions of the acidothermophilic archaeon Sulfolobus acidocaldarius DSM 639 with U(VI) were studied by using a combination of batch experiments, X-ray absorption spectroscopy (XAS), and time-resolved laser-induced fluorescence spectroscopy (TRLFS). We demonstrated that at pH 2 this archaeal strain possesses a low tolerance to U(VI) and that its growth is limited to a uranium concentration below 1.1 mM. At similarly high acidic conditions (pH 1.5 and 3.0), covering the physiological pH growth optimum of S. acidocaldarius, at which U(VI) is soluble and highly toxic, rapid accumulation of the radionuclide by the cells of the strain occurred. About half of the uranium binding capacity was reached by the strain after an incubation of five minutes and nearly total saturation of the binding sites was achieved after 30 min. Both, EXAFS- and TRLF-spectroscopic analyses showed that the accumulated U(VI) was complexed mainly through organic phosphate groups. The EXAFS measurements revealed that U(VI) is coordinated to the organic phosphate ligands of the archaeal cells in a monodentate binding mode with an average U-P bond distance of 3.60 {+-} 0.02 A (orig.)

  4. Dynamic properties of the Sulfolobus CRISPR/Cas and CRISPR/Cmr systems when challenged with vector-borne viral and plasmid genes and protospacers

    DEFF Research Database (Denmark)

    Guðbergsdóttir, Sóley Ruth; Deng, Ling; Chen, Zhengjun

    2011-01-01

    The adaptive immune CRISPR/Cas and CRISPR/Cmr systems of the crenarchaeal thermoacidophile Sulfolobus were challenged by a variety of viral and plasmid genes, and protospacers preceded by different dinucleotide motifs. The genes and protospacers were constructed to carry sequences matching...... individual spacers of CRISPR loci, and a range of mismatches were introduced. Constructs were cloned into vectors carrying pyrE/pyrF genes and transformed into uracil auxotrophic hosts derived from Sulfolobus solfataricus P2 or Sulfolobus islandicus REY15A. Most constructs, including those carrying different...... protospacer mismatches, yielded few viable transformants. These were shown to carry either partial deletions of CRISPR loci, covering a broad spectrum of sizes and including the matching spacer, or deletions of whole CRISPR/Cas modules. The deletions occurred independently of whether genes or protospacers...

  5. A novel interference mechanism by a type IIIB CRISPR-Cmr module in Sulfolobus

    DEFF Research Database (Denmark)

    Deng, Ling; Garrett, Roger Antony; Shah, Shiraz Ali

    2013-01-01

    Recent studies on CRISPR-based adaptive immune systems have revealed extensive structural and functional diversity of the interference complexes which often coexist intracellularly. The archaeon Sulfolobus islandicus REY15A encodes three interference modules, one of type IA and two of type IIIB...... targeting. A rationale is provided for the intracellular coexistence of the different interference systems in S.¿islandicus REY15A which cooperate functionally by sharing a single Cas6 protein for crRNA processing and utilize crRNA products from identical CRISPR spacers....

  6. CRISPR families of the crenarchaeal genus Sulfolobus: bidirectional transcription and dynamic properties.

    Science.gov (United States)

    Lillestøl, Reidun K; Shah, Shiraz A; Brügger, Kim; Redder, Peter; Phan, Hien; Christiansen, Jan; Garrett, Roger A

    2009-04-01

    Clusters of regularly interspaced short palindromic repeats (CRISPRs) of Sulfolobus fall into three main families based on their repeats, leader regions, associated cas genes and putative recognition sequences on viruses and plasmids. Spacer sequence matches to different viruses and plasmids of the Sulfolobales revealed some bias particularly for family III CRISPRs. Transcription occurs on both strands of the five repeat-clusters of Sulfolobus acidocaldarius and a repeat-cluster of the conjugative plasmid pKEF9. Leader strand transcripts cover whole repeat-clusters and are processed mainly from the 3'-end, within repeats, yielding heterogeneous 40-45 nt spacer RNAs. Processing of the pKEF9 leader transcript occurred partially in spacers, and was incomplete, probably reflecting defective repeat recognition by host enzymes. A similar level of transcripts was generated from complementary strands of each chromosomal repeat-cluster and they were processed to yield discrete approximately 55 nt spacer RNAs. Analysis of the partially identical repeat-clusters of Sulfolobus solfataricus strains P1 and P2 revealed that spacer-repeat units are added upstream only when a leader and certain cas genes are linked. Downstream ends of the repeat-clusters are conserved such that deletions and recombination events occur internally.

  7. Crystal structure of the S. solfataricus archaeal exosome reveals conformational flexibility in the RNA-binding ring.

    Directory of Open Access Journals (Sweden)

    Changrui Lu

    Full Text Available BACKGROUND: The exosome complex is an essential RNA 3'-end processing and degradation machinery. In archaeal organisms, the exosome consists of a catalytic ring and an RNA-binding ring, both of which were previously reported to assume three-fold symmetry. METHODOLOGY/PRINCIPAL FINDINGS: Here we report an asymmetric 2.9 A Sulfolobus solfataricus archaeal exosome structure in which the three-fold symmetry is broken due to combined rigid body and thermal motions mainly within the RNA-binding ring. Since increased conformational flexibility was also observed in the RNA-binding ring of the related bacterial PNPase, we speculate that this may reflect an evolutionarily conserved mechanism to accommodate diverse RNA substrates for degradation. CONCLUSION/SIGNIFICANCE: This study clearly shows the dynamic structures within the RNA-binding domains, which provides additional insights on mechanism of asymmetric RNA binding and processing.

  8. Comparative genome sequence analysis of Sulfolobus acidocaldarius and 9 other isolates of its genus for factors influencing codon and amino acid usage.

    Science.gov (United States)

    Nayak, Kinshuk Chandra

    2013-01-15

    In the present study, major constraints for codon and amino acid usage of Sulfolobus acidocaldarius, Sulfolobus solfataricus, Sulfolobus tokodali, Sulfolobus islandis and 6 other isolates from islandicus species of genus Sulfolobus were investigated. Correspondence analysis revealed high significant correlation between the major trend of synonymous codon usage and gene expression level, as assessed by the "Codon Adaptation Index" (CAI). There is a significant negative correlation between Nc (Effective number of codons) and CAI demonstrating role of codon bias as an important determinant of codon usage. The significant correlation between major trend of synonymous codon usage and GC3s (G+C at third synonymous position) indicated dominant role of mutational bias in codon usage pattern. The result was further supported from SCUO (synonymous codon usage order) analysis. The amino acid usage was found to be significantly influenced by aromaticity and hydrophobicity of proteins. However, translational selection which causes a preference for codons that are most rapidly translated by current tRNA with multiple copy numbers was not found to be highly dominating for all studied isolates. Notably, 26 codons that were found to be optimally used by genes of S. acidocaldarius at higher expression level and its comparative analysis with 9 other isolates may provide some useful clues for further in vivo genetic studies on this genus.

  9. Genetic engineering of sulfur-degrading Sulfolobus

    Energy Technology Data Exchange (ETDEWEB)

    Ho, N.W.Y.

    1991-01-01

    The objectives of the proposed research is to first establish a plasmid-mediated genetic transformation system for the sulfur degrading Sulfolobus, and then to clone and overexpress the genes encoding the organic-sulfur-degrading enzymes from Sulfolobus- as well as from other microorganisms, to develop a Sulfolobus-based microbial process for the removal of both organic and inorganic sulfur from coal.

  10. A novel integrative expression vector for Sulfolobus species.

    Science.gov (United States)

    Choi, Kyoung-Hwa; Hwang, Sungmin; Yoon, Naeun; Cha, Jaeho

    2014-11-28

    With the purpose of facilitating the process of stable strain generation, a shuttle vector for integration of genes via a double recombination event into two ectopic sites on the Sulfolobus acidocaldarius chromosome was constructed. The novel chromosomal integration and expression vector pINEX contains a pyrE gene from S. solfataricus P2 (pyrE(sso)) as an auxotrophic selection marker, a multiple cloning site with histidine tag, the internal sequences of malE and malG for homologous recombination, and the entire region of pGEM-T vector, except for the multiple cloning region, for propagation in E. coli. For stable expression of the target gene, an α-glucosidase-producing strain of S. acidocaldarius was generated employing this vector. The malA gene (saci_1160) encoding an α-glucosidase from S. acidocaldarius fused with the glutamate dehydrogenase (gdhA(saci)) promoter and leader sequence was ligated to pINEX to generate pINEX_malA. Using the "pop-in" and "pop-out" method, the malA gene was inserted into the genome of MR31 and correct insertion was verified by colony PCR and sequencing. This strain was grown in YT medium without uracil and purified by His-tag affinity chromatography. The α-glucosidase activity was confirmed by the hydrolysis of pNPαG. The pINEX vector should be applicable in delineating gene functions in this organism.

  11. Structure of the dimeric form of CTP synthase from Sulfolobus solfataricus

    DEFF Research Database (Denmark)

    Lauritsen, Iben; Willemoës, Martin; Jensen, Kaj Frank;

    2011-01-01

    CTP synthase catalyzes the last committed step in de novo pyrimidine-nucleotide biosynthesis. Active CTP synthase is a tetrameric enzyme composed of a dimer of dimers. The tetramer is favoured in the presence of the substrate nucleotides ATP and UTP; when saturated with nucleotide, the tetramer c...

  12. The architects of crenarchaeal chromatin : A biophysical characterization of chromatin proteins from Sulfolobus solfataricus

    NARCIS (Netherlands)

    Driessen, Rosalie Paula Catharina

    2014-01-01

    Understanding of chromatin organization and compaction in Archaea is currently limited. The genome of several megabasepairs long is folded by a set of small chromatin proteins to fit into the micron-sized cell. A first step in understanding archaeal chromatin organization is to study the action of i

  13. Bioaccumulation of U(VI) by Sulfolobus acidocaldarius under moderate acidic conditions

    Energy Technology Data Exchange (ETDEWEB)

    Reitz, T.; Merroun, M.L.; Rossberg, A.; Steudtner, R.; Selenska-Pobell, S. [Helmholtz-Zentrum Dresden-Rossendorf, Dresden (Germany). Inst. of Radiochemistry

    2011-07-01

    U(VI) accumulation by the acidothermophilic archaeon Sulfolobus acidocaldarius at a moderate acidic pH of 4.5 was investigated. This pH value is relevant for some heavy metal and uranium polluted environments where populations of S. acidocaldarius were found to persist. We demonstrate that U(VI) is rapidly complexed by the archaeal cells. A combination of X-ray absorption spectroscopy and time-resolved laser-induced fluorescence spectroscopy revealed that at pH 4.5 organic phosphate and carboxylic groups are involved in the U(VI) complexation. These results are in contrast to those published for most bacteria which at this pH precipitate U(VI) mainly in inorganic uranyl phosphate phases. As demonstrated by TEM only a limited part of the added U(VI) was biomineralized extracellularly in the case of the studied archaeon. Most of the U(VI) accumulates were localized in a form of intracellular deposits which were associated with the inner side of the cytoplasma membrane. Observed differences in U(VI) bioaccumulation between the studied archaeon and bacteria can be explained by the significant differences in their cell wall structures as well as by their different physiological characteristics. (orig.)

  14. Alterations of the transcriptome of Sulfolobus acidocaldarius by exoribonuclease aCPSF2.

    Science.gov (United States)

    Märtens, Birgit; Amman, Fabian; Manoharadas, Salim; Zeichen, Lukas; Orell, Alvaro; Albers, Sonja-Verena; Hofacker, Ivo; Bläsi, Udo

    2013-01-01

    Recent studies identified a 5´ to 3´ exoribonuclease termed Sso-RNase J in the crenarchaeon Sulfolobus solfataricus (Sso), which has been reclassified to the aCPSF2 (archaeal cleavage and polyadenylation specificity factor 2) group of β-CASP proteins. In this study, the Sso-aCPSF2 orthologue of Sulfolobus acidocaldarius (Saci-aCPSF2) was functionally characterized. Like Sso-aCPSF2, Saci-aCPSF2 degrades RNA with 5´ to 3´ directionality in vitro. To address the biological significance of Saci-aCPSF2, a deletion mutant was constructed, and the influence of Saci-aCPSF2 on the transcriptome profile was assessed employing high throughput RNA sequencing. This analysis revealed 560 genes with differential transcript abundance, suggesting a considerable role of this enzyme in RNA metabolism. In addition, bioinformatic analyses revealed several transcripts that are preferentially degraded at the 5´ end. This was exemplarily verified for two transcripts by Northern-blot analyses, showing for the first time that aCPSF2 proteins play a role in 5' to 3' directional mRNA decay in the crenarchaeal clade of Archaea.

  15. Alterations of the transcriptome of Sulfolobus acidocaldarius by exoribonuclease aCPSF2.

    Directory of Open Access Journals (Sweden)

    Birgit Märtens

    Full Text Available Recent studies identified a 5´ to 3´ exoribonuclease termed Sso-RNase J in the crenarchaeon Sulfolobus solfataricus (Sso, which has been reclassified to the aCPSF2 (archaeal cleavage and polyadenylation specificity factor 2 group of β-CASP proteins. In this study, the Sso-aCPSF2 orthologue of Sulfolobus acidocaldarius (Saci-aCPSF2 was functionally characterized. Like Sso-aCPSF2, Saci-aCPSF2 degrades RNA with 5´ to 3´ directionality in vitro. To address the biological significance of Saci-aCPSF2, a deletion mutant was constructed, and the influence of Saci-aCPSF2 on the transcriptome profile was assessed employing high throughput RNA sequencing. This analysis revealed 560 genes with differential transcript abundance, suggesting a considerable role of this enzyme in RNA metabolism. In addition, bioinformatic analyses revealed several transcripts that are preferentially degraded at the 5´ end. This was exemplarily verified for two transcripts by Northern-blot analyses, showing for the first time that aCPSF2 proteins play a role in 5' to 3' directional mRNA decay in the crenarchaeal clade of Archaea.

  16. Isolation and cultivation of Walsby's square archaeon

    NARCIS (Netherlands)

    Bolhuis, H; Poele, EMT; Rodriguez-Valera, F

    2004-01-01

    In 1980, A. E. Walsby described a square halophilic archaeon. This archaeon is of specific interest because of its unique shape and its abundance in hypersaline ecosystems, which suggests an important ecophysiological role. Ever since its discovery, the isolation and cultivation of 'Walsby's square

  17. Studies on structural stability of thermophilic Sulfolobus acidocaldarius ribosomes.

    Science.gov (United States)

    Yangala, Kalavathi; Suryanarayana, Tangirala

    2007-02-01

    Structural stability of thermophilic archaeon Sulfolobus acidocaldarius ribosomes, with respect their susceptibility to pancreatic RNase A and stability to temperature (deltaTm), on treatment with various stabilizing (polyamines) and destabilizing (sulfhydryl and intercalating) agents were studied and compared with mesophilic E. coli ribosomes, to understand the structural differences between thermophilic and mesophilic ribosomes. Thermophilic archaeal ribosomes and their subunits were 10-times less susceptible to pancreatic RNase A, compared to mesophilic ribosomes, showing the presence of strong and compact structural organization in them. Thermophilic ribosomes treated with destabilizing agents, such as sulfhydryl reagents [5,5'-Dithio-bis-(2-nitrobenzoic acid), N-ethylmaleimide and p-hydroxymercurybenzoate) and intercalating agents (ethidium bromide, EtBr) showed higher stability to RNase A, compared to similarly treated mesophilic ribosomes, indicating the unavailability of thiol-reactive groups and the presence of strong solvent inaccessible inner core. Higher stability of thermophilic ribosomes compared to mesophilic ribosomes to unfolding agents like urea further supported the presence of strong inner core particle. Thermophilic ribosomes treated with intercalating agents, such as EtBr were less susceptible to RNase A, though they bound to more reagent, showing the rigidity or resilience of their macromolecular structure to alterations caused by destabilizing agents. Overall, these results indicated that factors such as presence of strong solvent inaccessible inner core and rigidity of ribosome macromolecular structure contributed stability of thermophilic ribosomes to RNase A and other destabilizing agents, when compared to mesophilic ribosomes.

  18. Characterization of two β-decarboxylating dehydrogenases from Sulfolobus acidocaldarius.

    Science.gov (United States)

    Takahashi, Kento; Nakanishi, Fumika; Tomita, Takeo; Akiyama, Nagisa; Lassak, Kerstin; Albers, Sonja-Verena; Kuzuyama, Tomohisa; Nishiyama, Makoto

    2016-11-01

    Sulfolobus acidocaldarius, a hyperthermoacidophilic archaeon, possesses two β-decarboxylating dehydrogenase genes, saci_0600 and saci_2375, in its genome, which suggests that it uses these enzymes for three similar reactions in lysine biosynthesis through 2-aminoadipate, leucine biosynthesis, and the tricarboxylic acid cycle. To elucidate their roles, these two genes were expressed in Escherichia coli in the present study and their gene products were characterized. Saci_0600 recognized 3-isopropylmalate as a substrate, but exhibited slight and no activity for homoisocitrate and isocitrate, respectively. Saci_2375 exhibited distinct and similar activities for isocitrate and homoisocitrate, but no detectable activity for 3-isopropylmalate. These results suggest that Saci_0600 is a 3-isopropylmalate dehydrogenase for leucine biosynthesis and Saci_2375 is a dual function enzyme serving as isocitrate-homoisocitrate dehydrogenase. The crystal structure of Saci_0600 was determined as a closed-form complex that binds 3-isopropylmalate and Mg(2+), thereby revealing the structural basis for the extreme thermostability and novel-type recognition of the 3-isopropyl moiety of the substrate.

  19. Structural insight into substrate binding and catalysis of a novel 2-keto-3-deoxy-D-arabinonate dehydratase illustrates common mechanistic features of the FAH superfamily

    NARCIS (Netherlands)

    Brouns, S.J.J.; Barends, T.R.M.; Worm, P.; Akerboom, J.; Turnbull, A.P.; Salmon, L.; Oost, van der J.

    2008-01-01

    The archaeon Sulfolobus solfataricus converts d-arabinose to 2-oxoglutarate by an enzyme set consisting of two dehydrogenases and two dehydratases. The third step of the pathway is catalyzed by a novel 2-keto-3-deoxy-D-arabinonate dehydratase (KdaD). In this study, the crystal structure of the enzym

  20. Genes of de novo pyrimidine biosynthesis from the hyperthermoacidophilic crenarchaeote Sulfolobus acidocaldarius: novel organization in a bipolar operon.

    Science.gov (United States)

    Thia-Toong, Thia-Lin; Roovers, Martine; Durbecq, Virginie; Gigot, Daniel; Glansdorff, Nicolas; Charlier, Daniel

    2002-08-01

    Sequencing a 8,519-bp segment of the Sulfolobus acidocaldarius genome revealed the existence of a tightly packed bipolar pyrimidine gene cluster encoding the enzymes of de novo UMP synthesis. The G+C content of 35.3% is comparable to that of the entire genome, but intergenic regions exhibit a considerably lower percentage of strong base pairs. Coding regions harbor the classical excess of purines on the coding strand, whereas intergenic regions do not show this bias. Reverse transcription-PCR and primer extension experiments demonstrated the existence of two polycistronic messengers, pyrEF-orf8 and pyrBI-orf1-pyrCD-orf2-orf3-orf4, initiated from a pair of divergent and partially overlapping promoters. The gene order and the grouping in two wings of a bipolar operon constitute a novel organization of pyr genes that also occurs in the recently determined genome sequences of Sulfolobus solfataricus P2 and Sulfolobus tokodaii strain 7; the configuration appears therefore characteristic of Sulfolobus. The quasi-leaderless pyrE and pyrB genes do not bear a Shine-Dalgarno sequence, whereas the initiation codon of promoter-distal genes is preceded at an appropriate distance by a sequence complementary to the 3' end of 16S rRNA. The polycistronic nature of the pyr messengers and the existence of numerous overlaps between contiguous open reading frames suggests the existence of translational coupling. pyrB transcription was shown to be approximately twofold repressed in the presence of uracil. The mechanism underlying this modulation is as yet unknown, but it appears to be of a type different from the various attenuation-like mechanisms that regulate pyrB transcription in bacteria. In contrast, the pyrE-pyrB promoter/control region harbors direct repeats and imperfect palindromes reminiscent of target sites for the binding of a hypothetical regulatory protein(s).

  1. Genetic Studies on CRISPR-Cas Functions in Invader Defense in Sulfolobus islandicus

    DEFF Research Database (Denmark)

    Peng, Wenfang

    Archaea and bacteria contain CRISPR-Cas (clustered regularly interspaced short palindromic repeat-CRISPR-associated) systems that protect themselves against invasion by viruses and plasmids. There are three major types of CRISPR-Cas systems, type I, II and III, that are further divided...... into at least 11 subtypes. I employed Sulfolobus islandicus Rey15A as the model to study CRISPR mechanisms. The model archaeon encodes one subtype I-A (Cascade) and two subtype III-B (Cmr-α and Cmr-β) interference systems with no apparent redundancy in cas genes or in CRISPR systems, which is ideal for genetic...... analysis of cas gene function. Furthermore, a range of genetic tools have been developed for S. islandicus Rey15A in our laboratory and a plasmid interference assay has been successfully developed for testing CRISPR-directed DNA targeting activity, which have provided a solid basis for studying...

  2. Crystal structure of Sulfolobus acidocaldarius aspartate carbamoyltransferase in complex with its allosteric activator CTP.

    Science.gov (United States)

    De Vos, Dirk; Xu, Ying; Aerts, Tony; Van Petegem, Filip; Van Beeumen, Jozef J

    2008-07-18

    Aspartate carbamoyltransferase (ATCase) is a paradigm for allosteric regulation of enzyme activity. B-class ATCases display very similar homotropic allosteric behaviour, but differ extensively in their heterotropic patterns. The ATCase from the thermoacidophilic archaeon Sulfolobus acidocaldarius, for example, is strongly activated by its metabolic pathway's end product CTP, in contrast with Escherichia coli ATCase which is inhibited by CTP. To investigate the structural basis of this property, we have solved the crystal structure of the S. acidocaldarius enzyme in complex with CTP. Structure comparison reveals that effector binding does not induce similar large-scale conformational changes as observed for the E. coli ATCase. However, shifts in sedimentation coefficients upon binding of the bi-substrate analogue PALA show the existence of structurally distinct allosteric states. This suggests that the so-called "Nucleotide-Perturbation model" for explaining heterotropic allosteric behaviour, which is based on global conformational strain, is not a general mechanism of B-class ATCases.

  3. Pyrite oxidation by sulfolobus acidocaldarius

    Energy Technology Data Exchange (ETDEWEB)

    Tobita, M.; Yokozeki, M.; Nishikawa, N.; Kawakami, Y. (Institute of Research and Innovation, Kashiwa (Japan). Dept. of Biotechnology)

    1994-04-01

    Two strains of Sulfolobus acidocaldarius, a thermoacidophilic archaebacterium, were examined for their pyrite-oxidizing ability. S. acidocaldarius ATCC 33909 was shown to possess iron-oxidizing activity by ferrous sulfate oxidizing experiments, but S. acidocaldarius No. 7 did not have it. Pyrite-oxidizing rate of S. acidocaldarius ATCC 33909 was 1.6-fold higher than that of strain 7 though they had a similar level of self-oxidizing ability. These results show that the iron-oxidizing activity accelerates pyrite oxidation.

  4. Characterization of Sac10a, a hyperthermophile DNA-binding protein from Sulfolobus acidocaldarius.

    Science.gov (United States)

    Edmondson, Stephen P; Kahsai, Mebrahtu A; Gupta, Ramesh; Shriver, John W

    2004-10-19

    Sac10a is a member of a group of basic DNA-binding proteins thought to be important in chromatin structure and regulation in the archaeon Sulfolobus. We describe here the isolation, gene identification, and biophysical characterization of native Sac10a. The protein exists as a 23.8 kDa homodimer at pH 7 and unfolds with a T degrees of 122 degrees C. Dissociation of the dimer into folded globular subunits is promoted by decreased pH and salt concentration. Thermal unfolding of the monomeric subunits occurred with two transitions, indicating two independent domains. The dimer demonstrated a high affinity for duplex poly(dAdT) with a K(D) of 5 x 10(-)(10) M and a site size of 17 bp (in 0.15 M KCl, pH 7), with only weak binding (K(D) > 5 x 10(-)(6) M) to poly(dA)-poly(dT), poly(dGdC), poly(dG)-poly(dC), and Escherichia coli DNA under similar conditions. Binding to poly(dAdT) resulted in distortions in the DNA duplex that were consistent with overwinding as indicated by inversion of the CD spectrum of the DNA. The monomeric subunits are predicted to adopt a winged helix DNA-binding motif which dimerizes through formation of a two-stranded coiled coil involving an extended C-terminal helix with more than four heptad repeats (about 45 A in length). This is the first example of the conserved archaeal transcription regulator domain COG3432 to be characterized. Sequences for homologous proteins containing both COG3432 and predicted coiled coil domains occur in the genomes of both crenarchaeota (Sulfolobus, Pyrobaculum, Aeropyrum) and euryarchaeota (Methanosarcina, Methanococcus, Archaeoglobus, Thermoplasma), with multiple genes in some species. Sac10a shows no sequence similarity to the other Sulfolobus chromatin proteins Sac7d, Sac8, Sso10b2, and Alba.

  5. The Sulfolobicin Genes of Sulfolobus acidocaldarius Encode Novel Antimicrobial Proteins

    NARCIS (Netherlands)

    Ellen, Albert F.; Rohulya, Olha V.; Fusetti, Fabrizia; Wagner, Michaela; Albers, Sonja-Verena; Driessen, Arnold J. M.

    2011-01-01

    Crenarchaea, such as Sulfolobus acidocaldarius and Sulfolobus tokodaii, produce antimicrobial proteins called sulfolobicins. These antimicrobial proteins inhibit the growth of closely related species. Here we report the identification of the sulfolobicin-encoding genes in S. acidocaldarius. The acti

  6. Incorporation of nucleoside probes opposite O⁶-methylguanine by Sulfolobus solfataricus DNA polymerase Dpo4: importance of hydrogen bonding.

    Science.gov (United States)

    Stornetta, Alessia; Angelov, Todor; Guengerich, F Peter; Sturla, Shana J

    2013-09-02

    O⁶-Methylguanine (O⁶-MeG) is a mutagenic DNA lesion, arising from the action of methylating agents on guanine (G) in DNA. Dpo4, an archaeal low-fidelity Y-family DNA polymerase involved in translesion DNA synthesis (TLS), is a model for studying how human Y-family polymerases bypass DNA adducts. Previous work showed that Dpo4-mediated dTTP incorporation is favored opposite O⁶-MeG rather than opposite G. However, factors influencing the preference of Dpo4 to incorporate dTTP opposite O⁶-MeG are not fully defined. In this study, we investigated the influence of structural features of incoming dNTPs on their enzymatic incorporation opposite O⁶-MeG in a DNA template. To this end, we utilized a new fluorescence-based primer extension assay to evaluate the incorporation efficiency of a panel of synthetic dNTPs opposite G or O⁶-MeG by Dpo4. In single-dNTP primer extension studies, the synthetic dNTPs were preferentially incorporated opposite G, relative to O⁶-MeG. Moreover, pyrimidine-based dNTPs were generally better incorporated than purine-based syn-conformation dNTPs. The results suggest that hydrophobicity of the incoming dNTP appears to have little influence on the process of nucleotide selection by Dpo4, with hydrogen bonding capacity being a major influence. Additionally, modifications at the C2-position of dCTP increase the selectivity for incorporation opposite O⁶-MeG without a significant loss of efficiency.

  7. A Bac Library and Paired-PCR Approach to Mapping and Completing the Genome Sequence of Sulfolobus Solfataricus P2

    DEFF Research Database (Denmark)

    She, Qunxin; Confalonieri, F.; Zivanovic, Y.;

    2000-01-01

    -productive because there was a high sequence bias in the cosmid and lambda libraries. Therefore, a new approach was devised for linking the sequenced regions which may be generally applicable. BAC libraries were constructed and terminal sequences of the clones were determined and used for both end mapping and PCR...... screening. The PCR approaches included a novel chromosome walking method termed “paired-PCR”. 21 gaps were filled by BAC end sequence analyses and 6 gaps were filled by PCR including three large ones by paired-PCR. The complete map revealed that 0.9 Mb remained to be sequenced and 34 BAC clones were...... selected for walking over small gaps and preparing template libraries for larger ones. It is concluded that an optimal strategy for sequencing microorganism genomes involves construction of a high-resolution physical map by BAC end analyses, PCR screening and paired-PCR chromosome walking after about half...

  8. Genetic engineering of sulfur-degrading Sulfolobus

    Energy Technology Data Exchange (ETDEWEB)

    Ho, N.W.Y. (Purdue Univ., Lafayette, IN (USA). Lab. of Renewable Resources Engineering)

    1991-01-01

    Recent studies have shown that some microorganisms can play a significant role in removing the sulfur compound from coal. Sulfolobus acidocaldarius and related species are such microorganisms. The objective of this project is to develop a genetic transformation system for Sulfolobus species so that they could become the ideal host to overproduce homologous and heterologous enzymes that are most effective for the removal of sulfur from coal, particularly organic sulfur. Last quarter, we have identified three chemicals that can inhibit the growth of S. Acidocaldarius. These chemicals can be part of the selection system for the development of a transformation system for S. acidocaldarius. Due to the fact that Sulfolobus shibatae B12 becomes increasingly more attractive as a host for housing genes encoding desulfurization enzymes, in this period we also studied the affect of these three chemicals to growth of S. shibatae B12. We found that S. shibatae B12 is also sensitive to these chemicals. This quarter we succeeded in the isolation and purification of the double-stranded DNA virus from S. shibatae B12. Furthermore, the individual EcoRI and BamH1 fragments of the virus have also been cloned into pUC19 plasmid. These plasmids will be used for the construction of the final E. coli-Sulfolobus shuttle vector. 5 Flurouracil (5FU) is one of the chemicals that inhibit growth of Sulfolobus. Resistance strain of S. acidocaldarius to 5FU has also been isolated. DNA from the 5FU resistance strain has also been isolated. 2 figs.

  9. Genetic engineering of sulfur-degrading Sulfolobus

    Energy Technology Data Exchange (ETDEWEB)

    Ho, N.W.Y. (Purdue Univ., Lafayette, IN (United States). Lab. of Renewable Resources Engineering)

    1991-01-01

    An effective microbial process would be economical for the removal or organic sulfur and the finely dispersed inorganic sulfur in coal, particularly the former. However, some microoganisms may have the enzymes to degrade organic sulfur but themselves are not suitable to be cultured under the conditions best for coal desulfurization. On the other hand, some microorganisms can grow very well under the conditions most suitable for coal desulfurization but do not contain the enzymes most suitable for coal desulfurization. We believe that we can adopt a new approach to solve this problem. In this approach, a microorganism that can grow vigorously under the conditions most suitable for coal desulfurization will be chosen as the host for actively carrying out the desulfurization process. An effective gene cloning system is expected to be established for the chosen species so that heterologous and homologous genes encoding most of the effective enzymes for coal desulfurization can be cloned on the highly copy-number plasmid or plasmid-like molecules. Species of Sulfolobus are microorganisms that can be actively cultured under the condition most favorable for coal desulfurization. In particular, one of the Sulfolobus species, S. shibatae B12, contains a double-stranded DNA virus which might be easily converted into a gene cloning system. The objective of this project is to develop an effective gene cloning system for some of the Sulfolobus species. During the first two quarters of this year, we have shown that gene cloning systems might be able to be established for at least two of the Sulfolobus species; S. acidocaldarius and S. shibatae B12. In this quarter, we report on: (1) The construction of potential cloning vectors for the transformation of both S. acidocaldarius and S. shibatae B12; (2) the isolation of promoters from S. Shibatae and S. acidocaldarius, which can also function as promoters in E. coli.

  10. Induction of the Sulfolobus shibatae virus SSV1 DNA replication by mitomycin C

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    The temperate virus SSV1 from the hyperthermophilic archaeon Sulfolobus shibatae provides a useful model system for the study of archaeal DNA replication. Southern hybridization showed that SSV1 existed primarily as a provirus in its host that was grown without shaking. Upon UV or mitomycin C induction, the cellular level of free SSV1 DNA increased drastically whereas that of integrated viral DNA remained unchanged. The results of mitomycin C induction were more reproducible than those of UV induction. We found that, when the cells that had been grown without shaking were shaken, the replication of SSV1 DNA was also induced. Based on our results, we developed a method for the induction of SSV1 DNA replication by mitomycin C. When the S. shibatae virus production was induced using this method, the cellular level of free SSV1 DNA started to increase 10 h after induction, and peaked after 12-15 h. A fully induced S. shibatae cell contained ~50 molecules of free SSV1 DNA. The development of this induction method and the description of the process of SSV1 DNA replication following induction are valuable to the analysis of the origin and mode of replication of the virus.

  11. Effect of physical constraints on the mechanisms of membrane fusion: bolaform lipid vesicles as model systems.

    OpenAIRE

    1996-01-01

    Bolaform lipid vesicles were used to study the effect of physical constraints on membrane fusion. In these vesicles the membrane is organized in a single monolayer, because of the presence of covalent bonds in its middle plane. Therefore, the formation of fusion intermediates is subject to higher energy barriers and greater geometrical constraints than is usual in bilayer membranes. Bolaform lipids were extracted from the thermophilic archaeon Sulfolobus solfataricus. These lipids can be divi...

  12. Genetic engineering of sulfur-degrading Sulfolobus. Final technical report, September 1, 1990--August 31, 1991

    Energy Technology Data Exchange (ETDEWEB)

    Ho, N.W.Y.

    1991-12-31

    The objectives of the proposed research is to first establish a plasmid-mediated genetic transformation system for the sulfur degrading Sulfolobus, and then to clone and overexpress the genes encoding the organic-sulfur-degrading enzymes from Sulfolobus- as well as from other microorganisms, to develop a Sulfolobus-based microbial process for the removal of both organic and inorganic sulfur from coal.

  13. Endogenous mutagenesis in recombinant sulfolobus plasmids.

    Science.gov (United States)

    Sakofsky, Cynthia J; Grogan, Dennis W

    2013-06-01

    Low rates of replication errors in chromosomal genes of Sulfolobus spp. demonstrate that these extreme thermoacidophiles can maintain genome integrity in environments with high temperature and low pH. In contrast to this genetic stability, we observed unusually frequent mutation of the β-D-glycosidase gene (lacS) of a shuttle plasmid (pJlacS) propagated in Sulfolobus acidocaldarius. The resulting Lac(-) mutants also grew faster than the Lac(+) parent, thereby amplifying the impact of the frequent lacS mutations on the population. We developed a mutant accumulation assay and corrections for the effects of copy number and differential growth for this system; the resulting measurements and calculations yielded a corrected rate of 5.1 × 10(-4) mutational events at the lacS gene per plasmid replication. Analysis of independent lacS mutants revealed three types of mutations: (i) G · C-to-A · T transitions, (ii) slipped-strand events, and (iii) deletions. These mutations were frequent in plasmid-borne lacS expressed at a high level but not in single-copy lacS in the chromosome or at lower levels of expression in a plasmid. Substitution mutations arose at only two of 12 potential priming sites of the DNA primase of the pRN1 replicon, but nearly all these mutations created nonsense (chain termination) codons. The spontaneous mutation rate of plasmid-borne lacS was 175-fold higher under high-expression than under low-expression conditions. The results suggest that important DNA repair or replication fidelity functions are impaired or overwhelmed in pJlacS, with results analogous to those of the "transcription-associated mutagenesis" seen in bacteria and eukaryotes.

  14. Electron cryotomography of ESCRT assemblies and dividing Sulfolobus cells suggests that spiraling filaments are involved in membrane scission.

    Science.gov (United States)

    Dobro, Megan J; Samson, Rachel Y; Yu, Zhiheng; McCullough, John; Ding, H Jane; Chong, Parkson Lee-Gau; Bell, Stephen D; Jensen, Grant J

    2013-08-01

    The endosomal-sorting complex required for transport (ESCRT) is evolutionarily conserved from Archaea to eukaryotes. The complex drives membrane scission events in a range of processes, including cytokinesis in Metazoa and some Archaea. CdvA is the protein in Archaea that recruits ESCRT-III to the membrane. Using electron cryotomography (ECT), we find that CdvA polymerizes into helical filaments wrapped around liposomes. ESCRT-III proteins are responsible for the cinching of membranes and have been shown to assemble into helical tubes in vitro, but here we show that they also can form nested tubes and nested cones, which reveal surprisingly numerous and versatile contacts. To observe the ESCRT-CdvA complex in a physiological context, we used ECT to image the archaeon Sulfolobus acidocaldarius and observed a distinct protein belt at the leading edge of constriction furrows in dividing cells. The known dimensions of ESCRT-III proteins constrain their possible orientations within each of these structures and point to the involvement of spiraling filaments in membrane scission.

  15. Genomics and genetics of Sulfolobus islandicus LAL14/1, a model hyperthermophilic archaeon

    DEFF Research Database (Denmark)

    Jaubert, Carole; Danioux, Chloë; Oberto, Jacques

    2013-01-01

    common core genome of approximately 2 Mb and a long hyperplastic region containing most of the strain-specific genes. In LAL14/1, the latter region is enriched in insertion sequences, CRISPR (clustered regularly interspaced short palindromic repeats), glycosyl transferase genes, toxin-antitoxin genes...... and MITE (miniature inverted-repeat transposable elements). The tRNA genes of LAL14/1 are preferential targets for the integration of mobile elements but clusters of atypical genes (CAG) are also integrated elsewhere in the genome. LAL14/1 carries five CRISPR loci with 10 per cent of spacers matching...... perfectly or imperfectly the genomes of archaeal viruses and plasmids found in the Icelandic hot springs. Strikingly, the CRISPR_2 region of LAL14/1 carries an unusually long 1.9 kb spacer interspersed between two repeat regions and displays a high similarity to pING1-like conjugative plasmids. Finally, we...

  16. A synthetic arabinose-inducible promoter confers high levels of recombinant protein expression in hyperthermophilic archaeon Sulfolobus islandicus

    DEFF Research Database (Denmark)

    Peng, Nan; Deng, Ling; Mei, Yuxia;

    2012-01-01

    levels of target gene expression. More strikingly, N-terminal amino acid sequencing of recombinant proteins unraveled that the protein synthesized from pEXA-N-lacS lacked the designed 6×His tag and that translation initiation did not start at the ATG codon of the fusion gene. Instead, it started...

  17. Genomics and genetics of Sulfolobus islandicus LAL14/1, a model hyperthermophilic archaeon

    DEFF Research Database (Denmark)

    Jaubert, Carole; Danioux, Chloë; Oberto, Jacques;

    2013-01-01

    have developed a genetic system for S. islandicus LAL14/1 and created ¿pyrEF and ¿CRISPR_1 mutants using double cross-over and pop-in/pop-out approaches, respectively. Thus, LAL14/1 is a promising model to study virus-host interactions and the CRISPR/Cas defence mechanism in Archaea....... common core genome of approximately 2 Mb and a long hyperplastic region containing most of the strain-specific genes. In LAL14/1, the latter region is enriched in insertion sequences, CRISPR (clustered regularly interspaced short palindromic repeats), glycosyl transferase genes, toxin-antitoxin genes...... and MITE (miniature inverted-repeat transposable elements). The tRNA genes of LAL14/1 are preferential targets for the integration of mobile elements but clusters of atypical genes (CAG) are also integrated elsewhere in the genome. LAL14/1 carries five CRISPR loci with 10 per cent of spacers matching...

  18. Structural characterization of ether lipids from the archaeon Sulfolobus islandicus by high-resolution shotgun lipidomics

    DEFF Research Database (Denmark)

    Jensen, Sara Munk; Brandl, Martin; Treusch, Alexander H;

    2015-01-01

    -resolution Fourier transform mass spectrometry using an ion trap-orbitrap mass spectrometer. This analysis identified five clusters of molecular ions that matched ether lipids in the database with sub-ppm mass accuracy. To structurally characterize and validate the identities of the potential lipid species, we...

  19. The sulfolobicin genes of Sulfolobus acidocaldarius encode novel antimicrobial proteins.

    Science.gov (United States)

    Ellen, Albert F; Rohulya, Olha V; Fusetti, Fabrizia; Wagner, Michaela; Albers, Sonja-Verena; Driessen, Arnold J M

    2011-09-01

    Crenarchaea, such as Sulfolobus acidocaldarius and Sulfolobus tokodaii, produce antimicrobial proteins called sulfolobicins. These antimicrobial proteins inhibit the growth of closely related species. Here we report the identification of the sulfolobicin-encoding genes in S. acidocaldarius. The active sulfolobicin comprises two proteins that are equipped with a classical signal sequence. These proteins are secreted by the cells and found to be membrane vesicle associated. Gene inactivation studies demonstrate that both proteins are required for the bacteriostatic antimicrobial activity. Sulfolobicins constitute a novel class of antimicrobial proteins without detectable homology to any other protein.

  20. Genetic engineering of sulfur-degrading Sulfolobus

    Energy Technology Data Exchange (ETDEWEB)

    Ho, N.W.Y. (Purdue Univ., Lafayette, IN (USA). Lab. of Renewable Resources Engineering)

    1990-01-01

    Recent studies have shown that some microorganisms can play a significant role in removing the sulfur compounds from coal. Sulfolobus acidocaldarius is one such microorganism. Some microorganisms can remove only organic sulfur from coal, other can remove only inorganic sulfur from coal, but S. acidocaldarius seems to be able to remove both the organic and the inorganic sulfur from coal. Furthermore, S. acidocaldarius has been shown to be able to use the sulfur and carbon derived from coal as its sole carbon and energy source for growth. These properties make this microorganism unique for coal desulfurization. This project is aimed at applying recombinant DNA techniques to improve the capability of S. acidocaldarius for coal desulfurization, which includes making it the host for housing foreign genes that encode the most effective enzymes for coal desulfurization. Since there is no established vectors and procedures for introducing vectors into S. acidocaldarius and related microorganisms, the immediate goal is to establish a gene cloning system for this species. During the present quarter, the authors have studied a few systems which can be used as the potential selection mechanism for the selection of the desired transformants. In addition, they also analyzed the extracellular proteins from S. acidocaldarius as well as other potential organic sulfur removing species and also managed to obtain most strains and plasmids that are needed for this work. Results to date are given. 1 tab.

  1. Langmuir adsorption isotherm for sulfolobus acidocaldarius on coal particles

    Energy Technology Data Exchange (ETDEWEB)

    Chen, C.Y.; Skidmore, D.R.

    1987-03-01

    Sulfolobus acidocaldarius attachment to coal particles was observed in 20% coal slurry at 72 C and pH 2.0. The rate and extent of attachment were determined and the data fit the Langmuir adsorption model. The results compared qualitatively with selected adsorption data from other investigators for Thiobacillus ferrooxidans. 11 refs.

  2. UDP-sulfoquinovose formation by Sulfolobus acidocaldarius.

    Science.gov (United States)

    Zolghadr, Behnam; Gasselhuber, Bernhard; Windwarder, Markus; Pabst, Martin; Kracher, Daniel; Kerndl, Martina; Zayni, Sonja; Hofinger-Horvath, Andreas; Ludwig, Roland; Haltrich, Dietmar; Oostenbrink, Chris; Obinger, Christian; Kosma, Paul; Messner, Paul; Schäffer, Christina

    2015-03-01

    The UDP-sulfoquinovose synthase Agl3 from Sulfolobus acidocaldarius converts UDP-D-glucose and sulfite to UDP-sulfoquinovose, the activated form of sulfoquinovose required for its incorporation into glycoconjugates. Based on the amino acid sequence, Agl3 belongs to the short-chain dehydrogenase/reductase enzyme superfamily, together with SQD1 from Arabidopsis thaliana, the only UDP-sulfoquinovose synthase with known crystal structure. By comparison of sequence and structure of Agl3 and SQD1, putative catalytic amino acids of Agl3 were selected for mutational analysis. The obtained data suggest for Agl3 a modified dehydratase reaction mechanism. We propose that in vitro biosynthesis of UDP-sulfoquinovose occurs through an NAD(+)-dependent oxidation/dehydration/enolization/sulfite addition process. In the absence of a sulfur donor, UDP-D-glucose is converted via UDP-4-keto-D-glucose to UDP-D-glucose-5,6-ene, the structure of which was determined by (1)H and (13)C-NMR spectroscopy. During the redox reaction the cofactor remains tightly bound to Agl3 and participates in the reaction in a concentration-dependent manner. For the first time, the rapid initial electron transfer between UDP-D-glucose and NAD(+) could be monitored in a UDP-sulfoquinovose synthase. Deuterium labeling confirmed that dehydration of UDP-D-glucose occurs only from the enol form of UDP-4-keto-glucose. The obtained functional data are compared with those from other UDP-sulfoquinovose synthases. A divergent evolution of Agl3 from S. acidocaldarius is suggested.

  3. Transcriptome analysis of Sulfolobus solfataricus infected with two related fuselloviruses reveals novel insights into the regulation of CRISPR-Cas system

    DEFF Research Database (Denmark)

    Fusco, Salvatore; Liguori, Rossana; Limauro, Danila

    2015-01-01

    in a double-infected strain to explore both virus-host and virus-virus interactions. Whereas SSV1 did not induce major changes of the host gene expression, SSV2 elicited a strong host response, which includes the transcriptional activation of CRISPR loci and cas genes. As a consequence, a significant decrease...... of the SSV2 copy number has been observed, which in turn led to provirus-capture into the host chromosome. Results of this study have revealed novel aspects of the host-viral interaction in the frame of the CRISPR-response....

  4. Development of in vitro transposon assisted signal sequence trapping and its use in screening Bacillus halodurans C125 and Sulfolobus solfataricus P2 gene libraries

    DEFF Research Database (Denmark)

    Becker, F.; Schnorr, K.; Wilting, R.;

    2004-01-01

    To identify genes encoding extracytosolic proteins, a minitransposon, TnSig, containing a signal-less beta-lactamase ('bla) as reporter gene, was constructed and used for in vitro transposition of genomic libraries made in Escherichia coli. The 'bla gene was cloned into a bacteriophage MU...

  5. Thermodynamics of the ATPase cycle of GlcV, the nucleotide-binding domain of the glucose ABC transporter of Sulfolobus solfataricus

    NARCIS (Netherlands)

    Pretz, Monika G.; Albers, Sonja-Verena; Schuurman-Wolters, Gea; Tampe, Robert; Driessen, Arnold J. M.; van der Does, Chris

    2006-01-01

    ATP-binding cassette transporters drive the transport of substrates across the membrane by the hydrolysis of ATP. They typically have a conserved domain structure with two membrane-spanning domains that form the transport channel and two cytosolic nucleotide-binding domains ( NBDs) that energize the

  6. Development of in vitro transposon assisted signal sequence trapping and its use in screening Bacillus halodurans C125 and Sulfolobus solfataricus P2 gene libraries

    DEFF Research Database (Denmark)

    Becker, F.; Schnorr, K.; Wilting, R.

    2004-01-01

    To identify genes encoding extracytosolic proteins, a minitransposon, TnSig, containing a signal-less beta-lactamase ('bla) as reporter gene, was constructed and used for in vitro transposition of genomic libraries made in Escherichia coli. The 'bla gene was cloned into a bacteriophage MU minitra...

  7. Promiscuità enzimatica ed evoluzione molecolare:studio di una lattonasi da Sulfolobus solfataricus in grado di degradare agenti nervini

    OpenAIRE

    Merone, Luigia

    2010-01-01

    Si definisce promiscuità catalitica la capacità di un enzima di catalizzare reazioni aggiuntive al ruolo principale, che possono essere più o meno chimicamente differenti tra loro. Questa definizione si riferisce sia alla possibilità di catalizzare la trasformazione di substrati di natura diversa che di utilizzare differenti meccanismi di reazione e/o diversi siti catalitici1,2. L'enorme importanza legata allo studio della promiscuità enzimatica è sostanzialmente duplice. Da un punto di vista...

  8. Characterization of recombinantly expressed dihydroxy-acid dehydratase from Sulfobus solfataricus-A key enzyme for the conversion of carbohydrates into chemicals.

    Science.gov (United States)

    Carsten, Jörg M; Schmidt, Anja; Sieber, Volker

    2015-10-10

    Dihydroxyacid dehydratases (DHADs) are excellent biocatalysts for the defunctionalization of biomass. Here, we report on the recombinant production of DHAD from Sulfolobus solfataricus (SsDHAD) in E. coli and its characterization with special focus on activity toward non-natural substrates, thermo-stability, thermo-inactivation kinetics and activation capabilities and its application within multi-step cascades for chemicals production. Using a simple heat treatment of cell lysate as major purification step we achieved a specific activity of 4.4 units per gram cell mass toward the substrate d-gluconate. The optimal temperature and pH value for this reaction are 77°C and pH 6.2. The inhibitory concentration (IC50, 50% residual activity) of different alcohols was determined to be 15% (v/v) for ethanol, 4.5% (v/v) for butanol and 4% (v/v) for isobutanol. Besides d-gluconate and the natural substrate 2,3-dihydroxyisovalerate (DHIV) SsDHAD is able to convert the C3-sugar-acid d-glycerate to pyruvate, a reaction, which does not occur in natural metabolic pathways, with a specific activity of 10.7±0.4mU/mg. The specific activity of the enzyme can be increased 3-fold by incubation with 2-mercaptoethanol. The activation has no impact on temperature dependence, but modulates the thermo-inactivation tolerance at 50°C. The total turnover numbers for all of the three reactions was found to be 35.5×10(3)±1.0×10(3) for the conversion of d-gluconate to 2-keto-3-deoxygluconate (KDG), 28.2×10(3)±0.8×10(3) for DHIV to 2-ketovalerate (KIV) and 943±0.28×10(2) for d-glycerate to pyruvate. With activated SsDHAD these values could be further increased 5- and 4-fold for the d-gluconate and d-glycerate conversion, respectively.

  9. Transcriptomic analysis reveals how a lack of potassium ions increases Sulfolobus acidocaldarius sensitivity to pH changes.

    Science.gov (United States)

    Buetti-Dinh, Antoine; Dethlefsen, Olga; Friedman, Ran; Dopson, Mark

    2016-08-01

    Extremely acidophilic microorganisms (optimum growth pH of ≤3) maintain a near neutral cytoplasmic pH via several homeostatic mechanisms, including an inside positive membrane potential created by potassium ions. Transcriptomic responses to pH stress in the thermoacidophilic archaeon, Sulfolobus acidocaldarius were investigated by growing cells without added sodium and/or potassium ions at both optimal and sub-optimal pH. Culturing the cells in the absence of added sodium or potassium ions resulted in a reduced growth rate compared to full-salt conditions as well as 43 and 75 significantly different RNA transcript ratios, respectively. Differentially expressed RNA transcripts during growth in the absence of added sodium ions included genes coding for permeases, a sodium/proline transporter and electron transport proteins. In contrast, culturing without added potassium ions resulted in higher RNA transcripts for similar genes as a lack of sodium ions plus genes related to spermidine that has a general role in response to stress and a decarboxylase that potentially consumes protons. The greatest RNA transcript response occurred when S. acidocaldarius cells were grown in the absence of potassium and/or sodium at a sub-optimal pH. These adaptations included those listed above plus osmoregulated glucans and mechanosensitive channels that have previously been shown to respond to osmotic stress. In addition, data analyses revealed two co-expressed IclR family transcriptional regulator genes with a previously unknown role in the S. acidocaldarius pH stress response. Our study provides additional evidence towards the importance of potassium in acidophile growth at acidic pH.

  10. Recombination of synthetic oligonucleotides with prokaryotic chromosomes: substrate requirements of the Escherichia coli/lambdaRed and Sulfolobus acidocaldarius recombination systems.

    Science.gov (United States)

    Grogan, Dennis W; Stengel, Kristy R

    2008-09-01

    In order to reveal functional properties of recombination involving short ssDNAs in hyperthermophilic archaea, we evaluated oligonucleotide-mediated transformation (OMT) in Sulfolobus acidocaldarius and Escherichia coli as a function of the molecular properties of the ssDNA substrates. Unmodified ssDNAs as short as 20-22 nt yielded recombinants in both organisms, as did longer DNAs forming as few as 2-5 base pairs on one side of the genomic mutation. The two OMT systems showed similar responses to certain end modifications of the oligonucleotides, but E. coli was found to require a 5' phosphate on 5'-limited ssDNA whereas this requirement was not evident in S. acidocaldarius. The ability of both E. coli and S. acidocaldarius to incorporate short, mismatched ssDNAs into their genomes raises questions about the biological significance of this capability, including its phylogenetic distribution among microorganisms and its impact on genome stability. These questions seem particularly relevant for S. acidocaldarius, as this archaeon has natural competence for OMT, encodes no MutSL homologues and thrives under environmental conditions that accelerate DNA decomposition.

  11. Co-expression with RadA and the characterization of stRad55B, a RadA paralog from the hyperthermophilic crenarchaea Sulfolobus tokodaii

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    ST0838 (designed stRad55B) is one of the four RadA paralogs (or Rad55 homologues) in the genome of the hyperthermophilic crenarchaeon Sulfolobus tokodaii. The gene is induced by UV irradiation, sug-gesting that it is involved in DNA recombinational repair in this organism. However, this protein could not be expressed normally in vitro. In this study, thermostable and soluble stRad55B was obtained by co-expression with S. tokodaii RadA (stRadA) in E. coli, and the enzymatic properties were examined. It was found that stRad55B bound ssDNA preferentially and had a very weak ATPase activity that was not stimulated by DNA. The recombinant protein inhibited the strand exchange activity promoted by stRadA, indicating that stRad55B might be an inhibitor to the homologous recombination in this ar-chaeon. The results will be helpful for further functional and interaction analysis of RadA paralogs and for the understanding of the mechanism of recombinational repair in archaea.

  12. Co-expression with RadA and the characterization of stRad55B, a RadA paralog from the hyperthermophilic crenarchaea Sulfolobus tokodaii

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    ST0838 (designed stRad55B) is one of the four RadA paralogs (or Rad55 homologues) in the genome of the hyperthermophilic crenarchaeon Sulfolobus tokodaii. The gene is induced by UV irradiation, suggesting that it is involved in DNA recombinational repair in this organism. However, this protein could not be expressed normally in vitro. In this study, thermostable and soluble stRad55B was obtained by co-expression with S. tokodaii RadA (stRadA) in E. coli, and the enzymatic properties were examined. It was found that stRad55B bound ssDNA preferentially and had a very weak ATPase activity that was not stimulated by DNA. The recombinant protein inhibited the strand exchange activity promoted by stRadA, indicating that stRad55B might be an inhibitor to the homologous recombination in this archaeon. The results will be helpful for further functional and interaction analysis of RadA paralogs and for the understanding of the mechanism of recombinational repair in archaea.

  13. Biochemical and structural exploration of the catalytic capacity of Sulfolobus KDG aldolases

    NARCIS (Netherlands)

    Wolterink-van Loo, Suzanne; van Eerde, Andre; Siemerink, Marco A. J.; Akerboom, Jasper; Dijkstra, Bauke W.; van der Oost, John; Akerboom, S.P.

    2007-01-01

    Aldolases are enzymes with potential applications in biosynthesis, depending on their activity, specificity and stability. In the present study, the genomes of Sulfolobus species were screened for aldolases. Two new KDGA [2-keto-3-deoxygluconate (2-oxo-3-deoxygluconate) aldolases] from Sulfolobus ac

  14. Expanding and understanding the genetic toolbox of the hyperthermophilic genus Sulfolobus

    NARCIS (Netherlands)

    Wagner, Michaela; Berkner, Silvia; Ajon, Malgorzata; Driessen, Arnold J. M.; Lipps, Georg; Albers, Sonja-Verena

    2009-01-01

    Although Sulfolobus species are among the best studied archaeal micro-organisms, the development and availability of genetic tools has lagged behind. in the present paper, we discuss the latest progress in understanding recombination events of exogenous DNA into the chromosomes of Sulfolobus solfata

  15. Unmarked gene deletion and host-vector system for the hyperthermophilic crenarchaeon Sulfolobus islandicus

    DEFF Research Database (Denmark)

    Deng, Ling; Zhu, Haojun; Chen, Zhengjun

    2009-01-01

    , and unmarked lacS mutants were obtained by each method. A new alternative recombination mechanism, i.e., marker circularization and integration, was shown to operate in the latter method, which did not yield the designed deletion mutation. Subsequently, Sulfolobus-E. coli plasmid shuttle vectors were...

  16. Response of Haloalkaliphilic Archaeon Natronococcus Jeotgali RR17 to Hypergravity

    Science.gov (United States)

    Thombre, Rebecca S.; Bhalerao, Aniruddha R.; Shinde, Vinaya D.; Dhar, Sunil Kumar; Shouche, Yogesh S.

