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At least 19 recordsLinked to original sources
PubMed2000

The genome sequence of the thermoacidophilic scavenger Thermoplasma acidophilum.

Thermoplasma acidophilum is a thermoacidophilic archaeon that thrives at 59 degrees C and pH 2, which was isolated from self-heating coal refuse piles and solfatara fields. Species of the genus Thermoplasma do not possess a rigid cell wall, but are only delimited by a plasma membrane. Many macromolecular assemblies from Thermoplasma, primarily proteases and chaperones, have been pivotal in elucidating the structure and function of their more complex eukaryotic homologues. Our interest in protein folding and degradation led us to seek a more complete representation of the proteins involved in these pathways by determining the genome sequence of the organism. Here we have sequenced the 1,564,905-base-pair genome in just 7,855 sequencing reactions by using a new strategy. The 1,509 open reading frames identify Thermoplasma as a typical euryarchaeon with a substantial complement of bacteria-related genes; however, evidence indicates that there has been much lateral gene transfer between Thermoplasma and Sulfolobus solfataricus, a phylogenetically distant crenarchaeon inhabiting the same environment. At least 252 open reading frames, including a complete protein degradation pathway and various transport proteins, resemble Sulfolobus proteins most closely.

Archaeal Proteins↗
PubMed1999

The import pathway of human and Thermoplasma 20S proteasomes into HeLa cell nuclei is different from that of classical NLS-bearing proteins.

Wild-type proteasomes of human erythrocytes and the archaeon Thermoplasma acidophilum compete with each other for transport into nuclei of digitonin-permeabilized HeLa cells in the presence of an energy-regenerating system and rabbit reticulocyte lysate. 'NLS'-mutated Thermoplasma proteasomes were also able to compete with human proteasomes in the same assay, although with lower efficiency. Furthermore, in contrast to the other archaeal and bacterial cell lysates tested, the Thermoplasma cytosol efficiently supported nuclear import of human and Thermoplasma proteasomes. However, the same lysate could barely direct the nuclear transport of BSA-NLSsv40 peptide conjugates or the classical NLS-bearing protein, nucleoplasmin. Finally, additional importin alpha/beta significantly decreased the import efficiency of both human and Thermoplasma proteasomes. Taken together, these results suggest that nuclear import of proteasomes may use a novel pathway that is different from that of classical NLS-bearing proteins.

Animals↗
PubMed1992

Black lipid membranes of tetraether lipids from Thermoplasma acidophilum.

Black lipid membranes were formed of tetraether lipids from Thermoplasma acidophilum and compared to the bilayer forming lipids diphytanoylphosphatidylcholine and diphythanylglucosylglycerol. Bilayer-forming lipids varied in thickness of black lipid membranes due to the organic solvent used. Measurements of the specific membrane capacitance (Cm = 0.744 microF/cm2) showed that the membrane-spanning tetraether lipids from Thermoplasma acidophilum form a monolayer of a constant thickness of 2.5-3.0 nm no matter from which solvent. This finding corresponds to the results of Gliozzi et al. for the lipids of another archaebacterium, Sulfolobus solfataricus. Black lipid membranes were formed at room temperature with a torus from bilayer-forming lipids, however, the torus could also be formed by the tetraether-lipid itself at room temperature and at defined concentration. In these stable black lipid membranes, conductance was measured in the presence of valinomycin, nonactin, and gramicidin. At 10(-7) M concentration, valinomycin mediated higher conductance in membranes from tetraether lipids (200-1200 microS/cm2) than from bilayer-forming lipids (125-480 microS/cm2). Nonactin, at 10(-6) M concentration, mediated a 6-fold higher conductance in a tetraether lipid membrane than in a bilayer, whereas conductance, in the presence of 5 x 10(-11) M gramicidin could reach higher values in bilayers than in tetraether lipid monolayers of comparable thickness. Monensin did not increase the conductance of black lipid membranes from tetraether lipids under all conditions applied in our experiments. Poly(L-lysine) destroyed black lipid membranes. Lipopolysaccharides from Thermoplasma acidophilum were not able to form stable black lipid membranes by themselves. The lipopolysaccharide complexes from Thermoplasma acidophilum and from Escherichia coli decreased the valinomycin-mediated conductance of monolayer and bilayer membranes. This influence was stronger than that of the polysaccharide dextran.

