complete nucleotide sequence of the sulfolobus islandicus multicopy plasmid prn1.the complete sequence of the 5350-bp plasmid prn1 from the crenarchaeote sulfolobus islandicus has been determined. this plasmid is the first to be sequenced from this group of thermoacidophilic archaebacteria (archaea) and its high copy number and wide host range make it a good candidate for a cloning vector. prn1 contains several open reading frames, including one that spans over half the plasmid and has significant similarity to the helicase domain of viral primase proteins. directly upstream ...19968700967
biochemical and phylogenetic characterization of the dutpase from the archaeal virus sirv.the derived amino acid sequence from a 474-base pair open reading frame in the genome of the sulfolobus islandicus rod-shaped virus sirv shows striking similarity to bacterial dctp deaminases and to dutpases from eukaryotes, bacteria, poxviridae, and retroviridae. the putative gene was expressed in escherichia coli, and dutpase activity of the recombinant enzyme was demonstrated by hydrolysis of dutp to dump. deamination of dctp by the enzyme was not detected. phylogenetic analysis based on amin ...19989497317
conjugation in archaea: frequent occurrence of conjugative plasmids in sulfolobus.we describe five novel conjugative plasmids (cps) and two subfamilies, each comprising several closely related variants of cps isolated from colony-cloned strains of the extremely thermophilic, heterotrophic archaeon sulfolobus islandicus, which were obtained by plating of samples from icelandic solfataras after liquid enrichment. they are related to each other and to the previously described cp pnob8 from a japanese sulfolobus strain in that they share essential functions and limited similarity ...19989806856
sulfolobus islandicus plasmids prn1 and prn2 share distant but common evolutionary ancestry.the complete sequence of the plasmid prn2 from the thermoacidophile sulfolobus islandicus has been determined. the plasmid was found to be circular and 6959 bp in length. s. islandicus harbors another endogenous plasmid, prn1, and comparison of prn1 and prn2 revealed that these two plasmids are essentially homologous, although very distantly related. prn1 and prn2 share several stretches of highly conserved noncoding dna and three common open reading frames. two of these reading frames are likel ...19989827327
molecular analysis of pdl10 from acidianus ambivalens reveals a family of related plasmids from extremely thermophilic and acidophilic archaea.the 7598-bp plasmid pdl10 from the extremely thermophilic, acidophilic, and chemolithoautotrophic archaeon acidianus ambivalens was sequenced. it contains 10 open reading frames (orfs) organized in five putative operons. the deduced amino acid sequence of the largest orf (909 aa) showed similarity to bacterial rep proteins known from phages and plasmids with rolling-circle (rc) replication. from the comparison of the amino acid sequences, a novel family of rc rep proteins was defined. the pdl10 ...199910430561
control of dna topology during thermal stress in hyperthermophilic archaea: dna topoisomerase levels, activities and induced thermotolerance during heat and cold shock in sulfolobus.plasmid topology varies transiently in hyperthermophilic archaea during thermal stress. as in mesophilic bacteria, dna linking number (lk) increases during heat shock and decreases during cold shock. despite this correspondence, plasmid dna topology and proteins presumably involved in dna topological control in each case are different. plasmid dna in hyperthermophilic archaea is found in a topological form from relaxed to positively supercoiled in contrast to the negatively supercoiled state typ ...199910447886
the genetic element pssvx of the extremely thermophilic crenarchaeon sulfolobus is a hybrid between a plasmid and a virus.a new sulfolobus islandicus strain, rey15/4, harboured both a novel fusellovirus, ssv2, and a small plasmid, pssvx. the plasmid spread in s. solfataricus p1 together with the virus after infection with either the supernatant of a culture of rey15/4 or purified virus. spreading of the plasmid required co-transfection with either ssv2 or the related ssv1 as helpers. virus purified from rey15/4 constituted a mixture of two sizes of particles, one with the dimensions of a normal fusellovirus and the ...199910564466
a novel lipothrixvirus, sifv, of the extremely thermophilic crenarchaeon sulfolobus.we describe a novel lipothrixvirus, sifv, of the crenarchaeotal archaeon sulfolobus islandicus. sifv (s. islandicus filamentous virus) has a linear virion with a linear double-stranded dna genome. these two features coincide in several crenarchaeotal but not in any other viruses. the sifv core is formed by a zipper-like array of dna-associated protein subunits and is covered by a lipid envelope containing host lipids. we sequenced approximately 96% of the virus genome excepting the dna termini, ...200010662621
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