    2017-01-01

    The survival of archaeabacteria in extreme inhabitable environments on earth that challenge organismic survival is ubiquitously known. However, the studies related to the effect of hypergravity on the growth and proliferation of archaea are unprecedented. The survival of organisms in hypergravity and rocks in addition to resistance to cosmic radiations, pressure and other extremities is imperative to study the possibilities of microbial travel between planets and endurance in hyperaccelerative forces faced during ejection of rocks from planets. The current investigation highlights the growth of an extremophilic archaeon isolated from a rocky substrate in hypergravity environment. The haloalkaliphilic archaeon, Natronococcus jeotgali RR17 was isolated from an Indian laterite rock, submerged in the Arabian sea lining Coastal Maharashtra, India. The endolithic haloarchaeon was subjected to hypergravity from 56 - 893 X gusing acceleration generated by centrifugal rotation. The cells of N. jeotgali RR17 proliferated and demonstrated good growth in hypergravity (223 X g). This is the first report on isolation of endolithic haloarchaeon N. jeotgali RR17 from an Indian laterite rock and its ability to proliferate in hypergravity. The present study demonstrates the ability of microbial life to survive and proliferate in hypergravity. Thus the inability of organismic growth in hypergravity may no longer be a limitation for astrobiology studies related to habitability of substellar objects, brown dwarfs and other planetary bodies in the universe besides planet earth.

  17. Versatile genetic tool box for the crenarchaeote Sulfolobus acidocaldarius

    Directory of Open Access Journals (Sweden)

    Michaela eWagner

    2012-06-01

    Full Text Available For reverse genetic approaches inactivation or selective modification of genes are required to elucidate their putative function. Sulfolobus acidocaldarius is a thermoacidophilic Crenarchaeon which grows optimally at 76 °C and pH 3. As many antibiotics do not withstand these conditions the development of a genetic system in this organism is dependent on auxotrophies. Therefore we constructed a pyrE deletion mutant of S. acidocaldarius wild type strain DSM639 missing 322 bp called MW001. Using this strain as the base, we describe here different methods using single as well as double crossover events to obtain markerless deletion mutants, tag genes genomically and ectopically integrate foreign DNA into MW001. These methods enable us to construct single, double and triple deletions strains that can still be complemented with the pRN1 based expression vector. Taken together we have developed a versatile and robust genetic tool box for the crenarchaeote Sulfolobus acidocaldarius that will promote the study of unknown gene functions in this organism and makes this organism a suitable host for synthetic biology approaches.

  18. Energy metabolism of a thermoacidophilic archaebacterium,Sulfolobus acidocaldarius

    Science.gov (United States)

    Wakagi, Takayoshi; Oshima, Tairo

    1987-09-01

    To elucidate the phylogenic status of the archaebacterium and mechanisms of acidophily, membrane bound ATPase, cytochromes and NADH dehydrogenase of a thermoacidophilic archaebacterium,Sulfolobus acidocaldarius, were studied. Typea cytochrome was found in the membrane. The organism was sensitive to cyanide and azide, and though cytochromec is lacking in this organism, these respiratory poisons inhibited a terminal oxidase, when assayed with cytochromec from other sources. NADH dehydrogenase was highly purified from the crude extract of the cells. The enzyme was able to transfer electrons from NADH to caldariellaquinone, a unique benzothiophenequinone in the genusSulfolobus. Thus, the enzyme is a possible member of the respiratory chain. Membrane fraction contained two types of ATPase, one was active at neutral pH and slightly activated by sulfate; the other was an acid apyrase and inhibited by sulfate. Typical characteristics of F0F1ATPase could not be found in these enzymes. These results suggest that (1) the thermoacidophilic archaebacteria are phylogenically distant from both eubacteria and eukaryotes, (2) the archaebacterial thermoacidophiles can be classified in a different subgroup from methanogens and extreme halophiles, and (3) in spite of the aerobic nature of the organism, the energy yielding mechanisms appear quite unique, when compared to those of other aerobes and mitochondria.

  19. Characterization of the Sulfolobus host-SSV2 virus interaction

    DEFF Research Database (Denmark)

    Contursi, P.; Jensen, S.; Aucelli, T.

    2006-01-01

    The Sulfolobus spindle virus, SSV2, encodes a tyrosine integrase which furthers provirus formation in host chromosomes. Consistently with the prediction made during sequence analysis, integration was found to occur in the downstream half of the tRNAGly (CCC) gene. In this paper we report the find......The Sulfolobus spindle virus, SSV2, encodes a tyrosine integrase which furthers provirus formation in host chromosomes. Consistently with the prediction made during sequence analysis, integration was found to occur in the downstream half of the tRNAGly (CCC) gene. In this paper we report...... during the growth of the natural host REY15/4, the cellular content of SSV2 DNA remains fairly low throughout the incubation of the foreign host. The accumulation of episomal DNA in the former case cannot be traced to decreased packaging activity because of a simultaneous increase in the virus titre...... in the medium. In addition, the interaction between SSV2 and its natural host is characterized by the concurrence of host growth inhibition and the induction of viral DNA replication. When this virus-host interaction was investigated using S. islandicus REY15A, a strain which is closely related to the natural...

  20. Genomic evidence of rapid, global-scale gene flow in a Sulfolobus species.

    Science.gov (United States)

    Mao, Dominic; Grogan, Dennis

    2012-08-01

    Local populations of Sulfolobus islandicus diverge genetically with geographical separation, and this has been attributed to restricted transfer of propagules imposed by the unfavorable spatial distribution of acidic geothermal habitat. We tested the generality of genetic divergence with distance in Sulfolobus species by analyzing genomes of Sulfolobus acidocaldarius drawn from three populations separated by more than 8000 km. In sharp contrast to S. islandicus, the geographically diverse S. acidocaldarius genomes proved to be nearly identical. We could not link the difference in genome conservation between the two species to a corresponding difference in genome stability or ecological factors affecting propagule dispersal. The results provide the first evidence that genetic isolation of local populations does not result primarily from properties intrinsic to Sulfolobus and the severe discontinuity of its geothermal habitat, but varies with species, and thus may reflect biotic interactions that act after propagule dispersal.

  1. Microarray analysis in the archaeon Halobacterium salinarum strain R1.

    Directory of Open Access Journals (Sweden)

    Jens Twellmeyer

    Full Text Available BACKGROUND: Phototrophy of the extremely halophilic archaeon Halobacterium salinarum was explored for decades. The research was mainly focused on the expression of bacteriorhodopsin and its functional properties. In contrast, less is known about genome wide transcriptional changes and their impact on the physiological adaptation to phototrophy. The tool of choice to record transcriptional profiles is the DNA microarray technique. However, the technique is still rarely used for transcriptome analysis in archaea. METHODOLOGY/PRINCIPAL FINDINGS: We developed a whole-genome DNA microarray based on our sequence data of the Hbt. salinarum strain R1 genome. The potential of our tool is exemplified by the comparison of cells growing under aerobic and phototrophic conditions, respectively. We processed the raw fluorescence data by several stringent filtering steps and a subsequent MAANOVA analysis. The study revealed a lot of transcriptional differences between the two cell states. We found that the transcriptional changes were relatively weak, though significant. Finally, the DNA microarray data were independently verified by a real-time PCR analysis. CONCLUSION/SIGNIFICANCE: This is the first DNA microarray analysis of Hbt. salinarum cells that were actually grown under phototrophic conditions. By comparing the transcriptomics data with current knowledge we could show that our DNA microarray tool is well applicable for transcriptome analysis in the extremely halophilic archaeon Hbt. salinarum. The reliability of our tool is based on both the high-quality array of DNA probes and the stringent data handling including MAANOVA analysis. Among the regulated genes more than 50% had unknown functions. This underlines the fact that haloarchaeal phototrophy is still far away from being completely understood. Hence, the data recorded in this study will be subject to future systems biology analysis.

  2. Comparative lipid composition of heterotrophically and autotrophically grown Sulfolobus acidocaldarius.

    Science.gov (United States)

    Langworthy, T A

    1977-06-01

    Complex lipids from the thermoacidophilic facultative autotroph Sulfolobus acidocaldarius, as well as a strictly autotrophic isolate, were compared between cells grown on yeast extract and elemental sulfur. Lipids from both organisms grown autotrophically were nearly identical. Each contained about 15% neutral lipids, 35% glycolipids, and 50% acidic lipids. Glycolipids and acidic lipids contained C40H82-76-derived glycerol ether residues. Major glycolipids included the glycerol ether analogues of glucosyl galactosyl diglyceride (5%) and glucosyl polyol diglyceride (75%). Acidic lipids were comprised mainly of the glycerol ether analogues of phosphatidyl inositol (7%), inositolphosphoryl glucosyl polyol diglyceride (72%), and a partially characterized sulfate- and phosphate-containing derivative of glucosyl polyol diglyceride (13%). The lipids from cells grown heterotrophically were similar to those from autotrophically grown cells, except that the partially characterized acidic lipid was absent. In addition, the two glycolipids as well as the respective inositolphosphoryl derivatives were each present in nearly equal proportions.

  3. Factors affecting Archaeal Lipid Compositions of the Sulfolobus Species

    Science.gov (United States)

    He, L.; Han, J.; Wei, Y.; Lin, L.; Wei, Y.; Zhang, C.

    2010-12-01

    Temperature is the best known variable affecting the distribution of the archaeal glycerol dibiphytanyl glycerol tetraethers (GDGTs) in marine and freshwater systems. Other variables such as pH, ionic strength, or bicarbonate concentration may also affect archaeal GDGTs in terrestrial systems. Studies of pure cultures can help us pinpoint the specific effects these variables may have on archaeal lipid distribution in natural environments. In this study, three Sulfolobus species (HG4, HB5-2, HB9-6) isolated from Tengchong hot springs (pH 2-3, temperature 73-90°C) in China were used to investigate the effects of temperature, pH, substrate, and type of strain on the composition of GDGTs. Results showed that increase in temperature had negative effects on the relative contents of GDGT-0 (no cyclopentyl rings), GDGT-1 (one cyclopentyl ring), GDGT-2 and GDGT-3 but positive effects on GDGT-4, GDGT-4', GDGT-5 and GDGT-5'. Increase in pH, on the other hand, had negative effects on GDGT-0, GDGT-1, GDGT-4', GDGT-5 and GDGT-5', and positive effects on GDGT-3 and GDGT-4. GDGT-2 remained relatively constant with changing pH. When the HG4 was grown on different substrates, GDGT-5 was five time more abundant in sucrose-grown cultures than in yeast extract- or sulfur- grown cultures, suggesting that carbohydrates may stimulate the production of GDGT-5. For all three species, the ring index (average number of rings) of GDGTs correlated positively with incubation temperature. In HG4, ring index was much lower at optimal pH (3.5) than at other pH values. Ring index of HB5-2 or HB9-6 is higher than that of HG4, suggesting that speciation may affect the degree of cyclization of GDGT of the Sulfolobus. These results indicate that individual archaeal lipids respond differently to changes in environmental variables, which may be also species specific.

  4. Characterization of malate dehydrogenase from the hyperthermophilic archaeon Pyrobaculum islandicum.

    Science.gov (United States)

    Yennaco, Lynda J; Hu, Yajing; Holden, James F

    2007-09-01

    Native and recombinant malate dehydrogenase (MDH) was characterized from the hyperthermophilic, facultatively autotrophic archaeon Pyrobaculum islandicum. The enzyme is a homotetramer with a subunit mass of 33 kDa. The activity kinetics of the native and recombinant proteins are the same. The apparent K ( m ) values of the recombinant protein for oxaloacetate (OAA) and NADH (at 80 degrees C and pH 8.0) were 15 and 86 microM, respectively, with specific activity as high as 470 U mg(-1). Activity decreased more than 90% when NADPH was used. The catalytic efficiency of OAA reduction by P. islandicum MDH using NADH was significantly higher than that reported for any other archaeal MDH. Unlike other archaeal MDHs, specific activity of the P. islandicum MDH back-reaction also decreased more than 90% when malate and NAD(+) were used as substrates and was not detected with NADP(+). A phylogenetic tree of 31 archaeal MDHs shows that they fall into 5 distinct groups separated largely along taxonomic lines suggesting minimal lateral mdh transfer between Archaea.

  5. Isolation and Phylogenetic Analysis of Halophilic Archaeon AJ6

    Institute of Scientific and Technical Information of China (English)

    Xu Xiaohong; Wu Min; Cao Yi; Wu Yuehong; Zhang Ting

    2006-01-01

    Halophilic archaeon A J6 was isolated and purified from the Altun Mountain National Nature Reserve of the Xinjiang Uygur Autonomous Region.Strain AJ6 is a Gram-negative rod whose size is 0.2-0.6 by 1.6-4.2 μm,wherein a few cells are globular.The optimum salt concentration for its growth is 20% NaC1 and 0.6% Mg2+,and the optimum pH is 6.0-7.0.Morphological,physiological,and biochemical characteristics of strain AJ6 were observed.The 16S rRNA encoding gene (16S rDNA)sequence of strain A J6 was amplified by PCR,and its nucteotide sequence was determined subsequently."Clustalw"and"PHYLIP"software bags were used to analyze the 16S rDNA sequence;the homology was compared,and then the phylogenetic tree was established.The results indicate that strain AJ6 is a novel species of the genus Natrinema.The GenBank accession number of the 16S rDNA sequences of strain AJ6 is AY277584.

  6. Single gene insertion drives bioalcohol production by a thermophilic archaeon

    Energy Technology Data Exchange (ETDEWEB)

    Basen, M; Schut, GJ; Nguyen, DM; Lipscomb, GL; Benn, RA; Prybol, CJ; Vaccaro, BJ; Poole, FL; Kelly, RM; Adams, MWW

    2014-12-09

    Bioethanol production is achieved by only two metabolic pathways and only at moderate temperatures. Herein a fundamentally different synthetic pathway for bioalcohol production at 70 degrees C was constructed by insertion of the gene for bacterial alcohol dehydrogenase (AdhA) into the archaeon Pyrococcus furiosus. The engineered strain converted glucose to ethanol via acetate and acetaldehyde, catalyzed by the host-encoded aldehyde ferredoxin oxidoreductase (AOR) and heterologously expressed AdhA, in an energy-conserving, redox-balanced pathway. Furthermore, the AOR/AdhA pathway also converted exogenously added aliphatic and aromatic carboxylic acids to the corresponding alcohol using glucose, pyruvate, and/or hydrogen as the source of reductant. By heterologous coexpression of a membrane-bound carbon monoxide dehydrogenase, CO was used as a reductant for converting carboxylic acids to alcohols. Redirecting the fermentative metabolism of P. furiosus through strategic insertion of foreign genes creates unprecedented opportunities for thermophilic bioalcohol production. Moreover, the AOR/AdhA pathway is a potentially game-changing strategy for syngas fermentation, especially in combination with carbon chain elongation pathways.

  7. Structure of ST0929, a putative glycosyl transferase from Sulfolobus tokodaii.

    Science.gov (United States)

    Cielo, Charles B C; Okazaki, Seiji; Suzuki, Atsuo; Mizushima, Tsunehiro; Masui, Ryoji; Kuramitsu, Seiki; Yamane, Takashi

    2010-04-01

    The Sulfolobus tokodaii protein ST0929 shares close structural homology with S. acidocaldarius maltooligosyl trehalose synthase (SaMTSase), suggesting that the two enzymes share a common enzymatic mechanism. MTSase is one of a pair of enzymes that catalyze trehalose biosynthesis. The relative geometries of the ST0929 and SaMTSase active sites were found to be essentially identical. ST0929 also includes the unique tyrosine cluster that encloses the reducing-end glucose subunit in Sulfolobus sp. MTSases. The current structure provides insight into the structural basis of the increase in the hydrolase side reaction that is observed for mutants in which a phenylalanine residue is replaced by a tyrosine residue in the subsite +1 tyrosine cluster of Sulfolobus sp.

  8. Production of beta-xylanase and beta-xylosidase by the extremely halophilic archaeon Halorhabdus utahensis

    DEFF Research Database (Denmark)

    Wainø, M.; Ingvorsen, K.

    2003-01-01

    The extremely halophilic archaeon, Halorhabdus utahensis, isolated from the Great Salt Lake, Utah, produced beta-xylanase and beta-xylosidase activities. Both enzymes were active over a broad NaCl range from near zero to 30% NaCl when tested with culture broth. A broad NaCl optimum was observed......-xylosidase activity was optimal at 65degreesC. SDS-PAGE and zymogram techniques revealed the presence of two xylan-degrading proteins of approximately 45 and 67 kDa in culture supernatants. To our knowledge, this paper is the first report on hemicellulose-degrading enzymes produced by an extremely halophilic archaeon....

  9. An x-ray absorption spectroscopy study of Cd binding onto a halophilic archaeon

    Science.gov (United States)

    Showalter, Allison R.; Szymanowski, Jennifer E. S.; Fein, Jeremy B.; Bunker, Bruce A.

    2016-05-01

    X-ray absorption spectroscopy (XAS) and cadmium (Cd) isotherm experiments determine how Cd adsorbs to the surface of halophilic archaeon Halobacterium noricense. This archaeon, isolated from the Waste Isolation Pilot Plant (WIPP) near Carlsbad, New Mexico could be involved with the transport of toxic metals stored in the transuranic waste in the salt mine. The isotherm experiments show that adsorption is relatively constant across the tolerable pH range for H. noricense. The XAS results indicate that Cd adsorption occurs predominately via a sulfur site, most likely sulfhydryl, with the same site dominating all measured pH values.

  10. The Genome of Sulfolobus acidocaldarius, a Model Organism of the Crenarchaeota†

    OpenAIRE

    Chen, Lanming; Brügger, Kim; Skovgaard, Marie; Redder, Peter; She, Qunxin; Torarinsson, Elfar; Greve, Bo; Awayez, Mariana; Zibat, Arne; Klenk, Hans-Peter; Garrett, Roger A.

    2005-01-01

    Sulfolobus acidocaldarius is an aerobic thermoacidophilic crenarchaeon which grows optimally at 80°C and pH 2 in terrestrial solfataric springs. Here, we describe the genome sequence of strain DSM639, which has been used for many seminal studies on archaeal and crenarchaeal biology. The circular genome carries 2,225,959 bp (37% G+C) with 2,292 predicted protein-encoding genes. Many of the smaller genes were identified for the first time on the basis of comparison of three Sulfolobus genome se...

  11. CRISPR families of the crenarchaeal genus Sulfolobus: bidirectional transcription and dynamic properties

    DEFF Research Database (Denmark)

    Lillestøl, Reidun K; Shah, Shiraz Ali; Brügger, Kim

    2009-01-01

    Summary CRISPRs of Sulfolobus fall into three main families based on their repeats, leader regions, associated cas genes, and putative recognition sequences on viruses and plasmids. Spacer sequence matches to different viruses and plasmids of the Sulfolobales revealed some bias particularly...... for family III CRISPRs. Transcription occurs on both strands of the five repeat-clusters of Sulfolobus acidocaldarius and a repeat-cluster of the conjugative plasmid pKEF9. Leader strand transcripts cover whole repeat-clusters and are processed mainly from the 3'-end, within repeats, yielding heterogeneous...

  12. CRISPR associated diversity within a population of Sulfolobus islandicus.

    Directory of Open Access Journals (Sweden)

    Nicole L Held

    Full Text Available BACKGROUND: Predator-prey models for virus-host interactions predict that viruses will cause oscillations of microbial host densities due to an arms race between resistance and virulence. A new form of microbial resistance, CRISPRs (clustered regularly interspaced short palindromic repeats are a rapidly evolving, sequence-specific immunity mechanism in which a short piece of invading viral DNA is inserted into the host's chromosome, thereby rendering the host resistant to further infection. Few studies have linked this form of resistance to population dynamics in natural microbial populations. METHODOLOGY/PRINCIPAL FINDINGS: We examined sequence diversity in 39 strains of the archeaon Sulfolobus islandicus from a single, isolated hot spring from Kamchatka, Russia to determine the effects of CRISPR immunity on microbial population dynamics. First, multiple housekeeping genetic markers identify a large clonal group of identical genotypes coexisting with a diverse set of rare genotypes. Second, the sequence-specific CRISPR spacer arrays split the large group of isolates into two very different groups and reveal extensive diversity and no evidence for dominance of a single clone within the population. CONCLUSIONS/SIGNIFICANCE: The evenness of resistance genotypes found within this population of S. islandicus is indicative of a lack of strain dominance, in contrast to the prediction for a resistant strain in a simple predator-prey interaction. Based on evidence for the independent acquisition of resistant sequences, we hypothesize that CRISPR mediated clonal interference between resistant strains promotes and maintains diversity in this natural population.

  13. Attachment of Sulfolobus acidocaldarius cells on coal particles

    Energy Technology Data Exchange (ETDEWEB)

    Chiu-Yu Chen; Skidmore, D.R.

    1988-03-01

    Attachment of the thermophilic microorganism Sulfolobus acidocaldarius was studied on Kentucky No. 9 coal, an Ohio coal mixtures, Ohio No. 6, a European coal designated as Sulzer coal, activated carbon and iron pyrite to define the effects of initial cell density, prior acid leaching, pyrite content, and surface area on rate and extent of preleaching attachment. The results showed that the rate of preleaching attachment was so high that equilibrium was reached in less than 5 min in most cases. Attached cells increased in number with increasing initial cell density. Prior nitric acid leaching of coal reduced the extent of cell adsorption while hydrochloric acid leaching did not affect cell attachment. Some adsorption isotherms fit the Langmuir model. The specific adsorption (cells/cm/sup 3/ coal) increased with increasing surface areas measured by nitrogen adsorption. Pyrite gave the highest extent of cell attachment based on per unit surface area comparison. Adsorption isotherms of a pyrite-selective chemical adsorbate revealed that preleaching attachment of cells was affected further by altered surface properties of the coals and that the concept of selective attachment of cells on pyrite surfaces was also supported. 22 refs., 6 figs., 1 tab.

  14. Versatile Genetic Tool Box for the Crenarchaeote Sulfolobus acidocaldarius.

    Science.gov (United States)

    Wagner, Michaela; van Wolferen, Marleen; Wagner, Alexander; Lassak, Kerstin; Meyer, Benjamin H; Reimann, Julia; Albers, Sonja-Verena

    2012-01-01

    For reverse genetic approaches inactivation or selective modification of genes are required to elucidate their putative function. Sulfolobus acidocaldarius is a thermoacidophilic Crenarchaeon which grows optimally at 76°C and pH 3. As many antibiotics do not withstand these conditions the development of a genetic system in this organism is dependent on auxotrophies. Therefore we constructed a pyrE deletion mutant of S. acidocaldarius wild type strain DSM639 missing 322 bp called MW001. Using this strain as the starting point, we describe here different methods using single as well as double crossover events to obtain markerless deletion mutants, tag genes genomically and ectopically integrate foreign DNA into MW001. These methods enable us to construct single, double, and triple deletions strains that can still be complemented with the pRN1 based expression vector. Taken together we have developed a versatile and robust genetic tool box for the crenarchaeote S. acidocaldarius that will promote the study of unknown gene functions in this organism and makes it a suitable host for synthetic biology approaches.

  15. Formate hydrogenlyase in the hyperthermophilic archaeon, Thermococcus litoralis

    Directory of Open Access Journals (Sweden)

    Rákhely Gábor

    2008-06-01

    Full Text Available Abstract Background Thermococcus litoralis is a heterotrophic facultative sulfur dependent hyperthermophilic Archaeon, which was isolated from a shallow submarine thermal spring. It has been successfully used in a two-stage fermentation system, where various keratinaceous wastes of animal origin were converted to biohydrogen. In this system T. litoralis performed better than its close relative, P. furiosus. Therefore, new alternative enzymes involved in peptide and hydrogen metabolism were assumed in T. litoralis. Results An about 10.5 kb long genomic region was isolated and sequenced from Thermococcus litoralis. In silico analysis revealed that the region contained a putative operon consisting of eight genes: the fdhAB genes coding for a formate dehydrogenase and the mhyCDEFGH genes encoding a [NiFe] hydrogenase belonging to the group of the H2-evolving, energy-conserving, membrane-bound hydrogenases. Reverse transcription linked quantitative Real-Time PCR and Western blotting experiments showed that the expression of the fdh-mhy operon was up-regulated during fermentative growth on peptides and down-regulated in cells cultivated in the presence of sulfur. Immunoblotting and protein separation experiments performed on cell fractions indicated that the formate dehydrogenase part of the complex is associated to the membrane-bound [NiFe] hydrogenase. Conclusion The formate dehydrogenase together with the membrane-bound [NiFe] hydrogenase formed a formate hydrogenlyase (formate dehydrogenase coupled hydrogenase, FDH-MHY complex. The expression data suggested that its physiological role is linked to the removal of formate likely generated during anaerobic peptide fermentation.

  16. Production and characterization of a thermostable L-threonine dehydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus

    NARCIS (Netherlands)

    Machielsen, M.P.; Oost, van der J.

    2006-01-01

    The gene encoding a threonine dehydrogenase (TDH) has been identified in the hyperthermophilic archaeon Pyrococcus furiosus. The Pf-TDH protein has been functionally produced in Escherichia coli and purified to homogeneity. The enzyme has a tetrameric conformation with a molecular mass of ¿ 155 kDa.

  17. Argonaute of the archaeon Pyrococcus furiosus is a DNA-guided nuclease that targets cognate DNA

    NARCIS (Netherlands)

    Swarts, D.C.; Hegge, J.W.; Hinojo, Ismael; Shiimori, Masami; Ellis, Michael A.; Dumrongkulraksa, Justin; Terns, Rebecca M.; Terns, Michael P.; Oost, Van Der John

    2015-01-01

    Functions of prokaryotic Argonautes (pAgo) have long remained elusive. Recently, Argonautes of the bacteria Rhodobacter sphaeroides and Thermus thermophilus were demonstrated to be involved in host defense. The Argonaute of the archaeon Pyrococcus furiosus (PfAgo) belongs to a different branch in

  18. Molecular analysis of the UV-inducible pili operon from Sulfolobus acidocaldarius

    NARCIS (Netherlands)

    Wolferen, Marleen van; Ajon, Małgorzata; Driessen, Arnold J.M.; Albers, Sonja-Verena

    2013-01-01

    Upon ultraviolet (UV) stress, hyperthermophilic Sulfolobus species show a highly induced transcription of a gene cluster responsible for pili biogenesis: the UV-inducible pili operon (ups operon). This operon is involved in UV-induced pili assembly, cellular aggregation, and subsequent DNA exchange

  19. High Efficiency of Plating of the Thermophilic Sulfur-Dependent Archaebacterium Sulfolobus acidocaldarius

    OpenAIRE

    Lindström, E. Börje; Sehlin, H. Mikael

    1989-01-01

    A procedure for plating Sulfolobus acidocaldarius using Gelrite as the gelling agent is presented. The technique uses a supporting gel of 0.8% (wt/vol) Gelrite and an overlay soft gel of 0.4% (wt/vol) Gelrite, in which the colonies are grown. The plating efficiency was essentially 100%.

  20. Improving low-temperature activity of Sulfolobus acidocaldarius 2-keto-3-deoxygluconate aldolase

    NARCIS (Netherlands)

    Wolterink-van Loo, S.; Siemerink, M.A.J.; Perrakis, G.; Kaper, T.; Kengen, S.W.M.; Oost, van der J.

    2009-01-01

    Sulfolobus acidocaldarius 2-keto-3-deoxygluconate aldolase (SacKdgA) displays optimal activity at 95 degrees C and is studied as a model enzyme for aldol condensation reactions. For application of SacKdgA at lower temperatures, a library of randomly generated mutants was screened for improved synthe

  1. Development of a Simvastatin Selection Marker for a Hyperthermophilic Acidophile, Sulfolobus islandicus

    DEFF Research Database (Denmark)

    Zheng, Tao; Huang, Qihong; Zhang, Changyi;

    2012-01-01

    We report here a novel selectable marker for the hyperthermophilic crenarchaeon Sulfolobus islandicus. The marker cassette is composed of the sac7d promoter and the hmg gene coding for the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (P(sac7d)-hmg), which confers simvastatin resistance...

  2. Homologous recombination in Sulfolobus acidocaldarius: genetic assays and functional properties.

    Science.gov (United States)

    Grogan, Dennis W

    2009-02-01

    HR (homologous recombination) is expected to play important roles in the molecular biology and genetics of archaea, but, so far, few functional properties of archaeal HR have been measured in vivo. In the extreme thermoacidophile Sulfolobus acidocaldarius, a conjugational mechanism of DNA transfer enables quantitative analysis of HR between chromosomal markers. Early studies of this system indicated that HR occurred frequently between closely spaced mutations within the pyrE gene, and this result was later supported by various analyses involving defined point mutations and deletions. These properties of intragenic HR suggested a non-reciprocal mechanism in which donor sequences become incorporated into the recipient genome as short segments. Because fragmentation of donor DNA during cell-to-cell transfer could not be excluded from contributing to this result, subsequent analyses have focused on electroporation of selectable donor DNA directly into recipient strains. For example, S. acidocaldarius was found to incorporate synthetic ssDNA (single-stranded DNA) of more than approximately 20 nt readily into its genome. With respect to various molecular properties of the ssDNA substrates, the process resembled bacteriophage lambdaRed-mediated 'recombineering' in Escherichia coli. Another approach used electroporation of a multiply marked pyrE gene to measure donor sequence tracts transferred to the recipient genome in individual recombination events. Initial results indicate multiple discontinuous tracts in the majority of recombinants, representing a relatively broad distribution of tract lengths. This pattern suggests that properties of the HR process could, in principle, account for many of the apparent peculiarities of intragenic recombination initiated by S. acidocaldarius conjugation.

  3. Genome Sequence of "Candidatus Methanomassiliicoccus intestinalis" Issoire-Mx1, a Third Thermoplasmatales-Related Methanogenic Archaeon from Human Feces.

    Science.gov (United States)

    Borrel, Guillaume; Harris, Hugh M B; Parisot, Nicolas; Gaci, Nadia; Tottey, William; Mihajlovski, Agnès; Deane, Jennifer; Gribaldo, Simonetta; Bardot, Olivier; Peyretaillade, Eric; Peyret, Pierre; O'Toole, Paul W; Brugère, Jean-François

    2013-07-11

    "Candidatus Methanomassiliicoccus intestinalis" Issoire-Mx1 is a methanogenic archaeon found in the human gut and is a representative of the novel order of methanogens related to Thermoplasmatales. Its complete genome sequence is presented here.

  4. Thermostable and site-specific DNA binding of the gene product ORF56 from the Sulfolobus islandicus plasmid pRN1, a putative archael plasmid copy control protein

    Science.gov (United States)

    Lipps, Georg; Stegert, Mario; Krauss, Gerhard

    2001-01-01

    There is still a lack of information on the specific characteristics of DNA-binding proteins from hyperthermophiles. Here we report on the product of the gene orf56 from plasmid pRN1 of the acidophilic and thermophilic archaeon Sulfolobus islandicus. orf56 has not been characterised yet but low sequence similarily to several eubacterial plasmid-encoded genes suggests that this 6.5 kDa protein is a sequence-specific DNA-binding protein. The DNA-binding properties of ORF56, expressed in Escherichia coli, have been investigated by EMSA experiments and by fluorescence anisotropy measurements. Recombinant ORF56 binds to double-stranded DNA, specifically to an inverted repeat located within the promoter of orf56. Binding to this site could down-regulate transcription of the orf56 gene and also of the overlapping orf904 gene, encoding the putative initiator protein of plasmid replication. By gel filtration and chemical crosslinking we have shown that ORF56 is a dimeric protein. Stoichiometric fluorescence anisotropy titrations further indicate that ORF56 binds as a tetramer to the inverted repeat of its target binding site. CD spectroscopy points to a significant increase in ordered secondary structure of ORF56 upon binding DNA. ORF56 binds without apparent cooperativity to its target DNA with a dissociation constant in the nanomolar range. Quantitative analysis of binding isotherms performed at various salt concentrations and at different temperatures indicates that approximately seven ions are released upon complex formation and that complex formation is accompanied by a change in heat capacity of –6.2 kJ/mol. Furthermore, recombinant ORF56 proved to be highly thermostable and is able to bind DNA up to 85°C. PMID:11160922

  5. Sulfolobus Replication Factor C stimulates the activity of DNA Polymerase B1

    DEFF Research Database (Denmark)

    Xing, Xuanxuan; Zhang, Likui; Guo, Li;

    2014-01-01

    Replication factor C (RFC) is known to function in loading proliferating cell nuclear antigen (PCNA) onto primed DNA, allowing PCNA to tether DNA polymerase for highly processive DNA synthesis in eukaryotic and archaeal replication. In this report, we show that an RFC complex from...... with the ability of RFC to facilitate DNA binding by PolB1 through protein-protein interaction. These results suggest that Sulfolobus RFC may play a role in recruiting DNA polymerase for efficient primer extension, in addition to clamp loading, during DNA replication....... the hyperthermophilic archaea of the genus Sulfolobus physically interacts with DNA polymerase B1 (PolB1) and enhances both the polymerase and 3'-5' exonuclease activities of PolB1 in an ATP-independent manner. Stimulation of the PolB1 activity by RFC is independent of the ability of RFC to bind DNA but is consistent...

  6. Molecular analysis of the UV-inducible pili operon from Sulfolobus acidocaldarius.

    Science.gov (United States)

    van Wolferen, Marleen; Ajon, Małgorzata; Driessen, Arnold J M; Albers, Sonja-Verena

    2013-12-01

    Upon ultraviolet (UV) stress, hyperthermophilic Sulfolobus species show a highly induced transcription of a gene cluster responsible for pili biogenesis: the UV-inducible pili operon (ups operon). This operon is involved in UV-induced pili assembly, cellular aggregation, and subsequent DNA exchange between cells. As the system increases the fitness of Sulfolobus cells after UV light exposure, we assume that transfer of DNA takes place in order to repair UV-induced DNA damages via homologous recombination. Here, we studied all genes present in the ups cluster via gene deletion analysis with a focus on UpsX, a protein that shows no identifiable functional domains. UspX does not seem to be structurally essential for UV-induced pili formation and cellular aggregation, but appears to be important for efficient DNA transfer. In addition, we could show that pilin subunits UpsA and UpsB probably both function as major pilin subunits in the ups pili.

  7. Membrane homeoviscous adaptation in the piezo-hyperthermophilic archaeon Thermococcus barophilus

    Directory of Open Access Journals (Sweden)

    Anaïs eCario

    2015-10-01

    Full Text Available The archaeon Thermococcus barophilus, one of the most extreme members of hyperthermophilic piezophiles known thus far, is able to grow at temperatures up to 103°C and pressures up to 80MPa. We analyzed the membrane lipids of T. barophilus by HPLC-MS as a function of pressure and temperature. In contrast to previous reports, we show that under optimal growth conditions (40 MPa, 85°C the membrane spanning tetraether lipid GDGT-0 (sometimes called caldarchaeol is a major membrane lipid of T. barophilus together with archaeol. Increasing pressure and decreasing temperature lead to an increase of the proportion of archaeol and, reversely, a higher proportion of GDGT-0 is observed under low pressure and high temperature conditions. Noticeably, pressure and temperature fluctuations also impact the level of unsaturation of non-polar lipids with an irregular polyisoprenoid carbon skeleton (polyunsaturated lycopane derivatives, suggesting a structural role for these neutral lipids in the membrane of T. barophilus. Whether these apolar lipids insert in the membrane or not remains to be addressed. However, our results raise questions about the structure of the membrane in this archaeon and other archaeon harboring a mixture of di- and tetraether lipids.

  8. Highly enantioselective sec-alkyl sulfatase activity of Sulfolobus acidocaldarius DSM 639.

    Science.gov (United States)

    Wallner, Sabine R; Nestl, Bettina M; Faber, Kurt

    2004-12-23

    [reaction: see text] rac-sec-Alkyl sulfate esters 1a-4a were resolved in high enantioselectivities with E-values up to >200 using whole cells of aerobically grown Sulfolobus acidocaldarius DSM 639. The stereochemical course of this biohydrolysis was shown to proceed with strict inversion of configuration; thus, the preferred (R)-enantiomers were converted into the corresponding (S)-sec-alcohols to furnish a homochiral product mixture.

  9. The Identification of Sulfolobus Species from a Biocorrosion of Wastewater Pipes

    Directory of Open Access Journals (Sweden)

    Shakila Motamedi

    2011-01-01

    Full Text Available One of the species which has a prominent role in the biocorrosion of wastewater pipes is Sulfolobus bacterium. This bacterium is among the iron oxidizing bacteria. Sulfolobus is from the archae bacteria genus, which provides its needed energy by oxidation of sulfur in warm and acidic environments. In this research, the water sample exist in the water supply pipelines at Sarcheshmeh copper mine was studied, sulfur corrosive bacteria were separated and identified and their biochemical activity was researched and studied and it was found that the target species are in fact Sulfolobus archae bacteria which have different enzymatic activities. Due to these enzymatic activities, a considerable amount of sulfur is accumulated on the cell surface. Based on various incubation environments, as a result of sulfur accumulation, Sulfolubos archae bacteria were identified, and after doing isolation and purification methods, number of colonies have been decrease from 10-1 dilution to 10-10 dilution and in sample with 10-1 dilution sample 200 colonies has been reported and in plate with 10-10 dilution no colonies has been observed.

  10. NCBI nr-aa BLAST: CBRC-XTRO-01-3737 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-XTRO-01-3737 ref|NP_343011.1| Transporter (proton symporter) [Sulfolobus solfa...taricus P2] gb|AAK41801.1| Transporter (proton symporter) [Sulfolobus solfataricus P2] NP_343011.1 0.010 25% ...

  11. A method to accurately quantitate intensities of (32)P-DNA bands when multiple bands appear in a single lane of a gel is used to study dNTP insertion opposite a benzo[a]pyrene-dG adduct by Sulfolobus DNA polymerases Dpo4 and Dbh.

    Science.gov (United States)

    Sholder, Gabriel; Loechler, Edward L

    2015-01-01

    Quantitating relative (32)P-band intensity in gels is desired, e.g., to study primer-extension kinetics of DNA polymerases (DNAPs). Following imaging, multiple (32)P-bands are often present in lanes. Though individual bands appear by eye to be simple and well-resolved, scanning reveals they are actually skewed-Gaussian in shape and neighboring bands are overlapping, which complicates quantitation, because slower migrating bands often have considerable contributions from the trailing edges of faster migrating bands. A method is described to accurately quantitate adjacent (32)P-bands, which relies on having a standard: a simple skewed-Gaussian curve from an analogous pure, single-component band (e.g., primer alone). This single-component scan/curve is superimposed on its corresponding band in an experimentally determined scan/curve containing multiple bands (e.g., generated in a primer-extension reaction); intensity exceeding the single-component scan/curve is attributed to other components (e.g., insertion products). Relative areas/intensities are determined via pixel analysis, from which relative molarity of components is computed. Common software is used. Commonly used alternative methods (e.g., drawing boxes around bands) are shown to be less accurate. Our method was used to study kinetics of dNTP primer-extension opposite a benzo[a]pyrene-N(2)-dG-adduct with four DNAPs, including Sulfolobus solfataricus Dpo4 and Sulfolobus acidocaldarius Dbh. Vmax/Km is similar for correct dCTP insertion with Dpo4 and Dbh. Compared to Dpo4, Dbh misinsertion is slower for dATP (∼20-fold), dGTP (∼110-fold) and dTTP (∼6-fold), due to decreases in Vmax. These findings provide support that Dbh is in the same Y-Family DNAP class as eukaryotic DNAP κ and bacterial DNAP IV, which accurately bypass N(2)-dG adducts, as well as establish the scan-method described herein as an accurate method to quantitate relative intensity of overlapping bands in a single lane, whether generated

  12. Microbial desulfurization of coal by thermophilic archaea

    Energy Technology Data Exchange (ETDEWEB)

    Olsson, Gunnel

    1994-04-01

    The investigation was focused on the removal of pyrite as well as organic sulfur. One major objective was to identify and outline difficulties associated with microbial desulfurization of coal. The work has particularly been concentrated on the desulfurization environment of the microorganisms, the reprecipitation of dissolved sulfate as jarosite, the effect of microbial treatment on the properties of the coal and the comparison of different thermophilic archaea suggested for coal desulfurization. The investigated microorganisms were the thermophilic archaea Acidianus brierleyi, Sulfolobus acidocaldarius and Sulfolobus solfataricus and for comparison the mesophilic bacterium Thiobacillus ferrooxidans. The major part of the work has been done with Acidianus brierleyi. Compounds leached from coal may seriously affect the growth of microorganisms suggested for coal desulfurization. Degradation of pyritic sulfur with the used strains of S. solfataricus and S. acidocaldarius, was observed to be impossible. However, both the thermophilic archaeon Acidianus brierleyi and the mesophilic bacterium Thiobacillus ferrooxidans, were capable of degrading pure pyrite as well as pyrite from low-sulfur coals. Up to 85% removal of pyritic sulfur was obtained for coals when staring with a pyrite sulfur content of 0.5-0.7%. From kinetic studies, it was shown that A. brierleyi and T. ferrooxidans remove sulfur from coal at roughly the same rate, at least for the coals investigated in this study. However, the rate for microbial oxidation of pure pyrite was seen to be much higher for A. brierleyi than for T. ferroxidans. 62 refs, 16 figs, 5 tabs

  13. Dimethyl sulfoxide reduction by a hyperhermophilic archaeon Thermococcus onnurineus NA1 via a cysteine-cystine redox shuttle.

    Science.gov (United States)

    Choi, Ae Ran; Kim, Min-Sik; Kang, Sung Gyun; Lee, Hyun Sook

    2016-01-01

    A variety of microbes grow by respiration with dimethyl sulfoxide (DMSO) as an electron acceptor, and several distinct DMSO respiratory systems, consisting of electron carriers and a terminal DMSO reductase, have been characterized. The heterotrophic growth of a hyperthermophilic archaeon Thermococcus onnurineus NA1 was enhanced by the addition of DMSO, but the archaeon was not capable of reducing DMSO to DMS directly using a DMSO reductase. Instead, the archaeon reduced DMSO via a cysteine-cystine redox shuttle through a mechanism whereby cystine is microbially reduced to cysteine, which is then reoxidized by DMSO reduction. A thioredoxin reductase-protein disulfide oxidoreductase redox couple was identified to have intracellular cystine-reducing activity, permitting recycle of cysteine. This study presents the first example of DMSO reduction via an electron shuttle. Several Thermococcales species also exhibited enhanced growth coupled with DMSO reduction, probably by disposing of excess reducing power rather than conserving energy.

  14. CrRNA-Protospacer Recognition during CRISPR- Directed DNA Interference Sulfolobus islandicus REY 15A and Structural Studies of CRISPR Binding Proteins (CBP) of Crenarchaeon Sulfolobus

    DEFF Research Database (Denmark)

    Mousaei, Marzieh

    The CRISPR-Cas (clustered regularly interspaced short palindromic repeats and associated proteins) is one of the important known immune mechanisms in archaea and bacteria. This adaptive immune system degrades invading genetic elements and protects the cell. Amongst 3 main types I, II and III...... of CRISPR system, two types (I and III) are found in archaea. However, in Sulfolobus species, subtypes IA, I-D, and III-B, III-D and rarely III-A are found. The model organism used for interference and structural studies is S. islandicus REY15A which carries subtypes I-A and III-B (α and β). Besides CRISPR...... ribonucleoprotein complex which is involved directly in defense, there are some less- known parts of the system including CPBs (CRISPR repeat-binding proteins) which are suggested to play a role in transcription. In the first part of my thesis, I provide a brief introduction to archaea and viruses that infect...

  15. Circular chromosomal DNA in the sulfur-dependent archaebacterium Sulfolobus acidocaldarius.

    OpenAIRE

    Yamagishi, A; Oshima, T

    1990-01-01

    The shape of the chromosomal DNA of the sulfur-dependent archaebacterium Sulfolobus acidocaldarius was analyzed by the pulsed-field gel electrophoresis(PFGE). S.acidocaldarius DNA digested with Notl showed two DNA bands at around 1.0 Mbp and 2.1 Mbp. Notl-linking clones were isolated from the library of S.acidocaldarius chromosomal DNA. It contained two Notl sites. Both 1.0 and 2.1 Mbp DNA band separated by PFGE were hybridized with the two independent Notl-linking fragment. Each right and le...

  16. Biochemical evidence for the presence of two α-glucoside ABC-transport systems in the hyperthermophilic archaeon Pyrococcus furiosus

    NARCIS (Netherlands)

    Koning, Sonja M.; Konings, Wil N.; Driessen, Arnold J.M.

    2002-01-01

    The hyperthermophilic archaeon Pyrococcus furiosus can utilize different carbohydrates, such as starch, maltose and trehalose. Uptake of α-glucosides is mediated by two different, binding protein-dependent, ATP-binding cassette (ABC)-type transport systems. The maltose transporter also transports tr

  17. Positive selection for uracil auxotrophs of the sulfur-dependent thermophilic archaebacterium Sulfolobus acidocaldarius by use of 5-fluoroorotic acid.

    OpenAIRE

    Kondo, S; Yamagishi, A; Oshima, T

    1991-01-01

    Uracil auxotrophs of Sulfolobus acidocaldarius were positively selected by using 5-fluoroorotic acid. The wild-type strain was unable to grow in medium containing 5-fluoroorotic acid, whereas the mutants grew normally. Positive selection could be done for the auxotrophs. Mutants deficient in orotidine-5'-monophosphate pyrophosphorylase activity were isolated.

  18. Roles of the Y-family DNA polymerase Dbh in accurate replication of the Sulfolobus genome at high temperature.

    Science.gov (United States)

    Sakofsky, Cynthia J; Foster, Patricia L; Grogan, Dennis W

    2012-04-01

    The intrinsically thermostable Y-family DNA polymerases of Sulfolobus spp. have revealed detailed three-dimensional structure and catalytic mechanisms of trans-lesion DNA polymerases, yet their functions in maintaining their native genomes remain largely unexplored. To identify functions of the Y-family DNA polymerase Dbh in replicating the Sulfolobus genome under extreme conditions, we disrupted the dbh gene in Sulfolobus acidocaldarius and characterized the resulting mutant strains phenotypically. Disruption of dbh did not cause any obvious growth defect, sensitivity to any of several DNA-damaging agents, or change in overall rate of spontaneous mutation at a well-characterized target gene. Loss of dbh did, however, cause significant changes in the spectrum of spontaneous forward mutation in each of two orthologous target genes of different sequence. Relative to wild-type strains, dbh(-) constructs exhibited fewer frame-shift and other small insertion-deletion mutations, but exhibited more base-pair substitutions that converted G:C base pairs to T:A base pairs. These changes, which were confirmed to be statistically significant, indicate two distinct activities of the Dbh polymerase in Sulfolobus cells growing under nearly optimal culture conditions (78-80°C and pH 3). The first activity promotes slipped-strand events within simple repetitive motifs, such as mononucleotide runs or triplet repeats, and the second promotes insertion of C opposite a potentially miscoding form of G, thereby avoiding G:C to T:A transversions.

  19. Reconstitution of the Leucine Transport System of Lactococcus lactis into Liposomes Composed of Membrane-Spanning Lipids from Sulfolobus acidocaldarius

    NARCIS (Netherlands)

    in t Veld, Geertruida; Elferink, Maria; Driessen, Arnold J.M.; Konings, Wilhelmus

    1992-01-01

    The effect of bipolar tetraether lipids, extracted from the thermophilic archaebacterium Sulfolobus acidocaldarius, on the branched-chain amino acid transport system of the mesophilic bacterium Lactococcus lactis was investigated. Liposomes were prepared from mixtures of monolayer lipids and the bil

  20. Membrane homeoviscous adaptation in the piezo-hyperthermophilic archaeon Thermococcus barophilus.

    Science.gov (United States)

    Cario, Anaïs; Grossi, Vincent; Schaeffer, Philippe; Oger, Philippe M

    2015-01-01

    The archaeon Thermococcus barophilus, one of the most extreme members of hyperthermophilic piezophiles known thus far, is able to grow at temperatures up to 103°C and pressures up to 80 MPa. We analyzed the membrane lipids of T. barophilus by high performance liquid chromatography-mass spectrometry as a function of pressure and temperature. In contrast to previous reports, we show that under optimal growth conditions (40 MPa, 85°C) the membrane spanning tetraether lipid GDGT-0 (sometimes called caldarchaeol) is a major membrane lipid of T. barophilus together with archaeol. Increasing pressure and decreasing temperature lead to an increase of the proportion of archaeol. Reversely, a higher proportion of GDGT-0 is observed under low pressure and high temperature conditions. Noticeably, pressure and temperature fluctuations also impact the level of unsaturation of apolar lipids having an irregular polyisoprenoid carbon skeleton (unsaturated lycopane derivatives), suggesting a structural role for these neutral lipids in the membrane of T. barophilus. Whether these apolar lipids insert in the membrane or not remains to be addressed. However, our results raise questions about the structure of the membrane in this archaeon and other Archaea harboring a mixture of di- and tetraether lipids.

  1. Enrichment and Characterization of an Autotrophic Ammonia-Oxidizing Archaeon of Mesophilic Crenarchaeal Group I.1a from an Agricultural Soil

    NARCIS (Netherlands)

    Jung, M.Y.; Park, S.J.; Min, D.; Kim, J.S.; Rijpstra, W.I.C.; Sinninghe Damsté, J.S.; Kim, G.J.; Madsen, E.L.; Rhee, S.K.

    2011-01-01

    Soil nitrification is an important process for agricultural productivity and environmental pollution. Though one cultivated representative of ammonia-oxidizing Archaea from soil has been described, additional representatives warrant characterization. We describe an ammonia-oxidizing archaeon (strain

  2. Lipids of the ultra-thin square halophilic archaeon Haloquadratum walsbyi

    Directory of Open Access Journals (Sweden)

    Simona LoBasso

    2008-01-01

    Full Text Available The lipid composition of the extremely halophilic archaeon Haloquadratum walsbyi was investigated by thin-layer chromatography and electrospray ionization-mass spectrometry. The analysis of neutral lipids showed the presence of vitamin MK-8, squalene, carotene, bacterioruberin and several retinal isomers. The major polar lipids were phosphatidylglycerophosphate methyl ester, phosphatidylglycerosulfate, phosphatidylglycerol and sulfated diglycosyl diether lipid. Among cardiolipins, the tetra-phytanyl or dimeric phospholipids, only traces of bisphosphatidylglycerol were detected. When the cells were exposed to hypotonic medium, no changes in the membrane lipid composition occurred. Distinguishing it from other extreme halophiles of the Halobacteriaceae family, the osmotic stress did not induce the neo-synthesis of cardiolipins in H. walsbyi. The difference may depend on the three-laminar structure of the cell wall, which differs significantly from that of other Haloarchaea.

  3. Modulation of CRISPR locus transcription by the repeat-binding protein Cbp1 in Sulfolobus

    DEFF Research Database (Denmark)

    Deng, Ling; Kenchappa, Chandra Shekar; Peng, Xu

    2012-01-01

    CRISPR loci are essential components of the adaptive immune system of archaea and bacteria. They consist of long arrays of repeats separated by DNA spacers encoding guide RNAs (crRNA), which target foreign genetic elements. Cbp1 (CRISPR DNA repeat binding protein) binds specifically to the multiple...... direct repeats of CRISPR loci of members of the acidothermophilic, crenarchaeal order Sulfolobales. cbp1 gene deletion from Sulfolobus islandicus REY15A produced a strong reduction in pre-crRNA yields from CRISPR loci but did not inhibit the foreign DNA targeting capacity of the CRISPR/Cas system....... Conversely, overexpression of Cbp1 in S. islandicus generated an increase in pre-crRNA yields while the level of reverse strand transcripts from CRISPR loci remained unchanged. It is proposed that Cbp1 modulates production of longer pre-crRNA transcripts from CRISPR loci. A possible mechanism...

  4. Transcriptome changes in STSV2-infected Sulfolobus islandicus REY15A undergoing continuous CRISPR spacer acquisition

    DEFF Research Database (Denmark)

    León-Sobrino, Carlos; Kot, Witold P; Garrett, Roger A

    2016-01-01

    A transcriptome study was performed on Sulfolobus islandicus REY15A actively undergoing CRISPR spacer acquisition from the crenarchaeal monocaudavirus STSV2 in rich and basal media over a 6 day period. Spacer acquisition preceded strong host growth retardation, altered transcriptional activity...... of four different CRISPR-Cas modules and changes in viral copy numbers, and with significant differences in the two media. Transcript levels of proteins involved in the cell cycle were reduced, while those of DNA replication, DNA repair, transcriptional regulation, and some antitoxin-toxin pairs...... and transposases were unchanged or enhanced. Antisense RNAs were implicated in the transcriptional regulation of adaptation and interference modules of the type I-A CRISPR-Cas system and evidence was found for the occurrence of functional coordination between the single CRISPR-Cas adaptation module...

  5. Some Biochemical Properties of an Acido-Thermophilic Archae-Bacterium Sulfolobus Acidocaldarius

    Science.gov (United States)

    Oshima, Tairo; Ohba, Masayuki; Wagaki, Takayoshi

    1984-12-01

    To elucidate the phylogenic status of archaebacteria, some basic cellular components of an acido-thermophilic archaebacterium,Sulfolobus acidocaldarius, were studied. Poly(A) containing RNA was present in the cells, and performed the role of mRNA in a cell-free extract of reticulocyte or the archaebacteria. Poly(A) containing RNA was also found in other archaebacterial cells. The absence of cap structure was suggested in these RNAs. The cell-free protein synthesis using the archaebacterial extract was inhibited by anisomycin, a specific inhibitor for eukaryotic ribosomes. Two unique membrane-bound ATPases were detected. Based on resistance to H+-ATPase inhibitors, these enzymes seemed not to be F0F1-ATPase.

  6. Loss of genetic accuracy in mutants of the thermoacidophile Sulfolobus acidocaldarius

    Directory of Open Access Journals (Sweden)

    Greg D. Bell

    2002-01-01

    Full Text Available To investigate how hyperthermophilic archaea can propagate their genomes accurately, we isolated Sulfolobus acidocaldarius mutants exhibiting abnormally high rates of spontaneous mutation. Our isolation strategy involved enrichment for mutator lineages via alternating selections, followed by screening for the production of spontaneous, 5-fluoro-orotate-resistant mutants in micro-colonies. Several candidates were evaluated and found to have high frequencies of pyrE and pyrF mutation and reversion. Neither an increased efficiency of plating of mutants on selective medium, nor the creation of a genetically unstable pyrE allele, could be implicated as the cause of these high frequencies. The strains had elevated frequencies of other mutations, and exhibited certain phenotypic differences among themselves. A large increase in sensitivity to DNA-damaging agents was not observed, however. These properties generally resemble those of bacterial mutator mutants and suggest loss of functions specific to genetic accuracy.

  7. Kinetic elements in thermophilic-microbial leaching of sulfur from coal. [Sulfolobus acidocaldarius

    Energy Technology Data Exchange (ETDEWEB)

    Chen, C.C.Y.; Skidmore, D.R.

    1988-01-01

    A mechanism found suitable for pyritic sulfur removal from coal by mesophilic organisms has been modified and evaluated for the same reaction by the thermophile: Sulfolobus acidocaldarius. The mechanism as modified includes direct and indirect mechanistic elements whose magnitudes were determined experimentally at leaching conditions with and without microbes. Chemical leaching of pyrite by Fe(III) ions and the chemical oxidation of Fe(II) to Fe(III) were found to contribute significantly to overall conversions at thermophilic temperature (72/degree/C) but not at mesophilic temperature (25-37/degree/C). The model thus constructed fit overall sulfur leaching data well after suitable assumptions had been made for extent of cell attachment; gas (oxygen) transfer rates, sulfur accessibility, and sulfate reprecipitation rates. The resulting equation is useful for process simulation, control and design. Further work is needed to define additional sulfur leaching effects.

  8. Long-chain glycerol diether and polyol dialkyl glycerol triether lipids of Sulfolobus acidocaldarius.

    Science.gov (United States)

    Langworthy, T A; Mayberry, W R; Smith, P F

    1974-07-01

    Cells of Sulfolobus acidocaldarius contain about 2.5% total lipid on a dry-weight basis. Total lipid was found to contain 10.5% neutral lipid, 67.6% glycolipid, and 21.7% polar lipid. The lipids contained C(40)H(80) isopranol glycerol diethers. Almost no fatty acids were present. The glycolipids were composed of about equal amounts of the glycerol diether analogue of glucosyl galactosyl diglyceride and a glucosyl polyol glycerol diether. The latter compound contained an unidentified polyol attached by an ether bond to the glycerol diether. The polar lipids contained a small amount of sulfolipid, which appeared to be the monosulfate derivative of glucosyl polyol glycerol diether. About 40% of the lipid phosphorus was found in the diether analogue of phosphatidyl inositol. The remaining lipid phosphorus was accounted for by approximately equal amounts of two inositol monophosphate-containing phosphoglycolipids, inositolphosphoryl glucosyl galactosyl glycerol diether and inositolphosphoryl glucosyl polyol glycerol diether.

