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At least 325 records · Page 18Linked to original sources

PubMed1998

Characterization of homo-oligomeric complexes of alpha and beta chaperonin subunits from the acidothermophilic archaeon, Sulfolobus sp. strain 7.

The chaperonin from the acidothermophilic archaeon, Sulfolobus sp. Strain 7, is composed of two kinds of subunits designated as Scp alpha and Scp beta. In this study, we characterized the recombinant Scp alpha and Scp beta, which were separately expressed in Escherichia coli. Both of them were able to assemble to homo-oligomeric double-ring complexes, similar to subunits of group II chaperonins from Thermoplasma acidophilum and Thermococcus strain KS-1. Both complexes have no or at most trace ATPase activities. However, they could arrest spontaneous refolding of chemically denatured enzyme in the same way as the purified Sulfolobus chaperonin. We found that they dissociated in the presence of 15% ethanol to monomers, which spontaneously assembled to oligomers when concentrated in the absence of ethanol. Both the reconstituted homo-oligomers were unstable, and easily dissociated to monomers. Further structural and functional characterization is necessary to elucidate if these homo-oligomers exist and if so, their function in vivo.

3-Isopropylmalate Dehydrogenase↗

PubMed2002

Pyrococcus prefoldin stabilizes protein-folding intermediates and transfers them to chaperonins for correct folding.

A molecular chaperone prefoldin/GimC from the hyperthermophilic archaeum Pyrococcus horikoshii OT3 was characterized. Pyrococcus prefoldin protected porcine heart citrate synthase from thermal aggregation whereas each subunit alone afforded little protection. It also arrested the spontaneous refolding of acid-denatured green fluorescent protein and then transferred it not only to a group II chaperonin from the hyperthermophilic archaeum Thermococcus sp. strain KS-1, but also to a group I chaperonin from the thermophilic bacterium Thermus thermophilus HB8 for subsequent ATP dependent refolding.

Animals↗

PubMed2000

Maltose-binding protein from the hyperthermophilic bacterium Thermotoga maritima: stability and binding properties.

Recombinant maltose-binding protein from Thermotoga maritima (TmMBP) was expressed in Escherichia coli and purified to homogeneity, applying heat incubation of the crude extract at 75 degrees C. As taken from the spectral, physicochemical and binding properties, the recombinant protein is indistinguishable from the natural protein isolated from the periplasm of Thermotoga maritima. At neutral pH, TmMBP exhibits extremely high intrinsic stability with a thermal transition >105 degrees C. Guanidinium chloride-induced equilibrium unfolding transitions at varying temperatures result in a stability maximum at approximately 40 degrees C. At room temperature, the thermodynamic analysis of the highly cooperative unfolding equilibrium transition yields DeltaG(N-->U)=100(+/-5) kJ mol(-1 )for the free energy of stabilization. Compared to mesophilic MBP from E. coli as a reference, this value is increased by about 60 kJ mol(-1). At temperatures around the optimal growth temperature of T. maritima (t(opt) approximately 80 degrees C), the yield of refolding does not exceed 80 %; the residual 20 % are misfolded, as indicated by a decrease in stability as well as loss of the maltose-binding capacity. TmMBP is able to bind maltose, maltotriose and trehalose with dissociation constants in the nanomolar to micromolar range, combining the substrate specificities of the homologs from the mesophilic bacterium E. coli and the hyperthermophilic archaeon Thermococcus litoralis. Fluorescence quench experiments allowed the dissociation constants of ligand binding to be quantified. Binding of maltose was found to be endothermic and entropy-driven, with DeltaH(b)=+47 kJ mol(-1) and DeltaS(b)=+257 J mol(-1) K(-1). Extrapolation of the linear vant'Hoff plot to t(opt) resulted in K(d) approximately 0.3 microM. This result is in agreement with data reported for the MBPs from E. coli and T. litoralis at their respective optimum growth temperatures, corroborating the general observation that proteins under their specific physiological conditions are in corresponding states.

ATP-Binding Cassette Transporters↗

PubMed2000

Phage phi 29 DNA polymerase residues involved in the proper stabilisation of the primer-terminus at the 3'-5' exonuclease active site.