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
ping family of conjugative plasmids from the extremely thermophilic archaeon sulfolobus islandicus: insights into recombination and conjugation in crenarchaeota.a novel family of conjugative plasmids from sulfolobus comprising the active variants ping1, -4, and -6 and the functionally defective variants ping2 and -3, which require the help of an active variant for spreading, has been extensively characterized both functionally and molecularly. in view of the sparse similarity between bacterial and archaeal conjugation and the lack of a practical genetic system for sulfolobus, we compared the functions and sequences of these variants and the previously d ...200011092863
thermostable and site-specific dna binding of the gene product orf56 from the sulfolobus islandicus plasmid prn1, a putative archael plasmid copy control protein.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 escherichi ...200111160922
holliday junction resolving enzymes of archaeal viruses sirv1 and the final stages of genetic recombination, holliday junction resolving enzymes transform the four-way dna intermediate into two duplex dna molecules by introducing pairs of staggered nicks flanking the junction. this fundamental process is apparently common to cells from all three domains of life. two cellular resolving enzymes from extremely thermophilic representatives of both kingdoms of the domain archaea, the euryarchaeon pyrococcus furiosus and the crenarchaeon sulfolobus solfataricus, ...200111399079
independent replication of the plasmids prn1 and prn2 in the archaeon sulfolobus islandicus.the 5.4-kb and 6.9-kb plasmids prn1 and prn2 from the crenarchaeon sulfolobus islandicus are name-giving for a small family of archaeal plasmids. both plasmids have hitherto been supposed to be dependent on each other because they are always found together in their natural host. here we demonstrate that each of the plasmids can stably propagate and replicate on its own independent of the other plasmid. moreover, we could show that in vivo the plasmids bear tightly bound proteins.200111410356
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.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 symmetri ...200111812827
halocins and sulfolobicins: the emerging story of archaeal protein and peptide antibiotics.production of antibiotic peptides and proteins is a near-universal feature of living organisms regardless of phylogenetic classification. bacteriocins (proteinaceous antimicrobials from the domain bacteria) have been studied for over 75 years, and the eucaryocins (proteinaceous antimicrobials from the domain eucarya) since the early 1960s. however, one domain of organisms, the archaea, containing hyperthermophiles, extreme halophiles and the methanogens, is just beginning to be scrutinized for t ...200211938468
a novel type of replicative enzyme harbouring atpase, primase and dna polymerase activity.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, prima ...200312743045
structure of a bifunctional dna primase-polymerase.genome replication generally requires primases, which synthesize an initial oligonucleotide primer, and dna polymerases, which elongate the primer. primase and dna polymerase activities are combined, however, in newly identified replicases from archaeal plasmids, such as prn1 from sulfolobus islandicus. here we present a structure-function analysis of the prn1 primase-polymerase (prim-pol) domain. the crystal structure shows a central depression lined by conserved residues. mutations on one side ...200414730355
folding and association of an extremely stable dimeric protein from sulfolobus islandicus.orf56 is a plasmid-encoded protein from sulfolobus islandicus, which probably controls the copy number of the prn1 plasmid by binding to its own promotor. the protein showed an extremely high stability in denaturant, heat, and ph-induced unfolding transitions, which can be well described by a two-state reaction between native dimers and unfolded monomers. the homodimeric character of native orf56 was confirmed by analytical ultracentrifugation. far-uv circular dichroism and fluorescence spectros ...200414741218
the replication protein of the sulfolobus islandicus plasmid prn1.the thermoacidophile crenarchaeote sulfolobus ssp. is one of the best-studied archaea. cryptic and conjugative plasmids as well as viruses have been described for this genus. for the majority of the genetic elements only the genome sequence and the basic characteristics were determined. in contrast the fusellovirus ssv1 and the cryptic plasmid prn1, which is the smallest known genetic element of the crenarchaeota, have been studied in more detail. the three gene products of the plasmid prn1 have ...200415046580