  9. Studies on DNA Damage Response in Sulfolobus islandicus

    DEFF Research Database (Denmark)

    Han, Wenyuan

    global reactions known as DNA damage response (DDR). In Bacteria and Eukaryotes, the global reactions include a series of transcription regulations and protein post-translation modifications, which can activate DNA repair machineries, suppress cell division and delay DNA replication, and induce...... scattered light, damaged cell membrane and electron-dense area. During NQO and MMS treatment, degradation of chromatin proteins was coincided with DNA-less cell formation, suggesting their roles in protecting genomic DNA from massive degradation. Further, HU inhibited NQO-induced DSB formation and DNA...... damage response, suggesting the crucial roles of DSB in triggering DNA damage response. Then, NQO-induced DNA-less formation was impaired in the culture with retarded cell cycle, suggesting that DNA replication played an important role in DNA damage response in Sulfolobus. We also investigated the roles...

  10. Transcriptome changes in STSV2-infected Sulfolobus islandicus REY15A undergoing continuous CRISPR spacer acquisition

    DEFF Research Database (Denmark)

    León Sobrino, Carlos; Kot, W.P.; Garrett, Roger Antony

    2016-01-01

    and transposases were unchanged or enhanced. Antisense RNAs were implicated in the transcriptional regulation of adaptation and interference modules of the type I-A CRISPR-Cas system and evidence was found for the occurrence of functional coordination between the single CRISPR-Cas adaptation module......A transcriptome study was performed on Sulfolobus islandicus REY15A actively undergoing CRISPR spacer acquisition from the crenarchaeal monocaudavirus STSV2 in rich and basal media over a 6 day period. Spacer acquisition preceded strong host growth retardation, altered transcriptional activity...... of four different CRISPR-Cas modules and changes in viral copy numbers, and with significant differences in the two media. Transcript levels of proteins involved in the cell cycle were reduced, while those of DNA replication, DNA repair, transcriptional regulation, and some antitoxin-toxin pairs...

  11. Biochemical Properties of DNA Ligase From The Hyperthermophilic Archaeon Sulfolobus shibatae%极端嗜热古菌--芝田硫化叶菌DNA连接酶的生化性质

    Institute of Scientific and Technical Information of China (English)

    赖小勤; 黄力

    2005-01-01

    极端嗜热古菌--芝田硫化叶菌DNA连接酶(Ssh连接酶)的最适辅因子为ATP,在dATP存在时,该酶也能表现出较弱的连接活性.ATP或dATP都能够使该酶发生腺苷化,腺苷化的Ssh连接酶能够将腺苷基团转移至含切刻的DNA上.电泳迁移率改变实验表明,Ssh连接酶能够结合双链DNA,且与含切刻及不含切刻的DNA结合的亲和力相同,但不结合单链DNA.酵母双杂交实验显示,硫磺矿硫化叶菌(与芝田硫化叶菌亲缘关系很近)的DNA连接酶,与该菌所含的3个增殖细胞核抗原(PCNA)同源蛋白中的一个(PCNA-1)有相互作用,而与另外2个同源蛋白(PCNA-like和PCNA-2)则无相互作用.在古菌中高度保守的Sac10b蛋白家族成员Ssh10b能够激活Ssh连接酶的活性,而硫化叶菌中的主要染色体蛋白--7kuDNA结合蛋白(Ssh7)则对该酶活性没有影响.

  12. Genome-scale analysis of gene function in the hydrogenotrophic methanogenic archaeon Methanococcus maripaludis.

    Science.gov (United States)

    Sarmiento, Felipe; Mrázek, Jan; Whitman, William B

    2013-03-19

    A comprehensive whole-genome analysis of gene function by transposon mutagenesis and deep sequencing methodology has been implemented successfully in a representative of the Archaea domain. Libraries of transposon mutants were generated for the hydrogenotrophic, methanogenic archaeon Methanococcus maripaludis S2 using a derivative of the Tn5 transposon. About 89,000 unique insertions were mapped to the genome, which allowed for the classification of 526 genes or about 30% of the genome as possibly essential or strongly advantageous for growth in rich medium. Many of these genes were homologous to eukaryotic genes that encode fundamental processes in replication, transcription, and translation, providing direct evidence for their importance in Archaea. Some genes classified as possibly essential were unique to the archaeal or methanococcal lineages, such as that encoding DNA polymerase PolD. In contrast, the archaeal homolog to the gene encoding DNA polymerase B was not essential for growth, a conclusion confirmed by construction of an independent deletion mutation. Thus PolD, and not PolB, likely plays a fundamental role in DNA replication in methanococci. Similarly, 121 hypothetical ORFs were classified as possibly essential and likely play fundamental roles in methanococcal information processing or metabolism that are not established outside this group of prokaryotes.

  13. Proteomic Insights into Sulfur Metabolism in the Hydrogen-Producing Hyperthermophilic Archaeon Thermococcus onnurineus NA1

    Directory of Open Access Journals (Sweden)

    Yoon-Jung Moon

    2015-04-01

    Full Text Available The hyperthermophilic archaeon Thermococcus onnurineus NA1 has been shown to produce H2 when using CO, formate, or starch as a growth substrate. This strain can also utilize elemental sulfur as a terminal electron acceptor for heterotrophic growth. To gain insight into sulfur metabolism, the proteome of T. onnurineus NA1 cells grown under sulfur culture conditions was quantified and compared with those grown under H2-evolving substrate culture conditions. Using label-free nano-UPLC-MSE-based comparative proteomic analysis, approximately 38.4% of the total identified proteome (589 proteins was found to be significantly up-regulated (≥1.5-fold under sulfur culture conditions. Many of these proteins were functionally associated with carbon fixation, Fe–S cluster biogenesis, ATP synthesis, sulfur reduction, protein glycosylation, protein translocation, and formate oxidation. Based on the abundances of the identified proteins in this and other genomic studies, the pathways associated with reductive sulfur metabolism, H2-metabolism, and oxidative stress defense were proposed. The results also revealed markedly lower expression levels of enzymes involved in the sulfur assimilation pathway, as well as cysteine desulfurase, under sulfur culture condition. The present results provide the first global atlas of proteome changes triggered by sulfur, and may facilitate an understanding of how hyperthermophilic archaea adapt to sulfur-rich, extreme environments.

  14. Cloning and Characterization of an Alpha-amylase Gene from the Hyperthermophilic Archaeon Thermococcus Thioreducens

    Science.gov (United States)

    Bernhardsdotter, Eva C. M. J.; Pusey, Marc L.; Ng, Joseph D.; Garriott, Owen K.

    2004-01-01

    The gene encoding an extracellular a-amylase, TTA, from the hyperthermophilic archaeon Thermococcus thioreducens was cloned and expressed in Escherichia coli. Primary structural analysis revealed high similarity with other a-amylases from the Thermococcus and Pyrococcus genera, as well as the four highly conserved regions typical for a-amylases. The 1374 bp gene encodes a protein of 457 amino acids, of which 435 constitute the mature protein preceded by a 22 amino acid signal peptide. The molecular weight of the purified recombinant enzyme was estimated to be 43 kDa by denaturing gel electrophoresis. Maximal enzymatic activity of recombinant TTA was observed at 90 C and pH 5.5 in the absence of exogenous Ca(2+), and the enzyme was considerably stable even after incubation at 90 C for 2 hours. The thermostability at 90 and 102 C was enhanced in the presence of 5 mM Ca(2+). The extraordinarily high specific activity (about 7.4 x 10(exp 3) U/mg protein at 90 C, pH 5.5 with soluble starch as substrate) together with its low pH optimum makes this enzyme an interesting candidate for starch processing applications.

  15. Utilization of banana peel as a novel substrate for biosurfactant production by Halobacteriaceae archaeon AS65.

    Science.gov (United States)

    Chooklin, Chanika Saenge; Maneerat, Suppasil; Saimmai, Atipan

    2014-05-01

    In this study, biosurfactant-producing bacteria was evaluated for biosurfactant production by using banana peel as a sole carbon source. From the 71 strains screened, Halobacteriaceae archaeon AS65 produced the highest biosurfactant activity. The highest biosurfactant production (5.30 g/l) was obtained when the cells were grown on a minimal salt medium containing 35 % (w/v) banana peel and 1 g/l commercial monosodium glutamate at 30 °C and 200 rpm after 54 h of cultivation. The biosurfactant obtained by extraction with ethyl acetate showed high surface tension reduction (25.5 mN/m), a small critical micelle concentration value (10 mg/l), thermal and pH stability with respect to surface tension reduction and emulsification activity, and a high level of salt tolerance. The biosurfactant obtained was confirmed as a lipopeptide by using a biochemical test FT-IR, NMR, and mass spectrometry. The crude biosurfactant showed a broad spectrum of antimicrobial activity and had the ability to emulsify oil, enhance PAHs solubility, and oil bioremediation.

  16. Functional organization of a single nif cluster in the mesophilic archaeon Methanosarcina mazei strain Gö1.

    Science.gov (United States)

    Ehlers, Claudia; Veit, Katharina; Gottschalk, Gerhard; Schmitz, Ruth A

    2002-09-01

    The mesophilic methanogenic archaeon Methanosarcina mazei strain Gö1 is able to utilize molecular nitrogen (N2) as its sole nitrogen source. We have identified and characterized a single nitrogen fixation (nif) gene cluster in M. mazei Gö1 with an approximate length of 9 kbp. Sequence analysis revealed seven genes with sequence similarities to nifH, nifI1, nifI2, nifD, nifK, nifE and nifN, similar to other diazotrophic methanogens and certain bacteria such as Clostridium acetobutylicum, with the two glnB-like genes (nifI1 and nifI2) located between nifH and nifD. Phylogenetic analysis of deduced amino acid sequences for the nitrogenase structural genes of M. mazei Gö1 showed that they are most closely related to Methanosarcina barkeri nif2 genes, and also closely resemble those for the corresponding nif products of the gram-positive bacterium C. acetobutylicum. Northern blot analysis and reverse transcription PCR analysis demonstrated that the M. mazei nif genes constitute an operon transcribed only under nitrogen starvation as a single 8 kb transcript. Sequence analysis revealed a palindromic sequence at the transcriptional start site in front of the M. mazei nifH gene, which may have a function in transcriptional regulation of the nif operon.

  17. Minimal sulfur requirement for growth and sulfur-dependent metabolism of the hyperthermophilic archaeon Staphylothermus marinus

    Directory of Open Access Journals (Sweden)

    Xiaolei Hao

    2003-01-01

    Full Text Available Staphylothermus marinus is an anaerobic hyperthermophilic archaeon that uses peptides as carbon and energy sources. Elemental sulfur (S° is obligately required for its growth and is reduced to H2S. The metabolic functions and mechanisms of S° reduction were explored by examining S°-dependent growth and activities of key enzymes present in this organism. All three forms of S° tested—sublimed S°, colloidal S° and polysulfide—were used by S. marinus, and no other sulfur-containing compounds could replace S°. Elemental sulfur did not serve as physical support but appeared to function as an electron acceptor. The minimal S° concentration required for optimal growth was 0.05% (w/v. At this concentration, there appeared to be a metabolic transition from H2 production to S° reduction. Some enzymatic activities related to S°-dependent metabolism, including sulfur reductase, hydrogenase, glutamate dehydrogenase and electron transfer activities, were detected in cell-free extracts of S. marinus. These results indicate that S° plays an essential role in the heterotrophic metabolism of S. marinus. Reducing equivalents generated by the oxidation of amino acids from peptidolysis may be transferred to sulfur reductase and hydrogenase, which then catalyze the production of H2S and H2, respectively.

  18. Functional screening of hydrolytic activities reveals an extremely thermostable cellulase from a deep-sea archaeon

    Directory of Open Access Journals (Sweden)

    Benedikt eLeis

    2015-07-01

    Full Text Available Extreme habitats serve as a source of enzymes which are active under extreme conditions and are candidates for industrial applications. In this work, six large-insert mixed genomic libraries were screened for hydrolase activities in a broad temperature range (8 to 70 °C. Among a variety of hydrolytic activities, one fosmid clone, derived from a library of pooled isolates of hyperthermophilic archaea from deep sea vents, displayed hydrolytic activity on carboxymethyl cellulose substrate plates at 70 °C but not at lower temperatures. Sequence analysis of the fosmid insert revealed a gene encoding a novel glycoside hydrolase family 12 (GHF12 endo-1,4-β-glucanase, termed Cel12E. The enzyme shares 45 % sequence identity with a protein from the archaeon Thermococcus sp. AM4 and displays a unique multidomain architecture. Biochemical characterization of Cel12E revealed a remarkably thermostable protein, which appears to be of archaeal origin. The enzyme displayed maximum activity at 92 °C and was active on a variety of linear 1,4-β-glucans like carboxymethyl cellulose, β-glucan, lichenan, and phosphoric acid swollen cellulose. The protein is able to bind to various insoluble β-glucans. Product pattern analysis indicated that Cel12E is an endo-cleaving β-glucanase. Cel12E expands the toolbox of hyperthermostable archaeal cellulases with biotechnological potential.

  19. Natronorubrum texcoconense sp. nov., a haloalkaliphilic archaeon isolated from soil of the former lake Texcoco (Mexico).

    Science.gov (United States)

    Ruiz-Romero, Erick; Valenzuela-Encinas, César; López-Ramírez, María Patricia; de los Angeles Coutiño-Coutiño, María; Marsch, Rodolfo; Dendooven, Luc

    2013-02-01

    A new haloalkaliphilic archaeon, strain B4(T), was isolated from the former lake Texcoco in Mexico. The cells were Gram-negative, pleomorphic-shaped, pink to red pigmented and aerobic. Strain B4(T) required at least 2.5 M NaCl for growth, with optimum growth at 3.4 M NaCl. It was able to grow over a pH range of 7.5-10.0 and temperature of 25-50 °C, with optimal growth at pH 9 and 37 °C. Cells are lysed in hypotonic treatment with less than 1.3 M NaCl. The major polar lipids of strain B4(T) were phosphatidylglycerol and methyl-phosphatidylglycerophosphate. Phospholipids were detected, but not glycolipids. The nucleotide sequence of the 16S rRNA gene revealed that the strain B4(T) was phylogenetically related to members of the genus Natronorubrum. Sequence similarity with Natronorubrum tibetense was 96.28 %, with Natronorubrum sulfidifaciens 95.06 % and Natronorubrum sediminis 94.98 %. The G+C content of the DNA was 63.3 mol%. The name of Natronorubrum texcoconense sp. nov. is proposed. The type strain is B4(T) (=CECT 8067(T) = JCM 17497(T)).

  20. Natronobacterium texcoconense sp. nov., a haloalkaliphilic archaeon isolated from soil of a former lake.

    Science.gov (United States)

    Ruiz-Romero, Erick; Sánchez-López, Katia Berenice; de los Angeles Coutiño-Coutiño, María; González-Pozos, Sirenia; Bello-López, Juan Manuel; López-Ramírez, María Patricia; Ramírez-Villanueva, Daniel Alejandro; Dendooven, Luc

    2013-11-01

    A novel haloalkaliphilic archaeon, strain B23(T) was isolated from the former lake Texcoco in Mexico. The strain was Gram-stain-negative, the cells coccoid to ovoid rods, red pigmented and aerobic. Strain B23(T) grew in 1.7-4.3 M NaCl, at pH 6.5-9.5 and at 25-45 °C with optimal growth at 2.6-3.4 M NaCl, pH 7.5-8.5 and 37 °C. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain B23(T) was most closely related to Natronobacterium gregoryi SP2(T) with 97.3 % sequence similarity. The polar lipids of strain B23(T) were phosphatidylglycerol and several unidentified phospholipids. The G+C content of the DNA of the strain was 62.5 mol%. Levels of DNA-DNA relatedness between strain B23(T) and Natronobacterium gregoryi DSM 3393(T) was 32.3 %. The name Natronobacterium texcoconense sp. nov. is proposed. The type strain is B23(T) ( = CECT 8068(T) = JCM 17655(T)).

  1. Functional organization of a single nif cluster in the mesophilic archaeon Methanosarcina mazei strain Gö1

    Directory of Open Access Journals (Sweden)

    Claudia Ehlers

    2002-01-01

    Full Text Available The mesophilic methanogenic archaeon Methanosarcina mazei strain Gö1 is able to utilize molecular nitrogen (N2 as its sole nitrogen source. We have identified and characterized a single nitrogen fixation (nif gene cluster in M. mazei Gö1 with an approximate length of 9 kbp. Sequence analysis revealed seven genes with sequence similarities to nifH, nifI1, nifI2, nifD, nifK, nifE and nifN, similar to other diazotrophic methanogens and certain bacteria such as Clostridium acetobutylicum, with the two glnB-like genes (nifI1 and nifI2 located between nifH and nifD. Phylogenetic analysis of deduced amino acid sequences for the nitrogenase structural genes of M. mazei Gö1 showed that they are most closely related to Methanosarcina barkeri nif2 genes, and also closely resemble those for the corresponding nif products of the gram-positive bacterium C. acetobutylicum. Northern blot analysis and reverse transcription PCR analysis demonstrated that the M. mazei nif genes constitute an operon transcribed only under nitrogen starvation as a single 8 kb transcript. Sequence analysis revealed a palindromic sequence at the transcriptional start site in front of the M. mazei nifH gene, which may have a function in transcriptional regulation of the nif operon.

  2. High hydrostatic pressure increases amino acid requirements in the piezo-hyperthermophilic archaeon Thermococcus barophilus.

    Science.gov (United States)

    Cario, Anaïs; Lormières, Florence; Xiang, Xiao; Oger, Philippe

    2015-11-01

    We have established a defined growth medium for the piezophilic hyperthermophilic archaeon Thermococcus barophilus, which allows growth yields of ca. 10(8) cells/ml under both atmospheric and high hydrostatic pressure. Our results demonstrate a major impact of hydrostatic pressure on amino acid metabolism, with increases from 3 amino acids required at atmospheric pressure to 17 at 40 MPa. We observe in T. barophilus and other Thermococcales a similar discrepancy between the presence/absence of amino acid synthesis pathways and amino acid requirements, which supports the existence of alternate, but yet unknown, amino acid synthesis pathways, and may explain the low number of essential amino acids observed in T. barophilus and other Thermococcales. T. barophilus displays a strong metabolic preference for organic polymers such as polypeptides and chitin, which may constitute a more readily available resource of carbon and energy in situ in deep-sea hydrothermal vents. We hypothesize that the low energy yields of fermentation of organic polymers, together with energetic constraints imposed by high hydrostatic pressure, may render de novo synthesis of amino acids ecologically unfavorable. Induction of this metabolic switch to amino acid recycling can explain the requirement for non-essential amino acids by Thermococcales for efficient growth in defined medium.

  3. Dynamic Metabolite Profiling in an Archaeon Connects Transcriptional Regulation to Metabolic Consequences.

    Directory of Open Access Journals (Sweden)

    Horia Todor

    Full Text Available Previous work demonstrated that the TrmB transcription factor is responsible for regulating the expression of many enzyme-coding genes in the hypersaline-adapted archaeon Halobacterium salinarum via a direct interaction with a cis-regulatory sequence in their promoters. This interaction is abolished in the presence of glucose. Although much is known about the effects of TrmB at the transcriptional level, it remains unclear whether and to what extent changes in mRNA levels directly affect metabolite levels. In order to address this question, here we performed a high-resolution metabolite profiling time course during a change in nutrients using a combination of targeted and untargeted methods in wild-type and ΔtrmB strain backgrounds. We found that TrmB-mediated transcriptional changes resulted in widespread and significant changes to metabolite levels across the metabolic network. Additionally, the pattern of growth complementation using various purines suggests that the mis-regulation of gluconeogenesis in the ΔtrmB mutant strain in the absence of glucose results in low phosphoribosylpyrophosphate (PRPP levels. We confirmed these low PRPP levels using a quantitative mass spectrometric technique and found that they are associated with a metabolic block in de novo purine synthesis, which is partially responsible for the growth defect of the ΔtrmB mutant strain in the absence of glucose. In conclusion, we show how transcriptional regulation of metabolism affects metabolite levels and ultimately, phenotypes.

  4. Pyrobaculum calidifontis sp. nov., a novel hyperthermophilic archaeon that grows in atmospheric air

    Directory of Open Access Journals (Sweden)

    Taku Amo

    2002-01-01

    Full Text Available A novel, facultatively aerobic, heterotrophic hyperthermophilic archaeon was isolated from a terrestrial hot spring in the Philippines. Cells of the new isolate, strain VA1, were rod-shaped with a length of 1.5 to 10 μm and a width of 0.5 to 1.0 μm. Isolate VA1 grew optimally at 90 to 95 °C and pH 7.0 under atmospheric air. Oxygen served as a final electron acceptor under aerobic growth conditions, and vigorous shaking of the medium significantly enhanced growth. Elemental sulfur inhibited cell growth under aerobic growth conditions, whereas thiosulfate stimulated cell growth. Under anaerobic growth conditions, nitrate served as a final electron acceptor, but nitrite or sulfur-containing compounds such as elemental sulfur, thiosulfate, sulfate and sulfite could not act as final electron acceptors. The G+C content of the genomic DNA was 51 mol%. Phylogenetic analysis based on 16S rRNA sequences indicated that strain VA1 exhibited close relationships to species of the genus Pyrobaculum. A DNA–DNA hybridization study revealed a low level of similarity (≤ 18% between strain VA1 and previously described members of the genus Pyrobaculum. Physiological characteristics also indicated that strain VA1 was distinct from these Pyrobaculum species. Our results indicate that isolate VA1 represents a novel species, named Pyrobaculum calidifontis.

  5. Identification of several intracellular carbohydrate-degrading activities from the halophilic archaeon Haloferax mediterranei.

    Science.gov (United States)

    Pérez-Pomares, F; Díaz, S; Bautista, V; Pire, C; Bravo, G; Esclapez, J; Zafrilla, B; Bonete, María-José

    2009-07-01

    Three different amylolytic activities, designated AMY1, AMY2, and AMY3 were detected in the cytoplasm of the extreme halophilic archaeon Haloferax mediterranei grown in a starch containing medium. This organism had also been reported to excrete an alpha-amylase into the external medium in such conditions. The presence of these different enzymes which are also able to degrade starch may be related to the use of the available carbohydrates and maltodextrins, including the products obtained by the action of the extracellular amylase on starch that may be transported to the cytoplasm of the organism. The behavior of these intracellular hydrolytic enzymes on starch is reported here and compared with their extracellular counterpart. Two of these glycosidic activities (AMY1, AMY3) have also been purified and further characterized. As with other halophilic enzymes, they were salt dependent and displayed maximal activity at 3 M NaCl, and 50 degrees C. The purification steps and molecular masses have also been reported. The other activity (AMY2) was also detected in extracts from cells grown in media with glycerol instead of starch and in a yeast extract medium. This enzyme was able to degrade starch yielding small oligosaccharides and displayed similar halophilic behavior with salt requirement in the range 1.5-3 M NaCl.

  6. Characterization of the proteasome from the extremely halophilic archaeon Haloarcula marismortui

    Directory of Open Access Journals (Sweden)

    B. Franzetti

    2002-01-01

    Full Text Available A 20S proteasome, comprising two subunits α and β, was purified from the extreme halophilic archaeon Haloarcula marismortui, which grows only in saturated salt conditions. The three-dimensional reconstruction of the H. marismortui proteasome (Hm proteasome, obtained from negatively stained electron micrographs, is virtually identical to the structure of a thermophilic proteasome filtered to the same resolution. The stability of the Hm proteasome was found to be less salt-dependent than that of other halophilic enzymes previously described. The proteolytic activity of the Hm proteasome was investigated using the malate dehydrogenase from H. marismortui (HmMalDH as a model substrate. The HmMalDH denatures when the salt concentration is decreased below 2 M. Under these conditions, the proteasome efficiently cleaves HmMalDH during its denaturation process, but the fully denatured HmMalDH is poorly degraded. These in vitro experiments show that, at low salt concentrations, the 20S proteasome from halophilic archaea eliminates a misfolded protein.

  7. Genome-wide primary transcriptome analysis of H2-producing archaeon Thermococcus onnurineus NA1

    Science.gov (United States)

    Cho, Suhyung; Kim, Min-Sik; Jeong, Yujin; Lee, Bo-Rahm; Lee, Jung-Hyun; Kang, Sung Gyun; Cho, Byung-Kwan

    2017-01-01

    In spite of their pivotal roles in transcriptional and post-transcriptional processes, the regulatory elements of archaeal genomes are not yet fully understood. Here, we determine the primary transcriptome of the H2-producing archaeon Thermococcus onnurineus NA1. We identified 1,082 purine-rich transcription initiation sites along with well-conserved TATA box, A-rich B recognition element (BRE), and promoter proximal element (PPE) motif in promoter regions, a high pyrimidine nucleotide content (T/C) at the −1 position, and Shine-Dalgarno (SD) motifs (GGDGRD) in 5′ untranslated regions (5′ UTRs). Along with differential transcript levels, 117 leaderless genes and 86 non-coding RNAs (ncRNAs) were identified, representing diverse cellular functions and potential regulatory functions under the different growth conditions. Interestingly, we observed low GC content in ncRNAs for RNA-based regulation via unstructured forms or interaction with other cellular components. Further comparative analysis of T. onnurineus upstream regulatory sequences with those of closely related archaeal genomes demonstrated that transcription of orthologous genes are initiated by highly conserved promoter sequences, however their upstream sequences for transcriptional and translational regulation are largely diverse. These results provide the genetic information of T. onnurineus for its future application in metabolic engineering. PMID:28216628

  8. Dynamic Metabolite Profiling in an Archaeon Connects Transcriptional Regulation to Metabolic Consequences.

    Science.gov (United States)

    Todor, Horia; Gooding, Jessica; Ilkayeva, Olga R; Schmid, Amy K

    2015-01-01

    Previous work demonstrated that the TrmB transcription factor is responsible for regulating the expression of many enzyme-coding genes in the hypersaline-adapted archaeon Halobacterium salinarum via a direct interaction with a cis-regulatory sequence in their promoters. This interaction is abolished in the presence of glucose. Although much is known about the effects of TrmB at the transcriptional level, it remains unclear whether and to what extent changes in mRNA levels directly affect metabolite levels. In order to address this question, here we performed a high-resolution metabolite profiling time course during a change in nutrients using a combination of targeted and untargeted methods in wild-type and ΔtrmB strain backgrounds. We found that TrmB-mediated transcriptional changes resulted in widespread and significant changes to metabolite levels across the metabolic network. Additionally, the pattern of growth complementation using various purines suggests that the mis-regulation of gluconeogenesis in the ΔtrmB mutant strain in the absence of glucose results in low phosphoribosylpyrophosphate (PRPP) levels. We confirmed these low PRPP levels using a quantitative mass spectrometric technique and found that they are associated with a metabolic block in de novo purine synthesis, which is partially responsible for the growth defect of the ΔtrmB mutant strain in the absence of glucose. In conclusion, we show how transcriptional regulation of metabolism affects metabolite levels and ultimately, phenotypes.

  9. A transcription factor links growth rate and metabolism in the hypersaline adapted archaeon Halobacterium salinarum.

    Science.gov (United States)

    Todor, Horia; Dulmage, Keely; Gillum, Nicholas; Bain, James R; Muehlbauer, Michael J; Schmid, Amy K

    2014-09-01

    Co-ordinating metabolism and growth is a key challenge for all organisms. Despite fluctuating environments, cells must produce the same metabolic outputs to thrive. The mechanisms underlying this 'growth homeostasis' are known in bacteria and eukaryotes, but remain unexplored in archaea. In the model archaeon Halobacterium salinarum, the transcription factor TrmB regulates enzyme-coding genes in diverse metabolic pathways in response to glucose. However, H. salinarum is thought not to catabolize glucose. To resolve this discrepancy, we demonstrate that TrmB regulates the gluconeogenic production of sugars incorporated into the cell surface S-layer glycoprotein. Additionally, we show that TrmB-DNA binding correlates with instantaneous growth rate, likely because S-layer glycosylation is proportional to growth. This suggests that TrmB transduces a growth rate signal to co-regulated metabolic pathways including amino acid, purine, and cobalamin biosynthesis. Remarkably, the topology and function of this growth homeostatic network appear conserved across domains despite extensive alterations in protein components.

  10. Major and minor crRNA annealing sites facilitate low stringency DNA protospacer binding prior to Type I-A CRISPR-Cas interference in Sulfolobus

    DEFF Research Database (Denmark)

    Mousaei, Marzieh; Deng, Ling; She, Qunxin

    2016-01-01

    The stringency of crRNA-protospacer DNA base pair matching required for effective CRISPR-Cas interference is relatively low in crenarchaeal Sulfolobus species in contrast to that required in some bacteria. To understand its biological significance we studied crRNA-protospacer interactions...... in Sulfolobus islandicus REY15A which carries multiple, and functionally diverse, interference complexes. A range of mismatches were introduced into a vector-borne protospacer that was identical to spacer 1 of CRISPR locus 2, with a cognate CCN PAM sequence. Two important crRNA annealing regions were identified...

  11. C68 from the Sulfolobus islandicus plasmid-virus pSSVx is a novel member of the AbrB-like transcription factor family

    DEFF Research Database (Denmark)

    Contursi, Patrizia; D'Ambrosio, Katia; Pirone, Luciano;

    2011-01-01

    The genetic element pSSVx from Sulfolobus islandicus, strain REY15/4, is a hybrid between a plasmid and a fusellovirus. This plasmid-virus hybrid infects several species of the hyperthermophilic acidophilic crenarchaeon Sulfolobus. The open reading frame orfc68 of pSSVx encodes a 7.7 kDa protein ...... factors, such as AbrB from Bacillus subtilis. Nevertheless, C68 constitutes a novel representative of this family because it shows several peculiar structural and functional features....

  12. Complete Genome Sequence of the Hyperthermophilic Archaeon Pyrococcus sp. Strain ST04, Isolated from a Deep-Sea Hydrothermal Sulfide Chimney on the Juan de Fuca Ridge

    Science.gov (United States)

    Jung, Jong-Hyun; Lee, Ju-Hoon; Holden, James F.; Seo, Dong-Ho; Shin, Hakdong; Kim, Hae-Yeong; Kim, Wooki; Ryu, Sangryeol

    2012-01-01

    Pyrococcus sp. strain ST04 is a hyperthermophilic, anaerobic, and heterotrophic archaeon isolated from a deep-sea hydrothermal sulfide chimney on the Endeavour Segment of the Juan de Fuca Ridge in the northeastern Pacific Ocean. To further understand the distinct characteristics of this archaeon at the genome level (polysaccharide utilization at high temperature and ATP generation by a Na+ gradient), the genome of strain ST04 was completely sequenced and analyzed. Here, we present the complete genome sequence analysis results of Pyrococcus sp. ST04 and report the major findings from the genome annotation, with a focus on its saccharolytic and metabolite production potential. PMID:22843576

  13. Sulfolobus islandicus meta-populations in Yellowstone National Park hot springs.

    Science.gov (United States)

    Campbell, Kate M; Kouris, Angela; England, Whitney; Anderson, Rika E; McCleskey, R Blaine; Nordstrom, D Kirk; Whitaker, Rachel J

    2017-03-09

    Abiotic and biotic forces shape the structure and evolution of microbial populations. We investigated forces that shape the spatial and temporal population structure of Sulfolobus islandicus by comparing geochemical and molecular analysis from seven hot springs in five regions sampled over three years in Yellowstone National Park. Through deep amplicon sequencing, we uncovered 148 unique alleles at two loci whose relative frequency provides clear evidence for independent populations in different hot springs. Although geography controls regional geochemical composition and population differentiation, temporal changes in population were not explained by corresponding variation in geochemistry. The data suggest that the influence of extinction, bottleneck events, and/or selective sweeps within a spring and low migration between springs shape these populations. We suggest that hydrologic events such as storm events and surface snowmelt runoff destabilize smaller hot spring environments with smaller populations and result in high variation in the S. islandicus population over time. Therefore, physical abiotic features such as hot spring size and position in the landscape are important factors shaping the stability and diversity of the S. islandicus meta-population within Yellowstone National Park. This article is protected by copyright. All rights reserved.

  14. A kinetic assessment of substantial oxidation by sulfolobus acidocaldarius in pyrite dissolution

    Energy Technology Data Exchange (ETDEWEB)

    Vitaya, V.B.; Koizumi, J.; Toda, K. (King Mongkuts Institute of Technology, Bangkok (Thailand). Dept. of Chemical Engineering)

    1994-01-01

    The relative contributions of biological and chemical reactions to the total rate of pyrite oxidation in the presence of Sulfolobus acidocaldarius were studied on the basis of experimental work coupled with mathematical modeling. Kinetic constants for the individual reactions were determined in independent experiments. The specific growth rate of cells on the pyrite surface, which is the only unknown parameter, was assumed to be [mu](s) 0.1 h[sup -1] and justified by the agreement of the simulated results of a proposed model and the experimental results. The model includes: reversible adsorption, biological dissolution of pyrite by the adsorbed cells, chemical dissolution of pyrite accompanied by the reduction of ferric ions to ferrous ions, biochemical oxidation of ferrous ions to ferric ions by free cells, and change of the surface area of pyrite particles. It is suggested that the contributions of direct (biological) and indirect (chemical) reaction to the total rate of pyrite oxidation were in a ratio of 2:1.

  15. Improving low-temperature activity of Sulfolobus acidocaldarius 2-keto-3-deoxygluconate aldolase

    Directory of Open Access Journals (Sweden)

    Suzanne Wolterink-van Loo

    2009-01-01

    Full Text Available Sulfolobus acidocaldarius 2-keto-3-deoxygluconate aldolase (SacKdgA displays optimal activity at 95°C and is studied as a model enzyme for aldol condensation reactions. For application of SacKdgA at lower temperatures, a library of randomly generated mutants was screened for improved synthesis of 2-keto-3-deoxygluconate from pyruvate and glyceraldehyde at the suboptimal temperature of 50 °C. The single mutant SacKdgA-V193A displayed a threefold increase in activity compared with wild type SacKdgA. The increased specific activity at 40–60 °C of this mutant was observed, not only for the condensation of pyruvate with glyceraldehyde, but also for several unnatural acceptor aldehydes. The optimal temperature for activity of SacKdgA-V193A was lower than for the wild type enzyme, but enzymatic stability of the mutant was similar to that of the wild type, indicating that activity and stability were uncoupled. Valine193 has Van der Waals interactions with Lysine153, which covalently binds the substrate during catalysis. The mutation V193A introduced space close to this essential residue, and the increased activity of the mutant presumably resulted from increased flexibility of Lysine153. The increased activity of SacKdgA-V193A with unaffected stability demonstrates the potential for optimizing extremely thermostable aldolases for synthesis reactions at moderate temperatures.

  16. Absence of diauxie during simultaneous utilization of glucose and Xylose by Sulfolobus acidocaldarius.

    Science.gov (United States)

    Joshua, Chijioke J; Dahl, Robert; Benke, Peter I; Keasling, Jay D

    2011-03-01

    Sulfolobus acidocaldarius utilizes glucose and xylose as sole carbon sources, but its ability to metabolize these sugars simultaneously is not known. We report the absence of diauxie during growth of S. acidocaldarius on glucose and xylose as co-carbon sources. The presence of glucose did not repress xylose utilization. The organism utilized a mixture of 1 g/liter of each sugar simultaneously with a specific growth rate of 0.079 h(-1) and showed no preference for the order in which it utilized each sugar. The organism grew faster on 2 g/liter xylose (0.074 h(-1)) as the sole carbon source than on an equal amount of glucose (0.022 h(-1)). When grown on a mixture of the two carbon sources, the growth rate of the organism increased from 0.052 h(-1) to 0.085 h(-1) as the ratio of xylose to glucose increased from 0.25 to 4. S. acidocaldarius appeared to utilize a mixture of glucose and xylose at a rate roughly proportional to their concentrations in the medium, resulting in complete utilization of both sugars at about the same time. Gene expression in cells grown on xylose alone was very similar to that in cells grown on a mixture of xylose and glucose and substantially different from that in cells grown on glucose alone. The mechanism by which the organism utilized a mixture of sugars has yet to be elucidated.

  17. ESCRT-III mediated cell division in Sulfolobus acidocaldarius - a reconstitution perspective.

    Science.gov (United States)

    Härtel, Tobias; Schwille, Petra

    2014-01-01

    In the framework of synthetic biology, it has become an intriguing question what would be the minimal representation of cell division machinery. Thus, it seems appropriate to compare how cell division is realized in different microorganisms. In particular, the cell division system of Crenarchaeota lacks certain proteins found in most bacteria and Euryarchaeota, such as FtsZ, MreB or the Min system. The Sulfolobaceae family encodes functional homologs of the eukaryotic proteins vacuolar protein sorting 4 (Vps4) and endosomal sorting complex required for transport-III (ESCRT-III). ESCRT-III is essential for several eukaryotic pathways, e.g., budding of intraluminal vesicles, or cytokinesis, whereas Vps4 dissociates the ESCRT-III complex from the membrane. Cell Division A (CdvA) is required for the recruitment of crenarchaeal ESCRT-III proteins to the membrane at mid-cell. The proteins polymerize and form a smaller structure during constriction. Thus, ESCRT-III mediated cell division in Sulfolobus acidocaldarius shows functional analogies to the Z ring observed in prokaryotes like Escherichia coli, which has recently begun to be reconstituted in vitro. In this short perspective, we discuss the possibility of building such an in vitro cell division system on basis of archaeal ESCRT-III.

  18. Sa-Lrp from Sulfolobus acidocaldarius is a versatile, glutamine-responsive, and architectural transcriptional regulator.

    Science.gov (United States)

    Vassart, Amelia; Van Wolferen, Marleen; Orell, Alvaro; Hong, Ye; Peeters, Eveline; Albers, Sonja-Verena; Charlier, Daniel

    2013-02-01

    Sa-Lrp is a member of the leucine-responsive regulatory protein (Lrp)-like family of transcriptional regulators in Sulfolobus acidocaldarius. Previously, we demonstrated the binding of Sa-Lrp to the control region of its own gene in vitro. However, the function and cofactor of Sa-Lrp remained an enigma. In this work, we demonstrate that glutamine is the cofactor of Sa-Lrp by inducing the formation of octamers and increasing the DNA-binding affinity and sequence specificity. In vitro protein-DNA interaction assays indicate that Sa-Lrp binds to promoter regions of genes with a variety of functions including ammonia assimilation, transcriptional control, and UV-induced pili synthesis. DNA binding occurs with a specific affinity for AT-rich binding sites, and the protein induces DNA bending and wrapping upon binding, indicating an architectural role of the regulator. Furthermore, by analyzing an Sa-lrp deletion mutant, we demonstrate that the protein affects transcription of some of the genes of which the promoter region is targeted and that it is an important determinant of the cellular aggregation phenotype. Taking all these results into account, we conclude that Sa-Lrp is a glutamine-responsive global transcriptional regulator with an additional architectural role.

  19. Homologous recombination of exogenous DNA with the Sulfolobus acidocaldarius genome: properties and uses.

    Science.gov (United States)

    Kurosawa, Norio; Grogan, Dennis W

    2005-12-01

    In order to quantify recombination between exogenous DNA and the Sulfolobus acidocaldarius chromosome, we electroporated pyrE (uracil-auxtotrophic) recipient strains with functional pyrE sequences and counted Pyr+ transformants by direct plating. Certain culture and post-electroporation conditions increased the yield of Pyr+ recombinants from non-replicating pyrE plasmid, whereas cognate methylation of SuaI restriction sites in the plasmid decreased it. Recombination of linear DNAs with the S. acidocaldarius genome was proportional to the length of a limiting overlap, but even synthetic oligonucleotides produced reasonable numbers of recombinants with appropriate recipient strains. To investigate uses of this latter property, we electroporated an 18-bp pyrE deletion mutant with mixtures of synthetic oligonucleotides altering glycine-55 of the orotate phosphoribosyl transferase encoded by pyrE. Pyr+ transformants were recovered in which this codon was converted to each of the alternatives encoded by the oligonucleotide mixtures, thereby identifying five amino acid substitutions tolerated at this position of the thermostable enzyme.

  20. Insights into subunit interactions in the Sulfolobus acidocaldarius archaellum cytoplasmic complex.

    Science.gov (United States)

    Banerjee, Ankan; Neiner, Tomasz; Tripp, Patrick; Albers, Sonja-Verena

    2013-12-01

    Archaella are the archaeal motility structure that is the functional pendant of the bacterial flagellum but is assembled by a mechanism similar to that for type IV pili. Recently, it was shown by Banerjee et al. that FlaX, a crenarchaeal archaellum subunit from Sulfolobus acidocaldarius, forms a ring-like oligomer, and it was proposed that this ring may act as a static platform for torque generation in archaellum rotation [Banerjee A et al. (2012) J Biol Chem 287, 43322-43330]. Moreover, the hexameric crystal structure of FlaI was solved, and its dual function in the assembly and the rotation of the archaellum was demonstrated [Reindl S et al. (2013) Mol Cell 49, 1069-1082]. In this study, we show by biochemical and biophysical techniques that FlaX from S. acidocaldarius acts as a cytoplasmic scaffold in archaellum assembly, as it interacts with FlaI as well as with the recA family protein FlaH, the only cytoplasmic components of the archaellum. Interaction studies using various truncated versions of FlaI demonstrated that its N- and C-termini interact with FlaX. Moreover, using microscale thermophoresis, we show that FlaI, FlaX and FlaH interact with high affinities in the nanomolar range. Therefore, we propose that these three proteins form the cytoplasmic motor complex of the archaellum.

  1. Regioselective oxidation of lauric acid by CYP119, an orphan cytochrome P450 from Sulfolobus acidocaldarius.

    Science.gov (United States)

    Lim, Young-Ran; Eun, Chang-Yong; Park, Hyoung-Goo; Han, Songhee; Han, Jung-Soo; Cho, Kyoung Sang; Chun, Young-Jin; Kim, Donghak

    2010-03-01

    Archaebacteria Sulfolobus acidocaldarius contains the highly thermophilic cytochrome P450 enzyme (CYP119). CYP119 possesses stable enzymatic activity at up to 85 degrees C. However, this enzyme is still considered as an orphan P450 without known physiological function with endogenous or xenobiotic substrates. We characterized the regioselectivity of lauric acid by CYP119 using the auxiliary redox partner proteins putidaredoxin (Pd) and putidaredoxin reductase (PdR). Purified CYP119 protein showed a tight binding affinity to lauric acid (K(d)=1.1+/-0.1 microM) and dominantly hydroxylated (omega-1) position of lauric acid. We determined the steady-state kinetic parameters; k(cat) was 10.8 min(-1) and K(m) was 12 microM. The increased ratio to omega-hydroxylated production of lauric acid catalyzed by CYP119 was observed with increase in the reaction temperature. These studies suggested that the regioselectivity of CYP119 provide the critical clue for the physiological enzyme function in this thermophilic archaebacteria. In addition, regioselectivity control of CYP119 without altering its thermostability can lead to the development of novel CYP119-based catalysts through protein engineering.

  2. Structure and function of the adhesive type IV pilus of Sulfolobus acidocaldarius.

    Science.gov (United States)

    Henche, Anna-Lena; Ghosh, Abhrajyoti; Yu, Xiong; Jeske, Torsten; Egelman, Edward; Albers, Sonja-Verena

    2012-12-01

    Archaea display a variety of type IV pili on their surface and employ them in different physiological functions. In the crenarchaeon Sulfolobus acidocaldarius the most abundant surface structure is the aap pilus (archaeal adhesive pilus). The construction of in frame deletions of the aap genes revealed that all the five genes (aapA, aapX, aapE, aapF, aapB) are indispensible for assembly of the pilus and an impact on surface motility and biofilm formation was observed. Our analyses revealed that there exists a regulatory cross-talk between the expression of aap genes and archaella (formerly archaeal flagella) genes during different growth phases. The structure of the aap pilus is entirely different from the known bacterial type IV pili as well as other archaeal type IV pili. An aap pilus displayed 3 stranded helices where there is a rotation per subunit of ∼138° and a rise per subunit of ∼5.7 Å. The filaments have a diameter of ∼110 Å and the resolution was judged to be ∼9 Å. We concluded that small changes in sequence might be amplified by large changes in higher-order packing. Our finding of an extraordinary stability of aap pili possibly represents an adaptation to harsh environments that S. acidocaldarius encounters.

  3. Hydrolysis and transglycosylation activity of a thermostable recombinant beta-glycosidase from Sulfolobus acidocaldarius.

    Science.gov (United States)

    Park, Ah-Reum; Kim, Hye-Jung; Lee, Jung-Kul; Oh, Deok-Kun

    2010-04-01

    We expressed a putative beta-galactosidase from Sulfolobus acidocaldarius in Escherichia coli and purified the recombinant enzyme using heat treatment and Hi-Trap ion-exchange chromatography. The resultant protein gave a single 57-kDa band by SDS-PAGE and had a specific activity of 58 U/mg. The native enzyme existed as a dimer with a molecular mass of 114 kDa by gel filtration. The maximum activity of this enzyme was observed at pH 5.5 and 90 degrees C. The half-lives of the enzyme at 70, 80, and 90 degrees C were 494, 60, and 0.2 h, respectively. The hydrolytic activity with p-nitrophenyl(pNP) substrates followed the order p-nitrophenyl-beta-D-fucopyranoside > pNP-beta-D-glucopyranoside > pNP-beta-D-galactopyranoside > pNP-beta-D-mannopyranoside > pNP-beta-D-xylopyranoside, but not toward aryl-alpha-glycosides or pNP-beta-L-arabinofuranoside. Thus, the enzyme was actually a beta-glycosidase. The beta-glycosidase exhibited transglycosylation activity with pNP-beta-D-galactopyranoside, pNP-beta-D-glucopyranoside, and pNP-beta-D-fucopyranoside in decreasing order of activity, in the reverse order of its hydrolytic activity. The hydrolytic activity was higher toward cellobiose than toward lactose, but the transglycosylation activity was lower with cellobiose than with lactose.

  4. Sulfoquinovose synthase - an important enzyme in the N-glycosylation pathway of Sulfolobus acidocaldarius.

    Science.gov (United States)

    Meyer, Benjamin H; Zolghadr, Behnam; Peyfoon, Elham; Pabst, Martin; Panico, Maria; Morris, Howard R; Haslam, Stuart M; Messner, Paul; Schäffer, Christina; Dell, Anne; Albers, Sonja-Verena

    2011-12-01

    Recently, the Surface (S)-layer glycoprotein of the thermoacidophilic crenarchaeote Sulfolobus acidocaldarius was found to be N-glycosylated with a heterogeneous family of glycans, with the largest having a composition Glc(1)Man(2)GlcNAc(2) plus 6-sulfoquinovose. However, genetic analyses of genes involved in the N-glycosylation process in Crenarchaeota were missing so far. In this study we identify a gene cluster involved in the biosynthesis of sulfoquinovose and important for the assembly of the S-layer N-glycans. A successful markerless in-frame deletion of agl3 resulted in a decreased molecular mass of the S-layer glycoprotein SlaA and the flagellin FlaB, indicating a change in the N-glycan composition. Analyses with nanoLC ES-MS/MS confirmed the presence of only a reduced trisaccharide structure composed of Man(1) GlcNAc(2) , missing the sulfoquinovose, a mannose and glucose. Biochemical studies of the recombinant Agl3 confirmed the proposed function as a UDP-sulfoquinovose synthase. Furthermore, S. acidocaldarius cells lacking agl3 had a significantly lower growth rate at elevated salt concentrations compared with the background strain, underlining the importance of the N-glycosylation to maintain an intact and stable cell envelope, to enable the survival of S. acidocaldarius in its extreme environment.

  5. Deletion of cdvB paralogous genes of Sulfolobus acidocaldarius impairs cell division.

    Science.gov (United States)

    Yang, Nuan; Driessen, Arnold J M

    2014-03-01

    The majority of Crenarchaeota utilize the cell division system (Cdv) to divide. This system consists of three highly conserved genes, cdvA, cdvB and cdvC that are organized in an operon. CdvC is homologous to the AAA-type ATPase Vps4, involved in multivesicular body biogenesis in eukaryotes. CdvA is a unique archaeal protein that interacts with the membrane, while CdvB is homologous to the eukaryal Vps24 and forms helical filaments. Most Crenarcheota contain additional CdvB paralogs. In Sulfolobus acidocaldarius these are termed CdvB1-3. We have used a gene inactivation approach to determine the impact of these additional cdvB genes on cell division. Independent deletion mutants of these genes were analyzed for growth and protein localization. One of the deletion strains (ΔcdvB3) showed a severe growth defect on plates and delayed growth on liquid medium. It showed the formation of enlarged cells and a defect in DNA segregation. Since these defects are accompanied with an aberrant localization of CdvA and CdvB, we conclude that CdvB3 fulfills an important accessory role in cell division.

  6. Growth of extreme thermophile Sulfolobus acidocaldarius in a hyperbaric helium bioreactor

    Energy Technology Data Exchange (ETDEWEB)

    Sturm, F.J.; Hurwitz, S.A.; Deming, J.W.; Kelly, R.M.

    1987-01-01

    The relationship between pressure and temperature as it affects microbial growth and metabolism has been examined only for a limited number of bacterial species. Because many newly-discovered, extremely thermophilic bacteria have been isolated from pressurized environments, this relationship merits closer scrutiny. In this study, the extremely thermophilic bacterium, Sulfolobus acidocaldarius, was cultured successfully in a hyperbaric chamber containing helium and air enriched with 5% carbon dioxide. Over a pressure range of approximately 1-120 bar and a temperature range of 67-80/sup 0/C, growth was achieved in a heterotrophic medium with the air mixture at partial pressures up to 3.5 bar. Helium was used to obtain the final, desired incubation pressure. No significant growth was noted above 80/sup 0/C over the same range of hyperbaric pressures, or at 70/sup 0/C when pressure was applied hydrostatically. Growth experiments conducted under hyperbaric conditions may provide a means to study these bacteria under simulated in situ conditions and simultaneously avoid the complications associated with hydrostatic experiments. Results indicate that hyperbaric helium bioreactors will be important in the study of extremely thermophilic bacteria that are isolated from pressurized environments.

  7. Membrane-bound amylopullulanase is essential for starch metabolism of Sulfolobus acidocaldarius DSM639.

    Science.gov (United States)

    Choi, Kyoung-Hwa; Cha, Jaeho

    2015-09-01

    Sulfolobus acidocaldarius DSM639 produced an acid-resistant membrane-bound amylopullulanase (Apu) during growth on starch as a sole carbon and energy source. The physiological role of Apu in starch metabolism was investigated by the growth and starch degradation pattern of apu disruption mutant as well as biochemical properties of recombinant Apu. The Δapu mutant lost the ability to grow in minimal medium in the presence of starch, and the amylolytic activity observed in the membrane fraction of the wild-type strain was not detected in the Δapu mutant when the cells were grown in YT medium. The purified membrane-bound Apu initially hydrolyzed starch, amylopectin, and pullulan into various sizes of maltooligosaccharides, and then produced glucose, maltose, and maltotriose in the end, indicating Apu is a typical endo-acting glycoside hydrolase family 57 (GH57) amylopullulanase. The maltose and maltotriose observed in the culture medium during the exponential and stationary phase growth indicates that Apu is the essential enzyme to initially hydrolyze the starch into small maltooligosaccharides to be transported into the cell.

  8. Improving low-temperature activity of Sulfolobus acidocaldarius 2-keto-3-deoxygluconate aldolase.

    Science.gov (United States)

    Wolterink-van Loo, Suzanne; Siemerink, Marco A J; Perrakis, Georgios; Kaper, Thijs; Kengen, Servé W M; van der Oost, John

    2009-03-02

    Sulfolobus acidocaldarius 2-keto-3-deoxygluconate aldolase (SacKdgA) displays optimal activity at 95 degrees C and is studied as a model enzyme for aldol condensation reactions. For application of SacKdgA at lower temperatures, a library of randomly generated mutants was screened for improved synthesis of 2-keto-3-deoxygluconate from pyruvate and glyceraldehyde at the suboptimal temperature of 50 degrees C. The single mutant SacKdgA-V193A displayed a threefold increase in activity compared with wild type SacKdgA. The increased specific activity at 40-60 degrees C of this mutant was observed, not only for the condensation of pyruvate with glyceraldehyde, but also for several unnatural acceptor aldehydes. The optimal temperature for activity of SacKdgA-V193A was lower than for the wild type enzyme, but enzymatic stability of the mutant was similar to that of the wild type, indicating that activity and stability were uncoupled. Valine193 has Van der Waals interactions with Lysine153, which covalently binds the substrate during catalysis. The mutation V193A introduced space close to this essential residue, and the increased activity of the mutant presumably resulted from increased flexibility of Lysine153. The increased activity of SacKdgA-V193A with unaffected stability demonstrates the potential for optimizing extremely thermostable aldolases for synthesis reactions at moderate temperatures.

  9. Coupled TLC and MALDI-TOF/MS Analyses of the Lipid Extract of the Hyperthermophilic Archaeon Pyrococcus furiosus

    Directory of Open Access Journals (Sweden)

    Simona Lobasso

    2012-01-01

    Full Text Available The lipidome of the marine hyperthermophilic archaeon Pyrococcus furiosus was studied by means of combined thin-layer chromatography and MALDI-TOF/MS analyses of the total lipid extract. 80–90% of the major polar lipids were represented by archaeol lipids (diethers and the remaining part by caldarchaeol lipids (tetraethers. The direct analysis of lipids on chromatography plate showed the presence of the diphytanylglycerol analogues of phosphatidylinositol and phosphatidylglycerol, the N-acetylglucosamine-diphytanylglycerol phosphate plus some caldarchaeol lipids different from those previously described. In addition, evidence for the presence of the dimeric ether lipid cardiolipin is reported, suggesting that cardiolipins are ubiquitous in archaea.

  10. Apo and ligand-bound structures of ModA from the archaeon Methanosarcina acetivorans.

    Science.gov (United States)

    Chan, Sum; Giuroiu, Iulia; Chernishof, Irina; Sawaya, Michael R; Chiang, Janet; Gunsalus, Robert P; Arbing, Mark A; Perry, L Jeanne

    2010-03-01

    The trace-element oxyanion molybdate, which is required for the growth of many bacterial and archaeal species, is transported into the cell by an ATP-binding cassette (ABC) transporter superfamily uptake system called ModABC. ModABC consists of the ModA periplasmic solute-binding protein, the integral membrane-transport protein ModB and the ATP-binding and hydrolysis cassette protein ModC. In this study, X-ray crystal structures of ModA from the archaeon Methanosarcina acetivorans (MaModA) have been determined in the apoprotein conformation at 1.95 and 1.69 A resolution and in the molybdate-bound conformation at 2.25 and 2.45 A resolution. The overall domain structure of MaModA is similar to other ModA proteins in that it has a bilobal structure in which two mixed alpha/beta domains are linked by a hinge region. The apo MaModA is the first unliganded archaeal ModA structure to be determined: it exhibits a deep cleft between the two domains and confirms that upon binding ligand one domain is rotated towards the other by a hinge-bending motion, which is consistent with the 'Venus flytrap' model seen for bacterial-type periplasmic binding proteins. In contrast to the bacterial ModA structures, which have tetrahedral coordination of their metal substrates, molybdate-bound MaModA employs octahedral coordination of its substrate like other archaeal ModA proteins.

  11. Grappling archaea: ultrastructural analyses of an uncultivated, cold-loving archaeon and its biofilm

    Directory of Open Access Journals (Sweden)

    Alexandra ePerras

    2014-08-01

    Full Text Available Similarly to Bacteria, Archaea are microorganisms that interact with their surrounding environment in a versatile manner. To date, interactions based on cellular structure and surface appendages have mainly been documented using model systems of cultivable archaea under laboratory conditions. Here, we report on the microbial interactions and ultrastructural features of the uncultivated SM1 Euryarchaeon, which is highly dominant in its biotope. Therefore, biofilm samples taken from the Sippenauer Moor, Germany, were investigated via transmission electron microscopy (TEM; negative staining, thin-sectioning and scanning electron microscopy (SEM in order to elucidate the fine structures of the microbial cells and the biofilm itself. The biofilm consisted of small archaeal cocci (0.6 µm diameter, arranged in a regular pattern (1.2-2.0 µm distance from cell to cell, whereas each archaeon was connected to 6 other archaea on average. Extracellular polymeric substances (EPS were limited to the close vicinity of the archaeal cells, and specific cell surface appendages (hami, Moissl et al., 2005 protruded beyond the EPS matrix enabling microbial interaction by cell-cell contacts among the archaea and between archaea and bacteria. All analyzed hami revealed their previously described architecture of nano-grappling hooks and barb-wire basal structures. Considering the archaeal cell walls, the SM1 Euryarchaea exhibited a double-membrane, which has rarely been reported for members of this phylogenetic domain. Based on these findings, the current generalized picture on archaeal cell walls needs to be revisited, as archaeal cell structures are more complex and sophisticated than previously assumed, particularly when looking into the uncultivated majority.

  12. Citric acid cycle in the hyperthermophilic archaeon Pyrobaculum islandicum grown autotrophically, heterotrophically, and mixotrophically with acetate.

