Thermoacidophilic Archaebacterium Sulfolobus Acidocaldarius Research Articles

Stability and Rupture of Archaebacterial Cell Membrane: A Model Study

It is known that the thermoacidophilic archaebacterium Sulfolobus acidocaldarius can grow in hot springs at 65-80 degrees C and live in acidic environments (pH 2-3); however, the origin of its unusual thermal stability remains unclear. In this work, using a vesicle as a model, we study the thermal stability and rupture of archaebacterial cell membrane. We perform a simulation investigation of the structure-property relationship of monolayer membrane formed by bolaform lipids and compare it with that of bilayer membrane formed by monopolar lipids. The origin of the unusually thermal stability of archaebacterial cell and the mechanism for its rupture are presented in molecular details.

An authoring system for computer-assisted structured radiology reporting

A type II restriction endonuclease (SuaI) has been isolated from the thermoacidophilic archaebacterium Sulfolobusacidocaldarius. The enzyme is an isoschizomer of BspRI. It does not cut S. acidocaldarius DNA, as the recognition sequence GGCC in this DNA contains modified nucleotide(s). The enzyme is most active at 60–70°C and is highly thermostable.

Molecular modeling of archaebacterial bipolar tetraether lipid membranes

Membranes composed of glycerol dialkylnonitol tetraether (GDNT) lipids from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius have been studied by molecular modeling. GDNT membranes containing eight cyclopentane rings in the molecule are packed much tighter than those without rings. When containing eight cyclopentane rings, the β- d-galactosyl- d-glucose head-group of GDNT runs almost parallel to the membrane surface. However, when containing no rings, the head-group is oriented perpendicular to the membrane surface. Using molecular dynamics calculations, we have also conducted comparative studies of membrane packing between GDNT and various non-archaebacterial membranes. Compared to gel state dipalmitoylphosphatidylcholine (DPPC) and gel state distearoylphosphatidylcholine (DSPC) bilayers, the GDNT membrane with eight cyclopentane rings has a more negative interaction energy, thus a tighter membrane packing, while the GDNT without rings is less tightly packed than gel state DSPC. Based on the calculated interaction energies, the GDNT membranes (with and without rings) are much more tightly packed than DPhPC (an ester-linked diphytanyl PC) and DPhyPC (an ether-linked diphytanyl PC) bilayers. This suggests that the branched methyl group in the phytanyl chain is not the major contributor of the tight packing of GDNT membranes. The biological implication of this study is that the cyclopentane ring could increase GDNT membrane thermal stability. This explains why the number of cyclopentane rings in archaebacterial lipid increases with increasing growth temperature. Perhaps, through the ring-temperature compensation mechanism, the plasma membrane of thermoacidophilic archaebacteria is able to maintain a tight and rigid structure, consequently, a constant proton gradient between the extracelluar (pH 2.5) and intracelluar compartment (pH 6.5), over a wide range of growth temperatures.

Studies of Archaebacterial Bipolar Tetraether Liposomes by Perylene Fluorescence

Membrane packing and dynamics of bipolar tetraether liposomes composed of the polar lipid fraction E (PLFE) from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius have been studied by perylene fluorescence. At a probe-to-PLFE lipid ratio of 1:400, we have detected an unusual fluorescence intensity increase with increasing temperature, while the fluorescence lifetime changed little. As the ratio was decreased, the intensity anomaly was diminished. At 1:3200 and 1:6400, the anomaly disappeared. A remarkable perylene intensity anomaly was also observed in bilayers composed of saturated monopolar diester phosphatidylcholines at their main phase transition temperatures. These results suggest that the intensity anomaly may be due to probe aggregation caused by tight membrane packing. At the same probe-to-lipid ratio (1:400), however, 1,2-diphytanoyl- sn-glycero-3-phosphocholine (DPhPC) and 1,2-diphytanoyl- sn-glycero-3-phosphoglycerol (DPhPG) liposomes did not exhibit any intensity anomaly with increasing temperature. This suggests that DPhPC and DPhPG liposomes are more loosely packed than PLFE liposomes; thus the branched methyl groups are not the contributing factor of the tight membrane packing found in PLFE liposomes. Using a multiexcitation method, we have also determined the average ( R), in-plane ( R ip), and out-of-plane ( R op) rotational rates of perylene in PLFE liposomes at various temperatures (20–65°C). R and R ip, determined at two different probe-to-lipid ratios (1:400 and 1:3200), both undergo an abrupt increase when the temperature is elevated to ∼48°C. These data suggest that PLFE liposomes are rigid and tightly packed at low temperatures, but they begin to possess appreciable “membrane fluidity” at temperatures close to the minimum growth temperature (∼50°C) of thermoacidophilic archaebacteria.