transcription of the rod-shaped viruses sirv1 and sirv2 of the hyperthermophilic archaeon sulfolobus.the double-stranded dna genomes of the crenarchaeal rudiviruses sirv1 (32 kb) and sirv2 (35 kb) were previously sequenced. here we present results of the analysis of gene expression of these viruses at different time points after infection of the host cell, sulfolobus islandicus, and of the mapping of transcriptional start sites. transcription of both genomes starts simultaneously at multiple sites spread over the total length of the genome and from both strands. the earliest time point when vir ...200415516589
recombination shapes the natural population structure of the hyperthermophilic archaeon sulfolobus islandicus.although microorganisms make up the preponderance of the biodiversity on earth, the ecological and evolutionary factors that structure microbial populations are not well understood. we investigated the genetic structure of a thermoacidophilic crenarchaeal species, sulfolobus islandicus, using multilocus sequence analysis of six variable protein-coding loci on a set of 60 isolates from the mutnovsky region of kamchatka, russia. we demonstrate significant incongruence among gene genealogies and a ...200516093568
characterisation of the novel restriction endonuclease suii from sulfolobus islandicus.a restriction endonuclease activity from sulfolobus islandicus ren2h1 was purified by phosphocellulose and cation exchange chromatography. the enzyme cuts dna at the recognition site gcwgc as could be shown by restriction analysis of plasmids and short synthetic duplex dna. the cleavage occurs after the first guanosine base and is inhibited by 5-methyl-cytosine methylation. the restriction activity is salt-sensitive and has an optimal activity around 70 degrees c.200616896525
characterization of the sulfolobus host-ssv2 virus interaction.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 trna(gly) (ccc) gene. in this paper we report the findings of a comparative study of ssv2 physiology in the natural host, sulfolobus islandicus rey15/4, versus the foreign host, sulfolobus solfataricus, and provide evidence of differently regulated ssv2 l ...200616896526
crystallization and preliminary x-ray diffraction analysis of protein 14 from sulfolobus islandicus filamentous virus (sifv).a large-scale programme has been embarked upon aiming towards the structural determination of conserved proteins from viruses infecting hyperthermophilic archaea. here, the crystallization of protein 14 from the archaeal virus sifv is reported. this protein, which contains 111 residues (mw 13 465 da), was cloned and expressed in escherichia coli with an n-terminal his(6) tag and purified to homogeneity. the tag was subsequently cleaved and the protein was crystallized using peg 1000 or peg 4000 ...200616946470
a spreadable, non-integrative and high copy number shuttle vector for sulfolobus solfataricus based on the genetic element pssvx from sulfolobus islandicus.the pssvx genetic element from sulfolobus islandicus rey15/4 is a hybrid between a plasmid and a fusellovirus, able to be maintained in non-integrative form and to spread when the helper ssv2 virus is present in the cells. in this work, the satellite virus was engineered to obtain an escherichia coli-sulfolobus solfataricus shuttle vector for gene transfer and expression in s.solfataricus by fusing site-specifically the pssvx chromosome with an e.coli plasmid replicon and the ampicillin resistan ...200616971457
an active nonautonomous mobile element in sulfolobus islandicus the crenarchaeote sulfolobus islandicus ren1h1, a mobile element of 321 bp length has been shown to be active. it does not contain terminal inverted repeats and transposes by a replicative mechanism. this newly discovered element has been named smn1 (for sulfolobus miniature noninverted repeat transposable element).200717158679
characterization of the transcriptional activity of the cryptic plasmid prn1 from sulfolobus islandicus ren1h1 and regulation of its replication operon.the plasmid prn1 from sulfolobus islandicus ren1h1 belongs to the crenarchaeal plasmid family prn. the plasmids in this family encode three conserved proteins that participate in plasmid replication and copy number regulation, as suggested by biochemical characterization of the recombinant proteins. in order to deepen our understanding of the molecular biology of these plasmids, we investigated the transcriptional activity of the model plasmid prn1. we detected five major transcripts present at ...200717172324
small multicopy, non-integrative shuttle vectors based on the plasmid prn1 for sulfolobus acidocaldarius and sulfolobus solfataricus, model organisms of the (cren-)archaea.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 p ...200717576673
transcriptional analysis of the genetic element pssvx: differential and temporal regulation of gene expression reveals correlation between transcription and replication.pssvx from sulfolobus islandicus strain rey15/4 is a hybrid between a plasmid and a fusellovirus. a systematic study performed by a combination of northern blot analysis, primer extension, and reverse transcriptase pcr revealed the presence of nine major transcripts whose expression was differentially and temporally regulated over the growth cycle of s. islandicus. the map positions of the rnas as well as the clockwise and the anticlockwise directions of their transcription were determined. some ...200717586636