Three highly conserved amino acid residues have been characterised here as ssDNA ligands at the 3'-5' exonuclease active site of o29 DNA polymerase. The functional role of Tyr59, His61 and Phe69 residues of o29 DNA polymerase (belonging to Exo II motif, previously described as containing an invariant catalytic aspartate residue and two highly conserved ssDNA ligands) was assayed by biochemical analysis of six site-directed mutants at those residues. These studies revealed that the mutations introduced severely affected their ssDNA binding capacity and, as a consequence, the 3'-5' exonuclease activity on ssDNA substrates was also severely impaired, producing drastic defects in the maintenance of replication fidelity. Crystal structures of Klenow fragment of Pol Ik and Thermococcus gorgonarius DNA polymerase complexed with ssDNA at their 3'-5' exonuclease active sites revealed that residues Gln419 of the former, and Tyr209 of the latter, the counterparts of His61 of o29 DNA polymerase, are making contacts with the penultimate phosphodiester bond of ssDNA substrate. Here, the functional role of this residue is described.

Amino Acid Motifs↗

PubMed1990

Structure of the archaebacterial 7S RNA molecule.

The genes encoding the 7S RNAs of the archaebacteria Archaeoglobus fulgidus, Methanosarcina acetivorans, Sulfolobus, solfataricus, and Thermococcus celer have been isolated. All four genes occur as single genomic copies and are flanked by sequences containing potential signals for transcriptional promotion and termination. The genes encode RNA molecules approximately 300 nucleotides in length which conform strictly to a model of secondary structure common to all described archaebacterial 7S RNAs. Archaebacterial 7S RNAs exhibit a strong similarity to eukaryotic 7S RNAs in terms of overall secondary structure, while primary sequence conservation is limited to a specific structural domain of the molecule. This domain displays strong primary and secondary structural similarity to features of small eubacterial RNAs, including the small cytoplasmic (sc) RNA of Bacillus subtilis and the 4.5S RNA of Escherichia coli. Conservation of this structural domain among divergent RNA molecules across three kingdoms suggests that these RNAs are the descendants of a unique subcellular structure present before the divergence of the archaebacterial, eubacterial and eukaryotic kingdoms.

Archaea↗

PubMed1988

Aminoglycoside-induced mistranslation in thermophilic archaebacteria.

The effect of selected aminoglycoside antibiotics on the translational accuracy of poly(U) programmed ribosomes derived from the thermophilic archaebacteria Thermoplasma acidophilum, Sulfolobus solfataricus, Thermococcus celer and Desulfurococcus mobilis has been determined. Under optimum temperature and ionic conditions for polyphenylalanine synthesis, the four species investigated are found to be markedly diverse in their response to the miscoding-inducing action of aminoglycoside antibiotics. T. acidophilum is sensitive to all of the compounds tested except streptomycin; S. solfataricus responds to paromomycin and to hygromycin B; T. celer is only affected by neomycin, and D. mobilis is refractory to all drugs. The only feature shared by the four species under study, and by all archaebacteria so far investigated, is their complete insensitivity to streptomycin. The structural and phylogenetic implications of the remarkable diversity encountered among archaebacterial ribosomes in their susceptibility to aminoglycosides are discussed.

Anti-Bacterial Agents↗

PubMed2003

ADP-dependent glucokinase from the hyperthermophilic sulfate-reducing archaeon Archaeoglobus fulgidus strain 7324.