Electric Conductivity↗
PubMed2001

The intein of the Thermoplasma A-ATPase A subunit: structure, evolution and expression in E. coli.

BACKGROUND: Inteins are selfish genetic elements that excise themselves from the host protein during post translational processing, and religate the host protein with a peptide bond. In addition to this splicing activity, most reported inteins also contain an endonuclease domain that is important in intein propagation. RESULTS: The gene encoding the Thermoplasma acidophilum A-ATPase catalytic subunit A is the only one in the entire T. acidophilum genome that has been identified to contain an intein. This intein is inserted in the same position as the inteins found in the ATPase A-subunits encoding gene in Pyrococcus abyssi, P. furiosus and P. horikoshii and is found 20 amino acids upstream of the intein in the homologous vma-1 gene in Saccharomyces cerevisiae. In contrast to the other inteins in catalytic ATPase subunits, the T. acidophilum intein does not contain an endonuclease domain.T. acidophilum has different codon usage frequencies as compared to Escherichia coli. Initially, the low abundance of rare tRNAs prevented expression of the T. acidophilum A-ATPase A subunit in E. coli. Using a strain of E. coli that expresses additional tRNAs for rare codons, the T. acidophilum A-ATPase A subunit was successfully expressed in E. coli. CONCLUSIONS: Despite differences in pH and temperature between the E. coli and the T. acidophilum cytoplasms, the T. acidophilum intein retains efficient self-splicing activity when expressed in E. coli. The small intein in the Thermoplasma A-ATPase is closely related to the endonuclease containing intein in the Pyrococcus A-ATPase. Phylogenetic analyses suggest that this intein was horizontally transferred between Pyrococcus and Thermoplasma, and that the small intein has persisted in Thermoplasma apparently without homing.

Adenosine Triphosphatases↗
PubMed2001

Occurrence of D-Amino Acids and a pyridoxal 5'-phosphate-dependent aspartate racemase in the acidothermophilic archaeon, Thermoplasma acidophilum.

Free D-amino acid content in some archaea was investigated and D-forms of several amino acids were found in them. In the acidothermophilic archaeon, Thermoplasma acidophilum, the proportion of D-aspartate (D-Asp) to total Asp was as high as 39.7%. Crude extracts of Thermoplasma acidophilum had Asp-specific racemase activity that was pyridoxal 5'-phosphate (PLP)-dependent. The relative insensitivity to a SH-modifying reagent distinguished this activity from those of the PLP-independent Asp racemases found in other hyperthermophilic archaea (Matsumoto, M., et al., J. Bacteriol. 181, 6560-6563 1999). Thus, high levels of d-Asp should be produced by a new type(s) of Asp-specific racemase in Thermoplasma acidophilum, although the function of d-Asp in this archaeon remains unknown.

Alanine↗
PubMed1997

The thermosome: alternating alpha and beta-subunits within the chaperonin of the archaeon Thermoplasma acidophilum.

The thermosome of the archaeon Thermoplasma acidophilum is composed of two subunits, alpha and beta, which are arranged in two stacked, eight-membered rings. Electron cryo-microscopy in conjunction with image analysis revealed a 4-fold symmetry in the heterooligomeric alpha + beta thermosome isolated from Thermoplasma, but not in the homooligomeric alpha-only thermosome expressed in Escherichia coli. This indicates that alpha and beta-subunits alternate within the rings of the Thermoplasma thermosome rather than forming two different homooligomeric rings. In addition, a small subpopulation of 9-fold symmetric complexes was found among the recombinant alpha-only thermosomes, and a central mass most likely representing bound substrate molecules was observed in about half of the native and recombinant thermosome particles.

Adenosine Triphosphatases↗
PubMed2006

Structural analysis of the plasmid pTA1 isolated from the thermoacidophilic archaeon Thermoplasma acidophilum.