The structure of calditol isolated from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius

We have achieved for the first time the stereoselective chemical synthesis of a polyhydroxylated cyclopentanic compound which proved fully identical to a sample of calditol isolated from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius by Akihiko Sugai et al.

Low permeability of liposomal membranes composed of bipolar tetraether lipids from thermoacidophilic archaebacterium Sulfolobus acidocaldarius.

The physical origin of the extremely high thermal stability of tetraether liposomes composed of the polar lipid fraction E (PLFE) from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius has been investigated. The leakage rate of trapped 5, 6-carboxyfluorescein (5(6)CF) and the proton permeability in PLFE liposomes have been measured using fluorescence probe techniques in the temperature range of 25-85 degrees C. The results are compared with those obtained from nonarchaebacterial liposomes. Egg yolk phosphatidylglycerol (eggPG) and PLFE liposomes exhibit similar large negative zeta-potentials (-31 to -34 mV) and low permeability coefficients for 5(6)CF, indicating that membrane surface charge is responsible for the low leakage rate of 5(6)CF in PLFE liposomes. This assertion is confirmed by the observation of an increased leakage rate of 5(6)CF with decreasing membrane surface negative charge via varying the content of egg yolk phosphatidylcholine (eggPC) in eggPC/eggPG binary mixtures. Gel-state dipalmitoylphosphatidylcholine bilayers and PLFE liposomes exhibit similar permeability coefficients for 5(6)CF, suggesting that lipid packing also plays an important role in the low leakage rate of 5(6)CF. PLFE liposomes, especially those approximately 60 nm in diameter, are remarkably thermally stable in regard to proton permeability, which increases by less than 2 x 10(-10) cm/s from 25 to 82 degrees C. The proton permeability comparison of various liposomes reveals that the tight and rigid lipid packing is the major contributor of the extremely low proton permeation in PLFE liposomes; the inositol moiety and the branched methyl groups may also contribute, but to a much lesser extent.

Purification and characterization of thermostable maltooligosyl trehalose trehalohydrolase from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius.

A thermostable maltooligosyl trehalose trehalohydrolase from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius ATCC 33909 was purified from a cell-free extract to an electrophoretically pure state by successive column chromatographies on Sepabeads FP-DA13, Butyl-Toyopearl 650M, DEAE-Toyopearl 650S, Toyopearl HW-55S and Ultrogel AcA44. The enzyme had a molecular mass of 59,000 by SDS-polyacrylamide gel electrophoresis and a pI of 6.1 by gel isoelectrofocusing. The N-terminal amino acid of the enzyme was methionine. The enzyme showed the highest activity from pH 5.5 to 6.0 and at 75 degrees C, and was stable from pH 5.5 to 9.5 and up to 85 degrees C. The activity was inhibited by Hg2+, Cu2+, Fe2+, Pb2+, and Zn2+. The Km values of the enzyme for maltosyl trehalose, maltotriosyl trehalose, maltotetraosyl trehalose, and maltopentaosyl trehalose were 16.7 mM, 2.7 mM, 3.7 mM, and 4.9 mM, respectively.

Purification and characterization of thermostable maltooligosyl trehalose synthase from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius.

A thermostable maltooligosyl trehalose synthase was purified from a cell-free extract of the thermoacidophilic archaebacterium Sulfolobus acidocaldarius ATCC 33909 to an electrophoretically homogeneous state by successive column chromatography on Sepabeads FP-DA13, Butyl-Toyopearl 650M, DEAE-Toyopearl 650S, Ultrogel AcA44, and Mono Q. The enzyme had a molecular mass of 74,000 by SDS-polyacrylamide gel electrophoresis and a pI of 5.9 by gel isoelectrofocusing. The N-terminal amino acid of the enzyme was methionine. The enzyme showed the highest activity from pH 5.0 to 5.5 and at 75 degrees C, and was stable from pH 4.5 to 9.5 and up to 85 degrees C. The enzyme activity was inhibited by Hg2+ and Cu2+. The Kms of the enzyme for maltotetraose, maltopentaose, maltohexaose, maltoheptaose, and short chain amylose (DP 18) were 41.5 mM, 7.1 mM, 5.7 mM, 1.4 mM, and 0.6 mM, respectively.

Sulfolobus acidocaldarius terminal oxidase. A kinetic investigation and its structural interpretation.