The hyperthermophilic sulfate-reducing archaeon Archaeoglobus fulgidus strain 7324 has been shown to degrade starch via glucose using a modified Embden-Meyerhof pathway. The first enzyme of this pathway, ADP-dependent glucokinase, was purified 600-fold to homogeneity. The enzyme is a monomeric protein with an apparent molecular mass of 50 kDa. It had a temperature optimum at 83 degrees C and showed a significant thermostability up to 100 degrees C. The enzyme was highly specific for ADP and glucose as substrates; it did not use ATP, CDP, UDP, or GDP as phosphoryl donors, or mannose, fructose and fructose 6-phosphate as phosphoryl acceptors (at 80 degrees C). Only glucosamine was phosphorylated at significant rates. The apparent K(m) values for ADP and glucose (at 50 degrees C) were 0.07 mM and 0.78 mM, respectively; the apparent V(max) value was about 50 U/mg at 50 degrees C and 350 U/mg at 80 degrees C. Divalent cations were required for maximal activity; Mn(2+), Mg(2+ )and Ca(2+), which were most effective, could be replaced partially by Cu(2+), Ni(2+), Co(2+) and Zn(2+). The N-terminal amino acid sequence (42 amino acids) of ADP-dependent glucokinase was almost identical to that of ADP-dependent glucokinase from Thermococcus litoralis. In the genome of the closely related Archaeoglobus fulgidus strain VC16 a homologous gene for ADP-dependent glucokinase could not be identified.

Archaea↗

PubMed1996

Culturability and survival of an extreme thermophile isolated from deep-sea hydrothermal vents

The culturability of a strictly anaerobic, extremely thermophilic archaeon, Thermococcus peptonophilus (optimal growth temperature: 85° C), was studied during survival stages at various temperatures (98, 85, 70, and 4° C). Total cell number (determined by DAPI staining), active cells (rhodamine-stained cells), and culturable cells (using most-probable-number) were counted over time. The number of culturable cells decreased under each condition tested. The total number of cells significantly decreased only at temperatures close to the maximum for growth (98° C); at this temperature, the cells spontaneously lysed. Our results suggested that survival at 4° C in oxygenated waters might be a mechanism for the dispersion of extreme thermophiles in the ocean. In addition, we proved the existence of T. peptonophilus cells in several physiological states: culturable cells, active non-culturable cells, inactive non-culturable cells, and dead cells. Cell death was caused by cellular lysis.

Journal Article↗

PubMed1997

Purification and properties of an extremely thermostable NADP+-specific glutamate dehydrogenase from Archaeoglobus fulgidus.

NADP+-specific glutamate dehydrogenase (EC 1.4.1.4) was purified to homogeneity from the extremely thermophilic, strictly anaerobic, sulfate-reducing archaeon Archaeoglobus fulgidus strain 7324. The native enzyme (263 kDa) is composed of subunits of mol. mass 46 kDa, suggesting a hexameric structure. The temperature optimum for enzyme activity was > 95 degrees C. The enzyme was highly thermostable, having a half-life of 140 min at 100 degrees C. Potassium phosphate, KCl, and NaCl enhanced the thermal stability and increased the rate of activity three- to fourfold. The N-terminal 26-amino-acid sequence showed a high degree of similarity to glutamate dehydrogenases from Pyrococcus spp. and Thermococcus spp.

Amino Acid Sequence↗

PubMed2003

Expression of foreign proteins in Escherichia coli by fusing with an archaeal FK506 binding protein.

Improper protein-folding often results in inclusion-body formation in a protein expression system using Escherichia coli. To express such proteins in the soluble fraction of E. coli cytoplasm, we developed an expression system by fusing the target protein with an archaeal FK506 binding protein (FKBP). It has been reported that an archaeal FKBP from a hyperthermophilic archaeon, Thermococcus sp. KS-1 (TcFKBP18), possesses not only peptidyl-prolyl cis-trans isomerase activity, but also chaperone-like activity to enhance the refolding yield of an unfolded protein by suppressing irreversible protein aggregation. To study the effect of this fusion strategy with FKBP on the expression of foreign protein in E. coli, a putative rhodanese (thiosulfate sulfurtransferase) from a hyperthermophilic archaeon and two mouse antibody fragments were used as model target proteins. When they were expressed alone in E. coli, they formed insoluble aggregates. Their genes were designed to be expressed as a fusion protein by connecting them to the C-terminal end of TcFKBP18 with an oligopeptide containing a thrombin cleavage site. By fusing TcFKBP18, the expression of the target protein in the soluble fraction was significantly increased. The percentage of the soluble form in the expressed protein reached 10-28% of the host soluble proteins. After purification and protease digestion of the expressed antibody fragment-TcFKBP18 fusion protein, the cleaved antibody fragment (single-chain Fv) showed specific binding to the antigen in ELISA. This indicated that the expressed antibody fragment properly folded to the active form.