    Science.gov (United States)

    Hu, Yajing; Holden, James F

    2006-06-01

    The hyperthermophilic archaeon Pyrobaculum islandicum uses the citric acid cycle in the oxidative and reductive directions for heterotrophic and autotrophic growth, respectively, but the control of carbon flow is poorly understood. P. islandicum was grown at 95 degrees C autotrophically, heterotrophically, and mixotrophically with acetate, H2, and small amounts of yeast extract and with thiosulfate as the terminal electron acceptor. The autotrophic growth rates and maximum concentrations of cells were significantly lower than those in other media. The growth rates on H2 and 0.001% yeast extract with and without 0.05% acetate were the same, but the maximum concentration of cells was fourfold higher with acetate. There was no growth with acetate if 0.001% yeast extract was not present, and addition of H2 to acetate-containing medium greatly increased the growth rates and maximum concentrations of cells. P. islandicum cultures assimilated 14C-labeled acetate in the presence of H2 and yeast extract with an efficiency of 55%. The activities of 11 of 19 enzymes involved in the central metabolism of P. islandicum were regulated under the three different growth conditions. Pyruvate synthase and acetate:coenzyme A (CoA) ligase (ADP-forming) activities were detected only in heterotrophically grown cultures. Citrate synthase activity decreased in autotrophic and acetate-containing cultures compared to the activity in heterotrophic cultures. Acetylated citrate lyase, acetate:CoA ligase (AMP forming), and phosphoenolpyruvate carboxylase activities increased in autotrophic and acetate-containing cultures. Citrate lyase activity was higher than ATP citrate synthase activity in autotrophic cultures. These data suggest that citrate lyase and AMP-forming acetate:CoA ligase, but not ATP citrate synthase, work opposite citrate synthase to control the direction of carbon flow in the citric acid cycle.

  13. The protein interaction network of a taxis signal transduction system in a Halophilic Archaeon

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    Schlesner Matthias

    2012-11-01

    Full Text Available Abstract Background The taxis signaling system of the extreme halophilic archaeon Halobacterium (Hbt. salinarum differs in several aspects from its model bacterial counterparts Escherichia coli and Bacillus subtilis. We studied the protein interactions in the Hbt. salinarum taxis signaling system to gain an understanding of its structure, to gain knowledge about its known components and to search for new members. Results The interaction analysis revealed that the core signaling proteins are involved in different protein complexes and our data provide evidence for dynamic interchanges between them. Fifteen of the eighteen taxis receptors (halobacterial transducers, Htrs can be assigned to four different groups depending on their interactions with the core signaling proteins. Only one of these groups, which contains six of the eight Htrs with known signals, shows the composition expected for signaling complexes (receptor, kinase CheA, adaptor CheW, response regulator CheY. From the two Hbt. salinarum CheW proteins, only CheW1 is engaged in signaling complexes with Htrs and CheA, whereas CheW2 interacts with Htrs but not with CheA. CheY connects the core signaling structure to a subnetwork consisting of the two CheF proteins (which build a link to the flagellar apparatus, CheD (the hub of the subnetwork, two CheC complexes and the receptor methylesterase CheB. Conclusions Based on our findings, we propose two hypotheses. First, Hbt. salinarum might have the capability to dynamically adjust the impact of certain Htrs or Htr clusters depending on its current needs or environmental conditions. Secondly, we propose a hypothetical feedback loop from the response regulator to Htr methylation made from the CheC proteins, CheD and CheB, which might contribute to adaptation analogous to the CheC/CheD system of B. subtilis.

  14. Development of New Modular Genetic Tools for Engineering the Halophilic Archaeon Halobacterium salinarum.

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    Rafael Silva-Rocha

    Full Text Available Our ability to genetically manipulate living organisms is usually constrained by the efficiency of the genetic tools available for the system of interest. In this report, we present the design, construction and characterization of a set of four new modular vectors, the pHsal series, for engineering Halobacterium salinarum, a model halophilic archaeon widely used in systems biology studies. The pHsal shuttle vectors are organized in four modules: (i the E. coli's specific part, containing a ColE1 origin of replication and an ampicillin resistance marker, (ii the resistance marker and (iii the replication origin, which are specific to H. salinarum and (iv the cargo, which will carry a sequence of interest cloned in a multiple cloning site, flanked by universal M13 primers. Each module was constructed using only minimal functional elements that were sequence edited to eliminate redundant restriction sites useful for cloning. This optimization process allowed the construction of vectors with reduced sizes compared to currently available platforms and expanded multiple cloning sites. Additionally, the strong constitutive promoter of the fer2 gene was sequence optimized and incorporated into the platform to allow high-level expression of heterologous genes in H. salinarum. The system also includes a new minimal suicide vector for the generation of knockouts and/or the incorporation of chromosomal tags, as well as a vector for promoter probing using a GFP gene as reporter. This new set of optimized vectors should strongly facilitate the engineering of H. salinarum and similar strategies could be implemented for other archaea.

  15. Thermoacidophilic archaea for pyrite oxidation in coal desulphurization

    Energy Technology Data Exchange (ETDEWEB)

    Larsson, Liselotte

    1995-10-01

    The desulfurization of low-sulfur coals has been demonstrated with the thermoacidophilic archaeon Acidianus brierleyi. A. brierleyi facilitates the removal of inorganic sulfur from coal and oxidizes mineral pyrite. The results imply that the mechanism behind microbial coal desulfurization and pyrite oxidation is a combination of biotic and abiotic leaching of pyrite. The extent of sulfur removal is dependent on the type of coal and is closely related to he amount of pyritic sulfur in the coal. Studies have shown that neither ash content nor heating value were dramatically affected by the microbial treatment. The use of the archaea Sulfolobus acidocaldarius and Sulfolobus solfataricus, as well as the mesophilic bacteria Thiobacillus ferrooxidans and several Pseudomonas species, has also been studied for coal desulfurization and mineral pyrite oxidation. The archaea and Pseudomonas species did not grow autotrophically on mineral pyrite neither did they oxidize pyrite in coal. The oxidation rate was, however, 5-10 times less than with A. brierleyi on mineral pyrite. The rate of sulfur removal from coal was in the same range as for A. brierleyi which indicates that different reactions are rate limiting in coal depyritization than in mineral pyrite oxidation. 133 refs, 18 figs, 3 tabs

  16. An archaeal immune system can detect multiple protospacer adjacent motifs (PAMs) to target invader DNA.

    Science.gov (United States)

    Fischer, Susan; Maier, Lisa-Katharina; Stoll, Britta; Brendel, Jutta; Fischer, Eike; Pfeiffer, Friedhelm; Dyall-Smith, Mike; Marchfelder, Anita

    2012-09-28

    The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system provides adaptive and heritable immunity against foreign genetic elements in most archaea and many bacteria. Although this system is widespread and diverse with many subtypes, only a few species have been investigated to elucidate the precise mechanisms for the defense of viruses or plasmids. Approximately 90% of all sequenced archaea encode CRISPR/Cas systems, but their molecular details have so far only been examined in three archaeal species: Sulfolobus solfataricus, Sulfolobus islandicus, and Pyrococcus furiosus. Here, we analyzed the CRISPR/Cas system of Haloferax volcanii using a plasmid-based invader assay. Haloferax encodes a type I-B CRISPR/Cas system with eight Cas proteins and three CRISPR loci for which the identity of protospacer adjacent motifs (PAMs) was unknown until now. We identified six different PAM sequences that are required upstream of the protospacer to permit target DNA recognition. This is only the second archaeon for which PAM sequences have been determined, and the first CRISPR group with such a high number of PAM sequences. Cells could survive the plasmid challenge if their CRISPR/Cas system was altered or defective, e.g. by deletion of the cas gene cassette. Experimental PAM data were supplemented with bioinformatics data on Haloferax and Haloquadratum.

  17. An unusual 5S rRNA, from Sulfolobus acidocaldarius, and its implications for a general 5S rRNA structure.

    OpenAIRE

    Stahl, D A; Luehrsen, K R; Woese, C R; Pace, N R

    1981-01-01

    The nucleotide sequence of the 5S ribosomal RNA of the thermoacidophilic archaebacterium Sulfolobus acidocaldarius was determined. The high degree of evident secondary structure in the molecule has implications for the common higher order structure of other 5S rRNAs, both bacterial and eukaryotic.

  18. Draft Genome Sequence of the Novel Thermoacidophilic Archaeon Acidianus copahuensis Strain ALE1, Isolated from the Copahue Volcanic Area in Neuquen, Argentina.

    Science.gov (United States)

    Urbieta, M Sofía; Rascovan, Nicolás; Castro, Camila; Revale, Santiago; Giaveno, M Alejandra; Vazquez, Martín; Donati, Edgardo R

    2014-05-08

    Acidianus copahuensis is a recently characterized thermoacidophilic archaeon isolated from the Copahue volcanic area in Argentina. Here, we present its draft genome sequence, in which we found genes involved in key metabolic pathways for developing under Copahue's extreme environmental conditions, such as sulfur and iron oxidation, carbon fixation, and metal tolerance.

  19. Expression and Characterization of a Thermostable Acyl-peptide Releasing Enzyme ST0779 from Sulfolobus tokodaii

    Institute of Scientific and Technical Information of China (English)

    LI Rong; ZHANG Fei; CAO Shu-gui; XIE Gui-qiu; GAO Ren-jun

    2012-01-01

    Acyl-peptide releasing enzyme(AARE) belongs to a serine peptidase family and catalyzes the NH2-terminal hydrolysis of Nα-acylpeptides to release Nα-acylated amino acids.ORF0779(ORF=open reading frame)from thermophilic archaea Sulfolobus tokodaii(ST0779) was cloned and expressed in E.coti BL21 and the expressed protein was identified as a thermostable AARE.The target protein could be optimally overexpressed in E.coli at 30 ℃ for 8 h with 0.1 mmol/L isopropyl β-dthiogalactoside(IPTG).The crude enzyme was heated at 70 ℃ for 30 min,and then the target protein could account for above 40% of the total protein.The purification fold was 27 and the enzyme showed both esterase activity and peptidase activity.The optimal temperature and pH for ST0779 were 70 ℃and 8.0 when Ac-Ala3 was used as substrate.The half-life of the enzyme(0.2 mg/mL) at 90 ℃ was about 16 h,indicating that the enzyme exhibits a favorable thermostability.The activity of ST0779 could still remain over 85% after being treated at 25 ℃ in different buffers with pH range from 6.0 to10.0 for 24 h,which indicates ST0779 is stable in neutral or slight alkali environment.Under neutral or slightly alkali conditions,the enzyme exhibits really high catalytic efficiency against acyl-peptide,and the optimal substrate is Ac-Ala3.Most metal ions have no inhibition effect on the activity of ST0779,while 4% activity of ST0779 is inhibited in the presence of K+.This enzyme was supposed to be applied in the analysis of protein sequencing and the synthesis of small peptides.

  20. Transfection Studies with Colloidal Systems Containing Highly Purified Bipolar Tetraether Lipids from Sulfolobus acidocaldarius.

    Science.gov (United States)

    Engelhardt, Konrad H; Pinnapireddy, Shashank Reddy; Baghdan, Elias; Jedelská, Jarmila; Bakowsky, Udo

    2017-01-01

    Lipid vectors are commonly used to facilitate the transfer of nucleic acids into mammalian cells. In this study, two fractions of tetraether lipids from the archaea Sulfolobus acidocaldarius were extracted and purified using different methods. The purified lipid fractions polar lipid fraction E (PLFE) and hydrolysed glycerol-dialkyl-nonitol tetraether (hGDNT) differ in their structures, charge, size, and miscibility from conventional lipids. Liposomes were prepared by mixing tetraether lipids with cholesterol (CH) and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) resulting in stable vectors for gene delivery. Lipoplexes were prepared by complexation of liposomes with a luciferase expressing plasmid (pCMV-luc) at certain nitrogen-to-phosphorus (N/P) ratios and optimised for the transient transfection of ovarian adenocarcinoma cells (SK-OV-3). Complexation efficacy was investigated by gel-red fluorescence assay. Biophysical properties, like size, surface charge, and morphology, were investigated by differential light scattering (DLS), atomic force microscopy (AFM), and scanning electron microscopy (Cryo-SEM), respectively, revealing structural differences between liposomes and lipoplexes. A range of stable transfecting agents containing tetraether lipids were obtained by incorporating 5 mol% of tetraether lipids. Lipoplexes showed a decrease in free gel-red with increasing N/P ratios indicating efficient incorporation of plasmid DNA (pDNA) and remarkable stability. Transfection experiments of the lipoplexes revealed successful and superior transfection of SK-OV-3 cell line compared to the commercially available DOTAP and branched polyethyleneimine (25 kDa bPEI).

  1. The Potent In Vitro Skin Permeation of Archaeosome Made from Lipids Extracted of Sulfolobus acidocaldarius

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    Eskandar Moghimipour

    2013-01-01

    Full Text Available Archaeosomes are a new generation of liposomes that exhibit higher stabilities under different conditions, such as high temperatures, alkaline or acidic pH, and presence of bile salts in comparison with liposomes, and can be used in biotechnology including drug, gene, and vaccine delivery. The objective of this study was to prepare archaeosomes using lipid extracted from Sulfolobus acidocaldarius and evaluate their physicochemical properties. The lipids were extracted from S. acidocaldarius and assayed by High Performance Thin-Layer Chromatography (HPTLC. Archaeosomes were prepared using film method and methylene blue was used as drug model. They were characterized for their vesicle size and Differential Scanning Calorimetry (DSC was used to investigate changes in their thermal behavior. The released amount of methylene blue was determined using a dialysis membrane and rat skin. HPTLC analysis of the extracted lipids showed that glycerol ether may be the major lipid with more than 78 percent probability. Results of particle size determination showed a mean size of 158.33 nm and the results of DSC indicated the possible interaction of methylene blue with lipids during the preparation of archaeosome. The addition of cholesterol significantly improved the encapsulation of methylene blue in the archaeosome so that the encapsulation efficiency was 61.66 ± 2.88%. The result of in vitro skin permeation showed that methylene blue could pass through skin model according to Peppas model and there was about 41.66% release after 6 h, whereas no release was observed through dialysis membrane. According to the results of the study, it is concluded that archaeosome may be successfully used as drug delivery system.

  2. Spectroscopic study on uranyl carboxylate complexes formed at the surface layer of Sulfolobus acidocaldarius.

    Science.gov (United States)

    Reitz, Thomas; Rossberg, Andre; Barkleit, Astrid; Steudtner, Robin; Selenska-Pobell, Sonja; Merroun, Mohamed L

    2015-02-14

    The complexation of U(vi) at the proteinaceous surface layer (S-layer) of the archaeal strain Sulfolobus acidocaldarius was investigated over a pH range from pH 1.5 to 6 at the molecular scale using time-resolved laser-induced fluorescence spectroscopy (TRLFS) and U L(III)-edge extended X-ray absorption fine structure (EXAFS). The S-layer, which represents the interface between the cell and its environment, is very stable against high temperatures, proteases, and detergents. This allowed the isolation and purification of S-layer ghosts (= empty cells) that maintain the size and shape of the cells. In contrast to many other microbial cell envelope compounds the studied S-layer is not phosphorylated, enabling the investigation of uranyl carboxylate complexes formed at microbial surfaces. The latter are usually masked by preferentially formed uranyl phosphate complexes. We demonstrated that at highly acidic conditions (pH 1.5 to 3) no uranium was bound by the S-layer. In contrast to that, at moderate acidic pH conditions (pH 4.5 and 6) a complexation of U(vi) at the S-layer via deprotonated carboxylic groups was stimulated. Titration studies revealed dissociation constants for the carboxylic groups of glutamic and aspartic acid residues of pK(a) = 4.78 and 6.31. The uranyl carboxylate complexes formed at the S-layer did not show luminescence properties at room temperature, but only under cryogenic conditions. The obtained luminescence maxima are similar to those of uranyl acetate. EXAFS spectroscopy demonstrated that U(vi) in these complexes is mainly coordinated to carboxylate groups in a bidentate binding mode. The elucidation of the molecular structure of these complexes was facilitated by the absence of phosphate groups in the studied S-layer protein.

  3. Transfection Studies with Colloidal Systems Containing Highly Purified Bipolar Tetraether Lipids from Sulfolobus acidocaldarius

    Directory of Open Access Journals (Sweden)

    Konrad H. Engelhardt

    2017-01-01

    Full Text Available Lipid vectors are commonly used to facilitate the transfer of nucleic acids into mammalian cells. In this study, two fractions of tetraether lipids from the archaea Sulfolobus acidocaldarius were extracted and purified using different methods. The purified lipid fractions polar lipid fraction E (PLFE and hydrolysed glycerol-dialkyl-nonitol tetraether (hGDNT differ in their structures, charge, size, and miscibility from conventional lipids. Liposomes were prepared by mixing tetraether lipids with cholesterol (CH and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP resulting in stable vectors for gene delivery. Lipoplexes were prepared by complexation of liposomes with a luciferase expressing plasmid (pCMV-luc at certain nitrogen-to-phosphorus (N/P ratios and optimised for the transient transfection of ovarian adenocarcinoma cells (SK-OV-3. Complexation efficacy was investigated by gel-red fluorescence assay. Biophysical properties, like size, surface charge, and morphology, were investigated by differential light scattering (DLS, atomic force microscopy (AFM, and scanning electron microscopy (Cryo-SEM, respectively, revealing structural differences between liposomes and lipoplexes. A range of stable transfecting agents containing tetraether lipids were obtained by incorporating 5 mol% of tetraether lipids. Lipoplexes showed a decrease in free gel-red with increasing N/P ratios indicating efficient incorporation of plasmid DNA (pDNA and remarkable stability. Transfection experiments of the lipoplexes revealed successful and superior transfection of SK-OV-3 cell line compared to the commercially available DOTAP and branched polyethyleneimine (25 kDa bPEI.

  4. The potent in vitro skin permeation of archaeosome made from lipids extracted of Sulfolobus acidocaldarius.

    Science.gov (United States)

    Moghimipour, Eskandar; Kargar, Mohammad; Ramezani, Zahra; Handali, Somayeh

    2013-01-01

    Archaeosomes are a new generation of liposomes that exhibit higher stabilities under different conditions, such as high temperatures, alkaline or acidic pH, and presence of bile salts in comparison with liposomes, and can be used in biotechnology including drug, gene, and vaccine delivery. The objective of this study was to prepare archaeosomes using lipid extracted from Sulfolobus acidocaldarius and evaluate their physicochemical properties. The lipids were extracted from S. acidocaldarius and assayed by High Performance Thin-Layer Chromatography (HPTLC). Archaeosomes were prepared using film method and methylene blue was used as drug model. They were characterized for their vesicle size and Differential Scanning Calorimetry (DSC) was used to investigate changes in their thermal behavior. The released amount of methylene blue was determined using a dialysis membrane and rat skin. HPTLC analysis of the extracted lipids showed that glycerol ether may be the major lipid with more than 78 percent probability. Results of particle size determination showed a mean size of 158.33 nm and the results of DSC indicated the possible interaction of methylene blue with lipids during the preparation of archaeosome. The addition of cholesterol significantly improved the encapsulation of methylene blue in the archaeosome so that the encapsulation efficiency was 61.66 ± 2.88%. The result of in vitro skin permeation showed that methylene blue could pass through skin model according to Peppas model and there was about 41.66% release after 6 h, whereas no release was observed through dialysis membrane. According to the results of the study, it is concluded that archaeosome may be successfully used as drug delivery system.

  5. Transfection Studies with Colloidal Systems Containing Highly Purified Bipolar Tetraether Lipids from Sulfolobus acidocaldarius

    Science.gov (United States)

    Pinnapireddy, Shashank Reddy; Baghdan, Elias; Jedelská, Jarmila

    2017-01-01

    Lipid vectors are commonly used to facilitate the transfer of nucleic acids into mammalian cells. In this study, two fractions of tetraether lipids from the archaea Sulfolobus acidocaldarius were extracted and purified using different methods. The purified lipid fractions polar lipid fraction E (PLFE) and hydrolysed glycerol-dialkyl-nonitol tetraether (hGDNT) differ in their structures, charge, size, and miscibility from conventional lipids. Liposomes were prepared by mixing tetraether lipids with cholesterol (CH) and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) resulting in stable vectors for gene delivery. Lipoplexes were prepared by complexation of liposomes with a luciferase expressing plasmid (pCMV-luc) at certain nitrogen-to-phosphorus (N/P) ratios and optimised for the transient transfection of ovarian adenocarcinoma cells (SK-OV-3). Complexation efficacy was investigated by gel-red fluorescence assay. Biophysical properties, like size, surface charge, and morphology, were investigated by differential light scattering (DLS), atomic force microscopy (AFM), and scanning electron microscopy (Cryo-SEM), respectively, revealing structural differences between liposomes and lipoplexes. A range of stable transfecting agents containing tetraether lipids were obtained by incorporating 5 mol% of tetraether lipids. Lipoplexes showed a decrease in free gel-red with increasing N/P ratios indicating efficient incorporation of plasmid DNA (pDNA) and remarkable stability. Transfection experiments of the lipoplexes revealed successful and superior transfection of SK-OV-3 cell line compared to the commercially available DOTAP and branched polyethyleneimine (25 kDa bPEI).

  6. Genome Analyses of Icelandic Strains of Sulfolobus islandicus, Model Organisms for Genetic and Virus-Host Interaction Studies

    DEFF Research Database (Denmark)

    Guo, Li; Brügger, Kim; Liu, Chao;

    2011-01-01

    The genomes of two Sulfolobus islandicus strains obtained from Icelandic solfataras were sequenced and analyzed. Strain REY15A is a host for a versatile genetic toolbox. It exhibits a genome of minimal size, is stable genetically, and is easy to grow and manipulate. Strain HVE10/4 shows a broad h...... conjugative plasmids, which have integrated at a few tRNA genes lacking introns. This provides a possible rationale for the presence of the introns.......The genomes of two Sulfolobus islandicus strains obtained from Icelandic solfataras were sequenced and analyzed. Strain REY15A is a host for a versatile genetic toolbox. It exhibits a genome of minimal size, is stable genetically, and is easy to grow and manipulate. Strain HVE10/4 shows a broad...... in gene content and gene order occur. These include gene clusters involved in specific metabolic pathways, multiple copies of VapBC antitoxin-toxin gene pairs, and in strain HVE10/4, a 50-kb region rich in glycosyl transferase genes. The variable region also contains most of the insertion sequence (IS...

  7. Influence of C-terminal tail deletion on structure and stability of hyperthermophile Sulfolobus tokodaii RNase HI.

    Science.gov (United States)

    Chen, Lin; Zhang, Ji-Long; Zheng, Qing-Chuan; Chu, Wen-Ting; Xue, Qiao; Zhang, Hong-Xing; Sun, Chia-Chung

    2013-06-01

    The C-terminus tail (G144-T149) of the hyperthermophile Sulfolobus tokodaii (Sto-RNase HI) plays an important role in this protein's hyperstabilization and may therefore be a good protein stability tag. Detailed understanding of the structural and dynamic effects of C-terminus tail deletion is required for gaining insights into the thermal stability mechanism of Sto-RNase HI. Focused on Sulfolobus tokodaii RNase HI (Sto-RNase HI) and its derivative lacking the C-terminal tail (ΔC6 Sto-RNase HI) (PDB codes: 2EHG and 3ALY), we applied molecular dynamics (MD) simulations at four different temperatures (300, 375, 475, and 500 K) to examine the effect of the C-terminal tail on the hyperstabilization of Sto-RNase HI and to investigate the unfolding process of Sto-RNase HI and ΔC6 Sto-RNase HI. The simulations suggest that the C-terminal tail has significant impact in hyperstabilization of Sto-RNase HI and the unfolding of these two proteins evolves along dissimilar pathways. Essential dynamics analysis indicates that the essential subspaces of the two proteins at different temperatures are non-overlapping within the trajectories and they exhibit different directions of motion. Our work can give important information to understand the three-state folding mechanism of Sto-RNase HI and to offer alternative strategies to improve the protein stability.

  8. Genome-wide transcriptional response of the archaeon Thermococcus gammatolerans to cadmium.

    Directory of Open Access Journals (Sweden)

    Arnaud Lagorce

    Full Text Available Thermococcus gammatolerans, the most radioresistant archaeon known to date, is an anaerobic and hyperthermophilic sulfur-reducing organism living in deep-sea hydrothermal vents. Knowledge of mechanisms underlying archaeal metal tolerance in such metal-rich ecosystem is still poorly documented. We showed that T. gammatolerans exhibits high resistance to cadmium (Cd, cobalt (Co and zinc (Zn, a weaker tolerance to nickel (Ni, copper (Cu and arsenate (AsO(4 and that cells exposed to 1 mM Cd exhibit a cellular Cd concentration of 67 µM. A time-dependent transcriptomic analysis using microarrays was performed at a non-toxic (100 µM and a toxic (1 mM Cd dose. The reliability of microarray data was strengthened by real time RT-PCR validations. Altogether, 114 Cd responsive genes were revealed and a substantial subset of genes is related to metal homeostasis, drug detoxification, re-oxidization of cofactors and ATP production. This first genome-wide expression profiling study of archaeal cells challenged with Cd showed that T. gammatolerans withstands induced stress through pathways observed in both prokaryotes and eukaryotes but also through new and original strategies. T. gammatolerans cells challenged with 1 mM Cd basically promote: 1 the induction of several transporter/permease encoding genes, probably to detoxify the cell; 2 the upregulation of Fe transporters encoding genes to likely compensate Cd damages in iron-containing proteins; 3 the induction of membrane-bound hydrogenase (Mbh and membrane-bound hydrogenlyase (Mhy2 subunits encoding genes involved in recycling reduced cofactors and/or in proton translocation for energy production. By contrast to other organisms, redox homeostasis genes appear constitutively expressed and only a few genes encoding DNA repair proteins are regulated. We compared the expression of 27 Cd responsive genes in other stress conditions (Zn, Ni, heat shock, γ-rays, and showed that the Cd transcriptional pattern is

  9. Thermal stability and biochemical properties of isocitrate dehydrogenase from the thermoacidophilic archaeon Thermoplasma acidophilum.

    Science.gov (United States)

    Stokke, Runar; Birkeland, Nils-Kåre; Steen, Ida Helene

    2007-03-01

    Isocitrate dehydrogenase [IDH; EC 1.1.1.42] from the thermoacidophilic archaeon Thermoplasma acidophilum (TaIDH) showed high thermal stability with an apparent melting temperature, T(m), of 82.2 and 84.5 degrees C at pH 7.5 and 5.8, respectively. Based on structural alignment of TaIDH with IDH from Aeropyrum pernix (ApIDH) and Archaeoglobus fulgidus (AfIDH) residues forming an aromatic cluster in the clasp-domain thought to strengthen the dimer interface in ApIDH and AfIDH were identified in the former enzyme. Moreover, TaIDH had a shortened N-terminus that may protect the enzyme from thermal denaturation. The enzyme activity of TaIDH was highest at 70 degrees C. The pH-activity profile was bell-shaped with an optimum shifted to a lower pH compared to AfIDH. The activity of TaIDH was influenced by changes in pH with a three-fold reduction in activity when the pH was shifted from the pH-optimum at 7.5 to pH 5.8. However, the specific activity at pH 5.8 was still high when compared with AfIDH. The reduction in activity at pH 5.8 was not due to instability of the enzyme as the T(m) of TaIDH was higher at pH 5.8 than at 7.5 and the enzyme retained 91% of its activity after incubation at 1 h at pH 5 and 60 degrees C. The difference in the pH-profile of TaIDH in comparison with AfIDH may thus be related to the pK(a)s of their catalytic residues involved in the initial proton abstraction and the final proton donation during the catalysis of oxidative decarboxylation of isocitrate to 2-oxoglutarate and reduced coenzyme.

  10. Different glycosyltransferases are involved in lipid glycosylation and protein N-glycosylation in the halophilic archaeon Haloferax volcanii.

    Science.gov (United States)

    Naparstek, Shai; Vinagradov, Evguenii; Eichler, Jerry

    2010-07-01

    Both the lipid and the protein components of biological membranes can be modified by the covalent addition of polysaccharides. Whereas eukaryal and bacterial pathways of lipid and protein glycosylation are relatively well defined, considerably less is known of the parallel processes in Archaea. Recent efforts have identified glycosyltransferases involved in N-glycosylation of the surface-layer glycoprotein of the halophilic archaeon Haloferax volcanii. In the present study, the involvement of these same glycosyltransferases in the biosynthesis of Hfx. volcanii glycolipids was considered by performing nuclear magnetic resonance analysis of the glycolipid fraction of Hfx. volcanii cells deleted of genes encoding those glycosyltransferases, as well as the oligosaccharyltransferase, AglB. The results reveal that different glycosyltransferases are involved in the biosynthesis of N-linked glycoproteins and glycolipids in Archaea.

  11. Adaptive engineering of a hyperthermophilic archaeon on CO and discovering the underlying mechanism by multi-omics analysis.

    Science.gov (United States)

    Lee, Seong Hyuk; Kim, Min-Sik; Lee, Jae-Hak; Kim, Tae Wan; Bae, Seung Seob; Lee, Sung-Mok; Jung, Hae Chang; Yang, Tae-Jun; Choi, Ae Ran; Cho, Yong-Jun; Lee, Jung-Hyun; Kwon, Kae Kyoung; Lee, Hyun Sook; Kang, Sung Gyun

    2016-03-15

    The hyperthermophilic archaeon Thermococcus onnurineus NA1 can grow and produce H2 on carbon monoxide (CO) and its H2 production rates have been improved through metabolic engineering. In this study, we applied adaptive evolution to enhance H2 productivity. After over 150 serial transfers onto CO medium, cell density, CO consumption rate and H2 production rate increased. The underlying mechanism for those physiological changes could be explained by using multi-omics approaches including genomic, transcriptomic and epigenomic analyses. A putative transcriptional regulator was newly identified to regulate the expression levels of genes related to CO oxidation. Transcriptome analysis revealed significant changes in the transcript levels of genes belonging to the categories of transcription, translation and energy metabolism. Our study presents the first genome-scale methylation pattern of hyperthermophilic archaea. Adaptive evolution led to highly enhanced H2 productivity at high CO flow rates using synthesis gas produced from coal gasification.

  12. Restoration of the di-myo-inositol-phosphate pathway in the piezo-hyperthermophilic archaeon Thermococcus barophilus.

    Science.gov (United States)

    Cario, Anaïs; Mizgier, Alex; Thiel, Axel; Jebbar, Mohamed; Oger, Phil M

    2015-11-01

    Most Thermococcales accumulate di-myo-inositol-phosphate (DIP) as an organic solute as a response to heat stress. We have studied the accumulation of this osmolyte in the high-hydrostatic pressure adapted hyperthermophile Thermococcus barophilus. We found no accumulation of DIP under any of the stress conditions tested, although this archaeon harbors the 3 DIP synthesis genes. Lack of synthesis is due to the lack of expression of TERMP_01135 coding for the second step of DIP synthesis. In contrast to other species, the T. barophilus synthesis operon is interrupted by a four gene locus, in reverse orientation. Restoring an operon like structure at the DIP locus restored DIP synthesis, but did not have an impact on growth characteristics, suggesting that other mechanisms have evolved in this organism to cope with heat stress.

  13. The Genome Sequence of the psychrophilic archaeon, Methanococcoides burtonii: the Role of Genome Evolution in Cold-adaptation

    Energy Technology Data Exchange (ETDEWEB)

    Allen, Michelle A.; Lauro, Federico M.; Williams, Timothy J.; Burg, Dominic; Siddiqui, Khawar S.; De Francisci, David; Chong, Kevin W.Y.; Pilak, Oliver; Chew, Hwee H.; De Maere, Matthew Z.; Ting, Lily; Katrib, Marilyn; Ng, Charmaine; Sowers, Kevin R.; Galperin, Michael Y.; Anderson, Iain J.; Ivanova, Natalia; Dalin, Eileen; Martinez, Michelle; Lapidus, Alla; Hauser, Loren; Land, Miriam; Thomas, Torsten; Cavicchioli, Ricardo

    2009-04-01

    Psychrophilic archaea are abundant and perform critical roles throughout the Earth's expansive cold biosphere. Here we report the first complete genome sequence for a psychrophilic methanogenic archaeon, Methanococcoides burtonii. The genome sequence was manually annotated including the use of a five tiered Evidence Rating system that ranked annotations from Evidence Rating (ER) 1 (gene product experimentally characterized from the parent organism) to ER5 (hypothetical gene product) to provide a rapid means of assessing the certainty of gene function predictions. The genome is characterized by a higher level of aberrant sequence composition (51%) than any other archaeon. In comparison to hyper/thermophilic archaea which are subject to selection of synonymous codon usage, M. burtonii has evolved cold adaptation through a genomic capacity to accommodate highly skewed amino acid content, while retaining codon usage in common with its mesophilic Methanosarcina cousins. Polysaccharide biosynthesis genes comprise at least 3.3% of protein coding genes in the genome, and Cell wall/membrane/envelope biogenesis COG genes are over-represented. Likewise, signal transduction (COG category T) genes are over-represented and M. burtonii has a high 'IQ' (a measure of adaptive potential) compared to many methanogens. Numerous genes in these two over-represented COG categories appear to have been acquired from {var_epsilon}- and {delta}-proteobacteria, as do specific genes involved in central metabolism such as a novel B form of aconitase. Transposases also distinguish M. burtonii from other archaea, and their genomic characteristics indicate they play an important role in evolving the M. burtonii genome. Our study reveals a capacity for this model psychrophile to evolve through genome plasticity (including nucleotide skew, horizontal gene transfer and transposase activity) that enables adaptation to the cold, and to the biological and physical changes that have

  14. Uracil phosphoribosyltransferase from the extreme thermoacidophilic archaebacterium Sulfolobus shibatae is an allosteric enzyme, activated by GTP and inhibited by CTP

    DEFF Research Database (Denmark)

    Linde, Lise; Jensen, Kaj Frank

    1996-01-01

    -fold without much effect on Km for the substrates. The concentration of GTP required for half-maximal activation was about 80 µM. CTP was a strong inhibitor and acted by raising the concentration of GTP needed for half-maximal activation of the enzyme. We conclude that uracil phosphoribosyltransferase......Uracil phosphoribosyltransferase, which catalyses the formation of UMP and pyrophosphate from uracil and 5-phosphoribosyl a-1-pyrophosphate (PRPP), was partly purified from the extreme thermophilic archaebacterium Sulfolobus shibatae. The enzyme required divalent metal ions for activity...... and it showed the highest activity at pH 6.4. The specific activity of the enzyme was 50-times higher at 95°C than at 37°C, but the functional half-life was short at 95°C. The activity of uracil phosphoribosyltransferase was strongly activated by GTP, which increased Vmax of the reaction by approximately 20...

  15. Functional characterization of the origin of replication of pRN1 from Sulfolobus islandicus REN1H1.

    Science.gov (United States)

    Joshua, Chijioke J; Perez, Luis D; Keasling, Jay D

    2013-01-01

    Plasmid pRN1 from Sulfolobus islandicus REN1H1 is believed to replicate by a rolling circle mechanism but its origin and mechanism of replication are not well understood. We sought to create minimal expression vectors based on pRN1 that would be useful for heterologous gene expression in S. acidocaldarius, and in the process improve our understanding of the mechanism of replication. We constructed and transformed shuttle vectors that harbored different contiguous stretches of DNA from pRN1 into S. acidocaldarius E4-39, a uracil auxotroph. A 232-bp region 3' of orf904 was found to be critical for pRN1 replication and is therefore proposed to be the putative origin of replication. This 232-bp region contains a 100-bp stem-loop structure believed to be the double-strand origin of replication. The loop of the 100-bp structure contains a GTG tri-nucleotide motif, a feature that was previously reported to be important for the primase activity of Orf904. This putative origin and the associated orf56 and orf904 were identified as the minimal replicon of pRN1 because transformants of plasmids lacking any of these three features were not recovered. Plasmids lacking orf904 and orf56 but harboring the putative origin were transformable when orf904 and orf56 were provided in-trans; a 75-bp region 5' of the orf904 start codon was found to be essential for this complementation. Detailed knowledge of the pRN1 origin of replication will broaden the application of the plasmid as a genetic tool for Sulfolobus species.

  16. Functional characterization of the origin of replication of pRN1 from Sulfolobus islandicus REN1H1.

    Directory of Open Access Journals (Sweden)

    Chijioke J Joshua

    Full Text Available Plasmid pRN1 from Sulfolobus islandicus REN1H1 is believed to replicate by a rolling circle mechanism but its origin and mechanism of replication are not well understood. We sought to create minimal expression vectors based on pRN1 that would be useful for heterologous gene expression in S. acidocaldarius, and in the process improve our understanding of the mechanism of replication. We constructed and transformed shuttle vectors that harbored different contiguous stretches of DNA from pRN1 into S. acidocaldarius E4-39, a uracil auxotroph. A 232-bp region 3' of orf904 was found to be critical for pRN1 replication and is therefore proposed to be the putative origin of replication. This 232-bp region contains a 100-bp stem-loop structure believed to be the double-strand origin of replication. The loop of the 100-bp structure contains a GTG tri-nucleotide motif, a feature that was previously reported to be important for the primase activity of Orf904. This putative origin and the associated orf56 and orf904 were identified as the minimal replicon of pRN1 because transformants of plasmids lacking any of these three features were not recovered. Plasmids lacking orf904 and orf56 but harboring the putative origin were transformable when orf904 and orf56 were provided in-trans; a 75-bp region 5' of the orf904 start codon was found to be essential for this complementation. Detailed knowledge of the pRN1 origin of replication will broaden the application of the plasmid as a genetic tool for Sulfolobus species.

  17. Major and minor crRNA annealing sites facilitate low stringency DNA protospacer binding prior to Type I-A CRISPR-Cas interference in Sulfolobus

    DEFF Research Database (Denmark)

    Mousaei, Marzieh; Deng, Ling; She, Qunxin;

    2016-01-01

    The stringency of crRNA-protospacer DNA base pair matching required for effective CRISPR-Cas interference is relatively low in crenarchaeal Sulfolobus species in contrast to that required in some bacteria. To understand its biological significance we studied crRNA-protospacer interactions...... in Sulfolobus islandicus REY15A which carries multiple, and functionally diverse, interference complexes. A range of mismatches were introduced into a vector-borne protospacer that was identical to spacer 1 of CRISPR locus 2, with a cognate CCN PAM sequence. Two important crRNA annealing regions were identified...... lacking type III-Bα and III-Bβ interference gene cassettes, which showed similar interference levels to those of the wild-type strain. Parallels are drawn to the involvement of two annealing sites for microRNAs on some eukaryal mRNAs which provide enhanced binding capacity and specificity. A biological...

  18. Purification and biochemical properties of a cytochrome bc complex from the aerobic hyperthermophilic archaeon Aeropyrum pernix

    OpenAIRE

    Kabashima Yoshiki; Sakamoto Junshi

    2011-01-01

    Abstract Background The bioenergetics of Archaea with respect to the evolution of electron transfer systems is very interesting. In contrast to terminal oxidases, a canonical bc1 complex has not yet been isolated from Archaea. In particular, c-type cytochromes have been reported only for a limited number of species. Results Here, we isolated a c-type cytochrome-containing enzyme complex from the membranes of the hyperthermophilic archaeon, Aeropyrum pernix, grown aerobically. The redox spectr...

  19. Microbial community profiling of the Chinoike Jigoku ("Blood Pond Hell") hot spring in Beppu, Japan: isolation and characterization of Fe(III)-reducing Sulfolobus sp. strain GA1.

    Science.gov (United States)

    Masaki, Yusei; Tsutsumi, Katsutoshi; Hirano, Shin-Ichi; Okibe, Naoko

    2016-09-01

    Chinoike Jigoku ("Blood Pond Hell") is located in the hot spring town of Beppu on the southern island of Kyushu in Japan, and is the site of a red-colored acidic geothermal pond. This study aimed to investigate the microbial population composition in this extremely acidic environment and to isolate/characterize acidophilic microorganism with metal-reducing ability. Initially, PCR (using bacteria- and archaea-specific primers) of environmental DNA samples detected the presence of bacteria, but not archaea. This was followed by random sequencing analysis, confirming the presence of wide bacterial diversity at the site (123 clones derived from 18 bacterial and 1 archaeal genera), including those closely related to known autotrophic and heterotrophic acidophiles (Acidithiobacillus sp., Sulfobacillus sp., Alicyclobacillus sp.). Nevertheless, successive culture enrichment with Fe(III) under micro-aerobic conditions led to isolation of an unknown archaeal organism, Sulfolobus sp. GA1 (with 99.7% 16S rRNA gene sequence identity with Sulfolobus shibatae). Unlike many other known Sulfolobus spp., strain GA1 was shown to lack sulfur oxidation ability. Strain GA1 possessed only minor Fe(II) oxidation ability, but readily reduced Fe(III) during heterotrophic growth under micro-aerobic conditions. Strain GA1 was capable of reducing highly toxic Cr(VI) to less toxic/soluble Cr(III), demonstrating its potential utility in bioremediation of toxic metal species.

  20. Disruption of the Operon Encoding Ehb Hydrogenase Limits AnabolicCO2 Assimilation in the Archaeon Methanococcus maripaludis

    Energy Technology Data Exchange (ETDEWEB)

    Porat, Iris; Kim, Wonduck; Hendrickson, Erik L.; Xia, Qiangwei; Zhang, Yi; Wang, Tiansong; Taub, Fred; Moore, Brian C.; Anderson, IainJ.; Hackett, Murray; Leigh, John A.; Whitman, William B.

    2006-02-01

    Methanococcus maripaludis is a mesophilic archaeon thatreduces CO2 to methane with H2 or formate as an energy source. Itcontains two membrane-bound energy-conserving hydrogenases, Eha and Ehb.To determine therole of Ehb, a deletion in the ehb operon wasconstructed to yield the mutant, strain S40. Growth of S40 was severelyimpaired in minimal medium. Both acetate and yeast extract were necessaryto restore growth to nearly wild-type levels, suggesting that Ehb wasinvolved in multiple steps in carbon assimilation. However, nodifferences in the total hydrogenase specific activities were foundbetween the wild type and mutant in either cell extracts ormembrane-purified fractions. Methanogenesis by resting cells withpyruvate as the electron donor was also reduced by 30 percent in S40,suggesting a defect in pyruvate oxidation. CO dehydrogenase/acetylcoenzyme A (CoA) synthase and pyruvate oxidoreductase had higher specificactivities in the mutant, and genes encoding these enzymes, as well asAMP-forming acetyl-CoA synthetase, were expressed at increased levels.These observations support a role for Ehb in anabolic CO2 assimilation inmethanococci.

  1. Unusual starch degradation pathway via cyclodextrins in the hyperthermophilic sulfate-reducing archaeon Archaeoglobus fulgidus strain 7324.

    Science.gov (United States)

    Labes, Antje; Schönheit, Peter

    2007-12-01

    The hyperthermophilic archaeon Archaeoglobus fulgidus strain 7324 has been shown to grow on starch and sulfate and thus represents the first sulfate reducer able to degrade polymeric sugars. The enzymes involved in starch degradation to glucose 6-phosphate were studied. In extracts of starch-grown cells the activities of the classical starch degradation enzymes, alpha-amylase and amylopullulanase, could not be detected. Instead, evidence is presented here that A. fulgidus utilizes an unusual pathway of starch degradation involving cyclodextrins as intermediates. The pathway comprises the combined action of an extracellular cyclodextrin glucanotransferase (CGTase) converting starch to cyclodextrins and the intracellular conversion of cyclodextrins to glucose 6-phosphate via cyclodextrinase (CDase), maltodextrin phosphorylase (Mal-P), and phosphoglucomutase (PGM). These enzymes, which are all induced after growth on starch, were characterized. CGTase catalyzed the conversion of starch to mainly beta-cyclodextrin. The gene encoding CGTase was cloned and sequenced and showed highest similarity to a glucanotransferase from Thermococcus litoralis. After transport of the cyclodextrins into the cell by a transport system to be defined, these molecules are linearized via a CDase, catalyzing exclusively the ring opening of the cyclodextrins to the respective maltooligodextrins. These are degraded by a Mal-P to glucose 1-phosphate. Finally, PGM catalyzes the conversion of glucose 1-phosphate to glucose 6-phosphate, which is further degraded to pyruvate via the modified Embden-Meyerhof pathway.

  2. A predicted geranylgeranyl reductase reduces the ω-position isoprene of dolichol phosphate in the halophilic archaeon, Haloferax volcanii.

    Science.gov (United States)

    Naparstek, Shai; Guan, Ziqiang; Eichler, Jerry

    2012-06-01

    In N-glycosylation in both Eukarya and Archaea, N-linked oligosaccharides are assembled on dolichol phosphate prior to transfer of the glycan to the protein target. However, whereas only the α-position isoprene subunit is saturated in eukaryal dolichol phosphate, both the α- and ω-position isoprene subunits are reduced in the archaeal lipid. The agents responsible for dolichol phosphate saturation remain largely unknown. The present study sought to identify dolichol phosphate reductases in the halophilic archaeon, Haloferax volcanii. Homology-based searches recognize HVO_1799 as a geranylgeranyl reductase. Mass spectrometry revealed that cells deleted of HVO_1799 fail to fully reduce the isoprene chains of H. volcanii membrane phospholipids and glycolipids. Likewise, the absence of HVO_1799 led to a loss of saturation of the ω-position isoprene subunit of C(55) and C(60) dolichol phosphate, with the effect of HVO_1799 deletion being more pronounced with C(60) dolichol phosphate than with C(55) dolichol phosphate. Glycosylation of dolichol phosphate in the deletion strain occurred preferentially on that version of the lipid saturated at both the α- and ω-position isoprene subunits.

  3. In vivo characterization of the homing endonuclease within the polB gene in the halophilic archaeon Haloferax volcanii.

    Directory of Open Access Journals (Sweden)

    Adit Naor

    Full Text Available Inteins are parasitic genetic elements, analogous to introns that excise themselves at the protein level by self-splicing, allowing the formation of functional non-disrupted proteins. Many inteins contain a homing endonuclease (HEN gene, and rely on its activity for horizontal propagation. In the halophilic archaeon, Haloferax volcanii, the gene encoding DNA polymerase B (polB contains an intein with an annotated but uncharacterized HEN. Here we examine the activity of the polB HEN in vivo, within its natural archaeal host. We show that this HEN is highly active, and able to insert the intein into both a chromosomal target and an extra-chromosomal plasmid target, by gene conversion. We also demonstrate that the frequency of its incorporation depends on the length of the flanking homologous sequences around the target site, reflecting its dependence on the homologous recombination machinery. Although several evolutionary models predict that the presence of an intein involves a change in the fitness of the host organism, our results show that a strain deleted for the intein sequence shows no significant changes in growth rate compared to the wild type.

  4. Carbonate precipitation by the thermophilic archaeon Archaeoglobus fulgidus: A model of carbon flow for an ancient microorganism

    Science.gov (United States)

    Robbins, L.L.; Van Cleave, K. A.; Ostrom, P.

    2008-01-01

    Microbial carbonate precipitation experiments were conducted using the archaeon bacteria Archaeoglobus fulgidus to determine chemical and isotopic fractionation of organic and inorganic carbon into mineral phases. Carbonate precipitation was induced in two different experiments using A. fulgidus to determine the relative abundance of organically derived carbon incorporated into carbonate minerals as well as to define any distinct phases or patterns that could be attributed to the precipitation process. One experiment used a medium containing 13C-depleted organic carbon and 13C-enriched inorganic carbon, and the other used a 14C-labeled organic carbon source. Results indicated that 0.9 - 24.8% organic carbon was incorporated into carbonates precipitated by A. fulgidus and that this process was mediated primarily by pH and CO2 emission from cells. Data showed that the carbon in the CO2 produced from this microorganism is incorporated into carbonates and that the rate at which precipitation occurs and the dynamics of the carbonate precipitation process are strongly mediated by the specific steps involved in the biochemical process for lactate oxidation by A. fulgidus.

  5. Carbonate precipitation by the thermophilic archaeon Archaeoglobus fulgidus: a model of carbon flow for an ancient microorganism

    Directory of Open Access Journals (Sweden)

    P. Ostrom

    2008-08-01

    Full Text Available Microbial carbonate precipitation experiments were conducted using the archaeon bacteria Archaeoglobus fulgidus to determine chemical and isotopic fractionation of organic and inorganic carbon into mineral phases. Carbonate precipitation was induced in two different experiments using A. fulgidus to determine the relative abundance of organically derived carbon incorporated into carbonate minerals as well as to define any distinct phases or patterns that could be attributed to the precipitation process. One experiment used a medium containing 13C-depleted organic carbon and 13C-enriched inorganic carbon, and the other used a 14C-labeled organic carbon source. Results indicated that 0.9–24.8% organic carbon was incorporated into carbonates precipitated by A. fulgidus and that this process was mediated primarily by pH and CO2 emission from cells. Data showed that the carbon in the CO2 produced from this microorganism is incorporated into carbonates and that the rate at which precipitation occurs and the dynamics of the carbonate precipitation process are strongly mediated by the specific steps involved in the biochemical process for lactate oxidation by A. fulgidus.

  6. Control of archaellation in Sulfolobus acidocaldarius: unravelling of the regulation of surface structure biosynthesis in Archaea begins.

    Science.gov (United States)

    Jarrell, Ken F

    2012-10-01

    Archaea have a variety of surface appendages including archaella (archaeal flagella), pili, hami and cannulae. While expected to be energetically expensive to express, studies focused on the regulation of such structures are nevertheless lacking. In the current issue of Molecular Microbiology, Reimann et al. (2012) identified a two-partner system called ArnA and ArnB in Sulfolobus acidocaldarius that interact strongly with each other and are repressors of archaella expression while also having an enhancing effect on the appearance of type IV pili. ArnA is a forkhead-associated domain-containing protein while ArnB is a von Willebrand domain-containing protein. Both proteins can be phosphorylated in vitro by S. acidocaldarius protein kinases. The repression of archaella expression is dependent on dephosphorylation of the Arn proteins. Deletions of arnA or arnB resulted in increased levels of archaella operon proteins and cells that were hypermotile due to increased archaellation. Direct effects of ArnA/ArnB on transcription from fla promoters were demonstrated using arnA and arnB deletion strains but only a modest increase in transcription was demonstrated in each mutant suggesting that the repression effect observed may be due to protein-protein interactions. This paper represents a significant step forward in our understanding of archaeal surface structure biogenesis.

  7. Archaeal signal transduction: impact of protein phosphatase deletions on cell size, motility, and energy metabolism in Sulfolobus acidocaldarius.

    Science.gov (United States)

    Reimann, Julia; Esser, Dominik; Orell, Alvaro; Amman, Fabian; Pham, Trong Khoa; Noirel, Josselin; Lindås, Ann-Christin; Bernander, Rolf; Wright, Phillip C; Siebers, Bettina; Albers, Sonja-Verena

    2013-12-01

    In this study, the in vitro and in vivo functions of the only two identified protein phosphatases, Saci-PTP and Saci-PP2A, in the crenarchaeal model organism Sulfolobus acidocaldarius were investigated. Biochemical characterization revealed that Saci-PTP is a dual-specific phosphatase (against pSer/pThr and pTyr), whereas Saci-PP2A exhibited specific pSer/pThr activity and inhibition by okadaic acid. Deletion of saci_pp2a resulted in pronounced alterations in growth, cell shape and cell size, which could be partially complemented. Transcriptome analysis of the three strains (Δsaci_ptp, Δsaci_pp2a and the MW001 parental strain) revealed 155 genes that were differentially expressed in the deletion mutants, and showed significant changes in expression of genes encoding the archaella (archaeal motility structure), components of the respiratory chain and transcriptional regulators. Phosphoproteome studies revealed 801 unique phosphoproteins in total, with an increase in identified phosphopeptides in the deletion mutants. Proteins from most functional categories were affected by phosphorylation, including components of the motility system, the respiratory chain, and regulatory proteins. In the saci_pp2a deletion mutant the up-regulation at the transcript level, as well as the observed phosphorylation pattern, resembled starvation stress responses. Hypermotility was also observed in the saci_pp2a deletion mutant. The results highlight the importance of protein phosphorylation in regulating essential cellular processes in the crenarchaeon S. acidocaldarius.

  8. Agl16, a thermophilic glycosyltransferase mediating the last step of N-Glycan biosynthesis in the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius.

    Science.gov (United States)

    Meyer, Benjamin H; Peyfoon, Elham; Dietrich, Carsten; Hitchen, Paul; Panico, Maria; Morris, Howard R; Dell, Anne; Albers, Sonja-Verena

    2013-05-01

    Recently, the S-layer protein of Sulfolobus acidocaldarius was shown to be N-linked with a tribranched hexasaccharide, composed of Man2Glc1GlcNAc2 and a sulfated sugar called sulfoquinovose. To identify genes involved in the biosynthesis and attachment of this glycan, markerless in-frame deletions of genes coding for predicted glycosyltransferases were created. The successful deletion of agl16, coding for a glycosyltransferase, resulted in the S-layer protein and archaellins having reduced molecular weights, as visualized by Coomassie staining or immunoblotting. This analysis indicated a change in the N-glycan composition. Nano-liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses confirmed that the glycan of the S-layer protein from the agl16 deletion mutant was a pentasaccharide, which was missing a terminal hexose residue. High-performance liquid chromatography (HPLC) analyses of the hydrolyzed N-glycan indicated that the missing hexose is a glucose residue. A physiological characterization of the agl16 deletion mutant revealed a significant effect on the growth at elevated salt concentrations. At 300 mM NaCl, the doubling time of the Δagl16 mutant was increased 2-fold compared to that of the background strain. Furthermore, the incomplete glycan structure of the Δagl16 deletion strain affected the assembly and function of the archaellum, as exemplified by semisolid Gelrite plate analysis, in which the motility is decreased according to the N-glycan size.

  9. Biochemical characterization of a recombinant short-chain NAD(H)-dependent dehydrogenase/reductase from Sulfolobus acidocaldarius.

    Science.gov (United States)

    Pennacchio, Angela; Giordano, Assunta; Pucci, Biagio; Rossi, Mosè; Raia, Carlo A

    2010-03-01

    The gene encoding a novel alcohol dehydrogenase that belongs to the short-chain dehydrogenases/reductases (SDRs) superfamily was identified in the aerobic thermoacidophilic crenarchaeon Sulfolobus acidocaldarius strain DSM 639. The saadh gene was heterologously overexpressed in Escherichia coli, and the protein (SaADH) was purified to homogeneity and characterized. SaADH is a tetrameric enzyme consisting of identical 28,978-Da subunits, each composed of 264 amino acids. The enzyme has remarkable thermophilicity and thermal stability, displaying activity at temperatures up to 75 degrees C and a 30-min half-inactivation temperature of ~90 degrees C, and shows good tolerance to common organic solvents. SaADH has a strict requirement for NAD(H) as the coenzyme, and displays a preference for the reduction of alicyclic, bicyclic and aromatic ketones and alpha-keto esters, but is poorly active on aliphatic, cyclic and aromatic alcohols, and shows no activity on aldehydes. The enzyme catalyses the reduction of alpha-methyl and alpha-ethyl benzoylformate, and methyl o-chlorobenzoylformate with 100% conversion to methyl (S)-mandelate [17% enantiomeric excess (ee)], ethyl (R)-mandelate (50% ee), and methyl (R)-o-chloromandelate (72% ee), respectively, with an efficient in situ NADH-recycling system which involves glucose and a thermophilic glucose dehydrogenase. This study provides further evidence supporting the critical role of the D37 residue in discriminating NAD(H) from NAD(P)H in members of the SDR superfamily.

  10. Thermococcus thioreducens sp. nov., a Novel Hyperthermophilic, Obligately Sulfur-Reducing Archaeon from a Deep-Sea Hydrothermal Vent

    Science.gov (United States)

    Pikuta, Elena V.; Marsic, Damien; Itoh, Takashi; Bej, Asim K.; Tang, Jane; Whitman, William B.; Ng, Joseph D.; Garriott, Owen K.; Hoover, Richard B.

    2007-01-01

    A hyperthermophilic, sulfur-reducing, organo-heterotrophic archaeon, strain OGL-20P(sup T), was isolated from 'black smoker' chimney material from the Rainbow hydrothermal vent site on the Mid-Atlantic Ridge (36.2degN, 33.9degW). The cells of strain OGL-20P(T) have an irregular coccoid shape and are motile with a single flagellum. Growth was observed within a pH range of 5.0-8.5 (optimum pH 7.0), an NaCl concentration range of 1-5%(w/v) (optimum 3%)and a temperature range of 55-94 C (optimum 83-85 C). The novel isolate is strictly anaerobic and obligately dependent upon elemental sulfur as an electron acceptor, but it does not reduce sulfate, sulfite, thiosulfate, Fe(III) or nitrate. Proteolysis products (peptone, bacto-tryptone, Casamino acids and yeast extract) are utilized as substrates during sulfur reduction. Strain OGL-20P(sup T) is resistant to ampicillin, chloram phenicol, kanamycin and gentamicin, but sensitive to tetracycline and rifampicin. The G + C content of the DNA is 52.9 mol% The 16S rRNA gene sequence analysis revealed that strain OGL-20P(sup T) is closely related to Thermococcus coalescens and related species, but no significant homology by DNA-DNA hybridization was observed between those species and the new isolate. On the basis of physiological and molecular properties of the new isolate, we conclude that strain OGL-20P(sup T) represents a new separate species within the genus Thermococcus, for which we propose the name Thermococcus thioreducens sp. nov. The type strain is OGL-20P(sup T) (=JCM 12859(exp T) = DSM 14981(exp T)=ATCC BAA-394(exp T)).