The thermoacidophilic archaebacterium Sulfolobus acidocaldarius possesses a very unusual terminal oxidase. We report original kinetic experiments on membranes of this microorganism carried out by stopped flow, using time-resolved optical spectroscopy combined with singular value decomposition analysis. The reduced-oxidized kinetic difference spectrum of the Sulfolobus membranes is characterized by three significant peaks in the visible region at 605, 586, and 560 nm. The 605-nm peak and part of the 586-nm peak (cytochrome aa3-type quinol oxidase) are reduced synchronously by both ascorbate plus N,N,N',N'-tetramethyl-p-phenylendiamine (TMPD) and dithionite, and they are very rapidly oxidized by molecular oxygen. A second pool of cytochromes seems to contribute to the 586-nm peak which is not reduced by ascorbate plus TMPD and reacts very slowly with dithionite. The b-type cytochromes (560 nm peak) are reduced by both reductants and are essentially "non-autoxidizable" at room temperature. Only one CO binding site with spectral features, kinetic properties, and ligand affinity not very dissimilar from those of mammalian cytochrome oxidase can be detected in the ascorbate-reduced membranes. On the contrary, a second CO binding site having unusual properties for aa3 terminal oxidases can be detected in the dithionite-reduced membranes.

Identification, Cloning, and Expression of the Gene for Adenylate Kinase from the Thermoacidophilic Archaebacterium Sulfolobus acidocaldarius

An adenylate kinase gene from a member of the archaebacterial kingdom, the thermoacidophilic archaebacterium (archaeon) Sulfolobus acidocaldarius, has been cloned and sequenced for the first time. Two degenerate oligonucleotide probes, based on the N-terminal amino acid sequence information, led to the amplification of a gene-specific DNA fragment, used to screen subgenomic libraries. Comparing the DNA-derived amino acid sequence of total 194 residues with those of known procaryotic and eucaryotic adenylate kinases revealed only a low degree of similarity, except for a glycine-rich region close to the N-terminus, the so-called P-loop. Using inducible expression systems catalytically active S. acidocaldarius adenylate kinase was produced in large amounts. Although the total length of the protein and the results of alignment procedures suggest a closer relation to eucaryotic than to procaryotic sequences, the arcbaebacterial enzyme may represent a novel class of adenylate kinases. This is corroborated by the finding that an antiserum against this protein does not cross-react with Escherichia coli nor yeast or rabbit adenylate kinases for example.

A physical map of the sulfur-dependent archaebacterium Sulfolobus acidocaldarius 7 chromosome

A chromosomal map of the sulfur-dependent thermoacidophilic archaebacterium Sulfolobus acidocaldarius 7 was constructed with four restriction enzymes: NotI, BssHII, RsrII, and EagI. The map indicated that the chromosome is a single circular DNA of 2,760 +/- 20 kb (mean +/- standard error of the mean). rRNA genes were also mapped. They were located at one site in the genome.

Characterization and purification of a membrane-bound archaebacterial pyrophosphatase from Sulfolobus acidocaldarius.

Plasma membranes of the thermoacidophilic archaebacterium Sulfolobus acidocaldarius (DSM 639) display a pyrophosphate-hydrolyzing activity [M. Lübben & G. Schäfer (1987) Eur. J. Biochem. 164, 533-540]. In our present work, we solubilized and purified this pyrophosphatase to homogeneity. It consists of a single subunit with a molecular mass of 17-18 kDa, forming an oligomer of 70 kDa under native conditions. Edman degradation revealed 30 amino acids of the N-terminus. The enzyme cleaves phosphoric-acid-anhydride bonds independently of monovalent or divalent cations. Temperature and pH optima of 75 degrees C and 3.5-3.7, respectively, characterize it as an ectoenzyme. Membrane lipids of Sulfolobus stimulate the activity. The dolichol-pyrophosphate-complexing peptide-antibiotic bacitracin inhibited growth of Sulfolobus. A possible function of the acid pyrophosphatase is the hydrolysis of dolichol pyrophosphate in connection with glycosylation reactions of membrane proteins.

An archaebacterial terminal oxidase combines core structures of two mitochondrial respiratory complexes.