Archaeal Proteins↗

PubMed1996

UV and ethyl methanesulfonate effects in hyperthermophilic archaea and isolation of auxotrophic mutants of Pyrococcus strains.

The lethal and mutagenic effects of ethyl methanesulfonate (EMS) and UV on nine archaeal strains belonging to each of the two described genera of Thermococcales, Pyrococcus and Thermococcus, were investigated. To test the efficiency of the EMS and UV mutagenesis under a variety of experimental conditions, we chose Pyrococcus abyssi strain GE5 as a model strain. We observed a strong induced mutagenicity in both cases, since the spontaneous mutation frequency (expressed as the frequency of resistance to 5-fluoroorotic acid) increased up to 150-fold with EMS and 400-fold with UV, after mutagen exposure. Although a heterogeneous response to the induced effects caused after EMS and UV exposures was detected for all the other sulfothermophilic archaea tested, an efficient mutagenicity of Pyrococcus-like isolates GE27, GE23, and GE9 was observed. Optimal procedures described for UV mutagenesis yielded a number of useful uracil auxotrophic mutant strains of Pyrococcus abyssi.

Archaea↗

PubMed1999

Dissimilatory reduction of Fe(III) by thermophilic bacteria and archaea in deep subsurface petroleum reservoirs of western siberia

Twenty-five samples of stratal fluids obtained from a high-temperature (60-84 degrees C) deep subsurface (1700-2500 m) petroleum reservoir of Western Siberia were investigated for the presence of dissimilatory Fe(III)-reducing microorganisms. Of the samples, 44% and 76% were positive for Fe(III) reduction with peptone and H2 respectively as electron donors. In most of these samples, the numbers of culturable thermophilic H2-utilizing iron reducers were in the order of 10-100 cells/ml. Nine strains of thermophilic anaerobic bacteria and archaea isolated from petroleum reservoirs were tested for their ability to reduce Fe(III). Eight strains belonging to the genera Thermoanaerobacter, Thermotoga, and Thermococcus were found capable of dissimilatory Fe(III) reduction, with peptone or H2 as electron donor and amorphous Fe(III) oxide as electron acceptor. These results demonstrated that Fe(III) reduction may be a common feature shared by a wide range of anaerobic thermophiles and hyperthermophiles in deep subsurface petroleum reservoirs.http://link.springer-ny. com/link/service/journals/00284/bibs/39n2p99.html

Journal Article↗

PubMed2002

Cloning and transcriptional analysis of the Thermoanaerobacter ethanolicus strain 39E maltose ABC transport system.

Thermoanaerobacter ethanolicus strain 39E is a Gram-positive thermophile that converts sugars resulting from plant carbohydrate polymer degradation into ethanol. A putative maltose ATP-binding cassette (ABC) transport operon was isolated with genes encoding for the integral membrane components (malF and malG); the ATP-binding protein (malK); and a partial gene for the maltose-binding protein (malE). This operon is unlike most other maltose transport operons, which do not contain a contiguous malK gene. Sequence analysis showed that the individual genes in the putative operon possessed a considerable range of similarities to their respective homologs in other eubacteria and archaea. MalK had 52% amino-acid identity and over 70% similarity with its homolog from the archaeon Thermococcus litoralis, while the membrane components and binding protein exhibited much less similarity with a range of other thermophilic eubacteria. Transcript was not detected in maltose-, glucose-, or xylose-grown cells using Northern blotting, but RT-PCR showed that malFGK were expressed in cells grown on maltose or xylose. Based on these results, the strain 39E maltose operon may be subject to glucose catabolite repression.

ATP-Binding Cassette Transporters↗

PubMed2004

Identification of archaea and some extremophilic bacteria using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry.