structure of the acidianus filamentous virus 3 and comparative genomics of related archaeal lipothrixviruses.four novel filamentous viruses with double-stranded dna genomes, namely, acidianus filamentous virus 3 (afv3), afv6, afv7, and afv8, have been characterized from the hyperthermophilic archaeal genus acidianus, and they are assigned to the betalipothrixvirus genus of the family lipothrixviridae. the structures of the approximately 2-mum-long virions are similar, and one of them, afv3, was studied in detail. it consists of a cylindrical envelope containing globular subunits arranged in a helical f ...200817942536
molecular biology of the prn1 plasmid from sulfolobus islandicus.the prn1 plasmid is a rather small multicopy plasmid which was isolated from a sulfolobus islandicus strain in 1993 by wolfram zillig and co-workers. sequence analysis of the genome sequence suggested that three conserved genes are important for plasmid replication. these genes code for two sequence-specific dna-binding proteins (orf56 and orf80) and for a large multifunctional replication protein (orf904). the protein orf904 has primase, dna polymerase and helicase activity. remarkably, the pri ...200919143599
genetic analyses in the hyperthermophilic archaeon sulfolobus islandicus.sulfolobus belongs to the hyperthermophilic archaea and it serves as a model organism to study archaeal molecular biology and evolution. in the last few years, we have focused on developing genetic systems for sulfolobus islandicus using pyref as a selection marker and versatile genetic tools have been developed, including methods for constructing gene knockouts and for identifying essential genes. these genetic tools enable us to conduct genetic analysis on the functions of the genes involved i ...200919143609
viral biogeography revealed by signatures in sulfolobus islandicus genomes.viruses are a driving force of microbial evolution. despite their importance, the evolutionary dynamics that shape diversity in viral populations are not well understood. one of the primary factors that define viral population structure is coevolution with microbial hosts. experimental models predict that the trajectory of coevolution will be determined by the relative migration rates of viruses and their hosts; however, there are no natural microbial systems in which both have been examined. th ...200919196276
synergy of nmr, computation, and x-ray crystallography for structural biology.nmr spectroscopy and x-ray crystallography are currently the two most widely applied methods for the determination of macromolecular structures at high resolution. more recently, significant advances have been made in algorithms for the de novo prediction of protein structure, and, in favorable cases, the predicted models agree extremely well with experimentally determined structures. here, we demonstrate a synergistic combination of nmr spectroscopy, de novo structure prediction, and x-ray crys ...200919368883
biogeography of the sulfolobus islandicus pan-genome.variation in gene content has been hypothesized to be the primary mode of adaptive evolution in microorganisms; however, very little is known about the spatial and temporal distribution of variable genes. through population-scale comparative genomics of 7 sulfolobus islandicus genomes from 3 locations, we demonstrate the biogeographical structure of the pan-genome of this species, with no evidence of gene flow between geographically isolated populations. the evolutionary independence of each pop ...200919435847
the crystal structure of orf14 from sulfolobus islandicus filamentous virus. 200919452552
unmarked gene deletion and host-vector system for the hyperthermophilic crenarchaeon sulfolobus islandicus.sulfolobus islandicus is being used as a model for studying archaeal biology, geo-biology and evolution. however, no genetic system is available for this organism. to produce an s. islandicus mutant suitable for genetic analyses, we screened for colonies with a spontaneous pyref mutation. one mutant was obtained containing only 233 bp of the original pyre sequence in the mutant allele and it was used as a host to delete the beta-glycosidase (lacs) gene. two unmarked gene deletion methods were em ...200919513584
structure, function, and targets of the transcriptional regulator svtr from the hyperthermophilic archaeal virus sirv1.we have characterized the structure and the function of the 6.6-kda protein svtr (formerly called gp08) from the rod-shaped virus sirv1, which infects the hyperthermophilic archaeon sulfolobus islandicus that thrives at 85 degrees c in hot acidic springs. the protein forms a dimer in solution. the nmr solution structure of the protein consists of a ribbon-helix-helix (rhh) fold between residues 13 and 56 and a disordered n-terminal region (residues 1-12). the structure is very similar to that of ...200919535331
