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sulfolobus hakonensis sp. nov., a novel species of acidothermophilic archaeon.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 conte ...19968934897
uracil-dna glycosylase activities in hyperthermophilic micro-organisms.hyperthermophiles exist in conditions which present an increased threat to the informational integrity of their dna, particularly by hydrolytic damage. as in mesophilic organisms, specific activities must exist to restore and protect this template function of dna. in this study we have demonstrated the presence of thermally stable uracil-dna glycosylase activities in seven hyperthermophiles; one bacterial: thermotoga maritima, and six archaeal: sulfolobus solfataricus, sulfolobus shibatae, sulfo ...19968837481
cca-adding enzymes and poly(a) polymerases are all members of the same nucleotidyltransferase superfamily: characterization of the cca-adding enzyme from the archaeal hyperthermophile sulfolobus shibatae.we describe the purification, cloning, and characterization of the cca-adding enzyme [atp(ctp):trna nucleotidyl transferase] from the thermophilic archaebacterium, sulfolobus shibatae. characterization of an archaeal cca-adding enzyme provides formal proof that the cca-adding activity is present in all three contemporary kingdoms. antibodies raised against recombinant, expressed sulfolobus cca-adding enzyme reacted specifically with the 48-kda protein and fully depleted all cca-adding activity f ...19968809016
an atypical topoisomerase ii from archaea with implications for meiotic recombination.type ii topoisomerases help regulate dna topology during transcription, replication and recombination by catalysing dna strand transfer through transient double-stranded breaks. all type ii topoisomerases described so far are members of a single protein family. we have cloned and sequenced the genes encoding the a and b subunits of topoisomerase ii from the archaeon sulfolobus shibatae. this enzyme is the first of a new family. it has no similarity with other type ii topoisomerases, except for t ...19979121560
uracil phosphoribosyltransferase from the extreme thermoacidophilic archaebacterium sulfolobus shibatae is an allosteric enzyme, activated by gtp and inhibited by ctp.uracil phosphoribosyltransferase, which catalyses the formation of ump and pyrophosphate from uracil and 5-phosphoribosyl alpha-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 degrees c than at 37 degrees c, but the functional half-life was short at 95 degrees c. the activity ...19968765224
isolation and characterization of flagella and flagellin proteins from the thermoacidophilic archaea thermoplasma volcanium and sulfolobus shibatae.isolated flagellar filaments of sulfolobus shibatae were 15 nm in diameter, and they were composed of two major flagellins which have m(r)s of 31,000 and 33,000 and which stained positively for glycoprotein. the flagellar filaments of thermoplasma volcanium were 12 nm in diameter and were composed of one major flagellin which has an m(r) of 41,000 and which also stained positively for glycoprotein. n-terminal amino acid sequencing indicated that 18 of the n-terminal 20 amino acid positions of th ...19968550530
pcr-mediated cloning and sequencing of the gene encoding glutamate dehydrogenase from the archaeon sulfolobus shibatae: identification of putative amino-acid signatures for extremophilic adaptation.highly degenerate oligodeoxyribonucleotides (oligos) were used to pcr amplify the most conserved region of the glutamate dehydrogenase (gdh)-encoding gene from the extreme thermophilic archaeon, sulfolobus shibatae. the amplified fragment was cloned and sequenced, and then used as a homologous probe to clone a genomic restriction fragment containing the near-complete gdha gene. the deduced amino acid (aa) sequence shows a very high degree of similarity with the aa sequence previously determined ...19948125334
acquired thermotolerance and heat shock proteins in thermophiles from the three phylogenetic domains.thermophilic organisms from each of the three phylogenetic domains (bacteria, archaea, and eucarya) acquired thermotolerance after heat shock. bacillus caldolyticus grown at 60 degrees c and heat shocked at 69 degrees c for 10 min showed thermotolerance at 74 degrees c, sulfolobus shibatae grown at 70 degrees c and heat shocked at 88 degrees c for 60 min showed thermotolerance at 95 degrees c, and thermomyces lanuginosus grown at 50 degrees c and heat shocked at 55 degrees c for 60 min showed th ...19947928981
the thermosome of thermoplasma acidophilum and its relationship to the eukaryotic chaperonin tric.a high molecular-mass protein complex from the archaebacterium thermoplasma acidophilum, referred to here as the 'thermosome', is built from two subunits (m(r) 58 and 60). the thermosome has been purified to homogeneity. the molecular mass of the native complex was determined to be 1061 +/- 30 da by scanning transmission electron microscopy. it shows a weak atpase activity and is able to bind denatured polypeptides. averages obtained from electron micrographs of negatively stained molecules in t ...19957867646
primary structure of the thermosome from thermoplasma acidophilum.the thermosome, a chaperonin from the archaebacterium thermoplasma acidophilum, consists of two subunits (m(r) 58,000 and 60,000) which assemble into a cylindrical complex of pseudo eight-fold rotational symmetry. the sequences of the two subunits are approximately 60% identical to each other and to tf55 from sulfolobus shibatae, and are 30-40% identical to the subunits of the tcp1 containing ring complex (tric) from the eukaryotic cytosol. a dendrogram of this family of chaperonins contains eig ...19957794526
cloning and functional analysis of the tata binding protein from sulfolobus shibatae.archaea (formerly archaebacteria) comprise a domain of life that is phylogenetically distinct from both eucarya and bacteria. here we report the cloning of a gene from the archaeon sulfolobus shibatae that encodes a protein with strong homology to the tata binding protein (tbp) of eukaryotes. sulfolobus shibatae tbp is, however, almost as diverged from other archaeal tbps that have been cloned as it is from eukaryotic tbps. dna binding studies indicate that s.shibatae tbp recognizes tata-like a- ...19957784182
small abundant dna binding proteins from the thermoacidophilic archaeon sulfolobus shibatae constrain negative dna supercoils.major dna binding proteins, designated ssh7, were purified from the thermoacidophilic archaeon sulfolobus shibatae. the ssh7 proteins have an apparent molecular mass of 6.5 kda and are similar to the 7-kda dna binding proteins from sulfolobus acidocaldarius and sulfolobus solfataricus in n-terminal amino acid sequence. the proteins constitute about 4.8% of the cellular protein. upon binding to dna, the ssh7 proteins constrain negative supercoils. at the tested ssh7/dna mass ratios (0 to 1.65), o ...19989573212