  11. Identification of key components in the energy metabolism of the hyperthermophilic sulfate reducing archaeon Archaeoglobus fulgidus by transcriptome analyses

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    William Peter eHocking

    2014-03-01

    Full Text Available Energy conservation by the pathway of dissimilatory sulfate reduction is present in a diverse group of prokaryotes, but is most comprehensively studied in Deltaproteobacteria. Herein, whole-genome microarray analyses where used to provide a model of the energy me-tabolism of the sulfate reducing archaeon Archaeoglobus fulgidus, based comparative analysis litoautotrophic growth with H2/CO2 and thiosulfate, and heterotrophic growth on lactate with sulfate or thiosulfate. Only 72 genes were expressed differentially between the cultures utiliz-ing sulfate or thiosulfate whereas 269 genes were affected by a shift in energy source. We identified co-located gene cluster encoding putative lactate dehydrogenases (lldD, dld, lldEFG, also present in sulfate reducing bacteria. These enzymes may take part in energy conservation in A. fulgidus by specifically linking lactate oxidation with APS reduction via the Qmo complex. High transcriptional levels of Fqo confirm an important role of F420H2 and menaquinone mediated electron transport chain during heterotrophic growth. A putative pe-riplasmic thiosulfate reductase was identified by specific up-regulation. Also, putative genes for transport of sulfate and sulfite are discussed. We present a model for hydrogen metabo-lism, based on the probable bifurcation reaction of the Mvh:Hdl hydrogenase, that may inhibit the utilization of Fdred for energy conservation. Rather, energy conservation is probably facili-tated via menaquinone to multiple membrane bound heterodisulfide reductase complexes and the enzyme DsrC – linking periplasmic hydrogenase (Vht to the cytoplasmic reduction of sulfite. The ambiguous roles of genes corresponding to fatty acid metabolism induced during growth with H2 are discussed. Putative co-assimilation of organic acids is favored over a homologues secondary carbon fixation pathway, although both mechanisms may contribute to conserve the amount of Fdred needed during autotrophic growth

  12. Microorganisms for desulphurization of coal: the influence of leaching compounds on their growth

    Energy Technology Data Exchange (ETDEWEB)

    Olsson, G.; Larsson, L.; Holst, O.; Karlsson, H.T. (Lund University (Sweden). Dept. of Chemical Engineering II)

    1989-10-01

    An investigation was undertaken to outline how microorganisms suggested for use in coal desulphurization are affected by compounds leached from coal. Three species of the genus Sulfolobus were studied. Sulfolobus acidocaldarius was seen to be affected by compounds leached from coal more negatively than Sulfolobus brierleyi and Sulfolobus solfataricus. It was observed that certain conditions during leaching, such as pH, temperature and leaching time, were important for the concentration of compounds leached from coal. It was seen that compounds leached from coal, in some cases, can improve the growth yield for the microorganisms. 7 refs., 8 figs., 2 tabs.

  13. Crystal structure of archaeal photolyase from Sulfolobus tokodaii with two FAD molecules: implication of a novel light-harvesting cofactor.

    Science.gov (United States)

    Fujihashi, Masahiro; Numoto, Nobutaka; Kobayashi, Yukiko; Mizushima, Akira; Tsujimura, Masanari; Nakamura, Akira; Kawarabayasi, Yutaka; Miki, Kunio

    2007-01-26

    UV exposure of DNA molecules induces serious DNA lesions. The cyclobutane pyrimidine dimer (CPD) photolyase repairs CPD-type - lesions by using the energy of visible light. Two chromophores for different roles have been found in this enzyme family; one catalyzes the CPD repair reaction and the other works as an antenna pigment that harvests photon energy. The catalytic cofactor of all known photolyases is FAD, whereas several light-harvesting cofactors are found. Currently, 5,10-methenyltetrahydrofolate (MTHF), 8-hydroxy-5-deaza-riboflavin (8-HDF) and FMN are the known light-harvesting cofactors, and some photolyases lack the chromophore. Three crystal structures of photolyases from Escherichia coli (Ec-photolyase), Anacystis nidulans (An-photolyase), and Thermus thermophilus (Tt-photolyase) have been determined; however, no archaeal photolyase structure is available. A similarity search of archaeal genomic data indicated the presence of a homologous gene, ST0889, on Sulfolobus tokodaii strain7. An enzymatic assay reveals that ST0889 encodes photolyase from S. tokodaii (St-photolyase). We have determined the crystal structure of the St-photolyase protein to confirm its structural features and to investigate the mechanism of the archaeal DNA repair system with light energy. The crystal structure of the St-photolyase is superimposed very well on the three known photolyases including the catalytic cofactor FAD. Surprisingly, another FAD molecule is found at the position of the light-harvesting cofactor. This second FAD molecule is well accommodated in the crystal structure, suggesting that FAD works as a novel light-harvesting cofactor of photolyase. In addition, two of the four CPD recognition residues in the crystal structure of An-photolyase are not found in St-photolyase, which might utilize a different mechanism to recognize the CPD from that of An-photolyase.

  14. Identification and characterization of MalA in the maltose/maltodextrin operon of Sulfolobus acidocaldarius DSM639.

    Science.gov (United States)

    Choi, Kyoung-Hwa; Hwang, Sungmin; Cha, Jaeho

    2013-04-01

    A putative maltose/maltodextrin operon was found in the Sulfolobus acidocaldarius DSM639 genome. The gene cluster consisted of 7 genes (malA, trmB, amyA, malG, malF, malE, and malK). Here, we report the identification of MalA, which is responsible for the hydrolysis of maltose or maltodextrin to glucose in S. acidocaldarius. The transcription level of malA was increased 3-fold upon the addition of maltose or starch to the medium. Moreover, the α-glucosidase activity for maltose as a substrate in cell extracts of S. acidocaldarius DSM639 was also 11- and 10-fold higher during growth in YT medium (Brock's mineral salts, 0.1% [wt/vol] tryptone, and 0.005% [wt/vol] yeast extract) containing maltose or starch, respectively, than during growth on other sugars. The gene encoding MalA was cloned and expressed in S. acidocaldarius. The enzyme purified from the organism was a dodecamer in its active state and showed strong maltose-hydrolyzing activity at 100°C and pH 5.0. MalA was remarkably thermostable, with half-lives of 33.8 h, 10.6 h, and 1.8 h at 95°C, 100°C, and 105°C, respectively. Substrate specificity and kinetic studies of MalA with maltooligosaccharides indicated that MalA efficiently hydrolyzed maltose to maltopentaose, which is a typical characteristic of GH31-type α-glucosidases. However, glycogen or starch was not hydrolyzed. Reverse transcription-PCR, sugar uptake, and growth studies of the wild-type DSM639 and ΔmalEFG mutant on different sugars demonstrated that MalA located in the mal operon gene cluster is involved in maltose and starch metabolism in S. acidocaldarius.

  15. Regulation of archaella expression by the FHA and von Willebrand domain-containing proteins ArnA and ArnB in Sulfolobus acidocaldarius.

    Science.gov (United States)

    Reimann, Julia; Lassak, Kerstin; Khadouma, Sunia; Ettema, Thijs J G; Yang, Nuan; Driessen, Arnold J M; Klingl, Andreas; Albers, Sonja-Verena

    2012-10-01

    The ability of microorganisms to sense and respond to sudden changes in their environment is often based on regulatory systems comprising reversible protein phosphorylation. The archaellum (former: archaeal flagellum) is used for motility in Archaea and therefore functionally analogous to the bacterial flagellum. In contrast with archaellum-mediated movement in certain members of the Euryarchaeota, this process, including its regulation, remains poorly studied in crenarchaeal organisms like Sulfolobus species. Recently, it was shown in Sulfolobus acidocaldarius that tryptone limiting conditions led to the induction of archaella expression and assembly. Here we have identified two proteins, the FHA domain-containing protein ArnA and the vWA domain-containing protein ArnB that are involved in regulating archaella expression in S. acidocaldarius. Both proteins are phosphorylated by protein kinases in vitro and interact strongly in vivo. Phenotypic analyses revealed that these two proteins are repressors of archaella expression. These results represent the first step in understanding the networks that underlie regulation of cellular motility in Crenarchaeota and emphasize the importance of protein phosphorylation in the regulation of cellular processes in the Archaea.

  16. BarR, an Lrp-type transcription factor in Sulfolobus acidocaldarius, regulates an aminotransferase gene in a β-alanine responsive manner.

    Science.gov (United States)

    Liu, Han; Orell, Alvaro; Maes, Dominique; van Wolferen, Marleen; Lindås, Ann-Christin; Bernander, Rolf; Albers, Sonja-Verena; Charlier, Daniel; Peeters, Eveline

    2014-05-01

    In archaea, nothing is known about the β-alanine degradation pathway or its regulation. In this work, we identify and characterize BarR, a novel Lrp-like transcription factor and the first one that has a non-proteinogenic amino acid ligand. BarR is conserved in Sulfolobus acidocaldarius and Sulfolobus tokodaii and is located in a divergent operon with a gene predicted to encode β-alanine aminotransferase. Deletion of barR resulted in a reduced exponential growth rate in the presence of β-alanine. Furthermore, qRT-PCR and promoter activity assays demonstrated that BarR activates the expression of the adjacent aminotransferase gene, but only upon β-alanine supplementation. In contrast, auto-activation proved to be β-alanine independent. Heterologously produced BarR is an octamer in solution and forms a single complex by interacting with multiple sites in the 170 bp long intergenic region separating the divergently transcribed genes. In vitro, DNA binding is specifically responsive to β-alanine and site-mutant analyses indicated that β-alanine directly interacts with the ligand-binding pocket. Altogether, this work contributes to the growing body of evidence that in archaea, Lrp-like transcription factors have physiological roles that go beyond the regulation of α-amino acid metabolism.

  17. Hyperthermophilic endoglucanase for in planta lignocellulose conversion

    Science.gov (United States)

    2012-01-01

    Background The enzymatic conversion of lignocellulosic plant biomass into fermentable sugars is a crucial step in the sustainable and environmentally friendly production of biofuels. However, a major drawback of enzymes from mesophilic sources is their suboptimal activity under established pretreatment conditions, e.g. high temperatures, extreme pH values and high salt concentrations. Enzymes from extremophiles are better adapted to these conditions and could be produced by heterologous expression in microbes, or even directly in the plant biomass. Results Here we show that a cellulase gene (sso1354) isolated from the hyperthermophilic archaeon Sulfolobus solfataricus can be expressed in plants, and that the recombinant enzyme is biologically active and exhibits the same properties as the wild type form. Since the enzyme is inactive under normal plant growth conditions, this potentially allows its expression in plants without negative effects on growth and development, and subsequent heat-inducible activation. Furthermore we demonstrate that the recombinant enzyme acts in high concentrations of ionic liquids and can therefore degrade α-cellulose or even complex cell wall preparations under those pretreatment conditions. Conclusion The hyperthermophilic endoglucanase SSO1354 with its unique features is an excellent tool for advanced biomass conversion. Here we demonstrate its expression in planta and the possibility for post harvest activation. Moreover the enzyme is suitable for combined pretreatment and hydrolysis applications. PMID:22928996

  18. Hyperthermophilic endoglucanase for in planta lignocellulose conversion

    Directory of Open Access Journals (Sweden)

    Klose Holger

    2012-08-01

    Full Text Available Abstract Background The enzymatic conversion of lignocellulosic plant biomass into fermentable sugars is a crucial step in the sustainable and environmentally friendly production of biofuels. However, a major drawback of enzymes from mesophilic sources is their suboptimal activity under established pretreatment conditions, e.g. high temperatures, extreme pH values and high salt concentrations. Enzymes from extremophiles are better adapted to these conditions and could be produced by heterologous expression in microbes, or even directly in the plant biomass. Results Here we show that a cellulase gene (sso1354 isolated from the hyperthermophilic archaeon Sulfolobus solfataricus can be expressed in plants, and that the recombinant enzyme is biologically active and exhibits the same properties as the wild type form. Since the enzyme is inactive under normal plant growth conditions, this potentially allows its expression in plants without negative effects on growth and development, and subsequent heat-inducible activation. Furthermore we demonstrate that the recombinant enzyme acts in high concentrations of ionic liquids and can therefore degrade α-cellulose or even complex cell wall preparations under those pretreatment conditions. Conclusion The hyperthermophilic endoglucanase SSO1354 with its unique features is an excellent tool for advanced biomass conversion. Here we demonstrate its expression in planta and the possibility for post harvest activation. Moreover the enzyme is suitable for combined pretreatment and hydrolysis applications.

  19. Stability against temperature and external agents of vesicles composed of archael bolaform lipids and egg PC.

    Science.gov (United States)

    Fan, Q; Relini, A; Cassinadri, D; Gambacorta, A; Gliozzi, A

    1995-11-22

    The bolaform lipid PLE extracted from the thermophilic archaeon Sulfolobus solfataricus and its mixtures with egg phosphatidyl-choline (egg PC) have been used to prepare sonicated vesicles. The leakage of entrapped calcein was continuously monitored by fluorescence dequenching. The half times of leakage have been used to compare vesicle stability under different conditions of temperature, lipid composition and presence of destabilizing agents like Ca2+ ions and poly(ethylene glycol) (PEG). It has been found that leakage is primarily modulated by the monopolar/bipolar lipid ratio. In particular, the half time of leakage for vesicles formed from a mixture of the polar lipid extract (PLE) and egg PC is characterized by a maximum at about 1:2 molar ratio. The free energy of mixing has been evaluated from pressure-area isotherms on monolayers at the air/water interface. The results indicate a non monotonous behaviour of the excess free energy of mixing as a function of the molar ratio and the occurrence of a minimum at a fixed molar ratio. The possible formation of a complex is discussed and compared with previous calorimetric measurements on similar compounds.

  20. Bipolar tetraether lipids: chain flexibility and membrane polarity gradients from spin-label electron spin resonance.

    Science.gov (United States)

    Bartucci, R; Gambacorta, A; Gliozzi, A; Marsh, D; Sportelli, L

    2005-11-15

    Membranes of thermophilic Archaea are composed of unique tetraether lipids in which C40, saturated, methyl-branched biphytanyl chains are linked at both ends to polar groups. In this paper, membranes composed of bipolar lipids P2 extracted from the acidothermophile archaeon Sulfolobus solfataricus are studied. The biophysical basis for the membrane formation and thermal stability is investigated by using electron spin resonance (ESR) of spin-labeled lipids. Spectral anisotropy and isotropic hyperfine couplings are used to determine the chain flexibility and polarity gradients, respectively. For comparison, similar measurements have been carried out on aqueous dispersions of diacyl reference lipid dipalmitoyl phosphatidylcholine and also of diphytanoyl phosphatidylcholine, which has methyl-branched chains. At a given temperature, the bolaform lipid chains are more ordered and less flexible than in normal bilayer membranes. Only at elevated temperatures (80 degrees C) does the flexibility of the chain environment in tetraether lipid assemblies approach that of fluid bilayer membranes. The height of the hydrophobic barrier formed by a monolayer of archaebacterial lipids is similar to that in conventional fluid bilayer membranes, and the permeability barrier width is comparable to that formed by a bilayer of C16 lipid chains. At a mole ratio of 1:2, the tetraether P2 lipids mix well with dipalmitoyl phosphatidylcholine lipids and stabilize conventional bilayer membranes. The biological as well as the biotechnological relevance of the results is discussed.

  1. Transcription start site associated RNAs (TSSaRNAs are ubiquitous in all domains of life.

    Directory of Open Access Journals (Sweden)

    Livia S Zaramela

    Full Text Available A plethora of non-coding RNAs has been discovered using high-resolution transcriptomics tools, indicating that transcriptional and post-transcriptional regulation is much more complex than previously appreciated. Small RNAs associated with transcription start sites of annotated coding regions (TSSaRNAs are pervasive in both eukaryotes and bacteria. Here, we provide evidence for existence of TSSaRNAs in several archaeal transcriptomes including: Halobacterium salinarum, Pyrococcus furiosus, Methanococcus maripaludis, and Sulfolobus solfataricus. We validated TSSaRNAs from the model archaeon Halobacterium salinarum NRC-1 by deep sequencing two independent small-RNA enriched (RNA-seq and a primary-transcript enriched (dRNA-seq strand-specific libraries. We identified 652 transcripts, of which 179 were shown to be primary transcripts (∼7% of the annotated genome. Distinct growth-associated expression patterns between TSSaRNAs and their cognate genes were observed, indicating a possible role in environmental responses that may result from RNA polymerase with varying pausing rhythms. This work shows that TSSaRNAs are ubiquitous across all domains of life.

  2. Essential and non-essential DNA replication genes in the model halophilic Archaeon, Halobacterium sp. NRC-1

    Directory of Open Access Journals (Sweden)

    DasSarma Shiladitya

    2007-06-01

    Full Text Available Abstract Background Information transfer systems in Archaea, including many components of the DNA replication machinery, are similar to those found in eukaryotes. Functional assignments of archaeal DNA replication genes have been primarily based upon sequence homology and biochemical studies of replisome components, but few genetic studies have been conducted thus far. We have developed a tractable genetic system for knockout analysis of genes in the model halophilic archaeon, Halobacterium sp. NRC-1, and used it to determine which DNA replication genes are essential. Results Using a directed in-frame gene knockout method in Halobacterium sp. NRC-1, we examined nineteen genes predicted to be involved in DNA replication. Preliminary bioinformatic analysis of the large haloarchaeal Orc/Cdc6 family, related to eukaryotic Orc1 and Cdc6, showed five distinct clades of Orc/Cdc6 proteins conserved in all sequenced haloarchaea. Of ten orc/cdc6 genes in Halobacterium sp. NRC-1, only two were found to be essential, orc10, on the large chromosome, and orc2, on the minichromosome, pNRC200. Of the three replicative-type DNA polymerase genes, two were essential: the chromosomally encoded B family, polB1, and the chromosomally encoded euryarchaeal-specific D family, polD1/D2 (formerly called polA1/polA2 in the Halobacterium sp. NRC-1 genome sequence. The pNRC200-encoded B family polymerase, polB2, was non-essential. Accessory genes for DNA replication initiation and elongation factors, including the putative replicative helicase, mcm, the eukaryotic-type DNA primase, pri1/pri2, the DNA polymerase sliding clamp, pcn, and the flap endonuclease, rad2, were all essential. Targeted genes were classified as non-essential if knockouts were obtained and essential based on statistical analysis and/or by demonstrating the inability to isolate chromosomal knockouts except in the presence of a complementing plasmid copy of the gene. Conclusion The results showed that ten

  3. Activities of methionine-γ-lyase in the acidophilic archaeon “Ferroplasma acidarmanus” strain fer1

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    Khan MA

    2013-04-01

    Full Text Available M A Khan,1 Madeline M López-Muñoz,2 Charles W Kaspar,3 Kai F Hung1 1Department of Biological Sciences, Eastern Illinois University, Charleston, IL, USA; 2Department of Biology, Universidad de Puerto Rico, Mayaguez, Puerto Rico; 3Bacteriology Department, University of Wisconsin, Madison, WI, USA Abstract: Biogeochemical processes on exposed pyrite ores result in extremely high levels of sulfuric acid at these locations. Acidophiles that thrive in these conditions must overcome significant challenges, including an environment with proton concentrations at pH 3 or below. The role of sulfur metabolism in the archaeon “Ferroplasma acidarmanus” strain fer1's ability to thrive in this environment was investigated due to its growth-dependent production of methanethiol, a volatile organic sulfur compound. Two putative sequences for methionine-γ-lyase (EC 4.4.1.11, an enzyme known to carry out α, γ-elimination on L-methionine to produce methanethiol, were identified in fer1. Bioinformatic analyses identified a conserved pyridoxal-5'-phosphate (PLP binding domain and a partially conserved catalytic domain in both putative sequences. Detection of PLP-dependent and L-methionine-dependent production of α-keto compounds and thiol groups in fer1 confirmed the presence of methionine-γ-lyase activity. Further, fer1 lysate was capable of processing related substrates, including D-methionine, L-cysteine, L-cystathionine, and L/D-homocysteine. When the two putative fer1 methionine-γ-lyase gene-coded proteins were expressed in Escherichia coli cells, one sequence demonstrated an ability to carry out α, γ-elimination activity, while the other exhibited γ-replacement activity. These fer1 methionine-γ-lyases also exhibited optimum pH, substrate specificity, and catalytic preferences that are different from methionine-γ-lyases from other organisms. These differences are discussed in the context of molecular phylogeny constructed using a maximum

  4. Identification of a novel amino acid racemase from a hyperthermophilic archaeon Pyrococcus horikoshii OT-3 induced by D-amino acids.

    Science.gov (United States)

    Kawakami, Ryushi; Ohmori, Taketo; Sakuraba, Haruhiko; Ohshima, Toshihisa

    2015-08-01

    To date, there have been few reports analyzing the amino acid requirement for growth of hyperthermophilic archaea. We here found that the hyperthermophilic archaeon Pyrococcus horikoshii OT-3 requires Thr, Leu, Val, Phe, Tyr, Trp, His and Arg in the medium for growth, and shows slow growth in medium lacking Met or Ile. This largely corresponds to the presence, or absence, of genes related to amino acid biosynthesis in its genome, though there are exceptions. The amino acid requirements were dramatically lost by addition of D-isomers of Met, Leu, Val, allo-Ile, Phe, Tyr, Trp and Arg. Tracer analysis using (14)C-labeled D-Trp showed that D-Trp in the medium was used as a protein component in the cells, suggesting the presence of D-amino acid metabolic enzymes. Pyridoxal 5'-phosphate (PLP)-dependent racemase activity toward Met, Leu and Phe was detected in crude extract of P. horikoshii and was enhanced in cells grown in the medium supplemented with D-amino acids, especially D-allo-Ile. The gene encoding the racemase was narrowed down to one open reading frame on the basis of enzyme purification from P. horikoshii cells, and the recombinant enzyme exhibited PLP-dependent racemase activity toward several amino acids, including Met, Leu and Phe, but not Pro, Asp or Glu. This is the first report showing the presence in a hyperthermophilic archaeon of a PLP-dependent amino acid racemase with broad substrate specificity that is likely responsible for utilization of D-amino acids for growth.

  5. Genetic determinants of PAM-dependent DNA targeting and pre-crRNA processing in Sulfolobus islandicus

    DEFF Research Database (Denmark)

    Peng, Wenfang; Li, Huan; Hallstrøm, Søren

    2013-01-01

    Bacteria and Archaea encode clustered, regularly interspaced, short palindromic repeat (CRISPR) systems to confer adaptive immunity to invasive viruses and plasmids. Recent studies of CRISPR systems revealed that diverse CRISPR-associated (Cas) interference modules often coexist in different...... organisms but functions of cas genes have not been dissected in any of these systems. The crenarchaeon Sulfolobus islandicus encodes three distinct CRISPR interference modules, including a type IA system and two type IIIB systems: Cmr-a and Cmr-ß. To study the genetic determinants of protospacer......-adjacent motif (PAM)-dependent DNA targeting activity and mature CRISPR RNA (crRNA) production in this organism, mutants deleting individual genes of the type IA system or removing each of other Cas modules were constructed. Characterization of these mutants revealed that Cas7, Cas5, Cas6, Cas3' and Cas3...

  6. AglB, catalyzing the oligosaccharyl transferase step of the archaeal N-glycosylation process, is essential in the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius.

    Science.gov (United States)

    Meyer, Benjamin H; Albers, Sonja-Verena

    2014-08-01

    Sulfolobus acidocaldarius, a thermo-acidophilic crenarchaeon which grows optimally at 76 °C and pH 3, exhibits an astonishing high number of N-glycans linked to the surface (S-) layer proteins. The S-layer proteins as well as other surface-exposed proteins are modified via N-glycosylation, in which the oligosaccharyl transferase AglB catalyzes the final step of the transfer of the glycan tree to the nascent protein. In this study, we demonstrated that AglB is essential for the viability of S. acidocaldarius. Different deletion approaches, that is, markerless in-frame deletion as well as a marker insertion were unsuccessful to create an aglB deletion mutant. Only the integration of a second aglB gene copy allowed the successful deletion of the original aglB.

  7. New genes encoding subunits of a cytochrome bc1-analogous complex in the respiratory chain of the hyperthermoacidophilic crenarchaeon Sulfolobus acidocaldarius.

    Science.gov (United States)

    Hiller, A; Henninger, T; Schäfer, G; Schmidt, C L

    2003-04-01

    The soxL gene from Sulfolobus acidocaldarius (DSM 639) encodes a Rieske iron-sulfur protein. In this study we report the identification of two open reading frames in its downstream region. The first one, named soxN, codes for a membrane protein bearing a resemblance to the b-type cytochromes of the cytochrome bc1 and b6f complexes. The protein is predicted to contain at least 10 transmembrane helices and features the two conserved histidine pairs coordinating the heme groups of these cytochromes. The second open reading frame, named odsN, encodes a soluble protein of unknown function. The genomic region displays a complex transcription pattern. Northern blot and RT-PCR analyses revealed the presence of mono- and bi-cistronic transcripts as well as a tri-cistronic transcript of soxL and cbsAB, encoding the mono-heme cytochrome b558/566. Phylogenetic analyses of the genes of the soxLN pair and of other archaeal gene pairs encoding Rieske iron-sulfur proteins and b-type cytochromes revealed an identical branching patterns for both protein families, suggesting an evolutionary link of these genes provided by the functional interaction of the proteins. On the basis of the findings of this study and the previously studied properties of the soxL and cbsA proteins, we propose the occurrence of a novel cytochrome bc1-analogous complex in the membranes of Sulfolobus, consisting of the cytochrome b homolog soxN, the Rieske protein soxL, the high potential cytochrome cbsA, as well as the non-redox-active subunits cbsB and odsN.

  8. Optimization of selective conditions for the selection of uracil auxotrophs of thermophilic archaea Sulfolobus tokodaii%超嗜热古菌Sulfolobus tokodaii尿嘧啶营养缺陷型筛选条件的最适化及初步筛选

    Institute of Scientific and Technical Information of China (English)

    黄奇洪; 申玉龙; 倪金凤

    2008-01-01

    超嗜热古菌Sulfolobus tokodaii隶属于古菌中的泉古菌(Crenarchaea),硫化叶菌属(Sulfolobus).野生型S.tokodaii*$尿嘧啶相关基因表达的乳清核苷酸转移酶和乳清苷单磷酸脱羧酶可以将5-氟乳清酸(5-FOA)转化成有毒物质5-氟尿嘧啶核苷酸,导致野生型S.tokodaii无法正常生长.根据此原理,通过对筛选条件如5-FOA的质量浓度、紫外诱变时间等的最适化,运用微生物的自发突变或对其进行紫外照射等诱变方法,初步筛选出S.tokodaii的尿嘧啶营养缺陷型菌株.

  9. Pyrite oxidation by thermophilic archaebacteria

    Energy Technology Data Exchange (ETDEWEB)

    Larsson, L.; Olsson, G.; Holst, O.; Karlsson, H.T. (Lund Univ. (Sweden))

    1990-03-01

    Three species of thermophilic archaebacteria of the genera Sulfolobus (Sulfolobus acidocaldarius and S. solfataricus) and Acidianus (Acidianus brierleyi) were tested for their ability to oxidize pyrite and to grow autotropbically on pyrite, to explore their potential for use in coal desulfurization. Only A. brierleyi was able to oxidize and grow autotrophically on pyrite. Jarosite was formed during the pyrite oxidation, resulting in the precipitation of sulfate and iron. The medium composition affected the extent of jarosite formation.

  10. An upstream activation element exerting differential transcriptional activation on an archaeal promoter

    DEFF Research Database (Denmark)

    Peng, Nan; Xia, Qiu; Chen, Zhengjun

    2009-01-01

    Summary Microorganisms can utilize different sugars as energy and carbon sources and the genes involved in sugar metabolism often exhibit highly regulated expression. To study cis-acting elements controlling arabinose-responsive expression in archaea, the promoter of the Sulfolobus solfataricus a...

  11. DNA polymerases BI and D from the hyperthermophilic archaeon Pyrococcus furiosus both bind to proliferating cell nuclear antigen with their C-terminal PIP-box motifs.

    Science.gov (United States)

    Tori, Kazuo; Kimizu, Megumi; Ishino, Sonoko; Ishino, Yoshizumi

    2007-08-01

    Proliferating cell nuclear antigen (PCNA) is the sliding clamp that is essential for the high processivity of DNA synthesis during DNA replication. Pyrococcus furiosus, a hyperthermophilic archaeon, has at least two DNA polymerases, polymerase BI (PolBI) and PolD. Both of the two DNA polymerases interact with the archaeal P. furiosus PCNA (PfuPCNA) and perform processive DNA synthesis in vitro. This phenomenon, in addition to the fact that both enzymes display 3'-5' exonuclease activity, suggests that both DNA polymerases work in replication fork progression. We demonstrated here that both PolBI and PolD functionally interact with PfuPCNA at their C-terminal PIP boxes. The mutant PolBI and PolD enzymes lacking the PIP-box sequence do not respond to the PfuPCNA at all in an in vitro primer extension reaction. This is the first experimental evidence that the PIP-box motif, located at the C termini of the archaeal DNA polymerases, is actually critical for PCNA binding to form a processive DNA-synthesizing complex.

  12. Manual annotation, transcriptional analysis, and protein expression studies reveal novel genes in the agl cluster responsible for N glycosylation in the halophilic archaeon Haloferax volcanii.

    Science.gov (United States)

    Yurist-Doutsch, Sophie; Eichler, Jerry

    2009-05-01

    While Eukarya, Bacteria, and Archaea are all capable of protein N glycosylation, the archaeal version of this posttranslational modification is the least understood. To redress this imbalance, recent studies of the halophilic archaeon Haloferax volcanii have identified a gene cluster encoding the Agl proteins involved in the assembly and attachment of a pentasaccharide to select Asn residues of the surface layer glycoprotein in this species. However, because the automated tools used for rapid annotation of genome sequences, including that of H. volcanii, are not always accurate, a reannotation of the agl cluster was undertaken in order to discover genes not previously recognized. In the present report, reanalysis of the gene cluster that includes aglB, aglE, aglF, aglG, aglI, and aglJ, which are known components of the H. volcanii protein N-glycosylation machinery, was undertaken. Using computer-based tools or visual inspection, together with transcriptional analysis and protein expression approaches, genes encoding AglP, AglQ, and AglR are now described.

  13. Genome sequence of Methanobacterium congolense strain Buetzberg, a hydrogenotrophic, methanogenic archaeon, isolated from a mesophilic industrial-scale biogas plant utilizing bio-waste.

    Science.gov (United States)

    Tejerizo, Gonzalo Torres; Kim, Yong Sung; Maus, Irena; Wibberg, Daniel; Winkler, Anika; Off, Sandra; Pühler, Alfred; Scherer, Paul; Schlüter, Andreas

    2017-02-16

    Methanogenic Archaea are of importance at the end of the anaerobic digestion (AD) chain for biomass conversion. They finally produce methane, the end-product of AD. Among this group of microorganisms, members of the genus Methanobacterium are ubiquitously present in anaerobic habitats, such as bioreactors. The genome of a novel methanogenic archaeon, namely Methanobacterium congolense Buetzberg, originally isolated from a mesophilic biogas plant, was completely sequenced to analyze putative adaptive genome features conferring competitiveness of this isolate within the biogas reactor environment. Sequencing and assembly of the M. congolense Buetzberg genome yielded a chromosome with a size of 2,451,457bp and a mean GC-content of 38.51%. Additionally, a plasmid with a size of 18,118bp, featuring a GC content of 36.05% was identified. The M. congolense Buetzberg plasmid showed no sequence similarities with the plasmids described previously suggesting that it represents a new plasmid type. Analysis of the M. congolense Buetzberg chromosome architecture revealed a high collinearity with the Methanobacterium paludis chromosome. Furthermore, annotation of the genome and functional predictions disclosed several genes involved in cell wall and membrane biogenesis. Compilation of specific genes among Methanobacterium strains originating from AD environments revealed 474 genetic determinants that could be crucial for adaptation of these strains to specific conditions prevailing in AD habitats.

  14. The RosR transcription factor is required for gene expression dynamics in response to extreme oxidative stress in a hypersaline-adapted archaeon

    Directory of Open Access Journals (Sweden)

    Sharma Kriti

    2012-07-01

    Full Text Available Abstract Background Previous work has shown that the hypersaline-adapted archaeon, Halobacterium salinarum NRC-1, is highly resistant to oxidative stress caused by exposure to hydrogen peroxide, UV, and gamma radiation. Dynamic alteration of the gene regulatory network (GRN has been implicated in such resistance. However, the molecular functions of transcription regulatory proteins involved in this response remain unknown. Results Here we have reanalyzed several existing GRN and systems biology datasets for H. salinarum to identify and characterize a novel winged helix-turn-helix transcription factor, VNG0258H, as a regulator required for reactive oxygen species resistance in this organism. This protein appears to be unique to the haloarchaea at the primary sequence level. High throughput quantitative growth assays in a deletion mutant strain implicate VNG0258H in extreme oxidative stress resistance. According to time course gene expression analyses, this transcription factor is required for the appropriate dynamic response of nearly 300 genes to reactive oxygen species damage from paraquat and hydrogen peroxide. These genes are predicted to function in repair of oxidative damage to proteins and DNA. In vivo DNA binding assays demonstrate that VNG0258H binds DNA to mediate gene regulation. Conclusions Together these results suggest that VNG0258H is a novel archaeal transcription factor that regulates gene expression to enable adaptation to the extremely oxidative, hypersaline niche of H. salinarum. We have therefore renamed VNG0258H as RosR, for reactive oxygen species regulator.

  15. Genomic Analysis of the Extremely Halophilic Archaeon Halobacterium noricense CBA1132 Isolated from Solar Salt That Is an Essential Material for Fermented Foods.

    Science.gov (United States)

    Lim, Seul Ki; Kim, Joon Yong; Song, Hye Seon; Kwon, Min-Sung; Lee, Jieun; Oh, Young Jun; Nam, Young-Do; Seo, Myung-Ji; Lee, Dong-Gi; Choi, Jong-Soon; Yoon, Changmann; Sohn, Eunju; Rahman, Md Arif-Ur; Roh, Seong Woon; Choi, Hak-Jong

    2016-08-28

    The extremely halophilic archaeon Halobacterium noricense is a member of the genus Halobacterium. Strain CBA1132 (= KCCM 43183, JCM 31150) was isolated from solar salt. The genome of strain CBA1132 assembled with 4 contigs, including three rRNA genes, 44 tRNA genes, and 3,208 open reading frames. Strain CBA1132 had nine putative CRISPRs and the genome contained genes encoding metal resistance determinants: copper-translocating P-type ATPase (CtpA), arsenical pump-driving ATPase (ArsA), arsenate reductase (ArsC), and arsenical resistance operon repressor (ArsR). Strain CBA1132 was related to Halobacterium noricense, with 99.2% 16S rRNA gene sequence similarity. Based on the comparative genomic analysis, strain CBA1132 has distinctly evolved; moreover, essential genes related to nitrogen metabolism were only detected in the genome of strain CBA1132 among the reported genomes in the genus Halobacterium. This genome sequence of Halobacterium noricense CBA1132 may be of use in future molecular biological studies.

  16. N-Glycosylation of the archaellum filament is not important for archaella assembly and motility, although N-Glycosylation is essential for motility in Sulfolobus acidocaldarius.

    Science.gov (United States)

    Meyer, Benjamin H; Birich, Anton; Albers, Sonja-Verena

    2015-11-01

    N-Glycosylation is one of the predominant posttranslational modifications, which is found in all three domains of life. N-Glycosylation has been shown to influence many biological aspects of proteins, like protein folding, stability or activity. In this study we demonstrate that the archaellum filament subunit FlaB of Sulfolobus acidocaldarius is N-glycosylated. Each of the six predicted N-Glycosylation sites within FlaB are modified with the attachment of an N-glycan. Although, it has been previously shown that N-Glycosylation is essential for motility in S. acidocaldarius, as defects in the N-Glycosylation process resulted in none or reduced motile cells, strains lacking one to all six N-Glycosylation sites within FlaB still remained motile. Deletion of the first five N-Glycosylation sites in FlaB did not significantly affect the motility, whereas removal of all six N-Glycosylation sites reduced motility by about 40%. Transmission electron microscopy analyses of non glycosylated and glycosylated archaellum filament revealed no structural change in length. Therefore N-Glycosylation does not appear to be important for the stability and assembly of the archaellum filament itself, but plays a role in other parts of the archaellum assembly.

  17. Investigation of the malE promoter and MalR, a positive regulator of the maltose regulon, for an improved expression system in Sulfolobus acidocaldarius.

    Science.gov (United States)

    Wagner, Michaela; Wagner, Alexander; Ma, Xiaoqing; Kort, Julia Christin; Ghosh, Abhrajyoti; Rauch, Bernadette; Siebers, Bettina; Albers, Sonja-Verena

    2014-02-01

    In this study, the regulator MalR (Saci_1161) of the TrmB family from Sulfolobus acidocaldarius was identified and was shown to be involved in transcriptional control of the maltose regulon (Saci_1660 to Saci_1666), including the ABC transporter (malEFGK), α-amylase (amyA), and α-glycosidase (malA). The ΔmalR deletion mutant exhibited a significantly decreased growth rate on maltose and dextrin but not on sucrose. The expression of the genes organized in the maltose regulon was induced only in the presence of MalR and maltose in the growth medium, indicating that MalR, in contrast to its TrmB and TrmB-like homologues, is an activator of the maltose gene cluster. Electrophoretic mobility shift assays revealed that the binding of MalR to malE was independent of sugars. Here we report the identification of the archaeal maltose regulator protein MalR, which acts as an activator and controls the expression of genes involved in maltose transport and metabolic conversion in S. acidocaldarius, and its use for improvement of the S. acidocaldarius expression system under the control of an optimized maltose binding protein (malE) promoter by promoter mutagenesis.

  18. Expanding the archaellum regulatory network - the eukaryotic protein kinases ArnC and ArnD influence motility of Sulfolobus acidocaldarius.

    Science.gov (United States)

    Hoffmann, Lena; Schummer, Andreas; Reimann, Julia; Haurat, Maria F; Wilson, Amanda J; Beeby, Morgan; Warscheid, Bettina; Albers, Sonja-V

    2017-02-01

    Expression of the archaellum, the archaeal-type IV pilus-like rotating motility structure is upregulated under nutrient limitation. This is controlled by a network of regulators, called the archaellum regulatory network (arn). Several of the components of this network in Sulfolobus acidocaldarius can be phosphorylated, and the deletion of the phosphatase PP2A results in strongly increased motility during starvation, indicating a role for phosphorylation in the regulation of motility. Analysis of the motility of different protein kinase deletion strains revealed that deletion of saci_0965, saci_1181, and saci_1193 resulted in reduced motility, whereas the deletion of saci_1694 resulted in hypermotility. Here ArnC (Saci_1193) and ArnD (Saci_1694) are characterized. Purified ArnC and ArnD phosphorylate serine and threonine residues in the C-terminus of the repressor ArnB. arnC is upregulated in starvation medium, whereas arnD is constitutively expressed. However, while differences in the expression and levels of flaB were observed in the ΔarnD strain during growth under rich conditions, under nutrient limiting conditions the ΔarnC and ΔarnD strains showed no large differences in the expression levels of the archaellum or of the studied regulators. This suggests that next to the regulation via the archaellum regulatory network additional regulatory mechanisms of expression and/or activity of the archaellum exist.

  19. Biochemical and structural characterization of recombinant short-chain NAD(H)-dependent dehydrogenase/reductase from Sulfolobus acidocaldarius highly enantioselective on diaryl diketone benzil.

    Science.gov (United States)

    Pennacchio, Angela; Sannino, Vincenzo; Sorrentino, Giosuè; Rossi, Mosè; Raia, Carlo A; Esposito, Luciana

    2013-05-01

    The gene encoding a novel alcohol dehydrogenase that belongs to the short-chain dehydrogenases/reductases superfamily was identified in the aerobic thermoacidophilic crenarchaeon Sulfolobus acidocaldarius strain DSM 639. The saadh2 gene was heterologously overexpressed in Escherichia coli, and the resulting protein (SaADH2) was purified to homogeneity and both biochemically and structurally characterized. The crystal structure of the SaADH2 NADH-bound form reveals that the enzyme is a tetramer consisting of identical 27,024-Da subunits, each composed of 255 amino acids. The enzyme has remarkable thermophilicity and thermal stability, displaying activity at temperatures up to 80 °C and a 30-min half-inactivation temperature of ∼88 °C. It also shows good tolerance to common organic solvents and a strict requirement for NAD(H) as the coenzyme. SaADH2 displays a preference for the reduction of alicyclic, bicyclic and aromatic ketones and α-ketoesters, but is poorly active on aliphatic, cyclic and aromatic alcohols, showing no activity on aldehydes. Interestingly, the enzyme catalyses the asymmetric reduction of benzil to (R)-benzoin with both excellent conversion (98 %) and optical purity (98 %) by way of an efficient in situ NADH-recycling system involving a second thermophilic ADH. The crystal structure of the binary complex SaADH2-NADH, determined at 1.75 Å resolution, reveals details of the active site providing hints on the structural basis of the enzyme enantioselectivity.

  20. Sulfolobus acidocaldarius UDG Can Remove dU from the RNA Backbone: Insight into the Specific Recognition of Uracil Linked with Deoxyribose

    Directory of Open Access Journals (Sweden)

    Gang-Shun Yi

    2017-01-01

    Full Text Available Sulfolobus acidocaldarius encodes family 4 and 5 uracil-DNA glycosylase (UDG. Two recombinant S. acidocaldarius UDGs (SacUDG were prepared and biochemically characterized using oligonucleotides carrying a deaminated base. Both SacUDGs can remove deoxyuracil (dU base from both double-stranded DNA and single-stranded DNA. Interestingly, they can remove U linked with deoxyribose from single-stranded RNA backbone, suggesting that the riboses on the backbone have less effect on the recognition of dU and hydrolysis of the C-N glycosidic bond. However, the removal of rU from DNA backbone is inefficient, suggesting strong steric hindrance comes from the 2′ hydroxyl of ribose linked to uracil. Both SacUDGs cannot remove 2,2′-anhydro uridine, hypoxanthine, and 7-deazaxanthine from single-stranded DNA and single-stranded DNA. Compared with the family 2 MUG, other family UDGs have an extra N-terminal structure consisting of about 50 residues. Removal of the 46 N-terminal residues of family 5 SacUDG resulted in only a 40% decrease in activity, indicating that the [4Fe-4S] cluster and truncated secondary structure are not the key elements in hydrolyzing the glycosidic bond. Combining our biochemical and structural results with those of other groups, we discussed the UDGs’ catalytic mechanism and the possible repair reactions of deaminated bases in prokaryotes.

  1. Sulfolobus acidocaldarius UDG Can Remove dU from the RNA Backbone: Insight into the Specific Recognition of Uracil Linked with Deoxyribose.

    Science.gov (United States)

    Yi, Gang-Shun; Wang, Wei-Wei; Cao, Wei-Guo; Wang, Feng-Ping; Liu, Xi-Peng

    2017-01-18

    Sulfolobus acidocaldarius encodes family 4 and 5 uracil-DNA glycosylase (UDG). Two recombinant S. acidocaldarius UDGs (SacUDG) were prepared and biochemically characterized using oligonucleotides carrying a deaminated base. Both SacUDGs can remove deoxyuracil (dU) base from both double-stranded DNA and single-stranded DNA. Interestingly, they can remove U linked with deoxyribose from single-stranded RNA backbone, suggesting that the riboses on the backbone have less effect on the recognition of dU and hydrolysis of the C-N glycosidic bond. However, the removal of rU from DNA backbone is inefficient, suggesting strong steric hindrance comes from the 2' hydroxyl of ribose linked to uracil. Both SacUDGs cannot remove 2,2'-anhydro uridine, hypoxanthine, and 7-deazaxanthine from single-stranded DNA and single-stranded DNA. Compared with the family 2 MUG, other family UDGs have an extra N-terminal structure consisting of about 50 residues. Removal of the 46 N-terminal residues of family 5 SacUDG resulted in only a 40% decrease in activity, indicating that the [4Fe-4S] cluster and truncated secondary structure are not the key elements in hydrolyzing the glycosidic bond. Combining our biochemical and structural results with those of other groups, we discussed the UDGs' catalytic mechanism and the possible repair reactions of deaminated bases in prokaryotes.

  2. Production of the rare ginsenosides compound K, compound Y, and compound Mc by a thermostable beta-glycosidase from Sulfolobus acidocaldarius.

    Science.gov (United States)

    Noh, Kyeong-Hwan; Oh, Deok-Kun

    2009-11-01

    The rare ginsenosides compound K, compound Y, and compound Mc were produced from the major ginsenosides Rb(1), Rb(2), Rc, and Rd by a thermostable beta-glycosidase from Sulfolobus acidocaldarius via three pathways: Rb(1)-->Rd-->compound K, Rb(2)-->compound Y-->compound K, and Rc-->compound Mc. Each of the ginsenosides was identified by high-performance liquid chromatography using standards and liquid chromatography-mass spectrometry based on their molecular weights. The catalytic efficiency of the enzyme for ginsenosides followed the order Rb(1) (4.8)>Rc (4.5)>Rd (1.0)>Rb(2) (0.77 mM(-1) min(-1)). The enzyme converted 1 mg/ml reagent-grade Rb(1), Rb(2), and Rc to 0.53 mg/ml compound K, 0.56 mg/ml compound Y, and 0.70 mg/ml compound Mc, respectively, at pH 5.5 and 85 degrees C after 180 min, corresponding to mole conversion yields of 94, 80, and 100% (mol/mol), respectively. The enzyme converted the major ginsenosides Rb(1), Rb(2), Rc, and Rd in 10% (w/v) ginseng root extract to the rare ginsenosides with a mole yield of 99% after 24 h. These results suggest that beta-glycosidase from S. acidocaldarius can be used to produce compound K, compound Y, and compound Mc.

  3. The S-Layer Glycoprotein of the Crenarchaeote Sulfolobus acidocaldarius Is Glycosylated at Multiple Sites with Chitobiose-Linked N-Glycans

    Directory of Open Access Journals (Sweden)

    Elham Peyfoon

    2010-01-01

    Full Text Available Glycosylation of the S-layer of the crenarchaea Sulfolobus acidocaldarius has been investigated using glycoproteomic methodologies. The mature protein is predicted to contain 31 N-glycosylation consensus sites with approximately one third being found in the C-terminal domain spanning residues L1004-Q1395. Since this domain is rich in Lys and Arg and therefore relatively tractable to glycoproteomic analysis, this study has focused on mapping its N-glycosylation. Our analysis identified nine of the 11 consensus sequence sites, and all were found to be glycosylated. This constitutes a remarkably high glycosylation density in the C-terminal domain averaging one site for each stretch of 30–40 residues. Each of the glycosylation sites observed was shown to be modified with a heterogeneous family of glycans, with the largest having a composition Glc1Man2GlcNAc2 plus 6-sulfoquinovose (QuiS, consistent with the tribranched hexasaccharide previously reported in the cytochrome b558/566 of S. acidocaldarius. S. acidocaldarius is the only archaeal species whose N-glycans are known to be linked via the chitobiose core disaccharide that characterises the N-linked glycans of Eukarya.

  4. The S-layer glycoprotein of the crenarchaeote Sulfolobus acidocaldarius is glycosylated at multiple sites with chitobiose-linked N-glycans.

    Science.gov (United States)

    Peyfoon, Elham; Meyer, Benjamin; Hitchen, Paul G; Panico, Maria; Morris, Howard R; Haslam, Stuart M; Albers, Sonja-Verena; Dell, Anne

    2010-09-29

    Glycosylation of the S-layer of the crenarchaea Sulfolobus acidocaldarius has been investigated using glycoproteomic methodologies. The mature protein is predicted to contain 31 N-glycosylation consensus sites with approximately one third being found in the C-terminal domain spanning residues L(1004)-Q(1395). Since this domain is rich in Lys and Arg and therefore relatively tractable to glycoproteomic analysis, this study has focused on mapping its N-glycosylation. Our analysis identified nine of the 11 consensus sequence sites, and all were found to be glycosylated. This constitutes a remarkably high glycosylation density in the C-terminal domain averaging one site for each stretch of 30-40 residues. Each of the glycosylation sites observed was shown to be modified with a heterogeneous family of glycans, with the largest having a composition Glc(1)Man(2)GlcNAc(2) plus 6-sulfoquinovose (QuiS), consistent with the tribranched hexasaccharide previously reported in the cytochrome b(558/566) of S. acidocaldarius. S. acidocaldarius is the only archaeal species whose N-glycans are known to be linked via the chitobiose core disaccharide that characterises the N-linked glycans of Eukarya.

  5. Archaeal viruses of the sulfolobales

    DEFF Research Database (Denmark)

    Erdmann, Susanne; Garrett, Roger Antony

    2015-01-01

    Infection of archaea with phylogenetically diverse single viruses, performed in different laboratories, has failed to activate spacer acquisition into host CRISPR loci. The first successful uptake of archaeal de novo spacers was observed on infection of Sulfolobus solfataricus P2 with an environm......Infection of archaea with phylogenetically diverse single viruses, performed in different laboratories, has failed to activate spacer acquisition into host CRISPR loci. The first successful uptake of archaeal de novo spacers was observed on infection of Sulfolobus solfataricus P2...... in CRISPR loci of Sulfolobus species from a second coinfecting conjugative plasmid or virus (Erdmann and Garrett, Mol Microbiol 85:1044-1056, 2012; Erdmann et al. Mol Microbiol 91:900-917, 2014). Here we describe, firstly, the isolation of archaeal virus mixtures from terrestrial hot springs...

  6. AcEST: BP914779 [AcEST

    Lifescience Database Archive (English)

    Full Text Available ULSO Thermosome subunit beta OS=Sulfolobus solfataricus Align length 45 Score (bit) 32.7 E-value 1.0 Report ...12,525 sequences; 148,809,765 total letters Searching..................................................done Score...SB_SULSO Thermosome subunit beta OS=Sulfolobus solfataricus GN=thsB PE=3 SV=2 Length = 554 Score = 32.7 bits... beta OS=Sulfolobus shibatae GN=thsB PE=1 SV=1 Length = 552 Score = 32.7 bits (73), Expect = 1.0 Identities .... (strain NBC37-1) GN=pdxJ PE=3 SV=1 Length = 258 Score = 29.6 bits (65), Expect = 8.8 Identities = 15/53 (2

  7. Enhancing Heat Tolerance of the Little Dogwood Cornus canadensis L. f. with Introduction of a Superoxide Reductase Gene from the Hyperthermophilic Archaeon Pyrococcus furiosus.

    Science.gov (United States)

    Geng, Xing-Min; Liu, Xiang; Ji, Mikyoung; Hoffmann, William A; Grunden, Amy; Xiang, Qiu-Yun J

    2016-01-01

    Production of reactive oxygen species (ROS) can be accelerated under various biotic and abiotic stresses causing lipid peroxidation, protein degradation, enzyme inactivation, and DNA damage. Superoxide reductase (SOR) is a novel antioxidant enzyme from Pyrococcus furiosus and is employed by this anaerobic hyperthermophilic archaeon for efficient detoxification of ROS. In this study, SOR was introduced into a flowering plant Cornus canadensis to enhance its heat tolerance and reduce heat induced damage. A fusion construct of the SOR gene and Green Fluorescent Protein gene (GFP) was introduced into C. canadensis using Agrobacterium-mediated transformation. Heat tolerance of the GFP-SOR expressing transgenic plants was investigated by observing morphological symptoms of heat injury and by examining changes in photosynthesis, malondialdehyde (MDA), and proline levels in the plants. Our results indicate that the expression of the P. furiosus SOR gene in the transgenic plants alleviated lipid peroxidation of cell membranes and photoinhibition of PS II, and decreased the accumulation of proline at 40°C. After a series of exposures to increasing temperatures, the SOR transgenic plants remained healthy and green whereas most of the non-transgenic plants dried up and were unable to recover. While it had previously been reported that expression of SOR in Arabidopsis enhanced heat tolerance, this is the first report of the successful demonstration of improved heat tolerance in a non-model plant resulting from the introduction of P. furiosus SOR. The study demonstrates the potential of SOR for crop improvement and that inherent limitations of plant heat tolerance can be ameliorated with P. furiosus SOR.

  8. Enhancing heat tolerance of the little dogwood Cornus canadensis L. f. with introduction of a superoxide reductase gene from the hyperthermophilic archaeon Pyrococcus furiosus

    Directory of Open Access Journals (Sweden)

    Xinmin eGeng

    2016-01-01

    Full Text Available Production of reactive oxygen species (ROS can be accelerated under various biotic and abiotic stresses causing lipid peroxidation, protein degradation, enzyme inactivation, and DNA damage. Superoxide reductase (SOR is a novel antioxidant enzyme from Pyrococcus furiosus and is employed by this anaerobic hyperthermophilic archaeon for efficient detoxification of ROS. In this study, SOR was introduced into a flowering plant Cornus canadensis to enhance its heat tolerance and reduce heat induced damage. A fusion construct of the SOR gene and Green Fluorescent Protein gene (GFP was introduced into C. canadensis using Agrobacterium-mediated transformation. Heat tolerance of the GFP-SOR expressing transgenic plants was investigated by observing morphological symptoms of heat injury and by examining changes in photosynthesis, malondialdehyde (MDA, and proline levels in the plants. Our results indicate that the expression of the P. furiosus SOR gene in the transgenic plants alleviated lipid peroxidation of cell membranes and photoinhibition of PS II, and decreased the accumulation of proline at 40°C. After a series of exposures to increasing temperatures, the SOR transgenic plants remained healthy and green whereas most of the non-transgenic plants dried up and were unable to recover. While it had previously been reported that expression of SOR in Arabidopsis enhanced heat tolerance, this is the first report of the successful demonstration of improved heat tolerance in a non-model plant resulting from the introduction of P. furiosus SOR. The study demonstrates the potential of SOR for crop improvement and that inherent limitations of plant heat tolerance can be ameliorated with P. furiosus SOR.

  9. MutS and MutL are dispensable for maintenance of the genomic mutation rate in the halophilic archaeon Halobacterium salinarum NRC-1.

    Directory of Open Access Journals (Sweden)

    Courtney R Busch

    Full Text Available BACKGROUND: The genome of the halophilic archaeon Halobacterium salinarum NRC-1 encodes for homologs of MutS and MutL, which are key proteins of a DNA mismatch repair pathway conserved in Bacteria and Eukarya. Mismatch repair is essential for retaining the fidelity of genetic information and defects in this pathway result in the deleterious accumulation of mutations and in hereditary diseases in humans. METHODOLOGY/PRINCIPAL FINDINGS: We calculated the spontaneous genomic mutation rate of H. salinarum NRC-1 using fluctuation tests targeting genes of the uracil monophosphate biosynthesis pathway. We found that H. salinarum NRC-1 has a low incidence of mutation suggesting the presence of active mechanisms to control spontaneous mutations during replication. The spectrum of mutational changes found in H. salinarum NRC-1, and in other archaea, appears to be unique to this domain of life and might be a consequence of their adaption to extreme environmental conditions. In-frame targeted gene deletions of H. salinarum NRC-1 mismatch repair genes and phenotypic characterization of the mutants demonstrated that the mutS and mutL genes are not required for maintenance of the observed mutation rate. CONCLUSIONS/SIGNIFICANCE: We established that H. salinarum NRC-1 mutS and mutL genes are redundant to an alternative system that limits spontaneous mutation in this organism. This finding leads to the puzzling question of what mechanism is responsible for maintenance of the low genomic mutation rates observed in the Archaea, which for the most part do not have MutS and MutL homologs.

  10. Disruption of a sugar transporter gene cluster in a hyperthermophilic archaeon using a host-marker system based on antibiotic resistance.

    Science.gov (United States)

    Matsumi, Rie; Manabe, Kenji; Fukui, Toshiaki; Atomi, Haruyuki; Imanaka, Tadayuki

    2007-04-01

    We have developed a gene disruption system in the hyperthermophilic archaeon Thermococcus kodakaraensis using the antibiotic simvastatin and a fusion gene designed to overexpress the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase gene (hmg(Tk)) with the glutamate dehydrogenase promoter. With this system, we disrupted the T. kodakaraensis amylopullulanase gene (apu(Tk)) or a gene cluster which includes apu(Tk) and genes encoding components of a putative sugar transporter. Disruption plasmids were introduced into wild-type T. kodakaraensis KOD1 cells, and transformants exhibiting resistance to 4 microM simvastatin were isolated. The transformants exhibited growth in the presence of 20 microM simvastatin, and we observed a 30-fold increase in intracellular HMG-CoA reductase activity. The expected gene disruption via double-crossover recombination occurred at the target locus, but we also observed recombination events at the hmg(Tk) locus when the endogenous hmg(Tk) gene was used. This could be avoided by using the corresponding gene from Pyrococcus furiosus (hmg(Pf)) or by linearizing the plasmid prior to transformation. While both gene disruption strains displayed normal growth on amino acids or pyruvate, cells without the sugar transporter genes could not grow on maltooligosaccharides or polysaccharides, indicating that the gene cluster encodes the only sugar transporter involved in the uptake of these compounds. The Deltaapu(Tk) strain could not grow on pullulan and displayed only low levels of growth on amylose, suggesting that Apu(Tk) is a major polysaccharide-degrading enzyme in T. kodakaraensis.