Thermoplasma acidophilum is a thermoacidophilic archaeon that grows optimally at pH1.8 and 56 degrees C and has no cell wall. Plasmid pTA1 was found in some strains of the species. We sequenced plasmid pTA1 and analyzed the open reading frames (ORFs). pTA1 was found to be a circular DNA molecule of 15,723 bp. Eighteen ORFs were found; none of the gene products except ORF1 had sequence similarity to known proteins. ORF1 showed similarity to Cdc6, which is involved in genome-replication initiation in Eukarya and Archaea. T. acidophilum has two Cdc6 homologues in the genome. The homologue found in pTA1 is most similar to Tvo3, one of the three Cdc6 homologues found in the genome of Thermoplasma volcanium, among all of the Cdc6 family proteins. The phylogenetic analysis suggested that plasmid pTA1 is possibly originated from the chromosomal DNA of Thermoplasma.

Amino Acid Sequence↗
PubMed1991

Expression and purification of plasmid-encoded Thermoplasma acidophilum citrate synthase from Escherichia coli.

The citrate synthase gene from the thermophilic archaebacterium Thermoplasma acidophilum was expressed in Escherichia coli, yielding an active product of the expected molecular weight. Manipulation of the citrate synthase gene in a series of pUC19 constructs showed that the presumed Thermoplasma ribosome binding site is recognized by the E. coli ribosome. A rapid purification of the expression product to homogeneity was achieved, based on the thermostability of Thermoplasma citrate synthase.

Base Sequence↗
PubMed2004

The Thermoplasma acidophilum Lon protease has a Ser-Lys dyad active site.

A gene with significant similarity to bacterial Lon proteases was identified during the sequencing of the genome of the thermoacidophilic archaeon Thermoplasma acidophilum. Protein sequence comparison revealed that Thermoplasma Lon protease (TaLon) is more similar to the LonB proteases restricted to Gram-positive bacteria than to the widely distributed bacterial LonA. However, the active site residues of the protease and ATPase domain are highly conserved in all Lon proteases. Using site-directed mutagenesis we show here that TaLon and EcLon, and probably all other Lon proteases, contain a Ser-Lys dyad active site. The TaLon active site mutants were fully assembled and, similar to TaLon wild-type, displayed an apparent molar mass of 430 kDa upon gelfiltration. This would be consistent with a hexameric complex and indeed electron micrographs of TaLon revealed ring-shaped particles, although of unknown symmetry. Comparison of the ATPase activity of Lon wild-type from Thermoplasma or Escherichia coli with respective protease active site mutants revealed differences in Km and V values. This suggests that in the course of protein degradation by wild-type Lon the protease domain might influence the activity of the ATPase domain.

Amino Acid Sequence↗
PubMed2004

Thermoplasma acidophilum TAA43 is an archaeal member of the eukaryotic meiotic branch of AAA ATPases.

Sequencing of the Thermoplasma acidophilum genome revealed a new gene, taa43 , which codes for a 43-kDa protein containing one AAA domain; we therefore termed it Thermoplasma AAA ATPase of 43 kDa (TAA43). Close homologs of TAA43 are found only in related Thermoplasmales, e.g. T. volcanium and Ferroplasma acidarmanus , but not in other Archaea. A detailed phylogenetic analysis showed that TAA43 and its homologs belong to the 'meiotic' branch of the AAA family. Although AAA proteins usually assemble into high-molecular-weight complexes, native TAA43 is predominantly dimeric except for a minor fraction eluting in the void volume of a sizing column. Wild-type and mutant TAA43 proteins were overexpressed in Escherichia coli , purified as dimers and characterized functionally. Since the canonical proteasome activating nucleotidase is not present in Thermoplasmales, TAA43 was tested for stimulation of proteasome activity, which was, however, not detected. Interestingly, immunoprecipitation analysis with TAA43 specific antibodies found a fraction of native TAA43 associated with Thermoplasma ribosomal proteins.

Adenosine Triphosphatases↗
PubMed1992

Subunit stoichiometry and three-dimensional arrangement in proteasomes from Thermoplasma acidophilum.