The operon coding for a respiratory quinol oxidase was cloned from thermoacidophilic archaebacterium Sulfolobus acidocaldarius. It contains three genes, soxA, soxB and soxC. The first two genes code for proteins related to the cytochrome c oxidase subunits II and I, respectively. soxC encodes a protein homologous to cytochrome b, which is a subunit of the mitochondrial and bacterial cytochrome c reductases and the chloroplast cytochrome b6f complex. soxA is preceded by a promoter and the genes are cotranscribed into a 4 kb mRNA. Their protein products form a complex which has been partially purified and has quinol oxidase activity. The reduced minus oxidized absorption spectrum of the complex has two maxima at 586 and 606 nm. The latter is typical of cytochrome c oxidase. The complex contains four haems A. Two haems belong to the 'cytochrome oxidase' part of the complex and two are probably bound to be apocytochrome b (SoxC) and responsible for the 586 nm absorption peak. The homology between the sox gene products and their mitochondrial counterparts suggests that energy conservation coupled to the quinol oxidation catalysed either by the Sulfolobus oxidase or two mitochondrial respiratory enzymes may have a similar mechanism.

Purification and characterisation of an archaebacterial succinate dehydrogenase complex from the plasma membrane of the thermoacidophile Sulfolobus acidocaldarius

A succinate dehydrogenase complex was isolated in a three-step purification from plasma membranes of the thermoacidophilic archaebacterium Sulfolobus acidocaldarius. It consists of four subunits: a, 66 kDa; b, 31 kDa; c, 28 kDa and d, 12.8 kDa. In the 141-kDa native protein, the four subunits are present in an equimolar stoichiometry. The complex contains acid-non-extractable flavin, iron and acid-labile sulphide. Maximal succinate dehydrogenase activities were recorded at pH 6.5, which coincides with the internal pH of Sulfolobus cells. The temperature optimum of 81 degrees C defines the Sulfolobus succinate dehydrogenase as a thermophilic enzyme complex. The Km for succinate was found to be 1.42 mM (55 degrees C). Similar to the mitochondrial soluble succinate dehydrogenase, this enzyme is capable of transferring electrons to artificial electron acceptors, for instance phenazine methosulfate, N,N,N',N'-tetramethyl-p-phenylenediamine and ferricyanide. In contrast to the mitochondrial succinate dehydrogenase, the archaebacterial enzyme reduces 1,4-dichloroindophenol also in the absence of phenazine methosulfate. Caldariella quinone, the physiological electron mediator in the Sulfolobus respiratory chain, was only slowly reduced under adjusted conditions. The succinate--phenazine methosulfate-(1,4-dichloroindophenol) oxidoreductase of the isolated complex was strongly inhibited by tetrachlorobenzoquinone. In plasma membranes the complex reduces molecular oxygen in a cyanide-sensitive reaction. Polyclonal Sulfolobus anti-a antibodies crossreacted with 66-67-kDa polypeptides from membranes of Thermoplasma acidophilium, Sulfolobus solfataricus and beef heart submitochondrial particles.

Gene for the ADP‐ribosylatable elongation factor 2 from the extreme thermoacidophilic archaebacterium Sulfolobus acidocaldarius

The gene coding for ADP-ribosylatable elongation factor 2 (EF-2) from the extreme thermoacidophilic archaebacterium Sulfolobus acidocaldarius has been cloned and its sequence is reported. Amino acid sequence comparisons showed that EF-2 from S. acidocaldarius is more closely related to eukaryotic EF-2 than to eubacterial EF-G. Consensus sequences are derived from comparison of a region around the unique amino acid diphthamide, which is the target for ADP-ribosylation by diphtheria toxin in archaebacteria and eukaryotes. The conserved positions are likely to constitute a recognition site for the toxin and the histidine-modifying enzymes. A single transcript of approximately the size of the EF-2 gene was observed in Northern blot experiments. Transcription initiation and termination signals were identified in the immediate vicinity of the respective translation start and stop codons of the gene. These results indicate that, in contrast to all prokaryotic EF-2 genes studied previously, the gene of S. acidocaldarius is not located within the streptomycin operon but is transcribed separately.