molecular cloning of the transcription factor tfiib homolog from sulfolobus shibatae.the archaea (archaebacteria) constitute a group of prokaryotes that are phylogenetically distinct from eucarya (eukaryotes) and bacteria (eubacteria). although archaea possess only one rna polymerase, evidence suggests that their transcriptional apparatus is similar to that of eucarya. for example, archaea contain a homolog of the tata-binding protein which interacts with the tata-box like a-box sequence upstream of many archaeal genes. here, we report the cloning of a sulfolobus shibatae gene t ...19957597084
cloning, sequencing and transcript analysis of the gene encoding formylmethanofuran: tetrahydromethanopterin formyltransferase from the hyperthermophilic methanothermus fervidus.the formylmethanofuran:tetrahydromethanopterin formyltransferase (ftr) from methanothermus fervidus was partially purified and its n-terminal amino acid sequence determined. using as probe a mixture of oligonucleotides derived from the ftr n-terminus, the corresponding gene (ftr) was cloned and sequenced. the ftr gene codes for 297 amino acids, corresponding to a molecular mass of 31,836 daltons, in contrast to the 41,000 daltons estimated for the protein by sodium dodecylsulphate-polyacrylamide ...19947506350
conformational cycle of the archaeosome, a tcp1-like chaperonin from sulfolobus shibatae.the major heat shock proteins in the archaeon sulfolobus shibatae are similar to the cytosolic eukaryotic chaperonin and form an 18-subunit bitoroidal complex. two sequence-related subunits constitute a functional complex, named the archaeosome. the archaeosome exists in two distinct conformational states that are part of chaperonin functional cycle. the closed archaeosome complex binds atp and forms an open complex. upon atp hydrolysis, the open complex dissociates into subunits. free subunits ...19957499406
a 37.5 kb region of yeast chromosome x includes the sme1, mef2, gsh1 and csd3 genes, a tcp-1-related gene, an open reading frame similar to the dal80 gene, and a trna(arg).the complete dna sequence of cosmid clone p59 comprising 37,549 bp derived from chromosome x was determined from an ordered set of subclones. the sequence contains 14 open reading frames (orfs) containing at least 100 consecutive sense codons. four of the orfs represent already known and sequenced yeast genes: b645 is identical to the sme1 gene encoding a protein kinase, required for induction of meiosis in yeast, d819 represents the mef2 gene probably encoding a second mitochondrial elongation ...19957483851
complete nucleotide sequence of the virus ssv1 of the archaebacterium sulfolobus shibatae.the dna sequence of the sulfolobus shibatae virus ssv1 is the first complete sequence of an archaebacterial virus genome. the viral dna is a closed double-stranded dna circle of 15465 bp. the features of the sequence, the positions of all 11 transcripts, the three characterized proteins, and the open reading frames are described.19911926776
heterologous in vitro transcription from two archaebacterial promoters.a cell-free extract of sulfolobus shibatae is able to specifically initiate transcription in vitro at the promoter of the plasmid-encoded gene for the major gas vesicle protein of halobacterium halobium and at the promoter for the transcript t4 of the temperate h. halobium phage phi h. the corresponding promoter from the virulent phage mutant phi hl1 yields enhanced transcription in the heterologous system, in agreement with strongly increased in vivo expression.19911906812
a molecular chaperone from a thermophilic archaebacterium is related to the eukaryotic protein t-complex polypeptide-1.there is evidence to suggest that components of archaebacteria are evolutionarily related to cognates in the eukaryotic cytosol. we postulated that the major heat-shock protein of the thermophilic archaebacterium, sulfolobus shibatae, is a molecular chaperone and that it is related to an as-yet unidentified chaperone component in the eukaryotic cytosol. acquired thermotolerance in s. shibatae correlates with the predominant synthesis of this already abundant protein, referred to as thermophilic ...19911836250
isolate b12, which harbours a virus-like element, represents a new species of the archaebacterial genus sulfolobus, sulfolobus shibatae, sp. nov.the sulfolobus isolate b12 and its endogenous virus-like element ssv1 have provided a fruitful system for detailed analysis of certain aspects of archaebacterial molecular biology, especially those concerning gene expression. in the course of clarifying this isolate's taxonomic position, we determined dna base composition, ability to grow autotrophically, nucleotide sequence of 16s ribosomal rna, and level of total genomic homology to other sulfolobus strains. although the results generally demo ...19901703758
reverse gyrase gene from sulfolobus shibatae b12: gene structure, transcription unit and comparative sequence analysis of the two domains.we cloned and sequenced a dna fragment from the thermophilic archaeal strain sulfolobus shibatae b12 that includes the gene topr encoding the reverse gyrase. the rna of the reverse gyrase gene was characterized indicating that the topr gene is fully functional in vivo. we showed by primer extension analysis that transcription of topr initiates 28 bp downstream from a consensus a-box promoter. in order to understand how this particular type i dna topoisomerase introduces positive superturns into ...19968972852
gene duplications in evolution of archaeal family b dna polymerases.all archaeal dna-dependent dna polymerases sequenced to date are homologous to family b dna polymerases from eukaryotes and eubacteria. presently, representatives of the euryarchaeote division of archaea appear to have a single family b dna polymerase, whereas two crenarchaeotes, pyrodictium occultum and sulfolobus solfataricus, each possess two family b dna polymerases. we have found the gene for yet a third family b dna polymerase, designated b3, in the crenarchaeote s. solfataricus p2. the en ...19979098062
an abundant dna binding protein from the hyperthermophilic archaeon sulfolobus shibatae affects dna supercoiling in a temperature-dependent fashion.the dna binding protein ssh10b, a member of the sac10b family, has been purified from the hyperthermophilic archaeon sulfolobus shibatae. ssh10b constitutes about 4% of the cellular protein. electrophoretic mobility shift assays showed that ssh10b first bound a double-stranded dna fragment with an estimated binding size of approximately approximately 12 bp, forming distinct shifts, until the dna was coated with the protein. binding of more ssh10b resulted in the formation of smears of lower mobi ...200010869069
chaperonin filaments: the archaeal cytoskeleton?chaperonins are high molecular mass double-ring structures composed of 60-kda protein subunits. in the hyperthermophilic archaeon sulfolobus shibatae the two chaperonin proteins represent approximately 4% of its total protein and have a combined intracellular concentration of >30 mg/ml. at concentrations >/= 0.5 mg/ml purified chaperonins form filaments in the presence of mg2+ and nucleotides. filament formation requires nucleotide binding (not hydrolysis), and occurs at physiological temperatur ...19979144246