Archaea and a number of groups of environmentally important bacteria, e.g., sulfate-reducing bacteria, anoxygenic phototrophs, and some thermophiles, are difficult to characterize using current methods developed for phenotypically differentiating heterotrophic bacteria. We have evaluated matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF-MS) as a rapid method for identifying different groups of extremophilic prokaryotes using a linear mass spectrometer (Micromass, UK). The instrument is designed to acquire mass-spectral patterns from prokaryotic cell-wall components between masses of 500 and 10,000 Da in a statistically robust manner and create a database that can be used for identification. We have tested 28 archaea (10 genera, 20 spp.) and 42 bacteria (25 genera, 37 spp.) and found that all species yield reproducible, unique mass-spectral profiles. As a whole, the profiles for the archaea had fewer peaks and showed less differentiation compared to the bacteria, perhaps reflecting fundamental differences in cell-wall structure. The halophilic archaea all had consistent patterns that showed little differentiation; however, the software was able to consistently distinguish Halobacterium salinarium, Halococcus dombrowski, and Haloarcula marismortui from one another, although it could not always correctly distinguish four strains of Hb. salinarium from one another. The method was able to reliably identify 10(5) cells of either Albidovulum inexpectatum or Thermococcus litoralis and could detect as low as 10(3) cells. We found that the matrix, alpha-cyano-4-hydroxy-cinnamic acid yielded better spectra for archaea than 5-chloro-2-mercapto-benzothiazole. Overall, the method was rapid, required a minimum of sample processing, and was capable of distinguishing and identifying a very diverse group of prokaryotes.

Archaea↗

PubMed2005

Cultivated anaerobic acidophilic/acidotolerant thermophiles from terrestrial and deep-sea hydrothermal habitats.

Metabolic and phylogenetic diversity of cultivated anaerobic microorganisms from acidic continental hot springs and deep-sea hydrothermal vents was studied by molecular and microbiological methods. Anaerobic organotrophic enrichment cultures growing at pH 3.5-4.0 and 60 or 85 degrees C with organic energy sources were obtained from samples of acidic hot springs of Kamchatka Peninsula (Pauzhetka, Moutnovski Volcano, Uzon Caldera) and Kunashir Island (South Kurils) as well as from the samples of chimneys of East Pacific Rise (13 degrees N). The analyses of clone libraries obtained from terrestrial enrichment cultures growing at 60 degrees C revealed the presence of archaea of genus Thermoplasma and bacteria of genus Thermoanaerobacter. Bacterial isolates from these enrichments were shown to belong to genera Thermoanaerobacter and Thermoanaerobacterium, being acidotolerant with the pH optimum for growth at 5.5-6.0 and the pH minimum at 3.0. At 85 degrees C, domination of thermoacidophilic archaea of genus Acidilobus in terrestrial enrichments was found by both molecular and microbiological methods. Five isolates belonging to this genus possessed some phenotypic features that were new for this genus, such as flagellation or the ability to grow on monosaccharides or disaccharides. Analyses of clone libraries from the deep-sea thermoacidophilic enrichment cultures showed that the representatives of the genus Thermococcus were present at both 60 and 85 degrees C. From the 60 degrees C deep-sea enrichment, a strain belonging to Thermoanaerobacter siderophilus was isolated. It grew optimally at pH 6.0 with the minimum pH for growth at 3.0 and with salinity optimum at 0-2.5% NaCl and the maximum at 7%, thus differing significantly from the type strain. These data show that fermentative degradation of organic matter may occur at low pH and wide temperature range in both terrestrial and deep-sea habitats and can be performed by acidophilic or acidotolerant thermophilic prokaryotes.

Archaea↗

PubMed2001

The biochemical properties and phylogenies of phosphofructokinases from extremophiles.

The enzyme phosphofructokinase (PFK) is a defining activity of the highly conserved glycolytic pathway, and is present in the domains Bacteria, Eukarya, and Archaea. PFK subtypes are now known that utilize either ATP, ADP, or pyrophosphate as the primary phosphoryl donor and share the ability to catalyze the transfer of phosphate to the 1-position of fructose-6-phosphate. Because of the crucial position in the glycolytic pathway of PFKs, their biochemical characteristics and phylogenies may play a significant role in elucidating the origins of glycolysis and, indeed, of metabolism itself. Despite the shared ability to phosphorylate fructose-6-phosphate, PFKs that have been characterized to date now fall into three sequence families: the PFKA family, consisting of the well-known higher eukaryotic ATP-dependent PFKs together with their ATP- and pyrophosphate-dependent bacterial cousins (including the crenarchaeal pyrophosphate-dependent PFK of Thermoprotetus tenax) and plant pyrophosphate-dependent phosphofructokinases; the PFKB family, exemplified by the minor ATP-dependent PFK activity of Escherichia coli (PFK 2), but which also includes at least one crenarchaeal enzyme in Aeropyrum pernix; and the tentatively named PFKC family, which contains the unique ADP-dependent PFKs from the euryarchaeal genera of Pyrococcus and Thermococcus, which are indicated by sequence analysis to be present also in the methanogenic species Methanococcus jannaschii and Methanosarcina mazei.