a unique virus release mechanism in the archaea.little is known about the infection cycles of viruses infecting cells from archaea, the third domain of life. here, we demonstrate that the virions of the archaeal sulfolobus islandicus rod-shaped virus 2 (sirv2) are released from the host cell through a mechanism, involving the formation of specific cellular structures. large pyramidal virus-induced protrusions transect the cell envelope at several positions, rupturing the s-layer; they eventually open out, thus creating large apertures through ...200919549825
exploitation of plant and archaeal viruses in bionanotechnology.cpmv (cowpea mosaic virus), a plant virus, is a naturally occurring sphere-like nanoparticle, and is used as a synthon and/or template in bionanoscience. the virions formed by cpmv can be regarded as programmable nanobuilding blocks with a diameter of approximately 30 nm. a range of molecules have been attached to this viral nanoscaffold, yielding stable nanoparticles that display multiple copies of the desired molecule. it has been shown that, in addition to surface amine groups, surface carbox ...200919614572
structure-based stability analysis of an extremely stable dimeric dna binding protein from sulfolobus islandicus.orf56 is a small and thermodynamically extremely stable dimeric protein from the archaeon sulfolobus islandicus. this dna binding protein is encoded on plasmid prn1 and possibly controls the copy number of the plasmid. we report the solution nmr structure as well as the crystal structure of orf56 comprising a ribbon-helix-helix fold. the homodimer consists of an antiparallel intersubunit beta-sheet and two alpha-helices per monomer, which is a common dna binding fold of plasmid- and phage-encode ...200919788170
an upstream activation element exerting differential transcriptional activation on an archaeal promoter.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 aras gene encoding an arabinose binding protein was characterized using an sulfolobus islandicus reporter gene system. the minimal active aras promoter (p(aras)) was found to be 59 nucleotides long and harboured ...200919818017
acidianus filamentous virus 1 coat proteins display a helical fold spanning the filamentous archaeal viruses lineage.acidianus filamentous virus 1 (afv1), a member of the lipothrixviridae family, infects the hyperthermophilic, acidophilic crenarchaeaon acidianus hospitalis. the virion, covered with a lipidic outer shell, is 9,100-a long and contains a 20.8-kb linear dsdna genome. we have identified the two major coat proteins of the virion (mcps; 132 and 140 amino acids). they bind dna and form filaments when incubated with linear dsdna. a c-terminal domain is identified in their crystal structure with a four- ...200919934032
the sulfolobus rod-shaped virus 2 encodes a prominent structural component of the unique virion release system in archaea.recently a unique mechanism of virion release was discovered in archaea, different from lysis and egress systems of bacterial and eukaryotic viruses. it involves formation of pyramidal structures on the host cell surface that rupture the s-layer and by opening outwards, create apertures through which mature virions escape the cell. here we present results of a protein analysis of sulfolobus islandicus cells infected with the rudivirus sirv2, which enable us to postulate sirv2-encoded protein p98 ...201020488501
revealing the essentiality of multiple archaeal pcna genes using a mutant propagation assay based on an improved knockout method.organisms belonging to the crenarchaeota lineage contain three proliferating cell nuclear antigen (pcna) subunits, while those in the euryarchaeota have only one, as for eukarya. to study the mechanism of archaeal sliding clamps, we sought to generate knockouts for each pcna gene in sulfolobus islandicus, a hyperthermophilic crenarchaeon, but failed with two conventional knockout methods. then, a new knockout scheme, known as marker insertion and target gene deletion (mid), was developed, with w ...201020705666
transcription termination in the plasmid/virus hybrid pssvx from sulfolobus islandicus.the pssvx from sulfolobus islandicus, strain rey15/4, is a hybrid between a plasmid and a fusellovirus. a systematic study previously performed revealed the presence of nine major transcripts, the expression of which was differentially and temporally regulated over the growth cycle of s. islandicus. in this study, two new transcripts were identified. then, 3' termini of all the rnas were mapped using adaptor rt-pcr and rnase protection assays, and termination/arrest positions were identified for ...201020734095
crispr associated diversity within a population of sulfolobus islandicus.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 hav ...201020927396