unidentified open reading frames in the genome of methanococcus jannaschii are similar in sequence to an archaebacterial gene for trna nucleotidyltransferase.the protein sequence of atp/ctp:trna nucleotidyltransferase (cca) from sulfolobus shibatae was used to search open reading frames in the genome of methanococcus jannaschii. translations of two unidentified open reading frames showed significant sequence similarity to portions of the sulfolobus cca protein. when the two open reading frames were joined together, the expanded open reading frame was similar in sequence to the entire sulfolobus cca protein and displayed features of the active site si ...19979169562
factor requirements for transcription in the archaeon sulfolobus shibatae.archaea (archaebacteria) constitute a domain of life that is distinct from bacteria (eubacteria) and eucarya (eukaryotes). although archaeal cells share many morphological features with eubacteria, their transcriptional apparatus is more akin to eukaryotic rna polymerases i, ii and iii than it is to eubacterial transcription systems. thus, in addition to possessing a 10 subunit rna polymerase and a homologue of the tata-binding protein (tbp), archaea possess a polypeptide termed tfb that is homo ...19979184236
ssv1-encoded site-specific recombination system in sulfolobus shibatae.we present evidence for the existence of a conservative site-specific recombination system in archaea by demonstrating integrative recombination of sulfolobus shibatae virus ssv1 dna with the host chromosome, catalysed by the ssv1-encoded integrase in vitro. the putative int gene of ssv1 was expressed in escherichia coli yielding a protein of about 39 kda. this protein alone efficiently recombined linear dna substrates containing chromosomal (atta) and viral (attp) attachment sites; recombinatio ...19938483447
hin-mediated inversion on positively supercoiled dna.hin recombinase requires negatively supercoiled dna for an efficient inversion. we have generated positively supercoiled plasmid dna using reverse gyrase from sulfolobus shibatae and subjected it to the hin-mediated inversion reaction. both hin and fis showed the same dna binding activity regardless of the superhelical handedness of the substrate plasmid. however, inversion activity on positively supercoiled dna was less than 1% of negatively supercoiled dna. assays designed to probe steps in in ...19979218487
purification and characterization of reverse gyrase from sulfolobus shibatae. its proteolytic product appears as an atp-independent topoisomerase.sulfolobus shibatae b12 is a thermophilic archaebacterium that contains an inducible virus named ssv1. the viral dna has been shown to be positively supercoiled before encapsidation. we have previously purified an archaebacterial dna topoisomerase from sulfolobus acidocaldarius dsm 639, reverse gyrase, likely responsible for this positive supercoiling reaction. in order to study an homogeneous system containing both reverse gyrase and one of its preferential substrate, ssv1 dna, we have purified ...19948106509
purification of a dna topoisomerase ii from the hyperthermophilic archaeon sulfolobus shibatae. a thermostable enzyme with both bacterial and eucaryal features.a type ii dna topoisomerase has been purified to homogeneity from the hyperthermophilic archaeon sulfolobus shibatae. the enzyme is composed of two subunits of 60 and 47 kda. it has a stokes radius of 69 a and has a sedimentation coefficient of 7.8 s which gives a calculated native molecular mass of approximately 230 kda, indicating a heterotetrameric structure. this enzyme is atp and mg2+ dependent and can relax both negatively and positively supercoiled dna, but presents no supercoiling activi ...19947961685
1h, 15n, and 13c resonance assignments and secondary structure of the ssh10b from hyperthermophilic archaeon sulfolobus shibatae. 200212018492
purification and molecular cloning of the group ii chaperonin from the acidothermophilic archaeon, sulfolobus sp. strain 7.to elucidate the structure and functional mechanism of the group ii chaperonin, molecular cloning of the gene for and purification of the group ii chaperonin from the thermoacidophilic archaeon sulfolobus sp. strain 7 were performed. the purified sulfolobus chaperonin exhibited weak atpase activity and arrested the spontaneous refolding of the thermophilic lactate dehydrogenase. however, the refolding could not be resumed by addition of atp. the chaperonin consists of two kinds of subunits, alph ...19979245723
molecular characterization of the alpha-glucosidase gene (mala) from the hyperthermophilic archaeon sulfolobus solfataricus.acidic hot springs are colonized by a diversity of hyperthermophilic organisms requiring extremes of temperature and ph for growth. to clarify how carbohydrates are consumed in such locations, the structural gene (mala) encoding the major soluble alpha-glucosidase (maltase) and flanking sequences from sulfolobus solfataricus were cloned and characterized. this is the first report of an alpha-glucosidase gene from the archaeal domain. mala is 2,083 bp and encodes a protein of 693 amino acids with ...19989495770
[is chaperonin from archaea, sulfolobus shibatae, cytoskeleton?]. 19989503984
cca addition by trna nucleotidyltransferase: polymerization without translocation?the cca-adding enzyme repairs the 3'-terminal cca sequence of all trnas. to determine how the enzyme recognizes trna, we probed critical contacts between trna substrates and the archaeal sulfolobus shibatae class i and the eubacterial escherichia coli class ii cca-adding enzymes. both ctp addition to trna-c and atp addition to trna-cc were dramatically inhibited by alkylation of the same trna phosphates in the acceptor stem and tpsic stem-loop. both enzymes also protected the same trna phosphate ...19989606201
chaperonin filaments: their formation and an evaluation of methods for studying them.chaperonins are multisubunit protein complexes that can be isolated from cells as high-molecular-weight structures that appear as double rings in the electron microscope. we recently discovered that chaperonin double rings isolated from the hyperthermophilic archaeon sulfolobus shibatae, when incubated at physiological temperatures in the presence of atp and mg2+, stacked into filaments; we hypothesized that these filaments are related to filaments seen inside s. shibatae cells and that chaperon ...19989681991
the cca-adding enzyme has a single active site.the cca-adding enzyme (trna nucleotidyltransferase) synthesizes and repairs the 3'-terminal cca sequence of trna. the eubacterial, eukaryotic, and archaeal cca-adding enzymes all share a single active-site signature motif, which identifies these enzymes as belonging to the nucleotidyltransferase superfamily. here we show that mutations at asp-53 or asp-55 of the sulfolobus shibatae signature sequence abolish addition of both c and a, demonstrating that a single active site is responsible for add ...19989792681