The proteasome or multicatalytic proteinase from the archaebacterium Thermoplasma acidophilum is a 700 kDa multisubunit protein complex. Unlike proteasomes from eukaryotic cells which are composed of 10-20 different subunits, the Thermoplasma proteasome is made of only two types of subunit, alpha and beta, which have molecular weights of 25.8 and 22.3 kDa, respectively. In this communication we present a three-dimensional stoichiometric model of the archaebacterial proteasome deduced from electron microscopic investigations. The techniques which we have used include image analysis of negatively stained single particles, image analysis of metal decorated small three-dimensional crystals after freeze-etching and STEM mass measurements of freeze-dried particles. The archaebacterial and eukaryotic proteasomes are almost identical in size and shape; the subunits are arranged in four rings which are stacked together such that they collectively form a barrel-shaped complex. According to a previous immunoelectron microscopic investigation, the alpha-subunits form the two outer rings of the stack, while the two rings composed of beta-subunits, which are supposed to carry the active sites, are sandwiched between them. Each of the alpha- and beta-rings contains seven subunits; hence the stoichiometry of the whole proteasome is alpha 14 beta 14 and the symmetry is 7-fold. Image simulation experiments indicate that the alpha- and beta-subunits are not in register along the cylinder axis; rather it appears that the beta-rings are rotated with respect to the alpha-rings by approximately 25 degrees. In contrast to some previous reports we have not been able to find stoichiometric amounts of RNA associated with highly purified proteolytically active proteasome preparations.

Crystallization↗
PubMed1996

The effect of cysteine-43 mutation on thermostability and kinetic properties of citrate synthase from Thermoplasma acidophilum.

In this study, we have substituted serine-43 by cysteine in the recombinant citrate synthase from a moderately thermophilic Archaeon Thermoplasma acidophilum, for site-specific attachment of labels and have investigated the effects of this mutation on the biochemical properties and thermal stability of the enzyme. Both wild-type and the mutant enzymes were purified to homogenity using affinity chromatography on Matrex Gel Red A. The mutant Thermoplasma citrate synthase is very similar to wild-type citrate synthase in its substrate and co-factor specificities, pH profile and thermal stability. The mutation, however, has decreased the enzyme activity. The newly introduced reactive sulphydryl group could be easily modified by DTNB and labelled with 4-chloro-7-sulphobenzofuran, without loss of any activity.

Citrate (si)-Synthase↗
PubMed1995

The proteasome from Thermoplasma acidophilum is neither a cysteine nor a serine protease.

The 20 S proteasome, found in eukaryotes and in the archaebacterium Thermoplasma acidophilum, forms the proteolytic core of the 26 S proteasome which is the central protease of the non-lysosomal protein degradation pathway. Inhibitor studies have indicated that the 20 S proteasome may be an unusual type of cysteine or serine protease and a recent study of the Thermoplasma beta subunit has indicated that it carries the proteolytic activity. We have attempted to obtain information on the nature of the active site by mutating the only cysteine, both histidines and two completely conserved aspartates in the archaebacterial complex as well as all serines of the beta subunit, without decreasing the catalytic activity of the enzyme to any significant extent. Indeed, mutation of the conserved aspartate in the beta subunit increased the activity of the proteasome threefold. We conclude that the proteasome is not a cysteine or serine protease.

Amino Acid Sequence↗
PubMed1993

Nucleotide sequence of a putative succinate dehydrogenase operon in Thermoplasma acidophilum.

28 amino acids from the N-terminal region of a putative terminal oxidase from the archaebacterium Thermoplasma acidophilum were determined by Edman degradation. On basis of this amino acid sequence a degenerated oligonucleotide was synthesized and used as a radioactive probe for Southern blot analysis of EcoRI digested genomic DNA. A 2.3 kb EcoRI fragment strongly hybridized to the probe and size selected genomic library from genomic DNA was constructed. Several clones scored positive by screening the library with the degenerated oligonucleotide, from which only one clone contained a EcoRI DNA fragment encoding the 28 amino acid sequence determined by protein sequencing. Sequence analysis revealed the presence of three genes in the typical arrangement of an operon. The first gene codes for a protein containing 11 cystein residues in an arrangement typical for Fe/S proteins. Protein sequence comparison revealed significant homologies to the fumarate reductase and succinate dehydrogenase of other bacteria. The two other genes encode small hydrophobic proteins probably serving as membrane anchor for the Thermoplasma acidophilum succinate dehydrogenase.