  11. Characterization of the archaeal thermophile Sulfolobus turreted icosahedral virus validates an evolutionary link among double-stranded DNA viruses from all domains of life.

    Science.gov (United States)

    Maaty, Walid S A; Ortmann, Alice C; Dlakić, Mensur; Schulstad, Katie; Hilmer, Jonathan K; Liepold, Lars; Weidenheft, Blake; Khayat, Reza; Douglas, Trevor; Young, Mark J; Bothner, Brian

    2006-08-01

    Icosahedral nontailed double-stranded DNA (dsDNA) viruses are present in all three domains of life, leading to speculation about a common viral ancestor that predates the divergence of Eukarya, Bacteria, and Archaea. This suggestion is supported by the shared general architecture of this group of viruses and the common fold of their major capsid protein. However, limited information on the diversity and replication of archaeal viruses, in general, has hampered further analysis. Sulfolobus turreted icosahedral virus (STIV), isolated from a hot spring in Yellowstone National Park, was the first icosahedral virus with an archaeal host to be described. Here we present a detailed characterization of the components forming this unusual virus. Using a proteomics-based approach, we identified nine viral and two host proteins from purified STIV particles. Interestingly, one of the viral proteins originates from a reading frame lacking a consensus start site. The major capsid protein (B345) was found to be glycosylated, implying a strong similarity to proteins from other dsDNA viruses. Sequence analysis and structural predication of virion-associated viral proteins suggest that they may have roles in DNA packaging, penton formation, and protein-protein interaction. The presence of an internal lipid layer containing acidic tetraether lipids has also been confirmed. The previously presented structural models in conjunction with the protein, lipid, and carbohydrate information reported here reveal that STIV is strikingly similar to viruses associated with the Bacteria and Eukarya domains of life, further strengthening the hypothesis for a common ancestor of this group of dsDNA viruses from all domains of life.

  12. Cloning, over expression and characterization of a new thermophilic and acidic a-amylase from Sulfolobus tokodaii strain 7 in Escherichia coil%Sulfolobus tokodaii strain 7高温酸性α-淀粉酶基因在大肠杆菌中克隆表达及其酶学性质

    Institute of Scientific and Technical Information of China (English)

    魏涛; 孙浩; 申玉龙; 毛多斌

    2013-01-01

    A gene (ST0817) encoding a putative thermophilic and acidic α-amylase from the thermophilic crenarchaeota Sulfolobus tokodaii strain 7 was cloned and functionally overexpressed in Escherichia coli.The recombinant enzyme was purified to homogeneity after heat treatment,Ni-NTA affinity and superdex-200 gel filtration.The relative molecular mass of α-amylase was 53.0 kDa on SDS-PAGE.The purified enzyme displayed optimal activity at 70 ℃ and pH 5.5.It had a half-life of 8 h at 85℃.The enzyme was found to have high pH stability and maintained about 50% of activity even after 120 min of treatment at pH 5.2.The enzymatic activities of the purified enzyme towards different substrates were listed from high to low as follows:amylose > soluble starch > amylopectin > β-cyclodextrin > glycogen > cyclodextrin > pullulan.The enzyme was found to have high tolerance to organic solvents,metal ions and detergents.%以超嗜热古菌Sulfolobus tokodaii strain 7基因组DNA为模板,通过PCR扩增高温酸性α-淀粉酶基因ST0817,将此基因克隆至表达载体pET15b,并转化大肠杆菌Escherichia coli BL21-CodenPlus(DE3)-RIL,获得重组大肠杆菌工程菌.通过热处理、镍柱亲和层析和分子筛层析,得到纯化重组酶,SDS-PAGE分析表明,该酶分子量为53.0 kDa.酶学性质研究表明,该酶最适温度和pH分别为75℃和5.5;具有较强的热稳定性和pH稳定性,在85℃处理8h保持50%左右活力,在pH 5.2处理120 min仍保持50%活力.此酶对不同底物水解活性不同,直连淀粉>可溶性淀粉>支链淀粉>β-极限糊精>糖原>环糊精>普鲁兰糖;该酶对有机溶剂、变性剂和金属离子具有一定抗性.

  13. Characterization of a Single-Stranded DNA-Binding-Like Protein from Nanoarchaeum equitans--A Nucleic Acid Binding Protein with Broad Substrate Specificity.

    Directory of Open Access Journals (Sweden)

    Marcin Olszewski

    Full Text Available SSB (single-stranded DNA-binding proteins play an essential role in all living cells and viruses, as they are involved in processes connected with ssDNA metabolism. There has recently been an increasing interest in SSBs, since they can be applied in molecular biology techniques and analytical methods. Nanoarchaeum equitans, the only known representative of Archaea phylum Nanoarchaeota, is a hyperthermophilic, nanosized, obligatory parasite/symbiont of Ignicoccus hospitalis.This paper reports on the ssb-like gene cloning, gene expression and characterization of a novel nucleic acid binding protein from Nanoarchaeum equitans archaeon (NeqSSB-like protein. This protein consists of 243 amino acid residues and one OB fold per monomer. It is biologically active as a monomer like as SSBs from some viruses. The NeqSSB-like protein displays a low sequence similarity to the Escherichia coli SSB, namely 10% identity and 29% similarity, and is the most similar to the Sulfolobus solfataricus SSB (14% identity and 32% similarity. The NeqSSB-like protein binds to ssDNA, although it can also bind mRNA and, surprisingly, various dsDNA forms, with no structure-dependent preferences as evidenced by gel mobility shift assays. The size of the ssDNA binding site, which was estimated using fluorescence spectroscopy, is 7 ± 1 nt. No salt-dependent binding mode transition was observed. NeqSSB-like protein probably utilizes a different model for ssDNA binding than the SSB proteins studied so far. This protein is highly thermostable; the half-life of the ssDNA binding activity is 5 min at 100 °C and melting temperature (T(m is 100.2 °C as shown by differential scanning calorimetry (DSC analysis.NeqSSB-like protein is a novel highly thermostable protein which possesses a unique broad substrate specificity and is able to bind all types of nucleic acids.

  14. Characterization of a Single-Stranded DNA-Binding-Like Protein from Nanoarchaeum equitans—A Nucleic Acid Binding Protein with Broad Substrate Specificity

    Science.gov (United States)

    Olszewski, Marcin; Balsewicz, Jan; Nowak, Marta; Maciejewska, Natalia; Cyranka-Czaja, Anna; Zalewska-Piątek, Beata; Piątek, Rafał; Kur, Józef

    2015-01-01

    Background SSB (single-stranded DNA-binding) proteins play an essential role in all living cells and viruses, as they are involved in processes connected with ssDNA metabolism. There has recently been an increasing interest in SSBs, since they can be applied in molecular biology techniques and analytical methods. Nanoarchaeum equitans, the only known representative of Archaea phylum Nanoarchaeota, is a hyperthermophilic, nanosized, obligatory parasite/symbiont of Ignicoccus hospitalis. Results This paper reports on the ssb-like gene cloning, gene expression and characterization of a novel nucleic acid binding protein from Nanoarchaeum equitans archaeon (NeqSSB-like protein). This protein consists of 243 amino acid residues and one OB fold per monomer. It is biologically active as a monomer like as SSBs from some viruses. The NeqSSB-like protein displays a low sequence similarity to the Escherichia coli SSB, namely 10% identity and 29% similarity, and is the most similar to the Sulfolobus solfataricus SSB (14% identity and 32% similarity). The NeqSSB-like protein binds to ssDNA, although it can also bind mRNA and, surprisingly, various dsDNA forms, with no structure-dependent preferences as evidenced by gel mobility shift assays. The size of the ssDNA binding site, which was estimated using fluorescence spectroscopy, is 7±1 nt. No salt-dependent binding mode transition was observed. NeqSSB-like protein probably utilizes a different model for ssDNA binding than the SSB proteins studied so far. This protein is highly thermostable; the half-life of the ssDNA binding activity is 5 min at 100°C and melting temperature (Tm) is 100.2°C as shown by differential scanning calorimetry (DSC) analysis. Conclusion NeqSSB-like protein is a novel highly thermostable protein which possesses a unique broad substrate specificity and is able to bind all types of nucleic acids. PMID:25973760

  15. Chromosome segregation in Archaea mediated by a hybrid DNA partition machine.

    Science.gov (United States)

    Kalliomaa-Sanford, Anne K; Rodriguez-Castañeda, Fernando A; McLeod, Brett N; Latorre-Roselló, Victor; Smith, Jasmine H; Reimann, Julia; Albers, Sonja V; Barillà, Daniela

    2012-03-06

    Eukarya and, more recently, some bacteria have been shown to rely on a cytoskeleton-based apparatus to drive chromosome segregation. In contrast, the factors and mechanisms underpinning this fundamental process are underexplored in archaea, the third domain of life. Here we establish that the archaeon Sulfolobus solfataricus harbors a hybrid segrosome consisting of two interacting proteins, SegA and SegB, that play a key role in genome segregation in this organism. SegA is an ortholog of bacterial, Walker-type ParA proteins, whereas SegB is an archaea-specific factor lacking sequence identity to either eukaryotic or bacterial proteins, but sharing homology with a cluster of uncharacterized factors conserved in both crenarchaea and euryarchaea, the two major archaeal sub-phyla. We show that SegA is an ATPase that polymerizes in vitro and that SegB is a site-specific DNA-binding protein contacting palindromic sequences located upstream of the segAB cassette. SegB interacts with SegA in the presence of nucleotides and dramatically affects its polymerization dynamics. Our data demonstrate that SegB strongly stimulates SegA polymerization, possibly by promoting SegA nucleation and accelerating polymer growth. Increased expression levels of segAB resulted in severe growth and chromosome segregation defects, including formation of anucleate cells, compact nucleoids confined to one half of the cell compartment and fragmented nucleoids. The overall picture emerging from our findings indicates that the SegAB complex fulfills a crucial function in chromosome segregation and is the prototype of a DNA partition machine widespread across archaea.

  16. Autodisplay of an archaeal γ-lactamase on the cell surface of Escherichia coli using Xcc_Est as an anchoring scaffold and its application for preparation of the enantiopure antiviral drug intermediate (-) vince lactam.

    Science.gov (United States)

    Wang, Jianjun; Zhao, Guogang; Zhang, Zhiwei; Liang, Qiulin; Min, Cong; Wu, Sheng

    2014-08-01

    At present, autotransporter protein mediated surface display has opened a new dimension in the development of whole-cell biocatalysts. Here, we report the identification of a novel autotransporter Xcc_Est from Xanthomonas campestris pv campestris 8004 by bioinformatic analysis and application of Xcc_Est as an anchoring motif for surface display of γ-lactamase (Gla) from thermophilic archaeon Sulfolobus solfataricus P2 in Escherichia coli. The localization of γ-lactamase in the cell envelope was monitored by Western blot, activity assay and flow cytometry analysis. Either the full-length or truncated Xcc_Est could efficiently transport γ-lactamase to the cell surface. Compared with the free enzyme, the displayed γ-lactamase exhibited optimum temperature of 30 °C other than 90 °C, with a substantial decrease of 60 °C. Under the preparation system, the engineered E. coli with autodisplayed γ-lactamase converted 100 g racemic vince lactam to produce 49.2 g (-) vince lactam at 30 °C within 4 h. By using chiral HPLC, the ee value of the produced (-) vince lactam was determined to be 99.5 %. The whole-cell biocatalyst exhibited excellent stability under the operational conditions. Our results indicate that the E. coli with surface displayed γ-lactamase is an efficient and economical whole cell biocatalyst for preparing the antiviral drug intermediate (-) vince lactam at mild temperature, eliminating expensive energy cost performed at high temperature.

  17. Strong Enrichment of Aromatic Residues in Binding Sites from a Charge-neutralized Hyperthermostable Sso7d Scaffold Library*

    Science.gov (United States)

    Kiefer, Jonathan D.; Srinivas, Raja R.; Lobner, Elisabeth; Tisdale, Alison W.; Mehta, Naveen K.; Yang, Nicole J.; Tidor, Bruce; Wittrup, K. Dane

    2016-01-01

    The Sso7d protein from the hyperthermophilic archaeon Sulfolobus solfataricus is an attractive binding scaffold because of its small size (7 kDa), high thermal stability (Tm of 98 °C), and absence of cysteines and glycosylation sites. However, as a DNA-binding protein, Sso7d is highly positively charged, introducing a strong specificity constraint for binding epitopes and leading to nonspecific interaction with mammalian cell membranes. In the present study, we report charge-neutralized variants of Sso7d that maintain high thermal stability. Yeast-displayed libraries that were based on this reduced charge Sso7d (rcSso7d) scaffold yielded binders with low nanomolar affinities against mouse serum albumin and several epitopes on human epidermal growth factor receptor. Importantly, starting from a charge-neutralized scaffold facilitated evolutionary adaptation of binders to differentially charged epitopes on mouse serum albumin and human epidermal growth factor receptor, respectively. Interestingly, the distribution of amino acids in the small and rigid binding surface of enriched rcSso7d-based binders is very different from that generally found in more flexible antibody complementarity-determining region loops but resembles the composition of antibody-binding energetic hot spots. Particularly striking was a strong enrichment of the aromatic residues Trp, Tyr, and Phe in rcSso7d-based binders. This suggests that the rigidity and small size of this scaffold determines the unusual amino acid composition of its binding sites, mimicking the energetic core of antibody paratopes. Despite the high frequency of aromatic residues, these rcSso7d-based binders are highly expressed, thermostable, and monomeric, suggesting that the hyperstability of the starting scaffold and the rigidness of the binding surface confer a high tolerance to mutation. PMID:27582495

  18. Characterization of the TrmB-like protein, PF0124, a TGM-recognizing global transcriptional regulator of the hyperthermophilic archaeon Pyrococcus furiosus.

    Science.gov (United States)

    Lee, Sung-Jae; Surma, Melanie; Seitz, Sabine; Hausner, Winfried; Thomm, Michael; Boos, Winfried

    2007-07-01

    The characterization of the transcriptional regulator TrmBL1 of the hyperthermophilic archaeon Pyrococcus furiosus, homologous to TrmB (transcriptional regulator of the maltose system), was studied. The genome of P. furiosus contains three TrmB paralogues. One of the TrmB-like proteins (TrmBL), PF0124 (TrmBL1), was analysed in more detail. It regulated the expression of the genes encoding enzymes of the glycolytic pathway as well as the maltodextrin (MD) ABC transporter. By molecular sieve chromatography, purified TrmBL1 behaved at ambient temperature as a tetramer of 148.8 kDa. In the presence of 1 mM maltotriose or 5 mM maltose TrmBL1 formed octamers. As shown by electrophoretic mobility shift assay (EMSA) TrmBL1 was found to bind the MD (maltodextrin ABC transport genes) promoter DNA with sixfold higher binding affinity (K(d) 0.2 microM) than to the trehalose/maltose ABC transporter (TM) promoter (K(d) 1.2 microM). Maltotriose and maltose interfered in these assays indicating inducer function. In vitro transcription assays using purified transcription components corroborated the data obtained with EMSA and showed inhibition of transcription of the MD promoter by TrmBL1. Recently, van de Werken et al. (FEMS Microbiol Lett 2006; 260: 69-76) identified TGM, a conserved sequence (Thermococcales-Glycolytic-Motif) upstream of genes encoding glycolytic enzymes and the MD ABC transporter. The position of TGM is invariably located downstream of the BRE-TATA box and overlapping the transcription start site on each promoter. By footprint analysis TrmBL1 was found to recognize the TGM sequence in several TGM-containing promoter sequences. We identified the recognition helix in TrmBL1 revealing tyrosine (Y49) to be essential for target DNA binding. However, the TGM motif was not essential for TrmBL1 binding. We conclude that TrmBL1 is a global sugar-sensing transcriptional regulator controlling the genes of transport systems and of sugar-metabolizing enzymes.

  19. Sugar utilization in the hyperthermophilic, sulfate-reducing archaeon Archaeoglobus fulgidus strain 7324: starch degradation to acetate and CO2 via a modified Embden-Meyerhof pathway and acetyl-CoA synthetase (ADP-forming).

    Science.gov (United States)

    Labes, A; Schönheit, P

    2001-11-01

    The hyperthermophilic, sulfate-reducing archaeon Archaeoglobus fulgidus strain 7324, rather than the type strain VC16, was found to grow on starch and sulfate as energy and carbon source. Fermentation products and enzyme activities were determined in starch-grown cells and compared to those of cells grown on lactate and sulfate. During exponential growth on starch, 1 mol of glucose-equivalent was incompletely oxidized with sulfate to approximately 2 mol acetate, 2 mol CO2 and 1 mol H2S. Starch-grown cells did not contain measurable amounts of the deazaflavin factor F420 (reducer A. fulgidus strain 7324 converts starch to acetate via a modified Embden-Meyerhof pathway and acetyl-CoA synthetase (ADP-forming). This is the first report of growth of a sulfate reducer on starch, i.e. on a polymeric sugar.

  20. CLONING AND EXPRESSION OF THE GENE ENCODING NOVEL a-AMYLASE FROM SULFOLOBUS SHIBATAE IN E. COLI%芝田硫化叶菌新型α-淀粉酶基因在大肠杆菌的克隆和表达

    Institute of Scientific and Technical Information of China (English)

    刘莉; 陈炜; 金城

    2000-01-01

    A novel α-amylase gene was amplified from Sulfolobus shibatae by using PCR technique.The amplified 1.7kb DNA fragment was inserted into an expression vector pBV220 to yield the recombinant plasmid pSBAM. The novel α-amylase gene in pSBAM was expressed in E. coli. The production of the novel α-amylase activity reached over 8 units/100mL of the culture. The molecular weight of this enzyme was about 61kD by SDS-PAGE. The expressed novel α-amylase protein in E.coli DHSα accounted for about 20 % of the total protein in the recombinant cell. The cooperative action of the novel α-amylase and the maltooligosyltrehalose synthase from Sulfolobus shibatae was investigated and trehalose was detected by using HPLC analysis when using amylose and partial starch hydrolysates as substrates.

  1. STABILITY AND PROTON-PERMEABILITY OF LIPOSOMES COMPOSED OF ARCHAEAL TETRAETHER LIPIDS

    NARCIS (Netherlands)

    Elferink, Marieke G.L.; Wit, Janny G. de; Driessen, Arnold J.M.; Konings, Wil N.

    1994-01-01

    Liposomes composed of tetraether lipids originating from the thermoacidophilic archaeon Sulfolobus acidocaldarius were analyzed for their stability and proton permeability from 20 degrees C up to 80 degrees C. At room temperature, these liposomes are considerably more stable and have a much lower pr

  2. Mapping posttranscriptional modifications in 5S ribosomal RNA by MALDI mass spectrometry

    DEFF Research Database (Denmark)

    Kirpekar, F; Douthwaite, S; Roepstorff, P

    2000-01-01

    and the archaeon Sulfolobus acidocaldarius, as well as the halophile archaea Halobacterium halobium and Haloarcula marismortui. One S. acidocaldarius posttranscriptional modification was identified and was further characterized by PSD as a methylation of cytidine32. The modified C is located in a region...

  3. Archaeal promoter architecture and mechanism of gene activation

    DEFF Research Database (Denmark)

    Peng, Nan; Ao, Xiang; Liang, Yun Xiang;

    2011-01-01

    Sulfolobus solfataricus and Sulfolobus islandicus contain several genes exhibiting D-arabinose-inducible expression and these systems are ideal for studying mechanisms of archaeal gene expression. At sequence level, only two highly conserved cis elements are present on the promoters: a regulatory...... element named ara box directing arabinose-inducible expression and the basal promoter element TATA, serving as the binding site for the TATA-binding protein. Strikingly, these promoters possess a modular structure that allows an essentially inactive basal promoter to be strongly activated. The invoked...

  4. 耐热嗜酸古细菌Sulfolobus acidocaldarius谷氨酰胺合成酶的表达调控和性质研究%The Expression Regulation and Characterization of Glutamine Synthetase From The Hyperthermoacidophilic Crenarcheon Sulfolobus acidocaldarius

    Institute of Scientific and Technical Information of China (English)

    殷志敏; 陈群英; 司马健; 吴一凡; 张双全

    2003-01-01

    古细菌Sulfolobus acidocaldarius细胞内谷氨酰胺合成酶的表达量随着培养条件的改变有较大差异,RNA印迹表明,该差异是在mRNA水平受到调控.经DEAE-Sepharose和Sephacryl S-300两步分离酶蛋白,SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)和凝胶过滤测定分子质量,表明该酶为12个相同亚基组成,分子质量为630 ku的多聚体.该酶的最佳pH为7.3;对羟胺、谷氨酰胺、ADP和Mn2+的Km值分别为3.5 mmol/L、1.3 mmol/L、0.15 mmol/L和0.24 mmol/L;其γ-谷氨酰转移酶活性和生物合成酶活性的最佳温度均为90℃.Arrhenius曲线表明,γ-谷氨酰转移酶的活化能为47 kJ/(mol*K),生物合成酶的活化能分别为29 kJ/(mol*K)(40~75℃)和10 kJ/(mol*K)(55~90℃).对该酶的抑制剂研究发现,与其他来源的谷氨酰胺合成酶不同,甘氨酸、L-丙氨酸能明显抑制 S.acidocaldarius谷氨酰胺合成酶的活性,而常规的抑制剂如L-色氨酸、L-组氨酸、5′-AMP却没有抑制作用,甘氨酸、L-丙氨酸的抑制作用为竞争性抑制,推断该酶的活性调节与绝大多数革兰氏阳性菌一样不受腺甘酰化的影响.

  5. Extensive lysine methylation in hyperthermophilic crenarchaea : potential implications for protein stability and recombinant enzymes

    OpenAIRE

    Botting, Catherine H.; Paul Talbot; Sonia Paytubi; White, Malcolm F

    2010-01-01

    In eukarya and bacteria, lysine methylation is relatively rare and is catalysed by sequence-specific lysine methyltransferases that typically have only a single-protein target. Using RNA polymerase purified from the thermophilic crenarchaeum Sulfolobus solfataricus, we identified 21 methyllysines distributed across 9 subunits of the enzyme. The modified lysines were predominantly in alpha-helices and showed no conserved sequence context. A limited survey of the Thermoproteus tenax proteome re...

  6. On the mechanism of loading the PCNA sliding clamp by RFC.

    Science.gov (United States)

    Dionne, Isabelle; Brown, Nicola J; Woodgate, Roger; Bell, Stephen D

    2008-04-01

    Sliding clamps play central roles in a broad range of DNA replication and repair processes. The clamps form circular molecules that must be opened and resealed around DNA by the clamp loader complex to fulfil their function. While most eukaryotes and many archea possess a homo-trimeric PCNA, the PCNA of Sulfolobus solfataricus is a heterotrimer. Here, we exploit the asymmetry of S. solfataricus PCNA to create a series of circularly permuted PCNA subunit fusions, thereby covalently closing defined interfaces within the heterotrimer. Using these concatamers, we investigate the requirements for loading the clamp onto DNA and reveal that a single defined interface within the heterotrimer is opened during the loading process. Subunit-specific interactions between S. solfataricus RFC clamp loader and PCNA permit us to superimpose our data upon the structure of yeast RFC-PCNA complex, thereby presenting a general model for PCNA loading by RFC in archaea and eukaryotes.

  7. 新疆罗布泊地区可培养嗜盐古菌多样性及其功能酶筛选%Biodiversity and functional enzymes of cultured halophilic archaeon in Lop Nur region

    Institute of Scientific and Technical Information of China (English)

    刘冰冰; 唐蜀昆; 明红; 何松涛; 聂国兴; 关统伟; 张利莉; 李文均

    2011-01-01

    [目的]探索新疆罗布泊地区高盐环境可培养嗜盐古菌的多样性及其功能酶应用潜力.[方法]采集罗布泊地区13份土样,用纯培养并结合基于16S rRNA基因系统发育分析的方法来研究样品中嗜盐古菌的多样性.按系统进化树的聚类关系,挑选出一些菌株进行盐度耐受及淀粉酶、蛋白酶、酯酶的酶活检测.[结果]从13份土样中共分离到56株嗜盐古菌,经16S rRNA基因克隆测序,通过MEGA 4.0构建N-J树分析,56株菌分布于嗜盐古菌的10个生效发表属和5个潜在新属.运用Shannon-Wiener方法计算其多样性指数为1.820.挑选17株嗜盐古菌所测试盐浓度实验结果表明这一批嗜盐古菌的大部分生长范围在10% -35%之间,最适盐浓度在20% -25%之间.不同酶活检测结果为:淀粉酶酶活率为70.6%,蛋白酶酶活率为35.3%,酯酶酶活率为82.4%.[结论]新疆罗布泊周边地区由于气候及地理位置的独特性,蕴藏丰富的嗜盐古菌资源.本实验所设计的分离方法对嗜盐古菌的分离是极其有效的,为进一步研究新疆罗布泊及周边地区嗜盐古菌资源提供了技术基础.盐度耐受实验结果验证在低盐环境中分离嗜盐古菌新物种的可行性.同时,嗜盐古菌的酶活比率较高且活性较强为进一步开发利用嗜盐古菌资源提供了理论依据.%[ Objective ] In order to explore the diversity of cultured halophilic archaeon from hypersaline environments in Lop Nur region and their potential application. [Methods] Total 13 soil samples were collected from Lop Nur regions. Halophilic archaea strains were isolated and identified by 16S rRNA gene sequence analysis. In addition, 17 strains were selected based on different branches in pylogenetic tree, and their salt concentration tolerance and amylase, protease, esterase activities were further detected by conventional methods. [ Results ] The 16S rRNA gene sequences of 56 selected strains were

  8. AglH, a thermophilic UDP-N-acetylglucosamine-1-phosphate:dolichyl phosphate GlcNAc-1-phosphotransferase initiating protein N-glycosylation pathway in Sulfolobus acidocaldarius, is capable of complementing the eukaryal Alg7.

    Science.gov (United States)

    Meyer, Benjamin H; Shams-Eldin, Hosam; Albers, Sonja-Verena

    2017-01-01

    AglH, a predicted UDP-GlcNAc-1-phosphate:dolichyl phosphate GlcNAc-1-phosphotransferase, is initiating the protein N-glycosylation pathway in the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius. AglH successfully replaced the endogenous GlcNAc-1-phosphotransferase activity of Alg7 in a conditional lethal Saccharomyces cerevisiae strain, in which the first step of the eukaryal protein N-glycosylation process was repressed. This study is one of the few examples of cross-domain complementation demonstrating a conserved polyprenyl phosphate transferase reaction within the eukaryal and archaeal domain like it was demonstrated for Methanococcus voltae (Shams-Eldin et al. 2008). The topology prediction and the alignment of the AglH membrane protein with GlcNAc-1-phosphotransferases from the three domains of life show significant conservation of amino acids within the different proposed cytoplasmic loops. Alanine mutations of selected conserved amino acids in the putative cytoplasmic loops II (D100), IV (F220) and V (F264) demonstrated the importance of these amino acids for cross-domain AlgH activity in in vitro complementation assays in S. cerevisiae. Furthermore, antibiotic treatment interfering directly with the activity of dolichyl phosphate GlcNAc-1-phosphotransferases confirmed the essentiality of N-glycosylation for cell survival.

  9. Crystal structures of a subunit of the formylglycinamide ribonucleotide amidotransferase, PurS, from Thermus thermophilus , Sulfolobus tokodaii and Methanocaldococcus jannaschii

    Energy Technology Data Exchange (ETDEWEB)

    Watanabe, Yuzo; Yanai, Hisaaki; Kanagawa, Mayumi; Suzuki, Sakiko; Tamura, Satoko; Okada, Kiyoshi; Baba, Seiki; Kumasaka, Takashi; Agari, Yoshihiro; Chen, Lirong; Fu, Zheng-Qing; Chrzas, John; Wang, Bi-Cheng; Nakagawa, Noriko; Ebihara, Akio; Masui, Ryoji; Kuramitsu, Seiki; Yokoyama, Shigeyuki; Sampei, Gen-ichi; Kawai, Gota

    2016-07-27

    The crystal structures of a subunit of the formylglycinamide ribonucleotide amidotransferase, PurS, fromThermus thermophilus,Sulfolobus tokodaiiandMethanocaldococcus jannaschiiwere determined and their structural characteristics were analyzed. For PurS fromT. thermophilus, two structures were determined using two crystals that were grown in different conditions. The four structures in the dimeric form were almost identical to one another despite their relatively low sequence identities. This is also true for all PurS structures determined to date. A few residues were conserved among PurSs and these are located at the interaction site with PurL and PurQ, the other subunits of the formylglycinamide ribonucleotide amidotransferase. Molecular-dynamics simulations of the PurS dimer as well as a model of the complex of the PurS dimer, PurL and PurQ suggest that PurS plays some role in the catalysis of the enzyme by its bending motion.

  10. Heterologous Expression of Enzymes for Trehalose Synthesis from Sulfolobus acidocaldarius in Bacillus subtilis%1株古细菌海藻糖合成酶系在枯草芽孢杆菌中的诱导表达

    Institute of Scientific and Technical Information of China (English)

    王珊瑛; 李由然; 顾正华; 张梁; 丁重阳; 石贵阳

    2016-01-01

    来源于古细菌嗜酸硫化叶菌(Sulfolobus acidocaldarius)的麦芽寡糖基海藻糖合成酶(maltooligosylterhalose synthase,MTSase)和麦芽寡糖基海藻糖水解酶(maltooligosyltrehalose trehalohydrolase,MTHase)联合作用,可以利用淀粉为底物生成海藻糖.本研究构建了6种枯草芽孢杆菌-大肠杆菌穿梭表达载体,将密码子优化后的MTSase和MTHase编码基因在木糖异构酶基因的启动子及其阻遏蛋白介导下实现了在枯草芽孢杆菌中的功能表达,木糖启动子分别来源于枯草芽孢杆菌(Bacillus subtilis)、巨大芽孢杆菌(Bacillus megaterium)、地衣芽孢杆菌(Bacillus licheniformis).在以5 g/L甘油为碳源,24 g/L蛋白胨为氮源时,菌体培养8h后加入终质量浓度为6g/L的木糖,37℃诱导16h后重组MTSase、MTHase产生联合作用,海藻糖转化率达到33.57%.实现了MTSase、MTHase在枯草芽孢杆菌中的功能表达.

  11. Complete conversion of major protopanaxadiol ginsenosides to compound K by the combined use of α-L-arabinofuranosidase and β-galactosidase from Caldicellulosiruptor saccharolyticus and β-glucosidase from Sulfolobus acidocaldarius.

    Science.gov (United States)

    Shin, Kyung-Chul; Oh, Hye-Jin; Kim, Baek-Joong; Oh, Deok-Kun

    2013-08-10

    The ginsenoside compound K has pharmaceutical activities, including anti-tumor, anti-inflammatory, anti-allergic, and hepatoprotective effects. To increase the production of compound K, the α-L-arabinofuranoside-hydrolyzing α-L-arabinofuranosidase (CS-abf) and/or the α-L-arabinopyranoside-hydrolyzing β-galactosidase from Caldicellulosiruptor saccharolyticus (CS-bgal) were mixed with the β-D-glucopyranoside-hydrolyzing β-glucosidase from Sulfolobus acidocaldarius (SA-bglu). The optimum conditions for the production of ginsenoside compound K from ginsenoside Rc or Rb₂, or from major protopanaxadiol ginsenosides in ginseng root extract were determined to be pH 6.0 and 75°C with 8 mg ml⁻¹ ginsenoside Rc, 8 mg ml⁻¹ Rb₂, or 10% (w/v) ginseng root extract; and 10.5 U ml⁻¹ CS-abf or CS-bgal supplemented with 4.5 U ml⁻¹ SA-bglu, or 10.5 U ml⁻¹ CS-abf and 10.5 U ml⁻¹ CS-bgal supplemented with 4.5 U ml⁻¹ SA-bglu, respectively. Under optimum conditions, ginsenosides Rc and Rb2, and major protopanaxadiol ginsenosides in ginseng root extract were completely converted to compound K after 12, 14, and 20 h, respectively, with the respective productivities of 388, 328, and 144 mg l⁻¹ h⁻¹. This is the first report of the complete conversion of major protopanaxadiol ginsenosides to compound K.

  12. Design of pH sensitive binding proteins from the hyperthermophilic Sso7d scaffold.

    Directory of Open Access Journals (Sweden)

    Nimish Gera

    Full Text Available We have engineered pH sensitive binding proteins for the Fc portion of human immunoglobulin G (hIgG (hFc using two different strategies - histidine scanning and random mutagenesis. We obtained an hFc-binding protein, Sso7d-hFc, through mutagenesis of the Sso7d protein from the hyperthermophilic archaeon Sulfolobus solfataricus; Sso7d-hFc was isolated from a combinatorial library of Sso7d mutants using yeast surface display. Subsequently, we identified a pH sensitive mutant, Sso7d-his-hFc, through systematic evaluation of Sso7d-hFc mutants containing single histidine substitutions. In parallel, we also developed a yeast display screening strategy to isolate a different pH sensitive hFc binder, Sso7d-ev-hFc, from a library of mutants obtained by random mutagenesis of a pool of hFc binders. In contrast to Sso7d-hFc, both Sso7d-his-hFc and Sso7d-ev-hFc have a higher binding affinity for hFc at pH 7.4 than at pH 4.5. The Sso7d-mutant hFc binders can be recombinantly expressed at high yield in E. coli and are monomeric in solution. They bind an epitope in the CH3 domain of hFc that has high sequence homology in all four hIgG isotypes (hIgG(1-4, and recognize hIgG(1-4 as well as deglycosylated hIgG in western blotting assays. pH sensitive hFc binders are attractive candidates for use in chromatography, to achieve elution of IgG under milder pH conditions. However, the surface density of immobilized hFc binders, as well as the avidity effect arising from the multivalent interaction of dimeric hFc with the capture surface, influences the pH dependence of dissociation from the capture surface. Therefore, further studies are needed to evaluate if the Sso7d mutants identified in this study are indeed useful as affinity ligands in chromatography.

  13. 连四硫酸盐存在下利用嗜酸热古菌Acidianus copahuensis 提高锌回收率%Improving zinc recovery by thermoacidophilic archaeon Acidianus copahuensis using tetrathionate

    Institute of Scientific and Technical Information of China (English)

    Camila CASTRO; Edgardo R. DONATI

    2016-01-01

    The attachment and bioleaching experiments were conducted to evaluate the zinc recovery from Hualilan ore by the thermoacidophilic archaeon Acidianus copahuensis. Cells of this species pregrown on tetrathionate showed higher capability of attachment to the ore than cells pregrown on other energy sources and such attachment seemed to be mediated by the product of extracellular polymeric substances. A. copahuensis achieved a successful bioleaching of the ore reaching 100% of zinc recovery when tetrathionate was added. Simultaneous addition of yeast extract and tetrathionate maintained the zinc extraction at higher rate. Zinc dissolution kinetics was controlled by chemical reaction in cultures with the external addition of tetrathionate but by the diffusion through a product layer of jarosite in the other cultures.%通过吸附和生物浸出实验考察利用嗜酸热古菌 Acidianus copahuensis 从 Hualian 矿中回收锌。经过在连四硫酸盐表面预处理的菌种具有比经其他能量供给剂预处理的菌种更强的矿物吸附能力,且此吸附能力可由所产生的体外聚合物调节。当加入连四硫酸盐时,用 A. copahuensis 生物浸取 Hualian 矿中的锌,其浸出率达100%。同时添加酵母和连四硫酸盐不仅能保持较高的锌浸出率,而且能加快浸出速率。添加连四硫酸盐后,培养基中锌的溶解动力学受化学反应控制;而在未添加连四硫酸盐培养基中,锌的溶解动力学受经过黄钾铁矾反应层的扩散控制。

  14. 耐热嗜酸古细菌谷氨酰胺合成酶基因的克隆、诱导表达和活性测定%The Cloning,Induction and Activity Assay of Glutamine Synthetase Gene From Thermoacidophilic Archaebacterium Sulfolobus acidocaldarius

    Institute of Scientific and Technical Information of China (English)

    殷志敏; 闫淑珍; 戴谷; 吴一凡; 张双全

    2002-01-01

    采用DEAE-Sepharose及Sephacryl S-300柱分离纯化了耐热嗜酸古细菌Sulfolobus acidocaldarius谷氨酰胺合成酶(E 6.3.1.2,Sac-GS). SDS-聚丙烯酰胺凝胶电泳检测分子质量为53 ku的单一条带.测定其N端氨基酸序列为PGLPKNEHEALEFLKSNNIKWVDLQ,与其他古细菌谷氨酰胺合成酶的氨基酸序列进行比较后,发现均存在ATP结合保守序列TFMPKP(I/L/F)(F/P/Y)(G/R).采用碱基的简并性设计出一对简并引物,以Sulfolobus acidocaldarius的基因组DNA为模板进行PCR扩增,获得780bp的DNA片段,经克隆测序后确定为谷氨酰胺合成酶基因片段,以地高辛标记的该片段为探针,将Sulfolobus acidocaldarius基因组DNA用不同的限制性内切酶分别进行单双酶组合切割,然后进行DNA印迹分析,选出了2.4 kb的BamHⅠ/HindⅢ双酶切片段,并克隆到pBluescript KS+质粒中,通过菌落原位杂交获得阳性克隆, 进行DNA全序列测定后,得到完整的1.5 kb的谷氨酰胺合成酶全基因序列,再将其克隆到pET3c载体中,在大肠杆菌BL21中进行诱导表达并进行相应的活性测定,其酶活力的最适温度为90℃.该酶在高温下所表现出的高活性充分说明其来源于耐热微生物.

  15. Hydrogenases from the Hyperthermophilic Archaeon Pyrococcus furiosus

    NARCIS (Netherlands)

    van Haaster, D.J.

    2007-01-01

    Hydrogenase is an electron-transfer protein and catalyses the simplest chemical redox reaction, the reversible two-electron oxidation of molecular hydrogen in aerobic and anaerobic microorganisms. A kinetic study of the hydrogen oxidation reaction by Fe-hydrogenase from Desulfovibrio vulgaris (Hilde

  16. Structural and Functional Characterization of an Archaeal Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated Complex for Antiviral Defense (CASCADE)

    DEFF Research Database (Denmark)

    Lintner, Nathanael G; Kerou, Melina; Brumfield, Susan K

    2011-01-01

    In response to viral infection, many prokaryotes incorporate fragments of virus-derived DNA into loci called clustered regularly interspaced short palindromic repeats (CRISPRs). The loci are then transcribed, and the processed CRISPR transcripts are used to target invading viral DNA and RNA....... The Escherichia coli "CRISPR-associated complex for antiviral defense" (CASCADE) is central in targeting invading DNA. Here we report the structural and functional characterization of an archaeal CASCADE (aCASCADE) from Sulfolobus solfataricus. Tagged Csa2 (Cas7) expressed in S. solfataricus co-purifies with Cas5......a-, Cas6-, Csa5-, and Cas6-processed CRISPR-RNA (crRNA). Csa2, the dominant protein in aCASCADE, forms a stable complex with Cas5a. Transmission electron microscopy reveals a helical complex of variable length, perhaps due to substoichiometric amounts of other CASCADE components. A recombinant Csa2...

  17. Pactamycin binding site on archaebacterial and eukaryotic ribosomes

    Energy Technology Data Exchange (ETDEWEB)

    Tejedor, F.; Amils, R.; Ballesta, J.P.G.

    1987-01-27

    The presence of a photoreactive acetophenone group in the protein synthesis inhibitor pactamycin and the possibility of obtaining active iodinated derivatives that retain full biological activity allow the antibiotic binding site on Saccharomyces cerevisiae and archaebacterium Sulfolobus solfataricus ribosomes to be photoaffinity labeled. Four major labeled proteins have been identified in the yeast ribosomes, i.e., YS10, YS18, YS21/24, and YS30, while proteins AL1a, AS10/L8, AS18/20, and AS21/22 appeared as radioactive spots in S. solfataricus. There seems to be a correlation between some of the proteins labeled in yeast and those previously reported in Escherichia coli indicating that the pactamycin binding sites of both species, which are in the small subunit close to the initiation factors and mRNA binding sites, must have similar characteristics.

  18. Extremophiles Survival to Simulated Space Conditions: An Astrobiology Model Study

    Science.gov (United States)

    Mastascusa, V.; Romano, I.; Di Donato, P.; Poli, A.; Della Corte, V.; Rotundi, A.; Bussoletti, E.; Quarto, M.; Pugliese, M.; Nicolaus, B.

    2014-09-01

    In this work we investigated the ability of four extremophilic bacteria from Archaea and Bacteria domains to resist to space environment by exposing them to extreme conditions of temperature, UV radiation, desiccation coupled to low pressure generated in a Mars' conditions simulator. All the investigated extremophilic strains (namely Sulfolobus solfataricus, Haloterrigena hispanica, Thermotoga neapolitana and Geobacillus thermantarcticus) showed a good resistance to the simulation of the temperature variation in the space; on the other hand irradiation with UV at 254 nm affected only slightly the growth of H. hispanica, G. thermantarcticus and S. solfataricus; finally exposition to Mars simulated condition showed that H. hispanica and G. thermantarcticus were resistant to desiccation and low pressure.

  19. Extremophiles survival to simulated space conditions: an astrobiology model study.

    Science.gov (United States)

    Mastascusa, V; Romano, I; Di Donato, P; Poli, A; Della Corte, V; Rotundi, A; Bussoletti, E; Quarto, M; Pugliese, M; Nicolaus, B

    2014-09-01

    In this work we investigated the ability of four extremophilic bacteria from Archaea and Bacteria domains to resist to space environment by exposing them to extreme conditions of temperature, UV radiation, desiccation coupled to low pressure generated in a Mars' conditions simulator. All the investigated extremophilic strains (namely Sulfolobus solfataricus, Haloterrigena hispanica, Thermotoga neapolitana and Geobacillus thermantarcticus) showed a good resistance to the simulation of the temperature variation in the space; on the other hand irradiation with UV at 254 nm affected only slightly the growth of H. hispanica, G. thermantarcticus and S. solfataricus; finally exposition to Mars simulated condition showed that H. hispanica and G. thermantarcticus were resistant to desiccation and low pressure.

  20. Sequential protein NMR assignments in the liquid state via sequential data acquisition

    Science.gov (United States)

    Wiedemann, Christoph; Bellstedt, Peter; Kirschstein, Anika; Häfner, Sabine; Herbst, Christian; Görlach, Matthias; Ramachandran, Ramadurai

    2014-02-01

    Two different NMR pulse schemes involving sequential 1H data acquisition are presented for achieving protein backbone sequential resonance assignments: (i) acquisition of 3D {HCCNH and HNCACONH} and (ii) collection of 3D {HNCOCANH and HNCACONH} chemical shift correlation spectra using uniformly 13C,15N labelled proteins. The sequential acquisition of these spectra reduces the overall experimental time by a factor of ≈2 as compared to individual acquisitions. The suitability of this approach is experimentally demonstrated for the C-terminal winged helix (WH) domain of the minichromosome maintenance (MCM) complex of Sulfolobus solfataricus.

  1. Microbial desulfurization of coal by Thiobacillus ferrooxidans and thermophilic archaea

    Energy Technology Data Exchange (ETDEWEB)

    Olsson, G.; Pott, B.-M.; Larsson, L.; Holst, O.; Karlsson, H.T. (Univ. of Lund, Lund (Sweden). Dept. of Chemical Engineering II, Chemical Center)

    1994-11-01

    Several different microorganisms have been suggested for desulfurization. In the present investigation, the thermophilic archaea [ital Acidianus brierleyi] (DSM 1651), [ital Sulfolobus acidocaldarius] (DSM 639) and [ital Sulfolobus solfataricus] (DSM 1616) were compared with the mesophyilic bacterium [ital Thiobacillus ferrooxidans] (DSM 583) concerning their capability of removing sulfur from coal. The desulfurization rate as well as the amount of sulfur removed by the microorganisms was studied. Two of the investigated microorganisms [ital Thiobacillus ferrooxidans] and [ital Acidianus brierleyi], were capable of oxidizing pure pyrite as well as oxidizing sulfur in coal. A kinetic analysis was performed assuming first order reactions. The rate constant for oxidation of pure pyrite by [ital A. brierleyi] was observed to be higher than for [ital T. ferrooxidans]. The values of the rate constants for sulfur removal from coal were comparable for the two microorganisms, but were higher than for oxidation of pure pyrite. 18 refs., 2 figs., 1 tab.

  2. The Effects of Temperature and Growth Phase on the Lipidomes of Sulfolobus islandicus and Sulfolobus tokodaii

    DEFF Research Database (Denmark)

    Jensen, Sara Munk; Neesgaard, Vinnie Lund; Skjoldbjerg, Sandra Landbo Nedergaard;

    2015-01-01

    The functionality of the plasma membrane is essential for all organisms. Adaption to high growth temperatures imposes challenges and Bacteria, Eukarya, and Archaea have developed several mechanisms to cope with these. Hyperthermophilic archaea have earlier been shown to synthesize tetraether...

  3. Isolation,Identification and Characterization of Extremely Halophilic C50 Carotenoid-Producing Archaeon%1株产C50类胡萝卜素极端嗜盐古菌的筛选鉴定及特性分析

    Institute of Scientific and Technical Information of China (English)

    刘良森; 邓元告; 隋丽英

    2014-01-01

    An extremely halophilic C50 carotenoid-producing red archaeon was isolated from the crystalli-zer ponds in solar saltworks.The isolated strain is Gram-negative and short rod.The optimum salinity and pH for growth is 250 and 7,respectively.Phenotypic and molecular analyses of this strain indicated that it belonged to extremely halophilic archaea genus Halorubrum and named Halorubrum Sp1 (16S rRNA Genbank registration number KF697239).UV-visible scanning spectrum showed that C50 carote-noid was the major pigments presented in this strain.Pigment accumulation was maximizing at pH 8. In the salinity range of 150~300,increasing salinity resulted in declined pigment accumulation.%从日晒盐场结晶池中筛选到1株产C50类胡萝卜素的红色极端嗜盐古菌。该菌株为革兰氏阴性菌,短棒状,最适生长盐度为250,最适生长pH 为7。表型鉴定方法结合16S rDNA序列分析判定,该菌属于极端嗜盐古菌盐红菌属 Halorubrum,命名为 Halorubrum sp.Sp1(16S rRNA Genbank 登录号为KF697239)。根据紫外-可见光扫描特征光谱,确定该菌株主要色素为 C50类胡萝卜素。pH 8时单位细胞色素积累量最大,在盐度150~300范围内随盐度升高,单位细胞色素积累量逐渐降低。

  4. Crenarchaeal biofilm formation under extreme conditions.

    Directory of Open Access Journals (Sweden)

    Andrea Koerdt

    Full Text Available BACKGROUND: Biofilm formation has been studied in much detail for a variety of bacterial species, as it plays a major role in the pathogenicity of bacteria. However, only limited information is available for the development of archaeal communities that are frequently found in many natural environments. METHODOLOGY: We have analyzed biofilm formation in three closely related hyperthermophilic crenarchaeotes: Sulfolobus acidocaldarius, S. solfataricus and S. tokodaii. We established a microtitre plate assay adapted to high temperatures to determine how pH and temperature influence biofilm formation in these organisms. Biofilm analysis by confocal laser scanning microscopy demonstrated that the three strains form very different communities ranging from simple carpet-like structures in S. solfataricus to high density tower-like structures in S. acidocaldarius in static systems. Lectin staining indicated that all three strains produced extracellular polysaccharides containing glucose, galactose, mannose and N-acetylglucosamine once biofilm formation was initiated. While flagella mutants had no phenotype in two days old static biofilms of S. solfataricus, a UV-induced pili deletion mutant showed decreased attachment of cells. CONCLUSION: The study gives first insights into formation and development of crenarchaeal biofilms in extreme environments.

  5. Harnessing type I and type III CRISPR-Cas systems for genome editing

    DEFF Research Database (Denmark)

    Li, Yingjun; Pan, Saifu; Zhang, Yan;

    2016-01-01

    CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated) systems are widespread in archaea and bacteria, and research on their molecular mechanisms has led to the development of genome-editing techniques based on a few Type II systems. However, there has not been any...... report on harnessing a Type I or Type III system for genome editing. Here, a method was developed to repurpose both CRISPR-Cas systems for genetic manipulation in Sulfolobus islandicus, a thermophilic archaeon. A novel type of genome-editing plasmid (pGE) was constructed, carrying an artificial mini...

  6. The Y-Family DNA Polymerase Dpo4 Uses a Template Slippage Mechanism To Create Single-Base Deletions

    Energy Technology Data Exchange (ETDEWEB)

    Y Wu; R Wilson; J Pata

    2011-12-31

    The Y-family polymerases help cells tolerate DNA damage by performing translesion synthesis, yet they also can be highly error prone. One distinctive feature of the DinB class of Y-family polymerases is that they make single-base deletion errors at high frequencies in repetitive sequences, especially those that contain two or more identical pyrimidines with a 5? flanking guanosine. Intriguingly, different deletion mechanisms have been proposed, even for two archaeal DinB polymerases that share 54% sequence identity and originate from two strains of Sulfolobus. To reconcile these apparent differences, we have characterized Dpo4 from Sulfolobus solfataricus using the same biochemical and crystallographic approaches that we have used previously to characterize Dbh from Sulfolobus acidocaldarius. In contrast to previous suggestions that Dpo4 uses a deoxynucleoside triphosphate (dNTP)-stabilized misalignment mechanism when creating single-base deletions, we find that Dpo4 predominantly uses a template slippage deletion mechanism when replicating repetitive DNA sequences, as was previously shown for Dbh. Dpo4 stabilizes the skipped template base in an extrahelical conformation between the polymerase and the little-finger domains of the enzyme. This contrasts with Dbh, in which the extrahelical base is stabilized against the surface of the little-finger domain alone. Thus, despite sharing a common deletion mechanism, these closely related polymerases use different contacts with the substrate to accomplish the same result.

  7. Alpha-amylase from the Hyperthermophilic Archaeon Thermococcus thioreducens

    Science.gov (United States)

    Bernhardsdotter, E. C. M. J.; Pusey, M. L.; Ng, M. L.; Garriott, O. K.

    2003-01-01

    Extremophiles are microorganisms that thrive in, from an anthropocentric view, extreme environments such as hot springs. The ability of survival at extreme conditions has rendered enzymes from extremophiles to be of interest in industrial applications. One approach to producing these extremozymes entails the expression of the enzyme-encoding gene in a mesophilic host such as E.coli. This method has been employed in the effort to produce an alpha-amylase from a hyperthermophile (an organism that displays optimal growth above 80 C) isolated from a hydrothermal vent at the Rainbow vent site in the Atlantic Ocean. alpha-amylases catalyze the hydrolysis of starch to produce smaller sugars and constitute a class of industrial enzymes having approximately 25% of the enzyme market. One application for thermostable alpha-amylases is the starch liquefaction process in which starch is converted into fructose and glucose syrups. The a-amylase encoding gene from the hyperthermophile Thermococcus thioreducens was cloned and sequenced, revealing high similarity with other archaeal hyperthermophilic a-amylases. The gene encoding the mature protein was expressed in E.coli. Initial characterization of this enzyme has revealed an optimal amylolytic activity between 85-90 C and around pH 5.3-6.0.

  8. Total chemical synthesis of a thermostable enzyme capable of polymerase chain reaction.

    Science.gov (United States)

    Xu, Weiliang; Jiang, Wenjun; Wang, Jiaxing; Yu, Linping; Chen, Ji; Liu, Xianyu; Liu, Lei; Zhu, Ting F

    2017-01-01

    Polymerase chain reaction (PCR) has been a defining tool in modern biology. Towards realizing mirror-image PCR, we have designed and chemically synthesized a mutant version of the 352-residue thermostable Sulfolobus solfataricus P2 DNA polymerase IV with l-amino acids and tested its PCR activity biochemically. To the best of our knowledge, this enzyme is the largest chemically synthesized protein reported to date. We show that with optimization of PCR conditions, the fully synthetic polymerase is capable of amplifying template sequences of up to 1.5 kb. The establishment of this synthetic route for chemically synthesizing DNA polymerase IV is a stepping stone towards building a d-enzyme system for mirror-image PCR, which may open up an avenue for the creation of many mirror-image molecular tools such as mirror-image systematic evolution of ligands by exponential enrichment.

  9. Surface Shear Viscosity and Phase Transitions of Monolayers at the Air-Water Interface

    Science.gov (United States)

    Relini, A.; Ciuchi, F.; Rolandi, R.

    1995-08-01

    The canal method has been employed to measure the in-plane steady shear viscosity of monolayers of bolaform lipids extracted from the membrane of the thermophilic microorganism Sulfolobus solfataricus. Monolayers were formed with the polar lipid extract (PLE), which is a mixture of several bolaform lipids, each one endowed with two nonequivalent polar headgroups. Viscosities were obtained from the measured flows by using the equation introduced by Joly; this equation contains a semiempirical parameter A, which takes into account the monolayer-subphase mechanical coupling. Measuring the flows for two different substances (PLE and oleic acid) and channel widths, the monolayer viscosities and the parameter A were determined at the same time. The analysis of the viscosity data according to the free area model shows evidences of the molecular conformational changes matching monolayer phase transitions.

  10. Extensive Lysine Methylation in Hyperthermophilic Crenarchaea: Potential Implications for Protein Stability and Recombinant Enzymes

    Directory of Open Access Journals (Sweden)

    Catherine H. Botting

    2010-01-01

    Full Text Available In eukarya and bacteria, lysine methylation is relatively rare and is catalysed by sequence-specific lysine methyltransferases that typically have only a single-protein target. Using RNA polymerase purified from the thermophilic crenarchaeum Sulfolobus solfataricus, we identified 21 methyllysines distributed across 9 subunits of the enzyme. The modified lysines were predominantly in α-helices and showed no conserved sequence context. A limited survey of the Thermoproteus tenax proteome revealed widespread modification with 52 methyllysines in 30 different proteins. These observations suggest the presence of an unusual lysine methyltransferase with relaxed specificity in the crenarchaea. Since lysine methylation is known to enhance protein thermostability, this may be an adaptation to a thermophilic lifestyle. The implications of this modification for studies and applications of recombinant crenarchaeal enzymes are discussed.

  11. Monitoring Acidophilic Microbes with Real-Time Polymerase Chain Reaction (PCR) Assays

    Energy Technology Data Exchange (ETDEWEB)

    Frank F. Roberto

    2008-08-01

    Many techniques that are used to characterize and monitor microbial populations associated with sulfide mineral bioleaching require the cultivation of the organisms on solid or liquid media. Chemolithotrophic species, such as Acidithiobacillus ferrooxidans and Leptospirillum ferrooxidans, or thermophilic chemolithotrophs, such as Acidianus brierleyi and Sulfolobus solfataricus can grow quite slowly, requiring weeks to complete efforts to identify and quantify these microbes associated with bioleach samples. Real-time PCR (polymerase chain reaction) assays in which DNA targets are amplified in the presence of fluorescent oligonucleotide primers, allowing the monitoring and quantification of the amplification reactions as they progress, provide a means of rapidly detecting the presence of microbial species of interest, and their relative abundance in a sample. This presentation will describe the design and use of such assays to monitor acidophilic microbes in the environment and in bioleaching operations. These assays provide results within 2-3 hours, and can detect less than 100 individual microbial cells.

  12. Structural and functional characterization of an archaeal clustered regularly interspaced short palindromic repeat (CRISPR)-associated complex for antiviral defense (CASCADE).

    Science.gov (United States)

    Lintner, Nathanael G; Kerou, Melina; Brumfield, Susan K; Graham, Shirley; Liu, Huanting; Naismith, James H; Sdano, Matthew; Peng, Nan; She, Qunxin; Copié, Valérie; Young, Mark J; White, Malcolm F; Lawrence, C Martin

    2011-06-17

    In response to viral infection, many prokaryotes incorporate fragments of virus-derived DNA into loci called clustered regularly interspaced short palindromic repeats (CRISPRs). The loci are then transcribed, and the processed CRISPR transcripts are used to target invading viral DNA and RNA. The Escherichia coli "CRISPR-associated complex for antiviral defense" (CASCADE) is central in targeting invading DNA. Here we report the structural and functional characterization of an archaeal CASCADE (aCASCADE) from Sulfolobus solfataricus. Tagged Csa2 (Cas7) expressed in S. solfataricus co-purifies with Cas5a-, Cas6-, Csa5-, and Cas6-processed CRISPR-RNA (crRNA). Csa2, the dominant protein in aCASCADE, forms a stable complex with Cas5a. Transmission electron microscopy reveals a helical complex of variable length, perhaps due to substoichiometric amounts of other CASCADE components. A recombinant Csa2-Cas5a complex is sufficient to bind crRNA and complementary ssDNA. The structure of Csa2 reveals a crescent-shaped structure unexpectedly composed of a modified RNA-recognition motif and two additional domains present as insertions in the RNA-recognition motif. Conserved residues indicate potential crRNA- and target DNA-binding sites, and the H160A variant shows significantly reduced affinity for crRNA. We propose a general subunit architecture for CASCADE in other bacteria and Archaea.