a reporter gene system for the hyperthermophilic archaeon sulfolobus solfataricus based on a selectable and integrative shuttle vector.sulfolobus solfataricus has developed into an important model organism for molecular and biochemical studies of hyperthermophilic archaea. although a number of in vitro systems have been established for the organism, efficient tools for genetic manipulations have not yet been available for any hyperthermophile. in this work, we have developed a stable and selectable shuttle vector based on the virus ssv1 of sulfolobus shibatae. we have introduced puc18 for propagation in escherichia coli and the ...200312787352
reconstitution of dna topoisomerase vi of the thermophilic archaeon sulfolobus shibatae from subunits separately overexpressed in escherichia coli.dna topoisomerase vi from the hyperthermophilic archaeon sulfolobus shibatae is the prototype of a novel family of type ii dna topoisomerases that share little sequence similarity with other type ii enzymes, including bacterial and eukaryal type ii dna topoisomerases and archaeal dna gyrases. dna topoisomerase vi relaxes both negatively and positively supercoiled dna in the presence of atp and has no dna supercoiling activity. the native enzyme is a heterotetramer composed of two subunits, a and ...19989801313
the identification and partial characterisation of a novel inducible extracellular thermostable esterase from the archaeon sulfolobus shibatae.extracellular esterases have so far only been reported in eubacteria, here we report the first identification and partial characterisation of a novel inducible extracellular esterase from the thermoacidophilic archaeon sulfolobus shibatae. this esterase exhibits remarkable stability to both acid and heat. esterase activity is induced by growth on a range of polyoxyethylenesorbitan (tween) compounds as sole carbon source. activity occurs over a wide temperature (25-99 degrees c) and ph (ph4.0-9.0 ...19957488139
enzymes from sulfolobus shibatae for the production of trehalose and glucose from starch.enzymes that convert starch and dextrins to alpha,alpha-trehalose and glucose were found in cell homogenates of the hyperthermophilic acidophilic archaeon sulfolobus shibatae dms 5389. three enzymes were purified and characterized. the first, the s. shibatae trehalosyl dextrin-forming enzyme (sstdfe), transformed starch and dextrins to the corresponding trehalosyl derivatives with an intramolecular transglycosylation process that converted the glucosidic linkage at the reducing end from alpha-1, ...19989827330
the 60 kda heat shock proteins in the hyperthermophilic archaeon sulfolobus shibatae.one of the most abundant proteins in the hyperthermophilic archaeon sulfolobus shibatae is the 59 kda heat shock protein (tf55) that is believed to form a homo-oligomeric double ring complex structurally similar to the bacterial chaperonins. we discovered a second protein subunit in the s. shibatae ring complex (referred to as alpha) that is stoichiometric with tf55 (renamed beta). the gene and flanking regions of alpha were cloned and sequenced and its inferred amino acid sequence has 54.4% ide ...19957473746
the particle ssv1 from the extremely thermophilic archaeon sulfolobus is a virus: demonstration of infectivity and of transfection with viral dna.the lemon-shaped "virus-like" particle ssv1 produced by the thermophilic archaeon sulfolobus shibatae has not previously been observed to infect any host. using a plaque assay suitable for the extreme growth conditions of this archaeon, we have shown infection of sulfolobus solfataricus by ssv1. upon infection, the viral genome was always found integrated into a trna gene of the host chromosome, a situation similar to that in s. shibatae, proving that site-specific integration is involved in est ...19921502176
elements of an archaeal promoter defined by mutational analysis.the sequence requirements for specific and efficient transcription from the 16s/23s rrna promoter of sulfolobus shibatae were analysed by point mutations and by cassette mutations using an in vitro transcription system. the examination of the box a-containing distal promoter element (dpe) showed the great importance of the ta sequence in the center of box a for transcription efficiency and the influence of the sequence upstream of box a on determining the distance between the dpe and the start s ...19921279520
sulfolobicins, specific proteinaceous toxins produced by strains of the extremely thermophilic archaeal genus sulfolobus.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 mem ...200010781574
archaeal cca-adding enzymes: central role of a highly conserved beta-turn motif in rna polymerization without translocation.the cca-adding enzyme (trna nucleotidyltransferase) builds and repairs the 3' end of trna. a single active site adds both ctp and atp, but the enzyme has no nucleic acid template, and trna does not translocate or rotate during c75 and a76 addition. we modeled the structure of the class i archaeal sulfolobus shibatae cca-adding enzyme on eukaryotic poly(a) polymerase and mutated residues in the vicinity of the active site. we found mutations that specifically affected c74, c75, or a76 addition, a ...200415590678
production of a new sucrose derivative by transglycosylation of recombinant sulfolobus shibatae beta-glycosidase.the gene encoding beta-glycosidase of the hyperthermophilic archaea sulfolobus shibatae (ssg) was expressed in escherichia coli. recombinant ssg (referred to as rssg hereafter) was efficiently purified, and its transglycosylation activity was tested with lactose as a donor and various sugars as acceptors. when sucrose was used as an acceptor, we found a distinct intermolecular transglycosylation product and confirmed its presence by tlc and high performance anion exchange chromatography (hpaec). ...200515797124
sulfolobus shibatae cca-adding enzyme forms a tetramer upon binding two trna molecules: a scrunching-shuttling model of cca specificity.the trna cca-adding enzyme adds cca stepwise to immature transfer rna molecules untemplated, but with high specificity. we examined the oligomerization state of the enzyme from sulfolobus shibatae and its binding to transfer rna molecules, using various biophysical and biochemical methods including size exclusion chromatography, multi-angle laser light scattering, small-angle x-ray scattering, and gel electrophoresis band mobility shift assay. the 48 kda monomer forms a stable salt- resistant di ...200011090289
glutamate dehydrogenase from the aerobic hyperthermophilic archaeon aeropyrum pernix k1: enzymatic characterization, identification of the encoding gene, and phylogenetic implications.nadp-dependent glutamate dehydrogenase (l-glutamate: nadp oxidoreductase, deaminating, ec 1.4.1.4) from the aerobic hyperthermophilic archaeon aeropyrum pernix k1 (jcm 9820) was purified to homogeneity for characterization. the enzyme retained its full activity on heating at 95 degrees c for 30 min, and the maximum activity in l-glutamate deamination was obtained around 100 degrees c. the enzyme showed a strict specificity for l-glutamate and nadp on oxidative deamination and for 2-oxoglutarate ...200011139075