Cytochrome aa3 from Sulfolobus acidocaldarius

The thermoacidophilic archaebacterium Sulfolobus acidocaldarius (DSM 369) extrudes protons when expending respiratory energy [Moll, R. & Schäfer, G. (1988) FEBS Lett. 232, 359-363]. Cytochromes of the membrane electron-transport systems are assumed to represent the proton pumps. Only a- and b-type cytochromes can be found; no c-type cytochromes are present. Of the two terminal oxidases [Anemüller, S. & Schäfer, G. (1989) FEBS Lett. 244, 451-455] one shows an absorption band at 604-605 nm, typical of cytochromes of the aa3 type. This hemoprotein has been solubilized from the membrane and purified to homogeneity. It exhibits distinct differences from known aa3-type oxidases. (a) It consists of a single polypeptide subunit of 38-40 kDa apparent molecular mass with two heme-a molecules and two copper ions. (b) In the oxidized state, absorption maxima are found at 421 nm and 597 nm, and in the reduced state at 439 nm and 601 nm; CO difference spectra suggest one heme to be a heme-a3 centre. (c) The redox potentials of the heme centres are +220 mV and +370 mV, respectively. (d) A high-spin heme signal at g = 6 is present in EPR spectra, which is more prominent than the low-spin heme signal at g = 3, the former already being present in the oxidized state. A signal at g = 2.1 may be due to one of the copper ions and is superimposed upon a minor free radical signal at g = 2. (e) Caldariella quinone was also isolated from the plasma membrane of Sulfolobus. Its redox midpoint potential at pH 6.5 was determined to be +100 (+/- 5) mV; spectral properties have also been determined. (f) The isolated aa3 preparation does not oxidize cytochrome c; however, it oxidizes N,N,N',N'-tetramethyl-1,4-phenylenediamine dihydrochloride as an artificial single-electron donor as well as reduced caldariella quinone, which is assumed to represent the natural substrate. The reaction is cyanide-sensitive and the product of oxygen reduction is water. (g) On the basis of the results obtained a novel type of cytochrome aa3 is postulated in this paper which oxidizes reduced quinones; its ability to act as a proton pump remains to be shown.

Electron transport and energy conservation in the archaebacteriumSulfolobus acidocaldarius

The bioenergetic properties of the thermoacidophilic archaebacterium Sulfolobus acidocaldarius are reviewed and discussed under the aspect whether this archaebacterium conserved energy by oxidative phosphorylation and how the involved catalysts are related to those from eubacteria and eukaryotes. The thermodynamic parameters contributing to the proton-motive force and the efficiency of proton pumping are presented. The major components of the electron transport system are identified and a novel type of heme-aa3 containing terminal oxidase is described, oxidizing reduced caldariella quinone. The properties of an F1-analogous ATPase and of a DCCD-binding proteolipid from the plasmamembrane of Sulfolobus are discussed as likely components of an F0F1-analogous ATP-synthase. The structural and functional properties of this and other archaebacterial ATPases are compared to each other and with respect to evolutionary relations.

Electron transport and energy conservation in the archaebacterium Sulfolobus acidocaldarius

The bioenergetic properties of the thermoacidophilic archaebacterium Sulfolobus acidocaldarius are reviewed and discussed under the aspect whether this archaebacterium conserved energy by oxidative phosphorylation and how the involved catalysts are related to those from eubacteria and eukaryotes. The thermodynamic parameters contributing to the proton-motive force and the efficiency of proton pumping are presented. The major components of the electron transport system are identified and a novel type of heme- aa 3 containing terminal oxidase is described, oxidizing reduced caldariella quinone. The properties of an F 1-analogous ATPase and of a DCCD-binding proteolipid from the plasmamembrane of Sulfolobus are discussed as likely components of an F 0 F 1-analogous ATP-synthase. The structural and functional properties of this and other archaebacterial ATPases are compared to each other and with respect to evolutionary relations.

The DNA polymerase from the archaebacterium Sulfolobus acidocaldarius: A thermophilic and thermoresistant enzyme which can perform automated polymerase chain reaction

A DNA polymerase purified from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius was used to perform automated DNA amplification at 70°C as well as site directed mutagenesis by Polymerase Chain Reaction (P.C.R.). The yield of amplification performed at optimum MgCl2 concentration for the Taq or the S. acidocaldarius DNA polymerase, for the same DNA target, was equivalent. The ability of S. acidocaldarius DNA polymerase to perform P.C.R. under less stringent requirement of MgCl2 concentration gives this enzyme a non-negligeable advantage over the Taq DNA polymerase.

Archaebacterial Citrate Synthases: The Enzymes from the Thermoacidophiles Sulfolobus acidocaldarius and Thermoplasma acidophilum Show pro-S Stereospecificity

Abstract Citrate synthase from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius was purified 365-fold to electrophoretic homogeneity. At 40 °C and pH 8.1 the homogeneous enzyme shows a specific activity of 73 units per mg, which corresponds to a turnover number of 44 sec-1. Citrate synthase from S. acidocaldarius shows pro-S stereospecificity, as is found with a partially purified preparation of the enzyme from Thermoplasma acidophilum, another thermoacidophilic archaebacterium.