  13. Structural and Functional Characterization of an Archaeal Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated Complex for Antiviral Defense (CASCADE)*

    Science.gov (United States)

    Lintner, Nathanael G.; Kerou, Melina; Brumfield, Susan K.; Graham, Shirley; Liu, Huanting; Naismith, James H.; Sdano, Matthew; Peng, Nan; She, Qunxin; Copié, Valérie; Young, Mark J.; White, Malcolm F.; Lawrence, C. Martin

    2011-01-01

    In response to viral infection, many prokaryotes incorporate fragments of virus-derived DNA into loci called clustered regularly interspaced short palindromic repeats (CRISPRs). The loci are then transcribed, and the processed CRISPR transcripts are used to target invading viral DNA and RNA. The Escherichia coli “CRISPR-associated complex for antiviral defense” (CASCADE) is central in targeting invading DNA. Here we report the structural and functional characterization of an archaeal CASCADE (aCASCADE) from Sulfolobus solfataricus. Tagged Csa2 (Cas7) expressed in S. solfataricus co-purifies with Cas5a-, Cas6-, Csa5-, and Cas6-processed CRISPR-RNA (crRNA). Csa2, the dominant protein in aCASCADE, forms a stable complex with Cas5a. Transmission electron microscopy reveals a helical complex of variable length, perhaps due to substoichiometric amounts of other CASCADE components. A recombinant Csa2-Cas5a complex is sufficient to bind crRNA and complementary ssDNA. The structure of Csa2 reveals a crescent-shaped structure unexpectedly composed of a modified RNA-recognition motif and two additional domains present as insertions in the RNA-recognition motif. Conserved residues indicate potential crRNA- and target DNA-binding sites, and the H160A variant shows significantly reduced affinity for crRNA. We propose a general subunit architecture for CASCADE in other bacteria and Archaea. PMID:21507944

  14. Structure and mechanism of the CMR complex for CRISPR-mediated antiviral immunity

    Science.gov (United States)

    Zhang, Jing; Rouillon, Christophe; Kerou, Melina; Reeks, Judith; Brugger, Kim; Graham, Shirley; Reimann, Julia; Cannone, Giuseppe; Liu, Huanting; Albers, Sonja-Verena; Naismith, James H; Spagnolo, Laura; White, Malcolm F

    2012-01-01

    Summary The prokaryotic Clusters of Regularly Interspaced Palindromic Repeats (CRISPR) system utilizes genomically-encoded CRISPR RNA (crRNA), derived from invading viruses and incorporated into ribonucleoprotein complexes with CRISPR-associated (CAS) proteins, to target and degrade viral DNA or RNA on subsequent infection. RNA is targeted by the CMR complex. In Sulfolobus solfataricus, this complex is composed of seven CAS protein subunits (Cmr1-7) and carries a diverse “payload” of targeting crRNA. The crystal structure of Cmr7 and low resolution structure of the complex are presented. S. solfataricus CMR cleaves RNA targets in an endonucleolytic reaction at UA dinucleotides. This activity is dependent on the 8-nucleotide repeat-derived 5′ sequence in the crRNA, but not on the presence of a proto-spacer associated motif (PAM) in the target. Both target and guide RNAs can be cleaved, although a single molecule of guide RNA can support the degradation of multiple targets. PMID:22227115

  15. Transcriptome-wide mapping of 5-methylcytidine RNA modifications in bacteria, archaea, and yeast reveals m5C within archaeal mRNAs.

    Directory of Open Access Journals (Sweden)

    Sarit Edelheit

    2013-06-01

    Full Text Available The presence of 5-methylcytidine (m(5C in tRNA and rRNA molecules of a wide variety of organisms was first observed more than 40 years ago. However, detection of this modification was limited to specific, abundant, RNA species, due to the usage of low-throughput methods. To obtain a high resolution, systematic, and comprehensive transcriptome-wide overview of m(5C across the three domains of life, we used bisulfite treatment on total RNA from both gram positive (B. subtilis and gram negative (E. coli bacteria, an archaeon (S. solfataricus and a eukaryote (S. cerevisiae, followed by massively parallel sequencing. We were able to recover most previously documented m(5C sites on rRNA in the four organisms, and identified several novel sites in yeast and archaeal rRNAs. Our analyses also allowed quantification of methylated m(5C positions in 64 tRNAs in yeast and archaea, revealing stoichiometric differences between the methylation patterns of these organisms. Molecules of tRNAs in which m(5C was absent were also discovered. Intriguingly, we detected m(5C sites within archaeal mRNAs, and identified a consensus motif of AUCGANGU that directs methylation in S. solfataricus. Our results, which were validated using m(5C-specific RNA immunoprecipitation, provide the first evidence for mRNA modifications in archaea, suggesting that this mode of post-transcriptional regulation extends beyond the eukaryotic domain.

  16. Structure and cell biology of archaeal virus STIV.

    Science.gov (United States)

    Fu, Chi-yu; Johnson, Johnson E

    2012-04-01

    Recent investigations of archaeal viruses have revealed novel features of their structures and life cycles when compared to eukaryotic and bacterial viruses, yet there are structure-based unifying themes suggesting common ancestral relationships among dsDNA viruses in the three kingdoms of life. Sulfolobus solfataricus and the infecting virus Sulfolobus turreted icosahedral virus (STIV) is one of the well-established model systems to study archaeal virus replication and viral-host interactions. Reliable laboratory conditions to propagate STIV and available genetic tools allowed structural characterization of the virus and viral components that lead to the proposal of common capsid ancestry with PRD1 (bacteriophage), Adenovirus (eukaryotic virus) and PBCV (chlorellavirus). Microarray and proteomics approaches systematically analyzed viral replication and the corresponding host responses. Cellular cryo-electron tomography and thin-section EM studies uncovered the assembly and maturation pathway of STIV and revealed dramatic cellular ultra-structure changes upon infection. The viral-induced pyramid-like protrusions on cell surfaces represent a novel viral release mechanism and previously uncharacterized functions in viral replication.

  17. Harnessing type I and type III CRISPR-Cas systems for genome editing

    DEFF Research Database (Denmark)

    Li, Yingjun; Pan, Saifu; Zhang, Yan

    2016-01-01

    CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated) systems are widespread in archaea and bacteria, and research on their molecular mechanisms has led to the development of genome-editing techniques based on a few Type II systems. However, there has not been any...... report on harnessing a Type I or Type III system for genome editing. Here, a method was developed to repurpose both CRISPR-Cas systems for genetic manipulation in Sulfolobus islandicus, a thermophilic archaeon. A novel type of genome-editing plasmid (pGE) was constructed, carrying an artificial mini-CRISPR...... and selectively retained as transformants. Using this strategy, different types of mutation were generated, including deletion, insertion and point mutations. We envision this method is readily applicable to different bacteria and archaea that carry an active CRISPR-Cas system of DNA interference provided...

  18. Hot and sweet: protein glycosylation in Crenarchaeota.

    Science.gov (United States)

    Meyer, Benjamin H; Albers, Sonja-Verena

    2013-02-01

    Every living cell is covered with a dense and complex array of covalently attached sugars or sugar chains. The majority of these glycans are linked to proteins via the so-called glycosylation process. Protein glycosylation is found in all three domains of life: Eukarya, Bacteria and Archaea. However, on the basis of the limit in analytic tools for glycobiology and genetics in Archaea, only in the last few years has research on archaeal glycosylation pathways started mainly in the Euryarchaeota Haloferax volcanii, Methanocaldococcus maripaludis and Methanococcus voltae. Recently, major steps of the crenarchaeal glycosylation process of the thermoacidophilic archaeon Sulfolobus acidocaldarius have been described. The present review summarizes the proposed N-glycosylation pathway of S. acidocaldarius, describing the phenotypes of the mutants disrupted in N-glycan biosynthesis as well as giving insights into the archaeal O-linked and glycosylphosphatidylinositol anchor glycosylation process.

  19. Global transcriptional regulator TrmB family members in prokaryotes.

    Science.gov (United States)

    Kim, Minwook; Park, Soyoung; Lee, Sung-Jae

    2016-10-01

    Members of the TrmB family act as global transcriptional regulators for the activation or repression of sugar ABC transporters and central sugar metabolic pathways, including glycolytic, gluconeogenic, and other metabolic pathways, and also as chromosomal stabilizers in archaea. As a relatively newly classified transcriptional regulator family, there is limited experimental evidence for their role in Thermococcales, halophilic archaeon Halobacterium salinarum NRC1, and crenarchaea Sulfolobus strains, despite being one of the extending protein families in archaea. Recently, the protein structures of Pyrococcus furiosus TrmB and TrmBL2 were solved, and the transcriptomic data uncovered by microarray and ChIP-Seq were published. In the present review, recent evidence of the functional roles of TrmB family members in archaea is explained and extended to bacteria.

  20. Phage display selection of tight specific binding variants from a hyperthermostable Sso7d scaffold protein library.

    Science.gov (United States)

    Zhao, Ning; Schmitt, Margaret A; Fisk, John D

    2016-04-01

    Antibodies, the quintessential biological recognition molecules, are not ideal for many applications because of their large size, complex modifications, and thermal and chemical instability. Identifying alternative scaffolds that may be evolved into tight, specific binding molecules with improved physical properties is of increasing interest, particularly for biomedical applications in resource-limited environments. Hyperthermophilic organisms, such as Sulfolobus solfataricus, are an attractive source of highly stable proteins that may serve as starting points for alternative molecular recognition scaffolds. We describe the first application of phage display to identify binding proteins based on the S. solfataricus protein Sso7d scaffold. Sso7d is a small cysteine-free DNA-binding protein (approximately 7 kDa, 63 amino acids), with a melting temperature of nearly 100 °C. Tight-binding Sso7d variants were selected for a diverse set of protein targets from a 10(10) member library, demonstrating the versatility of the scaffold. These Sso7d variants are able to discriminate among closely related human, bovine and rabbit serum albumins. Equilibrium dissociation constants in the nanomolar to low micromolar range were measured via competitive ELISA. Importantly, the Sso7d variants continue to bind their targets in the absence of the phage context. Furthermore, phage-displayed Sso7d variants retain their binding affinity after exposure to temperatures up to 70 °C. Taken together, our results suggest that the Sso7d scaffold will be a complementary addition to the range of non-antibody scaffold proteins that may be utilized in phage display. Variants of hyperthermostable binding proteins have potential applications in diagnostics and therapeutics for environments with extreme conditions of storage and deployment.

  1. Crystal structure and stereochemical studies of KD(P)G aldolase from Thermoproteus tenax.

    Science.gov (United States)

    Pauluhn, Anuschka; Ahmed, Hatim; Lorentzen, Esben; Buchinger, Sebastian; Schomburg, Dietmar; Siebers, Bettina; Pohl, Ehmke

    2008-07-01

    Carbon-carbon bond forming enzymes offer great potential for organic biosynthesis. Hence there is an ongoing effort to improve their biocatalytic properties, regarding availability, activity, stability, and substrate specificity and selectivity. Aldolases belong to the class of C-C bond forming enzymes and play important roles in numerous cellular processes. In several hyperthermophilic Archaea the 2-keto-3-deoxy-(6-phospho)-gluconate (KD(P)G) aldolase was identified as a key player in the metabolic pathway. The carbohydrate metabolism of the hyperthermophilic Crenarchaeote Thermoproteus tenax, for example, has been found to employ a combination of a variant of the Embden-Meyerhof-Parnas pathway and an unusual branched Entner-Doudoroff pathway that harbors a nonphosphorylative and a semiphosphorylative branch. The KD(P)G aldolase catalyzes the reversible cleavage of 2-keto-3-deoxy-6-phosphogluconate (KDPG) and 2-keto-3-deoxygluconate (KDG) forming pyruvate and glyceraldehyde 3-phosphate or glyceraldehyde, respectively. In T. tenax initial studies revealed that the pathway is specific for glucose, whereas in the thermoacidophilic Crenarchaeote Sulfolobus solfataricus the pathway was shown to be promiscuous for glucose and galactose degradation. The KD(P)G aldolase of S. solfataricus lacks stereo control and displays additional activity with 2-keto-3-deoxy-6-phosphogalactonate (KDPGal) and 2-keto-3-deoxygalactonate (KDGal), similar to the KD(P)G aldolase of Sulfolobus acidocaldarius. To address the stereo control of the T. tenax enzyme the formation of the two C4 epimers KDG and KDGal was analyzed via gas chromatography combined with mass spectroscopy. Furthermore, the crystal structure of the apoprotein was determined to a resolution of 2.0 A, and the crystal structure of the protein covalently linked to a pathway intermediate, namely pyruvate, was determined to 2.2 A. Interestingly, although the pathway seems to be specific for glucose in T. tenax the enzyme

  2. Global analysis of viral infection in an archaeal model system

    Directory of Open Access Journals (Sweden)

    Walid S. Maaty

    2012-12-01

    Full Text Available The origin and evolutionary relationship of viruses is poorly understood. This makes archaeal virus-host of particular interest because the hosts generally root near the base of phylogenetic trees, while some of the viruses have clear structural similarities to those that infect prokaryotic and eukaryotic cells. Despite the advantageous position for use in evolutionary studies, little is known about archaeal viruses or how they interact with their hosts, compared to viruses of bacteria and eukaryotes. In addition, many archaeal viruses have been isolated from extreme environments and present a unique opportunity for elucidating factors that are important for existence at the extremes.. In this article we focus on virus-host interactions using a proteomics approach to study Sulfolobus Turreted Icosahedral Virus (STIV infection of Sulfolobus solfataricus P2. Using cultures grown from the ATCC cell stock, a single cycle of STIV infection was sampled 6 times over a 72 hr period. More than 700 proteins were identified throughout the course of the experiments. Seventy one host proteins were found to change by nearly two-fold (p<0.05 with 40 becoming more abundant and 31 less abundant. The modulated proteins represent 30 different cell pathways and 14 COG groups. 2D gel analysis showed that changes in post translational modifications were a common feature of the affected proteins. The results from these studies showed that the prokaryotic antiviral adaptive immune system CRISPR associated proteins (CAS proteins were regulated in response to the virus infection. It was found that regulated proteins come from mRNAs with a shorter than average half-life. In addition, activity-based protein profiling (ABPP profiling on 2D gels showed caspase, hydrolase and tyrosine phosphatase enzyme activity labeling at the protein isoform level. Together, this data provides a more detailed global view of archaeal cellular responses to viral infection, demonstrates the

  3. Comparative analyses of the two proliferating cell nuclear antigens from the hyperthermophilic archaeon, Thermococcus kodakarensis.

    Science.gov (United States)

    Kuba, Yumani; Ishino, Sonoko; Yamagami, Takeshi; Tokuhara, Masahiro; Kanai, Tamotsu; Fujikane, Ryosuke; Daiyasu, Hiromi; Atomi, Haruyuki; Ishino, Yoshizumi

    2012-11-01

    The DNA sliding clamp is a multifunctional protein involved in cellular DNA transactions. In Archaea and Eukaryota, proliferating cell nuclear antigen (PCNA) is the sliding clamp. The ring-shaped PCNA encircles double-stranded DNA within its central hole and tethers other proteins on DNA. The majority of Crenarchaeota, a subdomain of Archaea, have multiple PCNA homologues, and they are capable of forming heterotrimeric rings for their functions. In contrast, most organisms in Euryarchaeota, the other major subdomain, have a single PCNA forming a homotrimeric ring structure. Among the Euryarchaeota whose genome is sequenced, Thermococcus kodakarensis is the only species with two genes encoding PCNA homologues on its genome. We cloned the two genes from the T. kodakarensis genome, and the gene products, PCNA1 and PCNA2, were characterized. PCNA1 stimulated the DNA synthesis reactions of the two DNA polymerases, PolB and PolD, from T. kodakarensis in vitro. PCNA2, however, only had an effect on PolB. We were able to disrupt the gene for PCNA2, whereas gene disruption for PCNA1 was not possible, suggesting that PCNA1 is essential for DNA replication. The sensitivities of the Δpcna2 mutant strain to ultraviolet irradiation (UV), methyl methanesulfonate (MMS) and mitomycin C (MMC) were indistinguishable from those of the wild-type strain.

  4. Domain topology of the DNA polymerase D complex from a hyperthermophilic archaeon Pyrococcus horikoshii.

    Science.gov (United States)

    Tang, Xiao-Feng; Shen, Yulong; Matsui, Eriko; Matsui, Ikuo

    2004-09-21

    Family D DNA polymerase (PolD) is a recently found DNA polymerase extensively existing in Euryarchaeota of Archaea. Here, we report the domain function of PolD in oligomerization and interaction with other proteins, which were characterized with the yeast two-hybrid (Y2H) and surface plasmon resonance (SPR) assays. A proliferating cell nuclear antigen, PhoPCNA, interacted with the N-terminus of the small subunit, DP1(1-200). Specific interaction between the remaining part of the small subunit, DP1(201-622), and the N-terminus of the large subunit, DP2(1-300), was detected by the Y2H assay. The SPR assay also indicated the intrasubunit interaction within the N-terminus, DP2(1-100), and the C-terminus, DP2(792-1163), of the large subunit. A synthetic 21 amino acid peptide corresponding to the sequence from cysteine cluster II, DP2(1290-1310), tightly interacted (a dissociation constant K(D) = 4.3 nM) with the N-terminus of the small subunit, DP1(1-200). Since the peptide could increase the 3'-5' exonuclease activity of DP1 [Shen et al. (2004) Nucleic Acids Res. 32, 158], the short region DP2(1290-1310) seems to play dual roles to form the PhoPolD complex and to regulate the 3'-5' exonuclease activity of DP1 through interaction with DP1(1-200). Furthermore, DP2(792-1163) containing the catalytic residues for DNA polymerization, Asp1122 and Asp1124, interacted with the intrasubunit domain, DP2(1-100), and the intersubunit domain, DP1(1-200). DP2(792-1163) probably forms the most important domain deeply involved in both the catalysis of DNA polymerization and stabilization of the PhoPolD complex through these multiple interactions.

  5. Physiological plasticity of the thermophilic ammonia oxidizing archaeon Nitrosocaldus yellowstonii in response to a changing environment

    Science.gov (United States)

    Jewell, T.; Johnson, A.; Gelsinger, D.; de la Torre, J. R.

    2012-12-01

    Our understanding of nitrogen biogeochemical cycling in high temperature environments underwent a dramatic revision with the discovery of ammonia oxidizing archaea (AOA). The importance of AOA to the global nitrogen cycle came to light when recent studies of marine AOA demonstrated the dominance of these organisms in the ocean microbiome and their role as producers of the greenhouse gas nitrous oxide (N2O). Understanding how AOA respond to fluctuating environments is crucial to fully comprehending their contribution to global biogeochemical cycling and climate change. In this study we use the thermophilic AOA Nitrosocaldus yellowstonii strain HL72 to explore the physiological plasticity of energy metabolism in these organisms. Previous studies have shown that HL72 grows autotrophically by aerobically oxidizing ammonia (NH3) to nitrite (NO2-). Unlike studies of marine AOA, we find that HL72 can grow over a wide ammonia concentration range (0.25 - 10 mM NH4Cl) with comparable generation times when in the presence of 0.25 to 4 mM NH4Cl. However, preliminary data indicate that amoA, the alpha subunit of ammonia monooxygenase (AMO), is upregulated at low ammonia concentrations (urea transporter. Urea ((NH2)2CO) is an organic compound ubiquitous to aquatic and soil habitats that, when hydrolyzed, forms NH3 and CO2. We examined urea as an alternate source of ammonia for the ammonia oxidation pathway. HL72 grows over a wide range of urea concentrations (0.25 - 10 mM) at rates comparable to growth on ammonia. In a substrate competition experiment HL72 preferentially consumed NH3 from NH4Cl when both substrates were provided in equal molar concentrations. However, the urease alpha subunit ureC was expressed in both the presence and absence of urea. One consequence of urea hydrolysis is consumption of intracellular protons during the reaction. As ammonia oxidation produces H+, leading to a decrease in pH, the hydrolysis of urea prior to ammonia oxidation may help alleviate metabolism-driven pH change in HL72. A survey of archaeal ureC sequences from metagenomic data covering a range of hydrothermal features revealed that ureolytic potential is common to many Nitrosocaldus-like organisms and is geographically widespread. Measurements of urea from siliceous circumneutral springs indicate that the concentrations are generally low, below 10 μM. One possible explanation for low steady state urea concentrations is high consumption rates by ureolytic organisms. This, combined with abiotic thermal degradation, may mask high fluxes of urea in microbial hot spring communities.

  6. A novel ammonia-oxidizing archaeon from wastewater treatment plant: Its enrichment, physiological and genomic characteristics

    Science.gov (United States)

    Li, Yuyang; Ding, Kun; Wen, Xianghua; Zhang, Bing; Shen, Bo; Yang, Yunfeng

    2016-03-01

    Ammonia-oxidizing archaea (AOA) are recently found to participate in the ammonia removal processes in wastewater treatment plants (WWTPs), similar to their bacterial counterparts. However, due to lack of cultivated AOA strains from WWTPs, their functions and contributions in these systems remain unclear. Here we report a novel AOA strain SAT1 enriched from activated sludge, with its physiological and genomic characteristics investigated. The maximal 16S rRNA gene similarity between SAT1 and other reported AOA strain is 96% (with “Ca. Nitrosotenuis chungbukensis”), and it is affiliated with Wastewater Cluster B (WWC-B) based on amoA gene phylogeny, a cluster within group I.1a and specific for activated sludge. Our strain is autotrophic, mesophilic (25 °C–33 °C) and neutrophilic (pH 5.0–7.0). Its genome size is 1.62 Mb, with a large fragment inversion (accounted for 68% genomic size) inside. The strain could not utilize urea due to truncation of the urea transporter gene. The lack of the pathways to synthesize usual compatible solutes makes it intolerant to high salinity (>0.03%), but could adapt to low salinity (0.005%) environments. This adaptation, together with possibly enhanced cell-biofilm attachment ability, makes it suitable for WWTPs environment. We propose the name “Candidatus Nitrosotenuis cloacae” for the strain SAT1.

  7. Active ammonia oxidizers in an acidic soil are phylogenetically closely related to neutrophilic archaeon.

    Science.gov (United States)

    Wang, Baozhan; Zheng, Yan; Huang, Rong; Zhou, Xue; Wang, Dongmei; He, Yuanqiu; Jia, Zhongjun

    2014-03-01

    All cultivated ammonia-oxidizing archaea (AOA) within the Nitrososphaera cluster (former soil group 1.1b) are neutrophilic. Molecular surveys also indicate the existence of Nitrososphaera-like phylotypes in acidic soil, but their ecological roles are poorly understood. In this study, we present molecular evidence for the chemolithoautotrophic growth of Nitrososphaera-like AOA in an acidic soil with pH 4.92 using DNA-based stable isotope probing (SIP). Soil microcosm incubations demonstrated that nitrification was stimulated by urea fertilization and accompanied by a significant increase in the abundance of AOA rather than ammonia-oxidizing bacteria (AOB). Real-time PCR analysis of amoA genes as a function of the buoyant density of the DNA gradient following the ultracentrifugation of the total DNA extracted from SIP microcosms indicated a substantial growth of soil AOA during nitrification. Pyrosequencing of the total 16S rRNA genes in the "heavy" DNA fractions suggested that archaeal communities were labeled to a much greater extent than soil AOB. Acetylene inhibition further showed that (13)CO2 assimilation by nitrifying communities depended solely on ammonia oxidation activity, suggesting a chemolithoautotrophic lifestyle. Phylogenetic analysis of both (13)C-labeled amoA and 16S rRNA genes revealed that most of the active AOA were phylogenetically closely related to the neutrophilic strains Nitrososphaera viennensis EN76 and JG1 within the Nitrososphaera cluster. Our results provide strong evidence for the adaptive growth of Nitrososphaera-like AOA in acidic soil, suggesting a greater metabolic versatility of soil AOA than previously appreciated.

  8. Variation of the virus-related elements within syntenic genomes of the hyperthermophilic archaeon aeropyrum

    DEFF Research Database (Denmark)

    Daifuku, Takashi; Yoshida, Takashi; Kitamura, Takayuki;

    2013-01-01

    having stable genomes, interference of synteny occurred with two proviruses, A. pernix spindle-shaped virus 1 (APSV1) and A. pernix ovoid virus 1 (APOV1), and clustered regularly interspaced short palindromic repeat (CRISPR) elements. Spacer sequences derived from the A. camini CRISPR showed significant...

  9. The ABC of ABC-transport in the hyperthermophilic archaeon Pyrococcus furiosus

    OpenAIRE

    Koning, S.

    2003-01-01

    Living organisms of our earth can be divided into two groups, the prokaryotes and the eukaryotes. Eukaryotic cells have a nucleus, a special compartment in the cell, where the genetic material, the DNA is located. The DNA in the prokaryotic cell is floating freely in the cell. The eukaryotes, that is where we belong to, together with animals, plants and fungi. Bacteria and archaea belong to the prokaryotes. Archaea resemble bacteria but in certain features they resemble more the eukaryotes. T...

  10. Activation of methanogenesis by cadmium in the marine archaeon Methanosarcina acetivorans.

    Directory of Open Access Journals (Sweden)

    Elizabeth Lira-Silva

    Full Text Available Methanosarcina acetivorans was cultured in the presence of CdCl(2 to determine the metal effect on cell growth and biogas production. With methanol as substrate, cell growth and methane synthesis were not altered by cadmium, whereas with acetate, cadmium slightly increased both, growth and methane rate synthesis. In cultures metabolically active, incubations for short-term (minutes with 10 µM total cadmium increased the methanogenesis rate by 6 and 9 folds in methanol- and acetate-grown cells, respectively. Cobalt and zinc but not copper or iron also activated the methane production rate. Methanogenic carbonic anhydrase and acetate kinase were directly activated by cadmium. Indeed, cells cultured in 100 µM total cadmium removed 41-69% of the heavy metal from the culture and accumulated 231-539 nmol Cd/mg cell protein. This is the first report showing that (i Cd(2+ has an activating effect on methanogenesis, a biotechnological relevant process in the bio-fuels field; and (ii a methanogenic archaea is able to remove a heavy metal from aquatic environments.

  11. The Alternative Route to Heme in the Methanogenic Archaeon Methanosarcina barkeri

    Directory of Open Access Journals (Sweden)

    Melanie Kühner

    2014-01-01

    Full Text Available In living organisms heme is formed from the common precursor uroporphyrinogen III by either one of two substantially different pathways. In contrast to eukaryotes and most bacteria which employ the so-called “classical” heme biosynthesis pathway, the archaea use an alternative route. In this pathway, heme is formed from uroporphyrinogen III via the intermediates precorrin-2, sirohydrochlorin, siroheme, 12,18-didecarboxysiroheme, and iron-coproporphyrin III. In this study the heme biosynthesis proteins AhbAB, AhbC, and AhbD from Methanosarcina barkeri were functionally characterized. Using an in vivo enzyme activity assay it was shown that AhbA and AhbB (Mbar_A1459 and Mbar_A1460 together catalyze the conversion of siroheme into 12,18-didecarboxysiroheme. The two proteins form a heterodimeric complex which might be subject to feedback regulation by the pathway end-product heme. Further, AhbC (Mbar_A1793 was shown to catalyze the formation of iron-coproporphyrin III in vivo. Finally, recombinant AhbD (Mbar_A1458 was produced in E. coli and purified indicating that this protein most likely contains two [4Fe-4S] clusters. Using an in vitro enzyme activity assay it was demonstrated that AhbD catalyzes the conversion of iron-coproporphyrin III into heme.

  12. Carbon isotope fractionation by the marine ammonia-oxidizing archaeon Nitrosopumilus maritimus

    OpenAIRE

    Könneke, Martin; Lipp, Julius Sebastian; Hinrichs, Kai-Uwe

    2012-01-01

    Abstract Ammonia-oxidizing archaea (AOA) are abundant and widely distributed microorganisms in aquatic and terrestrial habitats. By catalyzing the first and rate limiting step in nitrification, these chemolithoautotrophs play a significant role in the global nitrogen cycle and contribute to primary production. Here, the carbon isotopic fractionation relative to inorganic carbon source was determined for bulk biomass, biphytanes and polar lipid bound sugars of a marine AOA pure culture. Bu...

  13. Morphological and structural aspects of the extremely halophilic archaeon Haloquadratum walsbyi.

    Directory of Open Access Journals (Sweden)

    Matilde Sublimi Saponetti

    Full Text Available Ultrathin square cell Haloquadratum walsbyi from the Archaea domain are the most abundant microorganisms in the hypersaline water of coastal salterns and continental salt lakes. In this work, we explore the cell surface of these microorganisms using amplitude-modulation atomic-force microscopy in nearly physiological conditions. We demonstrate the presence of a regular corrugation with a periodicity of 16-20 nm attributed to the surface layer (S-layer protein lattice, striped domains asymmetrically distributed on the cell faces and peculiar bulges correlated with the presence of intracellular granules. Besides, subsequent images of cell evolution during the drying process indicate the presence of an external capsule that might correspond to the giant protein halomucin, predicted by the genome but never before observed by other microscopy studies.

  14. An intron within the 16S ribosomal RNA gene of the archaeon Pyrobaculum aerophilum

    Science.gov (United States)

    Burggraf, S.; Larsen, N.; Woese, C. R.; Stetter, K. O.

    1993-01-01

    The 16S rRNA genes of Pyrobaculum aerophilum and Pyrobaculum islandicum were amplified by the polymerase chain reaction, and the resulting products were sequenced directly. The two organisms are closely related by this measure (over 98% similar). However, they differ in that the (lone) 16S rRNA gene of Pyrobaculum aerophilum contains a 713-bp intron not seen in the corresponding gene of Pyrobaculum islandicum. To our knowledge, this is the only intron so far reported in the small subunit rRNA gene of a prokaryote. Upon excision the intron is circularized. A secondary structure model of the intron-containing rRNA suggests a splicing mechanism of the same type as that invoked for the tRNA introns of the Archaea and Eucarya and 23S rRNAs of the Archaea. The intron contains an open reading frame whose protein translation shows no certain homology with any known protein sequence.

  15. Defining the topology of the N-glycosylation pathway in the halophilic archaeon Haloferax volcanii.

    Science.gov (United States)

    Plavner, Noa; Eichler, Jerry

    2008-12-01

    In Eukarya, N glycosylation involves the actions of enzymes working on both faces of the endoplasmic reticulum membrane. The steps of bacterial N glycosylation, in contrast, transpire essentially on the cytoplasmic side of the plasma membrane, with only transfer of the assembled glycan to the target protein occurring on the external surface of the cell. For Archaea, virtually nothing is known about the topology of enzymes involved in assembling those glycans that are subsequently N linked to target proteins on the external surface of the cell. To remedy this situation, subcellular localization and topology predictive algorithms, protease accessibility, and immunoblotting, together with cysteine modification following site-directed mutagenesis, were enlisted to define the topology of Haloferax volcanii proteins experimentally proven to participate in the N-glycosylation process. AglJ and AglD, involved in the earliest and latest stages, respectively, of assembly of the pentasaccharide decorating the H. volcanii S-layer glycoprotein, were shown to present their soluble N-terminal domain, likely containing the putative catalytic site of each enzyme, to the cytosol. The same holds true for Alg5-B, Dpm1-A, and Mpg1-D, proteins putatively involved in this posttranslational event. The results thus point to the assembly of the pentasaccharide linked to certain Asn residues of the H. volcanii S-layer glycoprotein as occurring within the cell.

  16. Defining the Topology of the N-Glycosylation Pathway in the Halophilic Archaeon Haloferax volcanii▿

    OpenAIRE

    Plavner, Noa; Eichler, Jerry

    2008-01-01

    In Eukarya, N glycosylation involves the actions of enzymes working on both faces of the endoplasmic reticulum membrane. The steps of bacterial N glycosylation, in contrast, transpire essentially on the cytoplasmic side of the plasma membrane, with only transfer of the assembled glycan to the target protein occurring on the external surface of the cell. For Archaea, virtually nothing is known about the topology of enzymes involved in assembling those glycans that are subsequently N linked to ...

  17. Structural characterization of the N-linked pentasaccharide decorating glycoproteins of the halophilic archaeon Haloferax volcanii.

    Science.gov (United States)

    Kandiba, Lina; Lin, Chia-Wei; Aebi, Markus; Eichler, Jerry; Guerardel, Yann

    2016-07-01

    N-Glycosylation is a post-translational modification performed in all three domains of life. In the halophilic archaea Haloferax volcanii, glycoproteins such as the S-layer glycoprotein are modified by an N-linked pentasaccharide assembled by a series of Agl (archaeal glycosylation) proteins. In the present study, mass spectrometry (MS) and nuclear magnetic resonance spectroscopy were used to define the structure of this glycan attached to at least four of the seven putative S-layer glycoprotein N-glycosylation sites, namely Asn-13, Asn-83, Asn-274 and Asn-279. Such approaches detected a trisaccharide corresponding to glucuronic acid (GlcA)-β1,4-GlcA-β1,4-glucose-β1-Asn, a tetrasaccharide corresponding to methyl-O-4-GlcA-β-1,4-galacturonic acid-α1,4-GlcA-β1,4-glucose-β1-Asn, and a pentasaccharide corresponding to hexose-1,2-[methyl-O-4-]GlcA-β-1,4-galacturonic acid-α1,4-GlcA-β1,4-glucose-β1-Asn, with previous MS and radiolabeling experiments showing the hexose at the non-reducing end of the pentasaccharide to be mannose. The present analysis thus corrects the earlier assignment of the penultimate sugar as a methyl ester of a hexuronic acid, instead revealing this sugar to be a methylated GlcA. The assignments made here are in good agreement with what was already known of the Hfx. volcanii N-glycosylation pathway from previous genetic and biochemical efforts while providing new insight into the process.

  18. Protein modification in archaeon%古菌蛋白质修饰研究进展

    Institute of Scientific and Technical Information of China (English)

    卢化; 金城

    2014-01-01

    20世纪50年代中期,在古菌的表层(S-层)首次发现了糖蛋白;21世纪初又在空肠弯曲菌(Campylobacter jejuni)中发现了蛋白质N-糖基化修饰.由此,同行开始认识到,蛋白质的糖基化修饰广泛存在于古菌、细菌及真核生物三域中.近十年来,古菌蛋白质糖基化修饰的研究取得了进展,特别是古菌蛋白质N-糖基化修饰研究进展快速.但对古菌糖蛋白O-糖基化修饰和脂修饰的了解甚少.本文综述了古菌蛋白质糖基化修饰的研究进展.

  19. Crystallization of [Fe4S3]-ferredoxin from the hyperthermophile archaeon pyrococcus furiosus

    DEFF Research Database (Denmark)

    Nielsen, Michael Ericsson Skovbo; Harris, Pernille; Christensen, Hans Erik Mølager

    2003-01-01

    Recombinant Pyrococcus furiosus ferredoxin with a [Fe3S4]-cluster was crystallized through steps of optimization and X-ray diffraction data were collected from several crystal forms. Flat plate-like crystals were grown by hanging-drop vapour diffusion. The precipitant used was 30% PEG 400; the p...

  20. Pcal_1699, an extremely thermostable malate dehydrogenase from hyperthermophilic archaeon Pyrobaculum calidifontis.

    Science.gov (United States)

    Gharib, Ghazaleh; Rashid, Naeem; Bashir, Qamar; Gardner, Qura-Tul Ann Afza; Akhtar, Muhammad; Imanaka, Tadayuki

    2016-01-01

    Two malate dehydrogenase homologs, Pcal_0564 and Pcal_1699, have been found in the genome of Pyrobaculum calidifontis. The gene encoding Pcal_1699 consisted of 927 nucleotides corresponding to a polypeptide of 309 amino acids. To examine the properties of Pcal_1699, the structural gene was cloned, expressed in Escherichia coli and the purified gene product was characterized. Pcal_1699 was NADH specific enzyme exhibiting a high malate dehydrogenase activity (886 U/mg) at optimal pH (10) and temperature (90 °C). Unfolding studies suggested that urea could not induce complete unfolding and inactivation of Pcal_1699 even at a final concentration of 8 M; however, in the presence of 4 M guanidine hydrochloride enzyme structure was unfolded with complete loss of enzyme activity. Thermostability experiments revealed that Pcal_1699 is the most thermostable malate dehydrogenase, reported to date, retaining more than 90 % residual activity even after heating for 6 h in boiling water.

  1. The complete genome sequence of Haloferax volcanii DS2, a model archaeon.

    Directory of Open Access Journals (Sweden)

    Amber L Hartman

    Full Text Available BACKGROUND: Haloferax volcanii is an easily culturable moderate halophile that grows on simple defined media, is readily transformable, and has a relatively stable genome. This, in combination with its biochemical and genetic tractability, has made Hfx. volcanii a key model organism, not only for the study of halophilicity, but also for archaeal biology in general. METHODOLOGY/PRINCIPAL FINDINGS: We report here the sequencing and analysis of the genome of Hfx. volcanii DS2, the type strain of this species. The genome contains a main 2.848 Mb chromosome, three smaller chromosomes pHV1, 3, 4 (85, 438, 636 kb, respectively and the pHV2 plasmid (6.4 kb. CONCLUSIONS/SIGNIFICANCE: The completed genome sequence, presented here, provides an invaluable tool for further in vivo and in vitro studies of Hfx. volcanii.

  2. Identifying Potential Mechanisms Enabling Acidophily in the Ammonia-Oxidizing Archaeon

    NARCIS (Netherlands)

    Lehtovirta-Morley, L.E.; Sayavedra-Soto, L.A.; Gallois, N.; Schouten, S.; Stein, L.Y.; Prosser, J.I.; Nicol, G.W.

    2016-01-01

    Ammonia oxidation is the first and rate-limiting step in nitrification and is dominated by two distinct groups of microorganismsin soil: ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB). AOA are often more abundant than AOBand dominate activity in acid soils. The mechanism of amm

  3. The ABC of ABC-transport in the hyperthermophilic archaeon Pyrococcus furiosus

    NARCIS (Netherlands)

    Koning, S

    2003-01-01

    Living organisms of our earth can be divided into two groups, the prokaryotes and the eukaryotes. Eukaryotic cells have a nucleus, a special compartment in the cell, where the genetic material, the DNA is located. The DNA in the prokaryotic cell is floating freely in the cell. The eukaryotes, that i

  4. NADP-Dependent Aldehyde Dehydrogenase from Archaeon Pyrobaculum sp.1860: Structural and Functional Features

    Directory of Open Access Journals (Sweden)

    Ekaterina Yu. Bezsudnova

    2016-01-01

    Full Text Available We present the functional and structural characterization of the first archaeal thermostable NADP-dependent aldehyde dehydrogenase AlDHPyr1147. In vitro, AlDHPyr1147 catalyzes the irreversible oxidation of short aliphatic aldehydes at 60–85°С, and the affinity of AlDHPyr1147 to the NADP+ at 60°С is comparable to that for mesophilic analogues at 25°С. We determined the structures of the apo form of AlDHPyr1147 (3.04 Å resolution, three binary complexes with the coenzyme (1.90, 2.06, and 2.19 Å, and the ternary complex with the coenzyme and isobutyraldehyde as a substrate (2.66 Å. The nicotinamide moiety of the coenzyme is disordered in two binary complexes, while it is ordered in the ternary complex, as well as in the binary complex obtained after additional soaking with the substrate. AlDHPyr1147 structures demonstrate the strengthening of the dimeric contact (as compared with the analogues and the concerted conformational flexibility of catalytic Cys287 and Glu253, as well as Leu254 and the nicotinamide moiety of the coenzyme. A comparison of the active sites of AlDHPyr1147 and dehydrogenases characterized earlier suggests that proton relay systems, which were previously proposed for dehydrogenases of this family, are blocked in AlDHPyr1147, and the proton release in the latter can occur through the substrate channel.

  5. Quantitative proteome and transcriptome analysis of the archaeon Thermoplasma acidophilum cultured under aerobic and anaerobic conditions.

    Science.gov (United States)

    Sun, Na; Pan, Cuiping; Nickell, Stephan; Mann, Matthias; Baumeister, Wolfgang; Nagy, István

    2010-09-03

    A comparative proteome and transcriptome analysis of Thermoplasma acidophilum cultured under aerobic and anaerobic conditions has been performed. One-thousand twenty-five proteins were identified covering 88% of the cytosolic proteome. Using a label-free quantitation method, we found that approximately one-quarter of the identified proteome (263 proteins) were significantly induced (>2 fold) under anaerobic conditions. Thirty-nine macromolecular complexes were identified, of which 28 were quantified and 15 were regulated under anaerobiosis. In parallel, a whole genome cDNA microarray analysis was performed showing that the expression levels of 445 genes were influenced by the absence of oxygen. Interestingly, more than 40% of the membrane protein-encoding genes (145 out of 335 ORFs) were up- or down-regulated at the mRNA level. Many of these proteins are functionally associated with extracellular protein or peptide degradation or ion and amino acid transport. Comparison of the transcriptome and proteome showed only a weak positive correlation between mRNA and protein expression changes, which is indicative of extensive post-transcriptional regulatory mechanisms in T. acidophilum. Integration of transcriptomics and proteomics data generated hypotheses for physiological adaptations of the cells to anaerobiosis, and the quantitative proteomics data together with quantitative analysis of protein complexes provide a platform for correlation of MS-based proteomics studies with cryo-electron tomography-based visual proteomics approaches.

  6. Genetic examination of initial amino acid oxidation and glutamate catabolism in the hyperthermophilic archaeon Thermococcus kodakarensis.

    Science.gov (United States)

    Yokooji, Yuusuke; Sato, Takaaki; Fujiwara, Shinsuke; Imanaka, Tadayuki; Atomi, Haruyuki

    2013-05-01

    Amino acid catabolism in Thermococcales is presumed to proceed via three steps: oxidative deamination of amino acids by glutamate dehydrogenase (GDH) or aminotransferases, oxidative decarboxylation by 2-oxoacid:ferredoxin oxidoreductases (KOR), and hydrolysis of acyl-coenzyme A (CoA) by ADP-forming acyl-CoA synthetases (ACS). Here, we performed a genetic examination of enzymes involved in Glu catabolism in Thermococcus kodakarensis. Examination of amino acid dehydrogenase activities in cell extracts of T. kodakarensis KUW1 (ΔpyrF ΔtrpE) revealed high NADP-dependent GDH activity, along with lower levels of NAD-dependent activity. NADP-dependent activities toward Gln/Ala/Val/Cys and an NAD-dependent threonine dehydrogenase activity were also detected. In KGDH1, a gene disruption strain of T. kodakarensis GDH (Tk-GDH), only threonine dehydrogenase activity was detected, indicating that all other activities were dependent on Tk-GDH. KGDH1 could not grow in a medium in which growth was dependent on amino acid catabolism, implying that Tk-GDH is the only enzyme that can discharge the electrons (to NADP(+)/NAD(+)) released from amino acids in their oxidation to 2-oxoacids. In a medium containing excess pyruvate, KGDH1 displayed normal growth, but higher degrees of amino acid catabolism were observed compared to those for KUW1, suggesting that Tk-GDH functions to suppress amino acid oxidation and plays an anabolic role under this condition. We further constructed disruption strains of 2-oxoglutarate:ferredoxin oxidoreductase and succinyl-CoA synthetase. The two strains displayed growth defects in both media compared to KUW1. Succinate generation was not observed in these strains, indicating that the two enzymes are solely responsible for Glu catabolism among the multiple KOR and ACS enzymes in T. kodakarensis.

  7. Association of a multi-synthetase complex with translating ribosomes in the archaeon Thermococcus kodakarensis

    DEFF Research Database (Denmark)

    Raina, Medha; Elgamal, Sara; Santangelo, Thomas J;

    2012-01-01

    that components of the archaeal protein synthesis machinery associate into macromolecular assemblies in vivo and provide the potential to increase translation efficiency by limiting substrate diffusion away from the ribosome, thus facilitating rapid recycling of tRNAs. STRUCTURED SUMMARY OF PROTEIN INTERACTIONS...... with several other factors involved in protein synthesis, suggesting that MSCs may interact directly with translating ribosomes. In support of this hypothesis, the aminoacyl-tRNA synthetase (aaRS) activities of the MSC were enriched in isolated T. kodakarensis polysome fractions. These data indicate......)-triphosphatase 205, thiamine monophosphate kinase 179, pyruvate formate lyase family activating protein 298, 3-hydroxy-3-methylglutaryl-CoA reductase (mevanolate), N(2), N(2)-dimethylguanosine tRNA methyltransferase 145, N2, N2-dimethylguanosine tRNA methyltransferase 170, putative 5-methylcytosine restriction...

  8. Did group II intron proliferation in an endosymbiont-bearing archaeon create eukaryotes?

    Directory of Open Access Journals (Sweden)

    Poole Anthony M

    2006-12-01

    Full Text Available Abstract Martin & Koonin recently proposed that the eukaryote nucleus evolved as a quality control mechanism to prevent ribosome readthrough into introns. In their scenario, the bacterial ancestor of mitochondria was resident in an archaeal cell, and group II introns (carried by the fledgling mitochondrion inserted into coding regions in the archaeal host genome. They suggest that if transcription and translation were coupled, and because splicing is expected to have been slower than translation, the effect of insertion would have been ribosome readthrough into introns, resulting in production of aberrant proteins. The emergence of the nuclear compartment would thus have served to separate transcription and splicing from translation, thereby alleviating this problem. In this article, I argue that Martin & Koonin's model is not compatible with current knowledge. The model requires that group II introns would spread aggressively through an archaeal genome. It is well known that selfish elements can spread through an outbreeding sexual population despite a substantial fitness cost to the host. The same is not true for asexual lineages however, where both theory and observation argue that such elements will be under pressure to reduce proliferation, and may be lost completely. The recent introduction of group II introns into archaea by horizontal transfer provides a natural test case with which to evaluate Martin & Koonin's model. The distribution and behaviour of these introns fits prior theoretical expectations, not the scenario of aggressive proliferation advocated by Martin & Koonin. I therefore conclude that the mitochondrial seed hypothesis for the origin of eukaryote introns, on which their model is based, better explains the early expansion of introns in eukaryotes. The mitochondrial seed hypothesis has the capacity to separate the origin of eukaryotes from the origin of introns, leaving open the possibility that the cell that engulfed the ancestor of mitochondria was a sexually outcrossing eukaryote cell.

  9. Deletion of cdvB paralogous genes of Sulfolobus acidocaldarius impairs cell division

    NARCIS (Netherlands)

    Yang, Nuan; Driessen, Arnold J.M.

    2014-01-01

    The majority of Crenarchaeota utilize the cell division system (Cdv) to divide. This system consists of three highly conserved genes, cdvA, cdvB and cdvC that are organized in an operon. CdvC is homologous to the AAA-type ATPase Vps4, involved in multivesicular body biogenesis in eukaryotes. CdvA is

  10. Biological upgrading of coal liquids. [Thiobacillus thiooxidans, rhodococcus rhodochorous, Sulfolobus acidocaldarius

    Energy Technology Data Exchange (ETDEWEB)

    1992-01-01

    The purpose of this report is to present the results of culture selection studies for the removal of heteroatom compounds from coal liquids. A variety of pure cultures have been selected based upon a comprehensive literature review. In addition, cultures are being isolated from natural sources. Synthetic heteroatom compounds are presently being utilized in the degradation studies until the Environmental Assessment Questionnaire is approved. (VC)

  11. Investigation of the potential of Sulfolobus acidocaldarius to transform dibenzothiophene: Final report

    Energy Technology Data Exchange (ETDEWEB)

    Krawiec, S.; Montenecourt, B.S.

    1986-12-31

    Archaebacteria including S. acidocaldarius appear genetically unstable and while some cultures of S. acidocaldarius may have the capacity to transform DBT the results remain unclear. A legitimate interpretation from the observations presented in this Report is that the phenotypic character of S. acidocaldarius changed during the course of the project. Because the environment in which the organism was subcultured was constant, the differences in phenotype would appear to be genetic. Culturing in the presence of 2% pristane plus DBT may be a sufficiently powerful form of selection to account for the apparent shift from DBT-sensitivity to DBT-adaptation and then from DBT-adaptation to DBT-resistance. However, any genetic change might be enhanced by an astonishing hypervariability which may possess uniquely archaebacterial features. The evidence for hypermutability comes largely from the Halobacteria. Any desulfurization process involving an Archaebacterium should include explicit means of maintaining and demonstrating the phenotypic stability of the organism which mediates the desired process. The possibility that S. acidocaldarius can degrade DBT remains tentative. 24 refs., 22 figs., 5 tabs.

  12. ESCRT-III mediated cell division in Sulfolobus acidocaldarius –A reconstitution perspective

    Directory of Open Access Journals (Sweden)

    Tobias eHärtel

    2014-06-01

    Full Text Available In the framework of Synthetic Biology, it has become an intriguing question what would be the minimal representation of cell division machinery. Thus, it seems appropriate to compare how cell division is realized in different microorganisms. In particular, the cell division system of Crenarchaeota lacks certain proteins found in most bacteria and Euryarchaeota, such as FtsZ, MreB or the Min system. The Sulfolobaceae family encodes functional homologs of the eukaryotic proteins Vps4 and ESCRT-III. ESCRT-III is essential for several eukaryotic pathways, e.g. budding of intralumenal vesicles (ILVs, or cytokinesis, whereas Vps4 dissociates the ESCRT-III complex from the membrane. CdvA (Cell Division A is required for the recruitment of crenarchaeal ESCRT-III proteins to the membrane at mid-cell. The proteins polymerize and form a smaller structure during constriction. Thus, ESCRT-III mediated cell division in S. acidocaldarius shows functional analogies to the Z ring observed in prokaryotes like E. coli, which has recently begun to be reconstituted in vitro. In this short perspective, we discuss the possibility of building such an in vitro cell division system on basis of archaeal ESCRT-III.

  13. Strong Paramagnetism of Gold Nanoparticles Deposited on a Sulfolobus acidocaldarius S Layer

    Science.gov (United States)

    Bartolomé, J.; Bartolomé, F.; García, L. M.; Figueroa, A. I.; Repollés, A.; Martínez-Pérez, M. J.; Luis, F.; Magén, C.; Selenska-Pobell, S.; Pobell, F.; Reitz, T.; Schönemann, R.; Herrmannsdörfer, T.; Merroun, M.; Geissler, A.; Wilhelm, F.; Rogalev, A.

    2012-12-01

    Magnetic properties of Au nanoparticles deposited on an archaeal S layer are reported. X-ray magnetic circular dichroism and superconducting quantum interference device magnetometries demonstrate that the particles are strongly paramagnetic, without any indication of magnetic blocking down to 16 mK. The average magnetic moment per particle is Mpart=2.36(7) μB. This contribution originates at the particle’s Au 5d band, in which an increased number of holes with respect to the bulk value is observed. The magnetic moment per Au atom is 25 times larger than any measured in other Au nanoparticles or any other configurations up to date.

  14. Comparative study of the extracellular proteome of Sulfolobus species reveals limited secretion

    NARCIS (Netherlands)

    Ellen, Albert F.; Albers, Sonja-Verena; Driessen, Arnold J. M.

    2010-01-01

    Although a large number of potentially secreted proteins can be predicted on the basis of genomic distribution of signal sequence-bearing proteins, protein secretion in Archaea has barely been studied. A proteomic inventory and comparison of the growth medium proteins in three hyperthermoacidophiles

  15. Structural properties of prokaryotic promoter regions correlate with functional features.

    Directory of Open Access Journals (Sweden)

    Pieter Meysman

    Full Text Available The structural properties of the DNA molecule are known to play a critical role in transcription. In this paper, the structural profiles of promoter regions were studied within the context of their diversity and their function for eleven prokaryotic species; Escherichia coli, Klebsiella pneumoniae, Salmonella Typhimurium, Pseudomonas auroginosa, Geobacter sulfurreducens Helicobacter pylori, Chlamydophila pneumoniae, Synechocystis sp., Synechoccocus elongates, Bacillus anthracis, and the archaea Sulfolobus solfataricus. The main anchor point for these promoter regions were transcription start sites identified through high-throughput experiments or collected within large curated databases. Prokaryotic promoter regions were found to be less stable and less flexible than the genomic mean across all studied species. However, direct comparison between species revealed differences in their structural profiles that can not solely be explained by the difference in genomic GC content. In addition, comparison with functional data revealed that there are patterns in the promoter structural profiles that can be linked to specific functional loci, such as sigma factor regulation or transcription factor binding. Interestingly, a novel structural element clearly visible near the transcription start site was found in genes associated with essential cellular functions and growth in several species. Our analyses reveals the great diversity in promoter structural profiles both between and within prokaryotic species. We observed relationships between structural diversity and functional features that are interesting prospects for further research to yet uncharacterized functional loci defined by DNA structural properties.

  16. Spectroscopic investigation of the reaction mechanism of CopB-B, the catalytic fragment from an archaeal thermophilic ATP-driven heavy metal transporter.

    Science.gov (United States)

    Völlmecke, Christian; Kötting, Carsten; Gerwert, Klaus; Lübben, Mathias

    2009-11-01

    The mechanism of ATP hydrolysis of a shortened variant of the heavy metal-translocating P-type ATPase CopB of Sulfolobus solfataricus was studied. The catalytic fragment, named CopB-B, comprises the nucleotide binding and phosphorylation domains. We demonstrated stoichiometric high-affinity binding of one nucleotide to the protein (K(diss) 1-20 microm). Mg is not necessary for nucleotide association but is essential for the phosphatase activity. Binding and hydrolysis of ATP released photolytically from the caged precursor nitrophenylethyl-ATP was measured at 30 degrees C by infrared spectroscopy, demonstrating that phosphate groups are not involved in nucleotide binding. The hydrolytic kinetics was biphasic, and provides evidence for at least one reaction intermediate. Modelling of the forward reaction gave rise to three kinetic states connected by two intrinsic rate constants. The lower kinetic constant (k(1) = 4.7 x 10(-3) s(-1) at 30 degrees C) represents the first and rate-limiting reaction, probably reflecting the transition between the open and closed conformations of the domain pair. The subsequent step has a faster rate (k(2) = 17 x 10(-3) s(-1) at 30 degrees C), leading to product formation. Although the latter appears to be a single step, it probably comprises several reactions with presently unresolved intermediates. Based on these data, we suggest a model of the hydrolytic mechanism.

  17. COV2HTML: a visualization and analysis tool of bacterial next generation sequencing (NGS) data for postgenomics life scientists.

    Science.gov (United States)

    Monot, Marc; Orgeur, Mickael; Camiade, Emilie; Brehier, Clément; Dupuy, Bruno

    2014-03-01

    COV2HTML is an interactive web interface, which is addressed to biologists, and allows performing both coverage visualization and analysis of NGS alignments performed on prokaryotic organisms (bacteria and phages). It combines two processes: a tool that converts the huge NGS mapping or coverage files into light specific coverage files containing information on genetic elements; and a visualization interface allowing a real-time analysis of data with optional integration of statistical results. To demonstrate the scope of COV2HTML, the program was tested with data from two published studies. The first data were from RNA-seq analysis of Campylobacter jejuni, based on comparison of two conditions with two replicates. We were able to recover 26 out of 27 genes highlighted in the publication using COV2HTML. The second data comprised of stranded TSS and RNA-seq data sets on the Archaea Sulfolobus solfataricus. COV2HTML was able to highlight most of the TSSs from the article and allows biologists to visualize both TSS and RNA-seq on the same screen. The strength of the COV2HTML interface is making possible NGS data analysis without software installation, login, or a long training period. A web version is accessible at https://mmonot.eu/COV2HTML/ . This website is free and open to users without any login requirement.

  18. Characterization of Fatty Acids in Crenarchaeota by GC-MS and NMR

    Directory of Open Access Journals (Sweden)

    Timothy Hamerly

    2015-01-01

    Full Text Available Lipids composed of condensed isoprenyl units connected to glycerol backbones by ether linkages are a distinguishing feature of Archaea. Data suggesting that fatty acids with linear hydrocarbon chains are present in some Archaea have been available for decades. However, lack of genomic and biochemical evidence for the metabolic machinery required to synthesize and degrade fatty acids has left the field unclear on this potentially significant biochemical aspect. Because lipids are energy currency and cell signaling molecules, their presence in Archaea is significant for understanding archaeal biology. A recent large-scale bioinformatics analysis reignited the debate as to the importance of fatty acids in Archaea by presenting genetic evidence for the presence of enzymes required for anabolic and catabolic fatty acid metabolism across the archaeal domain. Here, we present direct biochemical evidence from gas chromatography-mass spectrometry (GC-MS and nuclear magnetic resonance (NMR spectroscopy for the presence of fatty acids in two members of the Crenarchaeota, Sulfolobus solfataricus and Ignicoccus hospitalis. This is the first report providing biochemical data for the existence of fatty acids in these Crenarchaeota, opening new discussions on energy balance and the potential for the discovery of new thermostable enzymes for industry.