thermoadaptation of a mesophilic hygromycin b phosphotransferase by directed evolution in hyperthermophilic archaea: selection of a stable genetic marker for dna transfer into sulfolobus solfataricus.a mutated version of the hygromycin b phosphotransferase (hph(mut)) gene from escherichia coli, isolated by directed evolution at 75 degrees c in transformants of a thermophilic strain of sulfolobus solfataricus, was characterized with respect to its genetic stability in both the original mesophilic and the new thermophilic hosts. this gene was demonstrated to be able to express the hygromycin b resistance phenotype and to be steadily maintained and propagated also in other, more thermophilic st ...200111453458
purification of archaeal chaperonin from sulfolobus shibatae. 200011484476
cleavage properties of an archaeal site-specific recombinase, the ssv1 integrase.ssv1 is a virus infecting the extremely thermophilic archaeon sulfolobus shibatae. the viral-encoded integrase is responsible for site-specific integration of ssv1 into its host genome. the recombinant enzyme was expressed in escherichia coli, purified to homogeneity, and its biochemical properties investigated in vitro. we show that the ssv1 integrase belongs to the tyrosine recombinases family and that tyr(314) is involved in the formation of a 3'-phosphotyrosine intermediate. the integrase cl ...200211875075
poly(c) synthesis by class i and class ii cca-adding enzymes.the cca-adding enzymes [atp(ctp):trna nucleotidyl transferases], which catalyze synthesis of the conserved cca sequence to the trna 3' end, are divided into two classes. recent studies show that the class ii escherichia coli cca-adding enzyme synthesizes poly(c) when incubated with ctp alone, but switches to synthesize cca when incubated with both ctp and atp. because the poly(c) activity can shed important light on the mechanism of the untemplated synthesis of cca, it is important to determine ...200211926813
biochemical characterization of an atp-dependent dna ligase from the hyperthermophilic crenarchaeon sulfolobus shibatae.a gene encoding a putative atp-dependent dna ligase was identified in the genome of the hyperthermophilic archaeon sulfolobus shibatae and expressed in escherichia coli. the 601 amino acid recombinant polypeptide was a monomeric protein capable of strand joining on a singly nicked dna substrate in the presence of atp ( k(m)=34 micro mu) and a divalent cation (mn(2+), mg(2+), or ca(2+)). datp was partially active in supporting ligation catalyzed by the protein, but gtp, ctp, utp, dgtp, dctp, dttp ...200212486455
the composition, structure and stability of a group ii chaperonin are temperature regulated in a hyperthermophilic archaeon.the hyperthermoacidophilic archaeon sulfolobus shibatae contains group ii chaperonins, known as rosettasomes, which are two nine-membered rings composed of three different 60 kda subunits (tf55 alpha, beta and gamma). we sequenced the gene for the gamma subunit and studied the temperature-dependent changes in alpha, beta and gamma expression, their association into rosettasomes and their phylogenetic relationships. alpha and beta gene expression was increased by heat shock (30 min, 86 degrees c) ...200312657051
archaeal diversity in two thermophilic chalcopyrite bioleaching reactors.this study used a culture-independent molecular approach to investigate the archaeal community composition of thermophilic bioleaching reactors. two culture samples, mtc-a and mtc-b, grown with different concentrations of chalcopyrite (cufes2), a copper sulfidic ore, at a temperature of 78 degrees c and ph 1.6 were studied. phylogenetic analysis of the 16s rrna genes revealed that both cultures consisted of archaea belonging to the sulfolobales. the 16s rrna gene clone library of mtc-a grown wit ...200617014503
intracellular localization of a group ii chaperonin indicates a membrane-related function.chaperonins are protein complexes that are believed to function as part of a protein folding system in the cytoplasm of the cell. we observed, however, that the group ii chaperonins known as rosettasomes in the hyperthermophilic archaeon sulfolobus shibatae, are not cytoplasmic but membrane associated. this association was observed in cultures grown at 60 degrees c and 76 degrees c or heat-shocked at 85 degrees c by using immunofluorescence microscopy and in thick sections of rapidly frozen cell ...200314673104
two-dimensional crystals of reconstituted beta-subunits of the chaperonin tf55 from sulfolobus shibatae.we have obtained 2-dimensional crystals of the beta-subunits of the chaperonin tf55 from sulfolobus shibatae reconstituted into oligomers in the absence of alpha-subunits. the subunits form rings with 9-fold rotational symmetry which arrange themselves in a trigonal lattice. from electron micrographs of negatively stained specimens we have calculated a projection map in plane group p312 showing the rings in top-view.19989920382
analysis of dna cleavage by reverse gyrase from sulfolobus shibatae b12.reverse gyrase is a type i-5' topoisomerase, which catalyzes a positive dna supercoiling reaction in vitro. to ascertain how this reaction takes places, we looked at the dna sequences recognized by reverse gyrase. we used linear dna fragments of its preferred substrate, the viral ssv1 dna, which has been shown to be positively supercoiled in vivo. the sulfolobus shibatae b12 strain, an ssv1 virus host, was chosen for production of reverse gyrase. this naturally occurring system (ssv1 dna-s. shib ...199910091589
a novel species of thermoacidophilic archaeon, sulfolobus yangmingensis sp. nov.a novel microbe was isolated from a geothermal vent in yang-ming national park in northern taiwan. this spherical microbe with mean cell diameter of 1.1 +/- 0.2 microns is a facultatively chemolithoautotrophic archaeon that grows on elemental sulfur and reduced sulfur compounds. the optimal ph and temperature for growth are 4.0 (ph range 2.0-6.0) and 80 degrees c (temperature range 65-95 degrees c). its membranes contain the lipids calditoglycerocaldarchaeol and caldarchaeol, which are common to ...199910555364
three conformations of an archaeal chaperonin, tf55 from sulfolobus shibatae.chaperonins are cylindrical, oligomeric complexes, essential for viability and required for the folding of other proteins. the groe (group i) subfamily, found in eubacteria, mitochondria and chloroplasts, have 7-fold symmetry and provide an enclosed chamber for protein subunit folding. the central cavity is transiently closed by interaction with the co-protein, groes. the most prominent feature specific to the group ii subfamily, found in archaea and in the eukaryotic cytosol, is a long insertio ...200010677283
type 2 isopentenyl diphosphate isomerase from a thermoacidophilic archaeon sulfolobus shibatae.although isopentenyl diphosphate-dimethylallyl diphosphate isomerase is thought to be essential for archaea because they use the mevalonate pathway, its corresponding activity has not been detected in any archaea. a novel type of the enzyme, which has no sequence similarity to the known, well-studied type of enzymes, was recently reported in some bacterial strains. in this study, we describe the cloning of a gene of a homologue of the novel bacterial isomerase from a thermoacidophilic archaeon s ...200415009187