evolutionary rates and gene dispensability associate with replication timing in the archaeon sulfolobus bacterial chromosomes, the position of a gene relative to the single origin of replication generally reflects its replication timing, how often it is expressed, and consequently, its rate of evolution. however, because some archaeal genomes contain multiple origins of replication, bias in gene dosage caused by delayed replication should be minimized and hence the substitution rate of genes should associate less with chromosome position. to test this hypothesis, six archaeal genomes from the g ...201020978102
dynamic properties of the sulfolobus crispr/cas and crispr/cmr systems when challenged with vector-borne viral and plasmid genes and protospacers.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 ...201021166892
c68 from the sulfolobus islandicus plasmid-virus pssvx is a novel member of the abrb-like transcription factor family.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 that does not show significant sequence homology with any protein with known three-dimensional structure. emsa (electrophoretic mobility-shift assay) experiments, dna footprinting and cd analyses indicat ...201121208189
archaeal promoter architecture and mechanism of gene activation.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 modula ...201121265754
genome analyses of icelandic strains of sulfolobus islandicus, model organisms for genetic and virus-host interaction studies.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 host range for exceptional crenarchaeal viruses and conjugative plasmids and was selected for studying their life cycles and host interactions. the genomes of strains rey15a and hve10/4 are 2.5 and 2.7 mb ...201121278296
simple and elegant design of a virion egress structure in archaea.some viruses of archaea use an unusual egress mechanism that involves the formation of virus-associated pyramids (vaps) on the host cell surface. at the end of the infection cycle, these structures open outward and create apertures through which mature virions escape from the cell. here we describe in detail the structure and composition of vaps formed by the sulfolobus islandicus rod-shaped virus 2 (sirv2) in cells of its hyperthermophilic archaeal host. we show that the vaps are stable and aut ...201121282609
crystallization and preliminary x-ray diffraction analysis of the hyperthermophilic sulfolobus islandicus lactonase.phosphotriesterase-like lactonases (plls) constitute an interesting family of enzymes that are of paramount interest in biotechnology with respect to their catalytic functions. as natural lactonases, they may act against pathogens such as-ápseudomonas aeruginosa by shutting down their quorum-sensing system (quorum quenching) and thus decreasing pathogen virulence. owing to-átheir promiscuous phosphotriesterase activity, which can inactivate toxic organophosphorus compounds such as pesticides and ...201121393842
exceptional virion release mechanism: one more surprise from archaeal viruses.virion release from the host cell is the final and essential step for completion of the viral life cycle and spread of virions in the environment. although for eukaryotic and bacterial viruses the egress mechanisms are reasonably well understood, this subject has not been studied in detail for archaeal viruses until recently. here we summarize available data on the extraordinary egress mechanism exploited by the sulfolobus islandicus rod-shaped virus sirv2 and the sulfolobus turreted icosahedral ...201121531608
characterization of sulfolobus islandicus rod-shaped virus 2 gp19, a single-strand specific endonuclease.the hyperthermophilic sulfolobus islandicus rod-shaped virus 2 (sirv2) encodes a 25-kda protein (sirv2gp19) annotated as a hypothetical protein with sequence homology to the recb nuclease superfamily. even though sirv2gp19 homologs are conserved throughout the rudivirus family and presumably play a role in the viral life cycle, sirv2gp19 has not been functionally characterized. to define the minimal requirements for activity, sirv2gp19 was purified and tested under varying conditions. sirv2gp19 ...201121667093
deletion of the topoisomerase iii gene in the hyperthermophilic archaeon sulfolobus islandicus results in slow growth and defects in cell cycle control.topoisomerase iii (topo iii), a type ia topoisomerase, is widespread in hyperthermophilic archaea. in order to interrogate the in-ávivo 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 than the wild-type strain, especially in a nutrient-poor medium. flow cytometry analysis revealed changes of the mutant in growth cycle characteristics including an increase in proportion of cells contai ...201121703549
exceptional thermal stability and organic solvent tolerance of an esterase expressed from a thermophilic host.a protein expression system recently developed for the thermophilic crenarchaeon sulfolobus islandicus was employed to produce recombinant protein for esta, a thermophilic esterase encoded in the same organism. large amounts of protein were readily obtained by an affinity protein purification, giving sisesta. upon escherichia coli expression, only the thioredoxin-tagged esta recombinant protein was soluble. the fusion protein was then purified, and removing the protein tag yielded ecsisesta. bot ...201121847512