  19. Structure of a dimeric crenarchaeal Cas6 enzyme with an atypical active site for CRISPR RNA processing

    Science.gov (United States)

    Reeks, Judith; Sokolowski, Richard D.; Graham, Shirley; Liu, Huanting; Naismith, James H.; White, Malcolm F.

    2013-01-01

    The competition between viruses and hosts is played out in all branches of life. Many prokaryotes have an adaptive immune system termed ‘CRISPR’ (clustered regularly interspaced short palindromic repeats) which is based on the capture of short pieces of viral DNA. The captured DNA is integrated into the genomic DNA of the organism flanked by direct repeats, transcribed and processed to generate crRNA (CRISPR RNA) that is loaded into a variety of effector complexes. These complexes carry out sequence-specific detection and destruction of invading mobile genetic elements. In the present paper, we report the structure and activity of a Cas6 (CRISPR-associated 6) enzyme (Sso1437) from Sulfolobus solfataricus responsible for the generation of unit-length crRNA species. The crystal structure reveals an unusual dimeric organization that is important for the enzyme's activity. In addition, the active site lacks the canonical catalytic histidine residue that has been viewed as an essential feature of the Cas6 family. Although several residues contribute towards catalysis, none is absolutely essential. Coupled with the very low catalytic rate constants of the Cas6 family and the plasticity of the active site, this suggests that the crRNA recognition and chaperone-like activities of the Cas6 family should be considered as equal to or even more important than their role as traditional enzymes. PMID:23527601

  20. Structure and regulatory role of the C-terminal winged helix domain of the archaeal minichromosome maintenance complex

    Science.gov (United States)

    Wiedemann, Christoph; Szambowska, Anna; Häfner, Sabine; Ohlenschläger, Oliver; Gührs, Karl-Heinz; Görlach, Matthias

    2015-01-01

    The minichromosome maintenance complex (MCM) represents the replicative DNA helicase both in eukaryotes and archaea. Here, we describe the solution structure of the C-terminal domains of the archaeal MCMs of Sulfolobus solfataricus (Sso) and Methanothermobacter thermautotrophicus (Mth). Those domains consist of a structurally conserved truncated winged helix (WH) domain lacking the two typical ‘wings’ of canonical WH domains. A less conserved N-terminal extension links this WH module to the MCM AAA+ domain forming the ATPase center. In the Sso MCM this linker contains a short α-helical element. Using Sso MCM mutants, including chimeric constructs containing Mth C-terminal domain elements, we show that the ATPase and helicase activity of the Sso MCM is significantly modulated by the short α-helical linker element and by N-terminal residues of the first α-helix of the truncated WH module. Finally, based on our structural and functional data, we present a docking-derived model of the Sso MCM, which implies an allosteric control of the ATPase center by the C-terminal domain. PMID:25712103

  1. Characterization of Fatty Acids in Crenarchaeota by GC-MS and NMR.

    Science.gov (United States)

    Hamerly, Timothy; Tripet, Brian; Wurch, Louie; Hettich, Robert L; Podar, Mircea; Bothner, Brian; Copié, Valérie

    2015-01-01

    Lipids composed of condensed isoprenyl units connected to glycerol backbones by ether linkages are a distinguishing feature of Archaea. Data suggesting that fatty acids with linear hydrocarbon chains are present in some Archaea have been available for decades. However, lack of genomic and biochemical evidence for the metabolic machinery required to synthesize and degrade fatty acids has left the field unclear on this potentially significant biochemical aspect. Because lipids are energy currency and cell signaling molecules, their presence in Archaea is significant for understanding archaeal biology. A recent large-scale bioinformatics analysis reignited the debate as to the importance of fatty acids in Archaea by presenting genetic evidence for the presence of enzymes required for anabolic and catabolic fatty acid metabolism across the archaeal domain. Here, we present direct biochemical evidence from gas chromatography-mass spectrometry (GC-MS) and nuclear magnetic resonance (NMR) spectroscopy for the presence of fatty acids in two members of the Crenarchaeota, Sulfolobus solfataricus and Ignicoccus hospitalis. This is the first report providing biochemical data for the existence of fatty acids in these Crenarchaeota, opening new discussions on energy balance and the potential for the discovery of new thermostable enzymes for industry.

  2. Solution Structure of Archaeoglobus fulgidis Peptidyl-tRNA Hydrolase(Pth2) Provides Evidence for an Extensive Conserved Family of Pth2 Enzymes in Archaea, Bacteria and Eukaryotes.

    Energy Technology Data Exchange (ETDEWEB)

    Powers, Robert; Mirkovic, Nebojsa; Goldsmith-Fischman, Sharon; Acton, Thomas; Chiang, Yiwen; Huang, Yuanpeng; Ma, LiChung; Rajan, Paranji K.; Cort, John R.; Kennedy, Michael A.; Liu, Jinfeng; Rost, Burkhard; Honig, Barry; Murray, Diana; Montelione, Gaetano

    2005-11-01

    The solution structure of protein AF2095 from the thermophilic archaea Archaeglobus fulgidis, a 123-residue (13.6 kDa) protein, has been determined by NMR methods. The structure of AF2095 is comprised of four a-helices and a mixed b-sheet consisting of four parallel and anti-parallel b-strands, where the a-helices sandwich the b-sheet. Sequence and structural comparison of AF2095 with proteins from Homo sapiens, Methanocaldococcus jannaschii and Sulfolobus solfataricus, reveals that AF2095 is a peptidyl-tRNA hydrolase (Pth2). This structural comparison also identifies putative catalytic residues and a tRNA interaction region for AF2095. The structure of AF2095 is also similar to the structure of protein TA0108 from archaea Thermoplasma acidophilum, which is deposited in the Protein Database but not functionally annotated. The NMR structure of AF2095 has been further leveraged to obtain good quality structural models for 55 other proteins. Although earlier studies have proposed that the Pth2 protein family is restricted to archeal and eukaryotic organisms, the similarity of the AF2095 structure to human Pth2, the conservation of key active-site residues, and the good quality of the resulting homology models demonstrate a large family of homologous Pth2 proteins that are conserved in eukaryotic, archaeal and bacterial organisms, providing novel insights in the evolution of the Pth and Pth2 enzyme families.

  3. Structure determination of archaea-specific ribosomal protein L46a reveals a novel protein fold

    Energy Technology Data Exchange (ETDEWEB)

    Feng, Yingang, E-mail: fengyg@qibebt.ac.cn [Shandong Provincial Key Laboratory of Energy Genetics, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, Shandong 266101 (China); Song, Xiaxia [Department of Biological Science and Engineering, School of Chemical and Biological Engineering, University of Science and Technology Beijing, Beijing 100083 (China); Lin, Jinzhong [National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101 (China); Xuan, Jinsong [Department of Biological Science and Engineering, School of Chemical and Biological Engineering, University of Science and Technology Beijing, Beijing 100083 (China); Cui, Qiu [Shandong Provincial Key Laboratory of Energy Genetics, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, Shandong 266101 (China); Wang, Jinfeng [National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101 (China)

    2014-07-18

    Highlights: • The archaea-specific ribosomal protein L46a has no homology to known proteins. • Three dimensional structure and backbone dynamics of L46a were determined by NMR. • The structure of L46a represents a novel protein fold. • A potential rRNA-binding surface on L46a was identified. • The potential position of L46a on the ribosome was proposed. - Abstract: Three archaea-specific ribosomal proteins recently identified show no sequence homology with other known proteins. Here we determined the structure of L46a, the most conserved one among the three proteins, from Sulfolobus solfataricus P2 using NMR spectroscopy. The structure presents a twisted β-sheet formed by the N-terminal part and two helices at the C-terminus. The L46a structure has a positively charged surface which is conserved in the L46a protein family and is the potential rRNA-binding site. Searching homologous structures in Protein Data Bank revealed that the structure of L46a represents a novel protein fold. The backbone dynamics identified by NMR relaxation experiments reveal significant flexibility at the rRNA binding surface. The potential position of L46a on the ribosome was proposed by fitting the structure into a previous electron microscopy map of the ribosomal 50S subunit, which indicated that L46a contacts to domain I of 23S rRNA near a multifunctional ribosomal protein L7ae.

  4. Evolution and thermodynamics of the slow unfolding of hyperstable monomeric proteins

    Directory of Open Access Journals (Sweden)

    Koga Yuichi

    2010-07-01

    Full Text Available Abstract Background The unfolding speed of some hyperthermophilic proteins is dramatically lower than that of their mesostable homologs. Ribonuclease HII from the hyperthermophilic archaeon Thermococcus kodakaraensis (Tk-RNase HII is stabilized by its remarkably slow unfolding rate, whereas RNase HI from the thermophilic bacterium Thermus thermophilus (Tt-RNase HI unfolds rapidly, comparable with to that of RNase HI from Escherichia coli (Ec-RNase HI. Results To clarify whether the difference in the unfolding rate is due to differences in the types of RNase H or differences in proteins from archaea and bacteria, we examined the equilibrium stability and unfolding reaction of RNases HII from the hyperthermophilic bacteria Thermotoga maritima (Tm-RNase HII and Aquifex aeolicus (Aa-RNase HII and RNase HI from the hyperthermophilic archaeon Sulfolobus tokodaii (Sto-RNase HI. These proteins from hyperthermophiles are more stable than Ec-RNase HI over all the temperature ranges examined. The observed unfolding speeds of all hyperstable proteins at the different denaturant concentrations studied are much lower than those of Ec-RNase HI, which is in accordance with the familiar slow unfolding of hyperstable proteins. However, the unfolding rate constants of these RNases H in water are dispersed, and the unfolding rate constant of thermophilic archaeal proteins is lower than that of thermophilic bacterial proteins. Conclusions These results suggest that the nature of slow unfolding of thermophilic proteins is determined by the evolutionary history of the organisms involved. The unfolding rate constants in water are related to the amount of buried hydrophobic residues in the tertiary structure.

  5. Role of the N terminus in enzyme activity, stability and specificity in thermophilic esterases belonging to the HSL family.

    Science.gov (United States)

    Mandrich, Luigi; Merone, Luigia; Pezzullo, Margherita; Cipolla, Laura; Nicotra, Francesco; Rossi, Mosè; Manco, Giuseppe

    2005-01-21

    A superposition between the structures of Alicyclobacillus acidocaldarius esterase 2 (EST2) and Burkholderia cepacia lipase, the latter complexed with a phosphonate inhibitor, allowed us to hypothesize for the EST2 N terminus a role in restricting the access to the active site and therefore in modulating substrate specificity. In order to test this hypothesis we generated by site-directed mutagenesis some truncated versions of EST2 and its double mutant M211S/R215L (S/L) at the N terminus. In parallel, an analysis of the Sulfolobus solfataricus P2 genome allowed us to identify a gene coding for a putative esterase of the HSL family having a natural deletion of the corresponding region. The product of this gene and the above-mentioned EST2 mutants were expressed in Escherichia coli, purified and characterised. These studies support the notion that the N terminus affects substrate specificity other than several other enzyme parameters. Although the deletions afforded a tenfold and 550-fold decrease in catalytic efficiency towards the best substrate pNP-hexanoate at 50 degrees C for EST2 and S/L, respectively, the analysis of the specific activities with different triacylglycerols with respect to pNP-hexanoate showed that their ratios were higher for deleted versus non-deleted enzymes, on all tested substrates. In particular, the above ratios for glyceryl tridecanoate were 30-fold and 14-fold higher in S/L and EST2 deleted forms, respectively, compared with their full-length versions. This behaviour was confirmed by the analysis of the S.solfataricus esterase, which showed similar specific activities on pNP-hexanoate and triacylglycerols; in addition, higher activities on the latter substrates were observed in comparison with EST2, S/L and their deleted forms. Finally, a dramatic effect on thermophilicity and thermostability in the EST2 deleted forms was observed. This is the first report highlighting the importance of the "cap" domain in the HSL family, since the N

  6. Biochemical and Enzymatic Characterization of a Thermostable DNA Ligase Encoded by Thermophilic Acidophilic Archaebacterium Strain JP2%一种嗜热耐酸古细菌JP2菌株编码的热稳定DNA连接酶的生物化学及酶学特性研究

    Institute of Scientific and Technical Information of China (English)

    兰海燕; 刘纯; HENDRY PHIL

    2006-01-01

    A thermostable DNA ligase gene was identified, and the biochemical and enzymatic properties of the ligase were characterized from JP2 strain which was enriched from geothermally active sites in Papua New Guinea. The nucleotide and amino acid sequences showed much high identities compared with that of archeabacterium species Sulfolobus solfataricus and Sulfolobus shibatae,especially in the six conserved motif sequences, which are known to be closely related to the key function of ligase. Recombinant JP2 ligase showed high activity in nick-joining reaction. It was the most active when Mn2+ present as divalent metal cofactor rather than Mg2+ and Ca2+ etc.. Assay of thermostability over a range of temperatures showed that at 50~80℃ the enzyme displayed relative high activity. Further thermostability experiment indicated that the activity of JP2 ligase could last for a long time at 80℃ and 85℃,however, at 90℃ and 95℃, it became unstable quickly. An investigation on the acquired thermotolerance of recombinant JP2 ligase was done by applying a chaperonin known as TF55 in thermophile on JP2 ligase reaction. Result showed that TF55 could not help in improving thermostability of ligase at 85℃. The possible reason might be that at 85℃ in vitro, the chaperonin itself was denatured.%对分离自巴布亚新几内亚地热活跃区的一种嗜热耐酸古细菌--JP2菌株中的DNA连接酶基因进行了克隆、表达、纯化,并对其生物化学及酶学特性进行了研究.对其核酸及氨基酸序列的分析表明:JP2菌株的DNA连接酶与古细菌种Sulfolobus solfataricus和Sulfolobus shibatae的DNA连接酶具有很高的同源性,尤其在与功能紧密相关的6个保守结构基序的一致性更高.JP2连接酶表现出高的DNA缺口连接活性,在二价金属辅因子的选择方面,JP2连接酶更倾向于Mn2+离子而不是Mg2+、Ca2+及其他离子.不同温度时的热稳定性测试显示:JP2连接酶在50~80℃时为较适

  7. A novel type of replicative enzyme harbouring ATPase, primase and DNA polymerase activity

    Science.gov (United States)

    Lipps, Georg; Röther, Susanne; Hart, Christina; Krauss, Gerhard

    2003-01-01

    Although DNA replication is a process common in all domains of life, primase and replicative DNA polymerase appear to have evolved independently in the bacterial domain versus the archaeal/eukaryal branch of life. Here, we report on a new type of replication protein that constitutes the first member of the DNA polymerase family E. The protein ORF904, encoded by the plasmid pRN1 from the thermoacidophile archaeon Sulfolobus islandicus, is a highly compact multifunctional enzyme with ATPase, primase and DNA polymerase activity. Recombinant purified ORF904 hydrolyses ATP in a DNA-dependent manner. Deoxynucleotides are preferentially used for the synthesis of primers ∼8 nucleotides long. The DNA polymerase activity of ORF904 synthesizes replication products of up to several thousand nucleotides in length. The primase and DNA polymerase activity are located in the N-terminal half of the protein, which does not show homology to any known DNA polymerase or primase. ORF904 constitutes a new type of replication enzyme, which could have evolved indepen dently from the eubacterial and archaeal/eukaryal proteins of DNA replication. PMID:12743045

  8. Ultrasensitive biotin assay of a noncompetitive format in a homogeneous solution based on resonance energy transfer induced by a protein-protein interaction.

    Science.gov (United States)

    Ikeda, Tomohiro; Miyao, Hiroki; Sueda, Shinji

    2014-06-17

    Biotin is a water-soluble vitamin serving as a cofactor for several metabolic enzymes and plays crucial roles in every living cell. In the present study, we describe a noncompetitive assay for determination of biotin in a homogeneous solution. Our assay is based on a biotinylation reaction from archaeon Sulfolobus tokodaii. S. tokodaii biotinylation has a unique property that biotin protein ligase (BPL) forms a stable complex with its biotinylated substrate protein (BCCP). Determination of biotin was performed by monitoring the complexation reaction between BPL and BCCP through biotinylation, based on luminescence resonance energy transfer (LRET) from a Tb(3+) complex to fluorescein, where BPL and BCCP were labeled with a Tb(3+) complex and fluorescein, respectively. Our assay allows for ultrasensitive detection of biotin with a detection limit of approximately 1 pM (or 0.2 fmol in a 0.2 mL sample volume) by a simple procedure without use of radioactive materials or enzymatic signal amplification. In addition, owing to its noncompetitive format, our assay has a very wide measurement range of at least 3 orders of magnitude. Our assay is also beneficial as a model system for interaction analysis based on LRET.

  9. Chaperonin Polymers in Archaea: The Cytoskeleton of Prokaryotes?

    Science.gov (United States)

    Trent, J. D.; Kagawa, H. K.; Zaluzec, N. J.

    1997-07-01

    Chaperonins are protein complexes that play a critical role in folding nascent polypeptides under normal conditions and refolding damaged proteins under stress conditions. In all organisms these complexes are composed of evolutionarily conserved 60-kDa proteins arranged in double-ring structures with between 7 and 9 protein subunits per ring. These double ring structures are assumed to be the functional units in vivo, although they have never been observed inside cells. Here the authors show that the purified chaperonin from the hyperthermophilic archaeon Sulfolobus shibatae, which is closely related to chaperonins in eukaryotes, has a double ring structure at low concentrations (0.1 mg/ml), but at more physiological concentrations, the rings stack end to end to form polymers. The polymers are stable at physiological temperatures (75 C) and closely resemble structures observed inside unfixed S. shibatae cells. The authors suggest that in vivo chaperonin activity may be regulated by polymerization and that chaperonin polymers may act as a cytoskeleton-like structure in archaea and bacteria.

  10. Magnetic Au Nanoparticles on Archaeal S-Layer Ghosts as Templates

    Directory of Open Access Journals (Sweden)

    Sonja Selenska-Pobell

    2011-10-01

    Full Text Available Cell‐ghosts representing empty cells of the archaeon Sulfolobus acidocaldarius, consisting only of their highly ordered and unusually stable outermost proteinaceous surface layer (S‐layer, were used as templates for Au nanoparticles fabrication. The properties of these archaeal Au nanoparticles differ significantly from those produced earlier by us onto bacterial S‐layer sheets. The archaeal Au nanoparticles, with a size of about 2.5 nm, consist exclusively of metallic Au(0, while those produced on the bacterial S‐layer had a size of about 4 nm and represented a mixture of Au(0 and Au(III in the ratio of 40 to 60 %. The most impressive feature of the archaeal Au nanoparticles is that they are strongly paramagnetic, in contrast to the bacterial ones and also to bulk gold. SQUID magnetometry and XMCD measurements demonstrated that the archaeal Au nanoparticles possess a rather large magnetic moment of about 0.1 µB/atom. HR‐ TEM‐EDX analysis revealed that the archaeal Au nanoparticles are linked to the sulfur atoms of the thiol groups of the amino acid cysteine, characteristic only for archaeal S‐layers. This is the first study demonstrating the formation of such unusually strong magnetic Au nanoparticles on a non‐modified archaeal S‐layer.

  11. Speculations on the origin of life and thermophily: Review of available information on reverse gyrase suggests that hyperthermophilic procaryotes are not so primitive

    Science.gov (United States)

    Forterre, Patrick; Confalonier, Fabrice; Charbonnier, Franck; Duguet, Michel

    1995-06-01

    All present-day hyperthermophiles studied so far (eitherBacteria orArchaea) contain a unique DNA topoisomerase, reverse gyrase, which probably helps to stabilize genomic DNA at high temperature. Herein the data relating this enzyme is reviewed and discussed from the perspective of the nature of the last detectable common ancestor and the origin of life. The sequence of the gene encoding reverse gyrase from an archaeon,Sulfolobus acidocaldarius, suggests that this enzyme contains both a helicase and a topoisomerase domains (Confalonieriet al.,Proc. Natl. Acad. Sci., 1993, 90, 4735). Accordingly, it has been proposed that reverse gyrase originated by the fusion of DNA helicase and DNA topoisomerase genes. If reverse gyrase is essential for life at high temperature, its composite structure suggests that DNA helicases and topoisomerases appeared independently and first evolved in a mesophilic world. Such scenario contradicts the hypothesis that a direct link connects present day hyperthermophiles to a hot origin of life. We discuss different patterns for the early cellular evolution in which reverse gyrase appeared either before the emergence of the last common ancestor ofArchaea, Bacteria andEucarya, or in a lineage common to the two procaryotic domains. The latter scenario could explain why all today hyperthermophiles are procaryotes.

  12. Preservation of Archaeal Surface Layer Structure During Mineralization

    Science.gov (United States)

    Kish, Adrienne; Miot, Jennyfer; Lombard, Carine; Guigner, Jean-Michel; Bernard, Sylvain; Zirah, Séverine; Guyot, François

    2016-05-01

    Proteinaceous surface layers (S-layers) are highly ordered, crystalline structures commonly found in prokaryotic cell envelopes that augment their structural stability and modify interactions with metals in the environment. While mineral formation associated with S-layers has previously been noted, the mechanisms were unconstrained. Using Sulfolobus acidocaldarius a hyperthermophilic archaeon native to metal-enriched environments and possessing a cell envelope composed only of a S-layer and a lipid cell membrane, we describe a passive process of iron phosphate nucleation and growth within the S-layer of cells and cell-free S-layer “ghosts” during incubation in a Fe-rich medium, independently of metabolic activity. This process followed five steps: (1) initial formation of mineral patches associated with S-layer; (2) patch expansion; (3) patch connection; (4) formation of a continuous mineral encrusted layer at the cell surface; (5) early stages of S-layer fossilization via growth of the extracellular mineralized layer and the mineralization of cytosolic face of the cell membrane. At more advanced stages of encrustation, encrusted outer membrane vesicles are formed, likely in an attempt to remove damaged S-layer proteins. The S-layer structure remains strikingly well preserved even upon the final step of encrustation, offering potential biosignatures to be looked for in the fossil record.

  13. N-linked glycosylation in Archaea: a structural, functional, and genetic analysis.

    Science.gov (United States)

    Jarrell, Ken F; Ding, Yan; Meyer, Benjamin H; Albers, Sonja-Verena; Kaminski, Lina; Eichler, Jerry

    2014-06-01

    N-glycosylation of proteins is one of the most prevalent posttranslational modifications in nature. Accordingly, a pathway with shared commonalities is found in all three domains of life. While excellent model systems have been developed for studying N-glycosylation in both Eukarya and Bacteria, an understanding of this process in Archaea was hampered until recently by a lack of effective molecular tools. However, within the last decade, impressive advances in the study of the archaeal version of this important pathway have been made for halophiles, methanogens, and thermoacidophiles, combining glycan structural information obtained by mass spectrometry with bioinformatic, genetic, biochemical, and enzymatic data. These studies reveal both features shared with the eukaryal and bacterial domains and novel archaeon-specific aspects. Unique features of N-glycosylation in Archaea include the presence of unusual dolichol lipid carriers, the use of a variety of linking sugars that connect the glycan to proteins, the presence of novel sugars as glycan constituents, the presence of two very different N-linked glycans attached to the same protein, and the ability to vary the N-glycan composition under different growth conditions. These advances are the focus of this review, with an emphasis on N-glycosylation pathways in Haloferax, Methanococcus, and Sulfolobus.

  14. Preservation of Archaeal Surface Layer Structure During Mineralization.

    Science.gov (United States)

    Kish, Adrienne; Miot, Jennyfer; Lombard, Carine; Guigner, Jean-Michel; Bernard, Sylvain; Zirah, Séverine; Guyot, François

    2016-05-25

    Proteinaceous surface layers (S-layers) are highly ordered, crystalline structures commonly found in prokaryotic cell envelopes that augment their structural stability and modify interactions with metals in the environment. While mineral formation associated with S-layers has previously been noted, the mechanisms were unconstrained. Using Sulfolobus acidocaldarius a hyperthermophilic archaeon native to metal-enriched environments and possessing a cell envelope composed only of a S-layer and a lipid cell membrane, we describe a passive process of iron phosphate nucleation and growth within the S-layer of cells and cell-free S-layer "ghosts" during incubation in a Fe-rich medium, independently of metabolic activity. This process followed five steps: (1) initial formation of mineral patches associated with S-layer; (2) patch expansion; (3) patch connection; (4) formation of a continuous mineral encrusted layer at the cell surface; (5) early stages of S-layer fossilization via growth of the extracellular mineralized layer and the mineralization of cytosolic face of the cell membrane. At more advanced stages of encrustation, encrusted outer membrane vesicles are formed, likely in an attempt to remove damaged S-layer proteins. The S-layer structure remains strikingly well preserved even upon the final step of encrustation, offering potential biosignatures to be looked for in the fossil record.

  15. Chaperonin filaments: The archael cytoskeleton

    Energy Technology Data Exchange (ETDEWEB)

    Trent, J.D.; Kagawa, H.K.; Yaoi, Takuro; Olle, E.; Zaluzec, N.J.

    1997-08-01

    Chaperonins are multi-subunit double-ring complexed composed of 60-kDa proteins that are believed to mediate protein folding in vivo. The chaperonins in the hyperthermophilic archaeon Sulfolobus shibatae are composed of the organism`s two most abundant proteins, which represent 4% of its total protein and have an intracellular concentration of {ge} 3.0 mg/ml. At concentrations of 1.0 mg/ml, purified chaperonin proteins aggregate to form ordered filaments. Filament formation, which requires Mg{sup ++} and nucleotide binding (not hydrolysis), occurs at physiological temperatures under conditions suggesting filaments may exist in vivo. If the estimated 4,600 chaperonins per cell, formed filaments in vivo, they could create a matrix of filaments that would span the diameter of an average S. shibatae cell 100 times. Direct observations of unfixed, minimally treated cells by intermediate voltage electron microscopy (300 kV) revealed an intracellular network of filaments that resembles chaperonin filaments produced in vitro. The hypothesis that the intracellular network contains chaperonins is supported by immunogold analyses. The authors propose that chaperonin activity may be regulated in vivo by filament formation and that chaperonin filaments may serve a cytoskeleton-like function in archaea and perhaps in other prokaryotes.

  16. Harnessing Type I and Type III CRISPR-Cas systems for genome editing.

    Science.gov (United States)

    Li, Yingjun; Pan, Saifu; Zhang, Yan; Ren, Min; Feng, Mingxia; Peng, Nan; Chen, Lanming; Liang, Yun Xiang; She, Qunxin

    2016-02-29

    CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated) systems are widespread in archaea and bacteria, and research on their molecular mechanisms has led to the development of genome-editing techniques based on a few Type II systems. However, there has not been any report on harnessing a Type I or Type III system for genome editing. Here, a method was developed to repurpose both CRISPR-Cas systems for genetic manipulation in Sulfolobus islandicus, a thermophilic archaeon. A novel type of genome-editing plasmid (pGE) was constructed, carrying an artificial mini-CRISPR array and a donor DNA containing a non-target sequence. Transformation of a pGE plasmid would yield two alternative fates to transformed cells: wild-type cells are to be targeted for chromosomal DNA degradation, leading to cell death, whereas those carrying the mutant gene would survive the cell killing and selectively retained as transformants. Using this strategy, different types of mutation were generated, including deletion, insertion and point mutations. We envision this method is readily applicable to different bacteria and archaea that carry an active CRISPR-Cas system of DNA interference provided the protospacer adjacent motif (PAM) of an uncharacterized PAM-dependent CRISPR-Cas system can be predicted by bioinformatic analysis.

  17. New role of flavin as a general acid-base catalyst with no redox function in type 2 isopentenyl-diphosphate isomerase.

    Science.gov (United States)

    Unno, Hideaki; Yamashita, Satoshi; Ikeda, Yosuke; Sekiguchi, Shin-Ya; Yoshida, Norie; Yoshimura, Tohru; Kusunoki, Masami; Nakayama, Toru; Nishino, Tokuzo; Hemmi, Hisashi

    2009-04-03

    Using FMN and a reducing agent such as NAD(P)H, type 2 isopentenyl-diphosphate isomerase catalyzes isomerization between isopentenyl diphosphate and dimethylallyl diphosphate, both of which are elemental units for the biosynthesis of highly diverse isoprenoid compounds. Although the flavin cofactor is expected to be integrally involved in catalysis, its exact role remains controversial. Here we report the crystal structures of the substrate-free and complex forms of type 2 isopentenyl-diphosphate isomerase from the thermoacidophilic archaeon Sulfolobus shibatae, not only in the oxidized state but also in the reduced state. Based on the active-site structures of the reduced FMN-substrate-enzyme ternary complexes, which are in the active state, and on the data from site-directed mutagenesis at highly conserved charged or polar amino acid residues around the active site, we demonstrate that only reduced FMN, not amino acid residues, can catalyze proton addition/elimination required for the isomerase reaction. This discovery is the first evidence for this long suspected, but previously unobserved, role of flavins just as a general acid-base catalyst without playing any redox roles, and thereby expands the known functions of these versatile coenzymes.

  18. Molecular cloning and enzymological characterization of pyridoxal 5'-phosphate independent aspartate racemase from hyperthermophilic archaeon Thermococcus litoralis DSM 5473.

    Science.gov (United States)

    Washio, Tsubasa; Kato, Shiro; Oikawa, Tadao

    2016-09-01

    We succeeded in expressing the aspartate racemase homolog gene from Thermococcus litoralis DSM 5473 in Escherichia coli Rosetta (DE3) and found that the gene encodes aspartate racemase. The aspartate racemase gene consisted of 687 bp and encoded 228 amino acid residues. The purified enzyme showed aspartate racemase activity with a specific activity of 1590 U/mg. The enzyme was a homodimer with a molecular mass of 56 kDa and did not require pyridoxal 5'-phosphate as a coenzyme. The enzyme showed aspartate racemase activity even at 95 °C, and the activation energy of the enzyme was calculated to be 51.8 kJ/mol. The enzyme was highly thermostable, and approximately 50 % of its initial activity remained even after incubation at 90 °C for 11 h. The enzyme showed a maximum activity at a pH of 7.5 and was stable between pH 6.0 and 7.0. The enzyme acted on L-cysteic acid and L-cysteine sulfinic acid in addition to D- and L-aspartic acids, and was strongly inhibited by iodoacetic acid. The site-directed mutagenesis of the enzyme showed that the essential cysteine residues were conserved as Cys83 and Cys194. D-Forms of aspartic acid, serine, alanine, and valine were contained in T. litoralis DSM 5473 cells.

  19. Genome sequence of Halorhabdus tiamatea, the first archaeon isolated from a deep-sea anoxic brine lake.

    KAUST Repository

    Antunes, Andre

    2011-09-01

    We present the draft genome of Halorhabdus tiamatea, the first member of the Archaea ever isolated from a deep-sea anoxic brine. Genome comparison with Halorhabdus utahensis revealed some striking differences, including a marked increase in genes associated with transmembrane transport and putative genes for a trehalose synthase and a lactate dehydrogenase.

  20. Influence of Sulfobacillus thermosulfidooxidans on Initial Attachment and Pyrite Leaching by Thermoacidophilic Archaeon Acidianus sp. DSM 29099

    Directory of Open Access Journals (Sweden)

    Jing Liu

    2016-07-01

    Full Text Available At the industrial scale, bioleaching of metal sulfides includes two main technologies, tank leaching and heap leaching. Fluctuations in temperature caused by the exothermic reactions in a heap have a pronounced effect on the growth of microbes and composition of mixed microbial populations. Currently, little is known on the influence of pre-colonized mesophiles or moderate thermophiles on the attachment and bioleaching efficiency by thermophiles. The objective of this study was to investigate the interspecies interactions of the moderate thermophile Sulfobacillus thermosulfidooxidans DSM 9293T and the thermophile Acidianus sp. DSM 29099 during initial attachment to and dissolution of pyrite. Our results showed that: (1 Acidianus sp. DSM 29099 interacted with S. thermosulfidooxidansT during initial attachment in mixed cultures. In particular, cell attachment was improved in mixed cultures compared to pure cultures alone; however, no improvement of pyrite leaching in mixed cultures compared with pure cultures was observed; (2 active or inactivated cells of S. thermosulfidooxidansT on pyrite inhibited or showed no influence on the initial attachment of Acidianus sp. DSM 29099, respectively, but both promoted its leaching efficiency; (3 S. thermosulfidooxidansT exudates did not enhance the initial attachment of Acidianus sp. DSM 29099 to pyrite, but greatly facilitated its pyrite dissolution efficiency. Our study provides insights into cell-cell interactions between moderate thermophiles and thermophiles and is helpful for understanding of the microbial interactions in a heap leaching environment.

  1. Mutational analyses of the enzymes involved in the metabolism of hydrogen by the hyperthermophilic archaeon Pyrococcus furiosus

    Directory of Open Access Journals (Sweden)

    Gerrit J Schut

    2012-05-01

    Full Text Available Pyrococcus furiosus grows optimally near 100°C by fermenting carbohydrates to produce hydrogen (H2 or, if elemental sulfur (S0, is present hydrogen sulfide instead. It contains two cytoplasmic hydrogenases, SHI and SHII, that use NADP(H as an electron carrier, and a membrane bound hydrogenase (MBH, that utilizes the redox protein ferredoxin. We previously constructed deletion strains lacking SHI and/or SHII and showed that they exhibited no obvious phenotype. This study has now been extended to include biochemical analyses and growth studies using the ΔSHI and ΔSHII deletion strains together with strains lacking a functional MBH (ΔMbhL. Hydrogenase activities in cytoplasmic extracts of ΔSHII and the parent strain were similar but were much lower (<10% in the ΔSHI strain, and no activity was detected in the ΔSHIΔSHII double deletion strain, indicating that SHI is responsible for most of the cytoplasmic hydrogenase activity. In contrast, the ΔmbhL strain showed no growth in the absence of S0, confirming the hypothesis that, in the absence of S0, MBH is the only enzyme that can dispose of reductant (as H2 generated during sugar oxidation. The deletion strain devoid of all three hydrogenases also grew only in the presence of S0 and did not produce any detectable H2. When grown in the presence of limiting S0, both H2S and H2 were produced by the parent and ΔSHI/ΔSHII strains. A significant amount of H2 was also produced by the ΔmbhL strain, showing that SHI can produce H2 from NADPH in vivo, although this does not enable significant growth of ΔmbhL in the absence of S0. We propose that the physiological function of SHI is to recycle H2 and provide a link between external H2 and the intracellular pool of NADPH needed for biosynthesis. This likely has a distinct energetic advantage in the environment, but it is clearly not required for growth of the organism under the usual laboratory conditions. The function of SHII, however, remains unknown.

  2. Draft Genome Sequence of a Highly Flagellated, Fast-Swimming Archaeon, Methanocaldococcus villosus Strain KIN24-T80 (DSM 22612)

    KAUST Repository

    Thennarasu, Sugumar

    2013-07-11

    We report the draft genome sequence of a hyperthermophilic Methanocaldococcus villosus strain, KIN24-T80. The gene associated with its heavy flagellum formation was annotated in the 1.2-Mb draft genome sequence, and this strain may be a good model system to study the extensive functional role of flagella and their fast motor activity.

  3. Crystallization and preliminary X-ray diffraction analysis of L-threonine dehydrogenase (TDH) from the hyperthermophilic archaeon Thermococcus kodakaraensis.

    Science.gov (United States)

    Bowyer, A; Mikolajek, H; Wright, J N; Coker, A; Erskine, P T; Cooper, J B; Bashir, Q; Rashid, N; Jamil, F; Akhtar, M

    2008-09-01

    The enzyme L-threonine dehydrogenase catalyses the NAD(+)-dependent conversion of L-threonine to 2-amino-3-ketobutyrate, which is the first reaction of a two-step biochemical pathway involved in the metabolism of threonine to glycine. Here, the crystallization and preliminary crystallographic analysis of L-threonine dehydrogenase (Tk-TDH) from the hyperthermophilic organism Thermococcus kodakaraensis KOD1 is reported. This threonine dehydrogenase consists of 350 amino acids, with a molecular weight of 38 kDa, and was prepared using an Escherichia coli expression system. The purified native protein was crystallized using the hanging-drop vapour-diffusion method and crystals grew in the tetragonal space group P4(3)2(1)2, with unit-cell parameters a = b = 124.5, c = 271.1 A. Diffraction data were collected to 2.6 A resolution and preliminary analysis indicates that there are four molecules in the asymmetric unit of the crystal.

  4. Identification of the S-layer glycoproteins and their covalently linked glycans in the halophilic archaeon Haloarcula hispanica.

    Science.gov (United States)

    Lu, Hua; Lü, Yang; Ren, Jinwei; Wang, Zhongfu; Wang, Qian; Luo, Yuanming; Han, Jing; Xiang, Hua; Du, Yuguo; Jin, Cheng

    2015-11-01

    Haloarcula hispanica is one of members of the Halobacteriaceae, which displays particularly low restriction activity and is therefore important as one of the most tractable haloarchaea for archaeal genetic research. Although the Har. hispanica S-layer protein has been reported glycosylated, the S-layer glycoprotein and its glycosylation have not been investigated yet. In this study, the S-layer proteins of Har. hispanica were extracted and characterized. The S-layer was found containing two different glycoproteins which shared highly similar amino acid sequences. The genes coding for these two S-layer glycoproteins were found next to each other in the genome. Moreover, the N- and O-linked glycans were released from these two S-layer glycoproteins for structural determination. Based on the mass spectrometry and nuclear magnetic resonance, the N-glycan was determined as a branched trisaccharide containing a 225 Da residue corresponded to a 2-amino-6-sulfo-2, 6-dideoxy-quinovose, which was the first time that a naturally occurring form of sulfoquinovosamine was identified. Besides, the O-glycan was characterized as a Glcα-1,4-Gal disaccharide by mass spectrometry combined with monosaccharide composition analysis and glycosidase treatment. The determination of the N- and O-glycan structure will be helpful for studying the diverse protein glycosylation pathways in archaea utilizing H. hispanica as a new model.

  5. A Mesophilic, Autotrophic, Ammonia-Oxidizing Archaeon of Thaumarchaeal Group I.1a Cultivated from a Deep Oligotrophic Soil Horizon

    NARCIS (Netherlands)

    Jung, M.Y.; Park, S.J.; Kim, S.J.; Kim, J.G.; Sinninghe Damsté, J.S.; Jeon, C.O.; Rhee, S.K.

    2014-01-01

    Soil nitrification plays an important role in the reduction of soil fertility and in nitrate enrichment of groundwater. Various ammonia- oxidizing archaea (AOA) are considered to be members of the pool of ammonia-oxidizing microorganisms in soil. This study reports the discovery of a chemolithoautot

  6. Ser/Thr/Tyr protein phosphorylation in the archaeon Halobacterium salinarum--a representative of the third domain of life.

    Directory of Open Access Journals (Sweden)

    Michalis Aivaliotis

    Full Text Available In the quest for the origin and evolution of protein phosphorylation, the major regulatory post-translational modification in eukaryotes, the members of archaea, the "third domain of life", play a protagonistic role. A plethora of studies have demonstrated that archaeal proteins are subject to post-translational modification by covalent phosphorylation, but little is known concerning the identities of the proteins affected, the impact on their functionality, the physiological roles of archaeal protein phosphorylation/dephosphorylation, and the protein kinases/phosphatases involved. These limited studies led to the initial hypothesis that archaea, similarly to other prokaryotes, use mainly histidine/aspartate phosphorylation, in their two-component systems representing a paradigm of prokaryotic signal transduction, while eukaryotes mostly use Ser/Thr/Tyr phosphorylation for creating highly sophisticated regulatory networks. In antithesis to the above hypothesis, several studies showed that Ser/Thr/Tyr phosphorylation is also common in the bacterial cell, and here we present the first genome-wide phosphoproteomic analysis of the model organism of archaea, Halobacterium salinarum, proving the existence/conservation of Ser/Thr/Tyr phosphorylation in the "third domain" of life, allowing a better understanding of the origin and evolution of the so-called "Nature's premier" mechanism for regulating the functional properties of proteins.

  7. Energy conservation by oxidation of formate to carbon dioxide and hydrogen via a sodium ion current in a hyperthermophilic archaeon.

    Science.gov (United States)

    Lim, Jae Kyu; Mayer, Florian; Kang, Sung Gyun; Müller, Volker

    2014-08-05

    Thermococcus onnurineus NA1 is known to grow by the anaerobic oxidation of formate to CO2 and H2, a reaction that operates near thermodynamic equilibrium. Here we demonstrate that this reaction is coupled to ATP synthesis by a transmembrane ion current. Formate oxidation leads to H(+) translocation across the cytoplasmic membrane that then drives Na(+) translocation. The ion-translocating electron transfer system is rather simple, consisting of only a formate dehydrogenase module, a membrane-bound hydrogenase module, and a multisubunit Na(+)/H(+) antiporter module. The electrochemical Na(+) gradient established then drives ATP synthesis. These data give a mechanistic explanation for chemiosmotic energy conservation coupled to formate oxidation to CO2 and H2. Because it is discussed that the membrane-bound hydrogenase with the Na(+)/H(+) antiporter module are ancestors of complex I of mitochondrial and bacterial electron transport these data also shed light on the evolution of ion transport in complex I-like electron transport chains.

  8. ATP- and NAD+-dependent DNA ligases share an essential function in the halophilic archaeon Haloferax volcanii

    DEFF Research Database (Denmark)

    Zhao, A.; Gray, F. C; MacNeill, S. A.

    2006-01-01

    DNA ligases join the ends of DNA molecules during replication, repair and recombination. ATP-dependent ligases are found predominantly in the eukarya and archaea whereas NAD+-dependent DNA ligases are found only in the eubacteria and in entomopoxviruses. Using the genetically tractable halophile...

  9. The 1.5 resolution structure of the [Fe4S3]-ferredoxin from the hyperthermiphilic archaeon Pyrococcus furiosus

    DEFF Research Database (Denmark)

    Nielsen, Michael Ericsson Skovbo; Harris, Pernille; Ooi, Bee Lean;

    2004-01-01

    contains a double-conformation disulfide bond existing in a left-handed and a right-handed spiral conformation. The crystal packing reveals a beta-sheet interaction, which supports the suggestion that P. furiosus ferredoxin is a functional dimer. The extraordinary thermostability of P. furiosus ferredoxin...

  10. Histone and TK0471/TrmBL2 form a novel heterogeneous genome architecture in the hyperthermophilic archaeon Thermococcus kodakarensis.

    Science.gov (United States)

    Maruyama, Hugo; Shin, Minsang; Oda, Toshiyuki; Matsumi, Rie; Ohniwa, Ryosuke L; Itoh, Takehiko; Shirahige, Katsuhiko; Imanaka, Tadayuki; Atomi, Haruyuki; Yoshimura, Shige H; Takeyasu, Kunio

    2011-02-01

    Being distinct from bacteria and eukaryotes, Archaea constitute a third domain of living things. The DNA replication, transcription, and translation machineries of Archaea are more similar to those of eukaryotes, whereas the genes involved in metabolic processes show more similarity to their bacterial counterparts. We report here that TK0471/TrmB-like 2 (TrmBL2), in addition to histone, is a novel type of abundant chromosomal protein in the model euryarchaeon Thermococcus kodakarensis . The chromosome of T. kodakarensis can be separated into regions enriched either with histone, in which the genetic material takes on a “beads-on-a-string” appearance, or with TK0471/TrmBL2, in which it assumes a thick fibrous structure. TK0471/TrmBL2 binds to both coding and intergenic regions and represses transcription when bound to the promoter region. These results show that the archaeal chromosome is organized into heterogeneous structures and that TK0471/TrmBL2 acts as a general chromosomal protein as well as a global transcriptional repressor.

  11. Cloning and expression of the catalase-peroxidase gene from the hyperthermophilic archaeon Archaeoglobus fulgidus and characterization of the enzyme

    NARCIS (Netherlands)

    Kengen, S.W.M.; Bikker, F.; Vos, de W.M.; Oost, van der J.

    2001-01-01

    A putative perA gene from Archaeoglobus fulgidus was cloned and expressed in Escherichia coli BL21(DE3), and the recombinant catalase-peroxidase was purified to homogeneity. The enzyme is a homodimer with a subunit molecular mass of 85 kDa. UV-visible spectroscopic analysis indicated the presence of

  12. 嗜酸嗜热古菌Sulfolobus acidocaldarius编码尿嘧啶DNA糖苷酶表达,纯化与酶学特征%Cloning, expression, purification and characterization of two uracil-DNA glycosylases from Sulfolobus acidocaldarius

    Institute of Scientific and Technical Information of China (English)

    王婧; 易刚顺; 欧杰; 刘建华; 刘喜朋

    2015-01-01

    [目的]嗜高温微生物面临dC脱氨基生成dU损伤的巨大压力,鉴定嗜酸嗜热古菌S.acidocaldarius来源的尿嘧啶DNA糖苷酶(UDG)切除dU损伤的酶学活性.[方法]重组表达来源于S.acidocaldarius的Ⅳ和Ⅴ型UDG,经亲和纯化得到电泳纯重组蛋白.然后利用人工合成的dU (deoxyuracil)修饰寡核苷酸片段作为底物,体外鉴定两种重组UDG的酶学特性.[结果]来源于S.acidocaldarius的Ⅳ和Ⅴ型重组UDG具有相似的酶学特性.Ⅳ型UDG催化效率更高,比活性是Ⅴ型重组UDG的750倍左右.作为来自嗜热微生物的蛋白,S.acidocaldarius的Ⅳ和Ⅴ型UDG的最适反应温度为65-75℃.[结论]Ⅳ型UDG比Ⅴ型UDG水解dU碱基和脱氧核糖之间糖苷键的能力更强.

  13. Expression of Genes Involved in Iron and Sulfur Respiration in a Novel Thermophilic Crenarchaeon Isolated from Acid-Sulfate-Chloride Geothermal Systems

    Science.gov (United States)

    Kozubal, M.; Macur, R.; Inskeep, W. P.

    2007-12-01

    complex (thiosulfate:quinone oxidoreductase) related to the Acidianus ambivalens DOXAD complex and a sulfur reductase (SRE) complex related to one found in Sulfolobus solfataricus and Acidianus ambivalens. Here we report on the RNA expression of seven gene sequences from each of the above mentioned complexes for Metallosphaera strain MK1 grown aerobically on pyrite, sulfur, Fe(II)-ferrihydrite, and anaerobically with yeast extract and sulfur. In addition, expression studies are also compared to in situ samples collected from the geothermal Fe-mats.

  14. Functional Analysis of Homologous Recombination Repair Proteins HerA and NurA in the Thermophile Sulfolobus islandicus

    DEFF Research Database (Denmark)

    Huang, Qihong

    with the wild type strain. Microarray analysis showed that genes involved in cell division were down-regulated while the transcription of the genes implicated in chromosome resolution/segregation were also changed in the strain, suggesting that HerA overexpression impair DNA metabolism and resulted in mis...... in eukaryotes. As a third domain of life, archaea is considered as a chimaera between bacteria and eukaryotes. Its metabolic pathways and cell structures resemble those of bacteria, whereas the information processing is of the eukaryal type or more similar to their eukaryal counterparts. A number of archaea...... by microscopy and flow cytometry. DNA damaging agent assay showed that this strain is as sensitive as the wild type strain to methyl methanesulfonate (MMS) and cisplatin, while it exhibited higher sensitivity to hydroxyurea (HU), an agent revealed to cause G2 arrest in S. islandicus cells, compared...

  15. The saci_2123 gene of the hyperthermoacidophile Sulfolobus acidocaldarius encodes an ATP-binding cassette multidrug transporter

    NARCIS (Netherlands)

    Yang, Nuan; Driessen, Arnold J. M.

    2015-01-01

    Multidrug resistance (MDR) transporters are capable of secreting structurally and functionally unrelated toxic compounds from the cell. Among this group are ATP-binding cassette (ABC) transporters. These membrane proteins are typically arranged as either hetero- or homo-dimers of ABC half-transporte

  16. 通过染色体整合表达古菌乙酰化酶合成N-末端乙酰化胸腺肽β4%Biosynthesis of Nα-acetylated Thymosin β4 by Co-expressing an Archaeal Acetylase Integrated in Chromosome of Escherichia coli

    Institute of Scientific and Technical Information of China (English)

    曹赛; 谢达平; 周长林; 戴红梅; 孙旭; 颛孙丹丹; 和金周; 司信喜; 李树龙; 方宏清; 陈惠鹏

    2011-01-01

    Thymosin β4 (T(34), a 43-amino acid Na-acetylated peptide, has multiple significant biological functions. There are two bottlenecks to its biosynthesis: the difficulties in obtaining Na-acetylation and expressing small peptides. In this study we found that ssArdl, an archaeal acetylase from Sulfolobus solfataricus can acetylate the N-terminal residue Ser of T|34. A modified E. Coli BL21(DE3) with ssArdl-expressing cassette integrated in the locus of lpxM of chromosome by Red recombination was constructed and named as E. Coli BDA for Na-acetylation of proteins expressed in it. To obtain Na-acetylated T|34 efficiently, a fusion protein, Tβ4-Intein, was constructed, in which T(34 and a His tag were fused respectively to the N-terminus and the C-terminus of a smallest mini-intein, 136-amino acid Spl DnaX. After expression in E. Coli BDA and purification by Ni-Sepharose affinity chromatography, the fusion protein was induced by P-mercaptoethanol to release Na-acetylated Tβ4 through intein-mediated N-terminal cleavage. Three mutants of T|34 were prepared in the same way. All of Tβ4 and its three mutants have the ability to bind actin. This study laid a foundation for further investigation of the function and application of Tβ4.%胸腺肽β4(Tβ4)是N-末端乙酰化的43肽,具有多种重要生物学功能.其生物合成存在两大难点,即乙酰化修饰和小分子肽的表达.本研究发现来自古菌Sulfolobus solfataricus 的乙酰化酶ssArd1可以催化Tβ4的N-末端乙酰化修饰.利用Red同源重组技术将ssArd1 基因表达盒整合至E coli BL21(DE3)染色体的lpxM位点上,构建了可以实现Tβ4 N-末端乙酰化修饰的新型宿主E coli BDA.将Tβ4编码基因融合在改造的微型Spl DnaX Intein的N 端,并在Intein的C端添加His标签,构建了表达载体pET-Tβ4-Intein.在E.coli BDA中表达的融合蛋白,经镍亲和层析纯化后用β-巯基乙醇诱导融合蛋白切割释放小分子多肽,获得了

  17. Dynamic Conformational Change Regulates the Protein-DNA Recognition: An Investigation on Binding of a Y-Family Polymerase to Its Target DNA

    Science.gov (United States)

    Chu, Xiakun; Liu, Fei; Maxwell, Brian A.; Wang, Yong; Suo, Zucai; Wang, Haijun; Han, Wei; Wang, Jin

    2014-01-01

    Protein-DNA recognition is a central biological process that governs the life of cells. A protein will often undergo a conformational transition to form the functional complex with its target DNA. The protein conformational dynamics are expected to contribute to the stability and specificity of DNA recognition and therefore may control the functional activity of the protein-DNA complex. Understanding how the conformational dynamics influences the protein-DNA recognition is still challenging. Here, we developed a two-basin structure-based model to explore functional dynamics in Sulfolobus solfataricus DNA Y-family polymerase IV (DPO4) during its binding to DNA. With explicit consideration of non-specific and specific interactions between DPO4 and DNA, we found that DPO4-DNA recognition is comprised of first 3D diffusion, then a short-range adjustment sliding on DNA and finally specific binding. Interestingly, we found that DPO4 is under a conformational equilibrium between multiple states during the binding process and the distributions of the conformations vary at different binding stages. By modulating the strength of the electrostatic interactions, the flexibility of the linker, and the conformational dynamics in DPO4, we drew a clear picture on how DPO4 dynamically regulates the DNA recognition. We argue that the unique features of flexibility and conformational dynamics in DPO4-DNA recognition have direct implications for low-fidelity translesion DNA synthesis, most of which is found to be accomplished by the Y-family DNA polymerases. Our results help complete the description of the DNA synthesis process for the Y-family polymerases. Furthermore, the methods developed here can be widely applied for future investigations on how various proteins recognize and bind specific DNA substrates. PMID:25188490

  18. Structure and function of the archaeal exosome.

    Science.gov (United States)

    Evguenieva-Hackenberg, Elena; Hou, Linlin; Glaeser, Stefanie; Klug, Gabriele

    2014-01-01

    The RNA-degrading exosome in archaea is structurally very similar to the nine-subunit core of the essential eukaryotic exosome and to bacterial polynucleotide phosphorylase (PNPase). In contrast to the eukaryotic exosome, PNPase and the archaeal exosome exhibit metal ion-dependent, phosphorolytic activities and synthesize heteropolymeric RNA tails in addition to the exoribonucleolytic RNA degradation in 3' → 5' direction. The archaeal nine-subunit exosome consists of four orthologs of eukaryotic exosomal subunits: the RNase PH-domain-containing subunits Rrp41 and Rrp42 form a hexameric ring with three active sites, whereas the S1-domain-containing subunits Rrp4 and Csl4 form an RNA-binding trimeric cap on the top of the ring. In vivo, this cap contains Rrp4 and Csl4 in variable amounts. Rrp4 confers poly(A) specificity to the exosome, whereas Csl4 is involved in the interaction with the archaea-specific subunit of the complex, the homolog of the bacterial primase DnaG. The archaeal DnaG is a highly conserved protein and its gene is present in all sequenced archaeal genomes, although the exosome was lost in halophilic archaea and some methanogens. In exosome-containing archaea, DnaG is tightly associated with the exosome. It functions as an additional RNA-binding subunit with poly(A) specificity in the reconstituted exosome of Sulfolobus solfataricus and enhances the degradation of adenine-rich transcripts in vitro. Not only the RNA-binding cap but also the hexameric Rrp41-Rrp42 ring alone shows substrate selectivity and prefers purines over pyrimidines. This implies a coevolution of the exosome and its RNA substrates resulting in 3'-ends with different affinities to the exosome.

  19. Structure of the archaeal Cascade subunit Csa5: relating the small subunits of CRISPR effector complexes.

    Science.gov (United States)

    Reeks, Judith; Graham, Shirley; Anderson, Linzi; Liu, Huanting; White, Malcolm F; Naismith, James H

    2013-05-01

    The Cascade complex for CRISPR-mediated antiviral immunity uses CRISPR RNA (crRNA) to target invading DNA species from mobile elements such as viruses, leading to their destruction. The core of the Cascade effector complex consists of the Cas5 and Cas7 subunits, which are widely conserved in prokaryotes. Cas7 binds crRNA and forms the helical backbone of Cascade. Many archaea encode a version of the Cascade complex (denoted Type I-A) that includes a Csa5 (or small) subunit, which interacts weakly with the core proteins. Here, we report the crystal structure of the Csa5 protein from Sulfolobus solfataricus. Csa5 comprises a conserved α-helical domain with a small insertion consisting of a weakly conserved β-strand domain. In the crystal, the Csa5 monomers have multimerized into infinite helical threads. At each interface is a strictly conserved intersubunit salt bridge, deletion of which disrupts multimerization. Structural analysis indicates a shared evolutionary history among the small subunits of the CRISPR effector complexes. The same α-helical domain is found in the C-terminal domain of Cse2 (from Type I-E Cascade), while the N-terminal domain of Cse2 is found in Cmr5 of the CMR (Type III-B) effector complex. As Cmr5 shares no match with Csa5, two possibilities present themselves: selective domain loss from an ancestral Cse2 to create two new subfamilies or domain fusion of two separate families to create a new Cse2 family. A definitive answer awaits structural studies of further small subunits from other CRISPR effector complexes.

  20. Structure of the archaeal Cascade subunit Csa5

    Science.gov (United States)

    Reeks, Judith; Graham, Shirley; Anderson, Linzi; Liu, Huanting; White, Malcolm F.; Naismith, James H.

    2013-01-01

    The Cascade complex for CRISPR-mediated antiviral immunity uses CRISPR RNA (crRNA) to target invading DNA species from mobile elements such as viruses, leading to their destruction. The core of the Cascade effector complex consists of the Cas5 and Cas7 subunits, which are widely conserved in prokaryotes. Cas7 binds crRNA and forms the helical backbone of Cascade. Many archaea encode a version of the Cascade complex (denoted Type I-A) that includes a Csa5 (or small) subunit, which interacts weakly with the core proteins. Here, we report the crystal structure of the Csa5 protein from Sulfolobus solfataricus. Csa5 comprises a conserved α-helical domain with a small insertion consisting of a weakly conserved β-strand domain. In the crystal, the Csa5 monomers have multimerized into infinite helical threads. At each interface is a strictly conserved intersubunit salt bridge, deletion of which disrupts multimerization. Structural analysis indicates a shared evolutionary history among the small subunits of the CRISPR effector complexes. The same α-helical domain is found in the C-terminal domain of Cse2 (from Type I-E Cascade), while the N-terminal domain of Cse2 is found in Cmr5 of the CMR (Type III-B) effector complex. As Cmr5 shares no match with Csa5, two possibilities present themselves: selective domain loss from an ancestral Cse2 to create two new subfamilies or domain fusion of two separate families to create a new Cse2 family. A definitive answer awaits structural studies of further small subunits from other CRISPR effector complexes. PMID:23846216