evolutionary conservation and dna binding properties of the ssh7 proteins from sulfolobus shibatae.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. thes ...200218762890
a single catalytically active subunit in the multimeric sulfolobus shibatae cca-adding enzyme can carry out all three steps of cca addition.the cca-adding enzyme atp(ctp):trna nucleotidyltransferase builds and repairs the 3'-terminal cca sequence of trna. although this unusual rna polymerase has no nucleic acid template, it can construct the cca sequence one nucleotide at a time using ctp and atp as substrates. we found previously that trna does not translocate along the enzyme during cca addition (yue, d., weiner, a. m., and maizels, n. (1998) j. biol. chem. 273, 29693-29700) and that a single nucleotidyltransferase motif adds all ...200415265870
evolution of the family of prn plasmids and their integrase-mediated insertion into the chromosome of the crenarchaeon sulfolobus solfataricus.plasmid phen7 from sulfolobus islandicus was sequenced (7.83 kb) and shown to belong to the archaeal prn family, which includes plasmids prn1, prn2, pssvx and pdl10 that share a large conserved sequence region. phen7 is most closely related to prn1 in this conserved region. it also shares a large variant region containing several homologous genes with pdl10, which is absent from the other plasmids. the variant region is flanked by the sequence motif ttagaatggggattc and similar duplicated motifs ...200011054282
[overproduction and partial characterization of the ssh7a and ssh7b proteins from sulfolobus shibatae].the sulfolobus shibatae ssh7a and ssh7b proteins have been separately overproduced in escherichia coli and purified using a simple procedure which includes a heat treatment step. the recombinant and native ssh7 proteins are similar in the ability to bind both negatively supercoiled and relaxed dnas. in addition, the recombinant ssh7a resembles the native ssh7 protein in constraining negative dna supercoils. our data suggest that the two isoforms of ssh7 interact with dna in a similar fashion, an ...200012548955
new role of flavin as a general acid-base catalyst with no redox function in type 2 isopentenyl-diphosphate isomerase.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-diphosphat ...200919158086
[cloning and expression of the gene encoding novel alpha-amylase from sulfolobus shibatae in e. coli].a novel alpha-amylase gene was amplified from sulfolobus shibatae by using pcr technique. the amplified 1.7 kb dna fragment was inserted into an expression vector pbv220 to yield the recombinant plasmid psbam. the novel alpha-amylase gene in psbam was expressed in e. coli. the production of the novel alpha-amylase activity reached over 8 units/100 ml of the culture. the molecular weight of this enzyme was about 61 kd by sds-page. the expressed novel alpha-amylase protein in e. coli dh5 alpha acc ...200012549000
two conformations of archaeal ssh10b. the origin of its temperature-dependent interaction with dna.the dna-binding protein ssh10b from the hyperthermophilic archaeon sulfolobus shibatae is a member of the sac10b family, which has been speculated to be involved in the organization of the chromosomal dna in archaea. ssh10b affects the dna topology in a temperature dependent fashion that has not been reported for any other dna-binding proteins. heteronuclear nmr and site-directed mutagenesis were used to analyze the structural basis of the temperature-dependent ssh10b-dna interaction. the data a ...200314523014
codon optimization reveals critical factors for high level expression of two rare codon genes in escherichia coli: rna stability and secondary structure but not trna abundance.expression patterns in escherichia coli of two small archaeal proteins with a natural content of about 30% rare codons were analyzed. the proteins, a histone-like protein from sulfolobus shibatae (ssh10), and a glutaredoxin-like protein from methanobacterium thermoautotrophicum (mtgrx), were produced with expression plasmids encoding wild-type genes, codon-optimized synthetic, and gst-fusion genes. these constructs were expressed in bl21 (de3), its lyss derivative, and modified strains carrying ...200414672702
molecular cloning and characterization of a thermostable carboxylesterase from an archaeon, sulfolobus shibatae dsm5389: non-linear kinetic behavior of a hormone-sensitive lipase family enzyme.a gene coding for an esterase (sshesti, 915 bp in length) of the thermoacidophilic archaeon sulfolobus shibatae dsm5389 was cloned, sequenced, and overexpressed in escherichia coli jm109 cells as a soluble, catalytically active protein. the deduced amino acid sequence of sshesti was consistent with a protein containing 305 amino acid residues with a molecular mass of 33 kda. sequence comparison studies indicated that sshesti could be a member of the hormone-sensitive lipase family, in that it ha ...200416233734
inhibition of archaeal growth and dna topoisomerase vi activities by the hsp90 inhibitor radicicol.type ii dna topoisomerases have been classified into two families, topo iia and topo iib, based on structural and mechanistic dissimilarities. topo iia is the target of many important antibiotics and antitumoural drugs, most of them being inactive on topo iib. the effects and mode of action of topo iia inhibitors in vitro and in vivo have been extensively studied for the last twenty-five years. in contrast, studies of topo iib inhibitors were lacking. to document this field, we have studied two ...200515849317
in silico modeling of the type 2 idi enzymes of bacillus licheniformis, pseudomonas stutzeri, streptococcus pyogenes, and staphylococcus aureus for virtual screening of potential inhibitors of this therapeutic target.isopentenyl diphosphate isomerase is an essential enzyme in those living organisms such as pathogenic strains of streptococcus and staphylococcus genera which rely on the mevalonate pathway for the production of isoprenoids. the pathogens contain type 2 idi in contrast to human that contains type 1 idi. therefore, the type 2 idi may be a potential target for the therapy of some infectious diseases. in the current study, a virtual screening by docking was performed among 2000 chemicals from cococ ...201223280415
refolding of the hyperthermophilic protein ssh10b involves a kinetic dimeric intermediate.the alpha/beta-mixed dimeric protein ssh10b from the hyperthermophile sulfolobus shibatae is a member of the sac10b family that is thought to be involved in chromosomal organization or dna repair/recombination. the equilibrium unfolding/refolding of ssh10b induced by denaturants and heat was fully reversible, suggesting that ssh10b could serve as a good model for folding/unfolding studies of protein dimers. here, we investigate the folding/unfolding kinetics of ssh10b in detail by stopped-flow c ...200919002648