Amino Acid Sequence↗

PubMed1995

Determination of the [Fe4S4]Cys4 cluster geometry of Desulfovibrio africanus ferredoxin I by 1H NMR spectroscopy.

1D and 2D 1H NMR studies of the Fe4S4 cluster containing ferredoxin I from Desulfovibrio africanus have been carried out with the aim of determining the geometry of the cluster linkages with the 4 Cys side chains that bind the cluster. This required the Cys beta CH resonances of the oxidised protein to be sequence-specifically and stereo-specifically assigned, and this was accomplished by a combination of TOCSY and NOE measurements, allied to model building based on X-ray structures of related ferredoxins. An analysis of the estimated hyperfine shifts of the Cys beta CH resonances with a Karplus-type equation relating the shifts to iron-sulfur-beta carbon-beta proton dihedral angles, taken together with the relative relaxation rates of the two beta CH2 resonances, estimated from their linewidths, then allowed the iron-sulfur-beta-carbon-alpha-carbon dihedral angles to be determined. A novel representation of the NMR data is presented which shows that the cluster dihedral angles are uniquely determined by the NMR data. The analysis reveals that the dihedral angles for D. africanus ferredoxin I are similar to the corresponding angles of other ferredoxins even though there are differences in their 1H NMR spectra. The sequence-specific and stereospecific assignments have been extended by analogy to the related Fe4S4-containing D. gigas ferredoxin I, and the stereospecific assignments to the Fe4S4-containing Thermococcus litoralis ferredoxin.

Chemical Phenomena↗

PubMed1996

Tungsten in biological systems.

Tungsten (atomic number 74) and the chemically analogous and very similar metal molybdenum (atomic number 42) are minor yet equally abundant elements on this planet. The essential role of molybdenum in biology has been known for decades and molybdoenzymes are ubiquitous. Yet, it is only recently that a biological role for tungsten has been established in prokaryotes, although not as yet in eukaryotes. The best characterized organisms with regard to their metabolism of tungsten are certain species of hyperthermophilic archaea (Pyrococcus furiosus and Thermococcus litoralis), methanogens (Methanobacterium thermoautotrophicum and Mb. wolfei), Gram-positive bacteria (Clostridium thermoaceticum, C. formicoaceticum and Eubacterium acidaminophilum), Gram-negative anaerobes (Desulfovibrio gigas and Pelobacter acetylenicus) and Gram-negative aerobes (Methylobacterium sp. RXM). Of these, only the hyperthermophilic archaea appear to be obligately tungsten-dependent. Four different types of tungstoenzyme have been purified: formate dehydrogenase, formyl methanufuran dehydrogenase, acetylene hydratase, and a class of phylogenetically related oxidoreductases that catalyze the reversible oxidation of aldehydes. These are carboxylic reductase, and three ferredoxin-dependent oxidoreductases which oxidize various aldehydes, formaldehyde and glyceraldehyde 3-phosphate. All tungstoenzymes catalyze redox tungsten in these enzymes is bound by a pterin moiety similar to that found in molybdoenzymes. The first crystal structure of a tungsten- or pterin-containing enzyme, that of aldehyde ferredoxin oxidoreductase from P. furiosus, has revealed a catalytic site with one W atom coordinated to two pterin molecules which are themselves bridged by a magnesium ion. The geochemical, ecological, biochemical and phylogenetic basis for W- vs. Mo-dependent organisms is discussed.

Aldehyde Oxidoreductases↗