an expansion of age constraints for microbial clades that lack a conventional fossil record using phylogenomic dating.most microbial taxa lack a conventional microfossil or biomarker record, and so we currently have little information regarding how old most microbial clades and their associated traits are. building on the previously published oxygen age constraint, two new age constraints are proposed based on the ability of microbial clades to metabolize chitin and aromatic compounds derived from lignin. using the archaeal domain of life as a test case, phylogenetic analyses, along with published metabolic and ...201122105429
biochemical characterization of a structure-specific resolving enzyme from sulfolobus islandicus rod-shaped virus 2.sulfolobus islandicus rod shaped virus 2 (sirv2) infects the archaeon sulfolobus islandicus at extreme temperature (70°c-80°c) and acidity (ph 3). sirv2 encodes a holliday junction resolving enzyme (sirv2 hjr) that has been proposed as a key enzyme in sirv2 genome replication. the molecular mechanism for sirv2 hjr four-way junction cleavage bias, minimal requirements for four-way junction cleavage, and substrate specificity were determined. sirv2 hjr cleaves four-way dna junctions with a prefere ...201121858199
Development of a Simvastatin Selection Marker for a Hyperthermophilic Acidophile, Sulfolobus islandicus.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 to this crenarchaeon. The basic plasmid vector pSSR was constructed by substituting the pyrEF gene of the expression vector pSeSD for P(sac7d)-hmg with which the Sulfolobus expression plasmids pSSRlac ...201222081574
modulation of crispr locus transcription by the repeat-binding protein cbp1 in sulfolobus.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 ...201122139923
an archaeal crispr type iii-b system exhibiting distinctive rna targeting features and mediating dual rna and dna interference.crispr-cas systems provide a small rna-based mechanism to defend against invasive genetic elements in archaea and bacteria. to investigate the in vivo mechanism of rna interference by two type iii-b systems (cmr-α and cmr-β) in sulfolobus islandicus, a genetic assay was developed using plasmids carrying an artificial mini-crispr (ac) locus with a single spacer. after pac plasmids were introduced into different strains, northern analyses confirmed that mature crrnas were produced from the plasmid ...201425505143
identification and characterization of a highly conserved crenarchaeal protein lysine methyltransferase with broad substrate specificity.protein lysine methylation occurs extensively in the crenarchaeota, a major kingdom in the archaea. however, the enzymes responsible for this type of posttranslational modification have not been found. here we report the identification and characterization of the first crenarchaeal protein lysine methyltransferase, designated akmt, from the hyperthermophilic crenarchaeon sulfolobus islandicus. the enzyme was capable of transferring methyl groups to selected lysine residues in a substrate protein ...201223086207
structural and enzymatic characterization of the lactonase sislac from sulfolobus islandicus.a new member of the phosphotriesterase-like lactonases (pll) family from the hyperthermophilic archeon sulfolobus islandicus (sislac) has been characterized. sislac is a native lactonase that exhibits a high promiscuous phosphotriesterase activity. sislac thus represents a promising target for engineering studies, exhibiting both detoxification and bacterial quorum quenching abilities, including human pathogens such as pseudomonas aeruginosa.201223071703
virus-induced dormancy in the archaeon sulfolobus islandicus.we investigated the interaction between sulfolobus spindle-shaped virus (ssv9) and its native archaeal host sulfolobus islandicus. we show that upon exposure to ssv9, s. islandicus strain rjw002 has a significant growth delay where the majority of cells are dormant (viable but not growing) for 24 to 48 hours postinfection (hpi) compared to the growth of controls without virus. we demonstrate that in this system, dormancy (i) is induced by both active and inactive virus particles at a low multipl ...201525827422
the identification of a novel sulfolobus islandicus camp-like peptide points to archaeal microorganisms as cell factories for the production of antimicrobial molecules.pathogenic bacteria easily develop resistance to c onventional antibiotics so that even relatively new molecules are quickly losing efficacy. this strongly encourages the quest of new antimicrobials especially for the treatment of chronic infections. cationic antimicrobial peptides (camps) are small positively charged peptides with an amphipathic structure, active against gram-positive and gram-negative bacteria, fungi, as well as protozoa.201526338197