highly enantioselective stereo-inverting sec-alkylsulfatase activity of hyperthermophilic archaea.rac-sec-alkyl sulfate esters 1a-8a were resolved in low to excellent enantioselectivities with e-values up to >200 using whole cells of aerobically-grown hyperthermophilic sulfur-metabolizers, such as sulfolobus solfataricus dsm 1617, sulfolobus shibatae dsm 5389 and, most notably, sulfolobus acidocaldarius dsm 639. significantly enhanced selectivities were obtained using cells grown on sucrose-enriched brock-medium. the stereochemical course of this biohydrolysis was shown to proceed with stric ...200515999201
characterization of a thermophilic atp-dependent dna ligase from the euryarchaeon pyrococcus horikoshii.archaea encode a dna ligase composed of a c-terminal catalytic domain typical of atp-dependent ligases plus an n-terminal domain similar to that found in eukaryotic cellular and poxvirus dna ligases. all archaeal dna ligases characterized to date have atp-dependent adenylyltransferase and nick-joining activities. however, recent reports of dual-specificity atp/nad+ ligases in two thermococcus species and pyrococcus abyssi and an atp/adp ligase in aeropyrum pernix raise the prospect that certain ...200516199559
archaeal rna polymerase: the influence of the protruding stalk in crystal packing and preliminary biophysical analysis of the rpo13 subunit.we review recent results on the complete structure of the archaeal rnap (rna polymerase) enzyme of sulfolobus shibatae. we compare the three crystal forms in which this rnap packs (space groups p212121, p21212 and p21) and provide a preliminary biophysical characterization of the newly identified 13-subunit rpo13. the availability of different crystal forms for this rnap allows the analysis of the packing degeneracy and the intermolecular interactions that determine this degeneracy. we observe t ...201121265742
novel thermostable y-family polymerases: applications for the pcr amplification of damaged or ancient dnas.for many years, taq polymerase has served as the stalwart enzyme in the pcr amplification of dna. however, a major limitation of taq is its inability to amplify damaged dna, thereby restricting its usefulness in forensic applications. in contrast, y-family dna polymerases, such as dpo4 from sulfolobus solfataricus, can traverse a wide variety of dna lesions. here, we report the identification and characterization of five novel thermostable dpo4-like enzymes from acidianus infernus, sulfolobus sh ...200616488882
bioenergetic response of the extreme thermoacidophile metallosphaera sedula to thermal and nutritional stresses.the bioenergetic response of the extremely thermoacidophilic archaeon metallosphaera sedula to thermal and nutritional stresses was examined. continuous cultures (ph 2.0, 70(deg)c, and dilution rate of 0.05 h(sup-1)) in which the levels of casamino acids and ferrous iron in growth media were reduced by a step change of 25 to 50% resulted in higher levels of several proteins, including a 62-kda protein immunologically related to the molecular chaperone designated thermophilic factor 55 in sulfolo ...199516535051
acquired thermotolerance and stressed-phase growth of the extremely thermoacidophilic archaeon metallosphaera sedula in continuous culture.the response of an extremely thermoacidophilic archaeon, metallosphaera sedula (growth temperature range, 50 to 79(deg)c; optimum temperature, 74(deg)c; optimum ph, 2.0), to thermal stress was investigated by using a 10-liter continuous cultivation system. m. sedula, growing at 74(deg)c, ph 2.0, and a dilution rate of 0.04 hr(sup-1), was subjected to both abrupt and gradual temperature shifts in continuous culture to determine the responses of cell density levels and protein synthesis patterns. ...199716535631
[ssh10b2 differs from its paralogue ssh10b in cellular abundance and the ability to constrain dna supercoils].the ssh10b and ssh10b2 genes, a pair of distantly related paralogues in sulfolobus shibatae, encode members of the sac10b dna binding protein family in thermophilic archaea. it has been shown previously that ssh10b exists in abundance in s. shibatae and is capable of constraining negative dna supercoils, properties that are consistent with a speculated architectural role for the protein in chromosomal organization. in this study, the ssh10b2 gene was cloned and expressed in escherichia coli, and ...200616736601
[cloning and expression of the gene encoding maltoologosyl trehalose synthase from sulfolobus shibatae in e. coli].2.2 kb dna fragment encoding a novel enzyme, maltooligosyl trehalose synthase (mtsase) was amplified from sulfolobus shibatae by using pcr technique. the amplified 2.2 kb dna fragment was inserted into an expression vector, pbv220, to yield the recombinant plasmid psbgt1. mtsase gene in pbsgt1 was expressed in e. coli. the molecular weight of expressed mtsase detected by sds-page was about 74 kd, which is conformed with that deduced from nucleotide sequence. the expressed mtsase protein accounte ...200012548879
evolution of complex rna polymerases: the complete archaeal rna polymerase structure.the archaeal rna polymerase (rnap) shares structural similarities with eukaryotic rnap ii but requires a reduced subset of general transcription factors for promoter-dependent initiation. to deepen our knowledge of cellular transcription, we have determined the structure of the 13-subunit dna-directed rnap from sulfolobus shibatae at 3.35 a resolution. the structure contains the full complement of subunits, including rpog/rpb8 and the equivalent of the clamp-head and jaw domains of the eukaryoti ...200919419240
[purification and characterization of a dna helicase from the hyperthermophilic archaeon sulfolobus shibatae].a dna helicase from the hyperthermophilic archaeon sulfolobus shibatae has been purified to homogeneity by column chromatography on q seqharose, phosphocellulose p11, heparin agarose, superdex 200 and phenyl superose. the purified enzyme shows dna-stimulated atpase activity. the molecular mass of the enzyme is approximately 63 kd. the s. shibatae dna helcase is capable of unwinding a 70 bp duplex dna flanked by unpaired single-stranded tails at both ends. the helicase activity requires mg2+ and ...200112549089
pav1, the first virus-like particle isolated from a hyperthermophilic euryarchaeote, "pyrococcus abyssi".we describe the first virus-like particle of a hyperthermophilic euryarchaeote which was discovered in a strain of "pyrococcus abyssi" previously characterized in our laboratory. this particle, named pav1, is lemon-shaped (120 nm x 80 nm), with a short tail terminated by fibers, and resembles the virus ssv1, the type member of the fuselloviridae, isolated from sulfolobus shibatae. sensitivity of the virus-like particle to organic solvents and detergents suggested that the envelope of pav1 may co ...200312813083
probing the archaeal diversity of a mixed thermophilic bioleaching culture by tgge and fish.the archaeal community present in a sample of mixed thermophilic culture-b (mtc-b) from a laboratory-scale thermophilic bioleaching reactor was investigated by temperature gradient gel electrophoresis (tgge) and fluorescence in situ hybridisation (fish). both techniques were specifically adapted for use on native state bioleaching samples, with a view to establishing convenient means for monitoring culture composition. using the tgge protocol developed, the relative species composition of the th ...200919541445