Amino Acid Sequence↗
PubMed1990

Cloning and sequencing of the gene coding for the elongation factor 1 alpha from the archaebacterium Thermoplasma acidophilum.

The gene which encodes the elongation factor 1 alpha (EF-1 alpha) of the archaebacterium Thermoplasma acidophilum (tuf-gene) has been cloned and sequenced. The gene coding for elongation factor EF-2 was found downstream from the 3' end of the tuf-gene. Comparison of the predicted amino acid sequence of Thermoplasma EF-1 alpha with EF-1 alpha sequences of other organisms showed that the highest similarity values were found between T. acidophilum and Methanococcus vannielii.

Amino Acid Sequence↗
PubMedDate not supplied

Localization of a sequence motif complementary to the nuclear localization signal in proteasomes from Thermoplasma acidophilum by immunoelectron microscopy.

A sequence motif complementary to the nuclear localization signal (NLS) has been localized in proteasomes from Thermoplasma acidophilum by immunoelectron microscopy using sequence-specific antibodies. The antibodies were generated in two different ways: by immunization with a carrier-coupled peptide and by isolation of the sequence-specific antibody from an immune serum against native proteasomes using a peptide-affinity column. The sequence specificity of the isolated antibody was confirmed by a PEPSCAN-ELISA performed on overlapping nonapeptides deduced from the sequence of the alpha-subunit of the Thermoplasma proteasome. Compared to the antibody induced by the carrier-coupled peptide this antibody fraction showed a much higher affinity for native proteasomes. The attachment site of the Fab portion of the antibody to the proteasome was mapped by electron microscopy in conjunction with image processing. The antibody was found to bind to the periphery of the two outer "disks" of the proteasome complex formed by the alpha-subunits.

Amino Acid Sequence↗
PubMed1997

Identification of beta-L-gulose as the sugar moiety of the main polar lipid Thermoplasma acidophilum.

The main polar lipid (MPL) of Thermoplasma acidophilum has been purified and its structure determined. NMR, mass spectrometry, and capillary gas chromatography-mass spectrometry experiments have shown that the previously unidentified sugar moiety of MPL is the rare sugar L-gulose. MPL is thus a tetraether lipid with cyclopentane rings and head groups of phosphoglycerol, as previously reported, and beta-L-gulopyranose. Further, MPL is also the dominant lipid found in lipid extracts from another species of the Thermoplasma genus, T. volcanium, suggesting that L-gulose may represent a dominant sugar moiety of the polar lipids biosynthesized by this archaeobacterial genus. Minor phospholipids were tentatively identified as diether and hydroxydiether analogs of phosphatidylglycerol, and phosphatidylinositol.

Glycolipids↗
PubMed1997

Cloning, sequencing and expression of VAT, a CDC48/p97 ATPase homologue from the archaeon Thermoplasma acidophilum.

A member of the AAA family of Mg2(+)-ATPases from the archaeon Thermoplasma acidophilum has been cloned and expressed in Escherichia coli. The protein, VCP-like ATPase of Thermoplasma acidophilum (VAT), is a homologue of SAV from Sulfolobus acidocaldarius and CdcH of Halobacterium salinarium, and belongs to the CDC48/VCP/p97 subfamily. The deduced product of the vat gene is 745 residues long (Mr 83,000), which has an optimal Mg2(+)-ATPase activity at 70 degrees C. Electron microscopy shows the purified protein to form single and double homo-hexameric rings. Although the symmetry is different, the appearance of the complexes formed of two rings resembles the 20S proteasome and Hsp60/GroEL.

Adenosine Triphosphatases↗