protein-protein interactions leading to recruitment of the host dna sliding clamp by the hyperthermophilic sulfolobus islandicus rod-shaped virus 2.viruses infecting hyperthermophilic archaea typically do not encode dna polymerases, raising questions regarding their genome replication. here, using a yeast two-hybrid approach, we have assessed interactions between proteins of sulfolobus islandicus rod-shaped virus 2 (sirv2) and the host-encoded proliferating cell nuclear antigen (pcna), a key dna replication protein in archaea. five sirv2 proteins were found to interact with pcna, providing insights into the recruitment of host replisome for ...201424696494
massive activation of archaeal defense genes during viral infection.archaeal viruses display unusually high genetic and morphological diversity. studies of these viruses proved to be instrumental for the expansion of knowledge on viral diversity and evolution. the sulfolobus islandicus rod-shaped virus 2 (sirv2) is a model to study virus-host interactions in archaea. it is a lytic virus that exploits a unique egress mechanism based on the formation of remarkable pyramidal structures on the host cell envelope. using whole-transcriptome sequencing, we present here ...201323698312
virology. a virus that infects a hyperthermophile encapsidates a-form dna.extremophiles, microorganisms thriving in extreme environmental conditions, must have proteins and nucleic acids that are stable at extremes of temperature and ph. the nonenveloped, rod-shaped virus sirv2 (sulfolobus islandicus rod-shaped virus 2) infects the hyperthermophilic acidophile sulfolobus islandicus, which lives at 80°c and ph 3. we have used cryo-electron microscopy to generate a three-dimensional reconstruction of the sirv2 virion at ~4 angstrom resolution, which revealed a previousl ...201525999507
functional characterization of a conserved archaeal viral operon revealing single-stranded dna binding, annealing and nuclease activities.the majority of archaeal viral genes are of unknown function hindering our understanding of the virus life cycle and viral interactions with their host. here, we first describe functional characterization of orf131b (gp17) and orf436 (gp18) of sulfolobus islandicus rod-shaped virus 2 (sirv2), both encoding proteins of unknown function and forming an operon with orf207 (gp19). sirv2 gp17 was found to be a single-stranded dna (ssdna) binding protein different in structure from all previously chara ...201525828870
unveiling cell surface and type iv secretion proteins responsible for archaeal rudivirus entry.sulfolobus mutants resistant to archaeal lytic virus sulfolobus islandicus rod-shaped virus 2 (sirv2) were isolated, and mutations were identified in two gene clusters, cluster sso3138 to sso3141 and cluster sso2386 and sso2387, encoding cell surface and type iv secretion proteins, respectively. the involvement of the mutations in the resistance was confirmed by genetic complementation. blocking of virus entry into the mutants was demonstrated by the lack of early gene transcription, strongly su ...201424965447
first insights into the entry process of hyperthermophilic archaeal viruses.a decisive step in a virus infection cycle is the recognition of a specific receptor present on the host cell surface, subsequently leading to the delivery of the viral genome into the cell interior. until now, the early stages of infection have not been thoroughly investigated for any virus infecting hyperthermophilic archaea. here, we present the first study focusing on the primary interactions between the archaeal rod-shaped virus sulfolobus islandicus rod-shaped virus 2 (sirv2) (family rudiv ...201324089554
genomics and biology of rudiviruses, a model for the study of virus-host interactions in archaea.archaeal viruses, especially viruses that infect hyperthermophilic archaea of the phylum crenarchaeota, constitute one of the least understood parts of the virosphere. however, owing to recent substantial research efforts by several groups, archaeal viruses are starting to gradually reveal their secrets. in the present review, we summarize the current knowledge on one of the emerging model systems for studies on crenarchaeal viruses, the rudiviridae. we discuss the recent advances towards unders ...201323356326
insights into a viral lytic pathway from an archaeal virus-host system.archaeal host cells infected by sulfolobus turreted icosahedral virus (stiv) and sulfolobus islandicus rod-shaped virus 2 (sirv2) produce unusual pyramid-like structures on the cell surface prior to virus-induced cell lysis. this viral lysis process is distinct from known viral lysis processes associated with bacterial or eukaryal viruses. the stiv protein c92 and the sirv2 protein 98 are the only viral proteins required for the formation of the pyramid lysis structures of stiv and sirv2, respec ...201323221563
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.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 <30% dna-dna hybridization (ddh) values with the most closely related known sulfolobus species (i.e., sulfolobus islandicus and sulfolobus solfataricus), suggesting that it represents a novel sulfolobus spe ...201627965637
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