ssh10b, a conserved thermophilic archaeal protein, binds rna in vivo.proteins of the sac10b family, which is highly conserved among hyperthermophilic archaea, have been regarded as dna-binding proteins. based on their in vitro dna-binding properties, these proteins are thought to be involved in chromosomal organization or dna repair/recombination. we show that ssh10b, a member of the sac10b family from sulfolobus shibatae, bound with similar affinities to double-stranded dna, single-stranded dna and rna in vitro. however, the protein was exclusively bound to rna ...200314651642
the rosettazyme: a synthetic cellulosome.cellulose is an attractive feedstock for biofuel production because of its abundance, but the cellulose polymer is extremely stable and its constituent sugars are difficult to access. in nature, extracellular multi-enzyme complexes known as cellulosomes are among the most effective ways to transform cellulose to useable sugars. cellulosomes consist of a diversity of secreted cellulases and other plant cell-wall degrading enzymes bound to a protein scaffold. these scaffold proteins have cohesin m ...200919559062
protein-dna interactions at the sulfolobus spindle-shaped virus-1 (ssv1) t5 and t6 gene promoters.ultraviolet irradiation upregulates transcription from the sulfolobus spindle-shaped virus 1 (ssv-1) t5, t(ind), and t6 genes promoters and also triggers viral dna replication, but nothing is known about the proteins involved in this process. a notable feature of t5 and t6 promoters is that they contain 4 copies of a highly conserved dna sequence 5'-atagatagagt-3'; 2 copies of this repeat are found in tandem upstream of the a-box, whereas 2 additional tandem copies span the initiator region from ...200718026228
enzymatic characterization and substrate specificity of thermostable beta-glycosidase from hyperthermophilic archaea, sulfolobus shibatae, expressed in e. coli.enzymatic properties and substrate specificity of recombinant beta-glycosidases from a hyperthermophilic archaeon, sulfolobus shibatae (rssg), were analyzed. rssg showed its optimum temperature and ph at 95 degrees c and ph 5.0, respectively. thermal inactivation of rssg showed that its half-life of enzymatic activity at 75 degrees c was 15 h whereas it drastically decreased to 3.9 min at 95 degrees c. the addition of 10 mm of mncl2 enhanced the hydrolysis activity of rssg up to 23% whereas most ...200718050949
the flagellar filament structure of the extreme acidothermophile sulfolobus shibatae b12 suggests that archaeabacterial flagella have a unique and common symmetry and design.archaea, constituting a third domain of life between eubacteria and eukarya, characteristically inhabit extreme environments. they swim by rotating flagellar filaments that are phenomenologically and functionally similar to those of eubacteria. however, biochemical, genetic and structural evidence has pointed to significant differences but even greater similarity to eubacterial type iv pili. here we determined the three-dimensional symmetry and structure of the flagellar filament of the acidothe ...200818068187
a stabilizing alpha/beta-hydrophobic core greatly contributes to hyperthermostability of archaeal [p62a]ssh10b.the hyperthermophilic ssh10b from sulfolobus shibatae is a member of the sac10b family, which has been postulated to play a role in chromosomal organization in archaea. ssh10b is capable of significantly constraining negative dna supercoils at elevated temperatures. in this study, the solution structure of the dimeric p62a mutant ssh10b ([p62a]ssh10b) was determined by multidimensional nmr spectroscopy. the backbone 15n dynamics, h/d exchange with and without the denaturant gdmscn, and chemical ...200818821773
the structure of f-pili.exchange of dna between bacteria involves conjugative pili. while the prevailing view has been that f-pili are completely retracted before single-stranded dna is passed from one cell to another, it has recently been reported that the f-pilus, in addition to establishing the contact between mating cells, serves as a channel for passing dna between spatially separated cells during conjugation. the structure and function of f-pili are poorly understood. they are built from a single subunit having o ...200918992755
an intrinsically fluorescent recognition ligand scaffold based on chaperonin protein and semiconductor quantum-dot conjugates.genetic engineering of a novel protein-nanoparticle hybrid system with great potential for biosensing applications and for patterning of various types of nanoparticles is described. the hybrid system is based on a genetically modified chaperonin protein from the hyperthermophilic archaeon sulfolobus shibatae. this chaperonin is an 18-subunit double ring, which self-assembles in the presence of mg ions and atp. described here is a mutant chaperonin (his-beta-loopless, hbll) with increased access ...200919334012
cloning and characterization of glycogen-debranching enzyme from hyperthermophilic archaeon sulfolobus shibatae.a gene encoding a putative glycogen-debranching enzyme in sulfolobus shibatae (abbreviated as ssgde) was cloned and expressed in escherichia coli. the recombinant enzyme was purified to homogeneity by heat treatment and ni-nta affinity chromatography. the recombinant ssgde was extremely thermostable, with an optimal temperature at 85 degrees c. the enzyme had an optimum ph of 5.5 and was highly stable from ph 4.5 to 6.5. the substrate specificity of ssgde suggested that it possesses characterist ...200718051301
thermal unfolding of the archaeal dna and rna binding protein ssh10.the reversible thermal unfolding of the archaeal histone-like protein ssh10b from the extremophile sulfolobus shibatae was studied using differential scanning calorimetry and circular dichroism spectroscopy. analytical ultracentrifugation and gel filtration showed that ssh10b is a stable dimer in the ph range 2.5-7.0. thermal denaturation data fit into a two-state unfolding model, suggesting that the ssh10 dimer unfolds as a single cooperative unit with a maximal melting temperature of 99.9 degr ...200818571501
role of the sulfolobus shibatae viral t6 initiator in conferring promoter strength and in influencing transcription start site selection.archaeal promoters contain a tata-box, an adjacent upstream tfb-recognition element (bre), and a downstream initiator (inr) region from which transcription originates. while the contribution of a-box and bre to promoter strength is well established, the role of dna sequences within the inr region and its vicinity on transcription efficiency and start site selection remains unclear. here, i demonstrate using the strong sulfolobus shibatae viral t6 promoter that either substitution of its natural ...200617215906
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