Extreme Thermophilic Archaebacterium Research Articles

Comparative growth studies of the extreme thermophile Sulfolobus acidocaldarius in submerged and solidified substrate cultures

An attempt was made, for the first time, to exploit cultures on solidified substrates (SSC) as an alternative to submerged cultures (SmC) for growing extremophilic micro-organisms. The extreme thermophilic archaebacterium Sulfolobus acidocaldarius was grown on a number of carbon sources and, in all experiments, biomass yields and growth rates were always higher in SSC than in the corresponding SmC. Inoculum age significantly affected growth characteristics on both types of fermentation. Heavy growth of the micro-organism in SSC was observed on low-cost carbon sources such as starch. Wheat bran significantly enhanced growth characteristics when used to supplement starch media. The results of this work show that cultures on solid surfaces could be a promising alternative method for growing extreme thermophiles.

Uracil phosphoribosyltransferase from the extreme thermoacidophilic archaebacterium Sulfolobus shibatae is an allosteric enzyme, activated by GTP and inhibited by CTP

Uracil phosphoribosyltransferase, which catalyses the formation of UMP and pyrophosphate from uracil and 5-phosphoribosyl α-1-pyrophosphate (PRPP), was partly purified from the extreme thermophilic archaebacterium Sulfolobus shibatae. The enzyme required divalent metal ions for activity and it showed the highest activity at pH 6.4. The specific activity of the enzyme was 50-times higher at 95°C than at 37°C, but the functional half-life was short at 95°C. The activity of uracil phosphoribosyltransferase was strongly activated by GTP, which increased V max of the reaction by approximately 20-fold without much effect on K m for the substrates. The concentration of GTP required for half-maximal activation was about 80 μM. CTP was a strong inhibitor and acted by raising the concentration of GTP needed for half-maximal activation of the enzyme. We conclude that uracil phosphoribosyltransferase from S. shibatae is an allosteric enzyme which is activated by a purine nucleotide and inhibited by a pyrimidine nucleotide as seen for several enzymes in the pyrimidine nucleotide biosynthetic pathway of Escherichia coli, but not observed before for any phosphoribosyltransferase.

Crystallization and Preliminary X-ray Analysis of an NAD+ -dependent Alcohol Dehydrogenase from the Extreme Thermophilic Archaebacterium Sulfolobus solfataricus

An NAD+ -dependent alcohol dehydrogenase from the extreme thermophilic archaebacterium Sulfolobus solfataricus has been crystallized in the holo-enzyme and apo-enzyme forms. Crystals of the holo-enzyme grow from 2-methyl-2,4-pentanediol at pH 8·4 with the addition of NADH and at pH 7·0 with the addition of NADH and dimethyl sulphoxide. Crystals of the apo-enzyme grow at pH 6·3 from a mixture of polyethylene glycol 4000 and propan-2-ol. The holo-enzyme crystallizes in C 2 with a dimer in the asymmetric unit, however the crystals are twinned and unsuitable for data collection. The apo-enzyme crystallizes in I 4122 (a = 126·82 Å, b = 118·95 Å) with a monomer in the asymmetric unit, and the single crystals diffract to 2·8 Å.

Crystallization and Preliminary X-ray Analysis of the β-Galactosidase from the Extreme Thermophilic Archaebacterium Sulfolobus solfataricus

The β-galactosidase from the extreme thermophilic archaebacterium Sulfolobus solfataricus has been crystallized from polyethylene glycol 4000 in the presence of sodium acetate and acetate buffer at pH 4·6. The protein crystallizes in P3121 or P3221 (a = 169·4, c = 98·29) and the crystals diffract beyond 2·5 Å. The measured crystal density (≈ 1·28 g/cm3) is consistent with the presence of a tetramer (molecular mass 240 kDa) in the asymmetric unit. The specific volume of the crystals is 1·7 Å3/Da, indicating a solvent content by volume of only 27%, which is amongst the lowest values observed for protein crystals, and indicates virtual close-packaging of the tetramers.

Purification and characterization of a thermostable carboxypeptidase from the extreme thermophilic archaebacterium Sulfolobus solfataricus.

A carboxypeptidase was purified to electrophoretic homogeneity from the thermoacidophilic archaebacterium Sulfolobus solfataricus. Molecular masses assessed by SDS/PAGE and gel filtration were 42 kDa and 170 kDa, respectively, which points to a tetrameric structure for the molecule. An isoelectric point of 5.9 was also determined. The enzyme was proven to be a metalloprotease, as shown by the inhibitory effects exerted by EDTA and o-phenanthroline; furthermore, dialysis against EDTA led to a complete loss of activity, which could be restored by addition of Zn2+ in the micromolar range, and, to a lesser extent, by Co2+. The enzyme was endowed with a broad substrate specificity, as shown by its ability to release basic, acidic and aromatic amino acids from the respective benzoylglycylated and benzyloxycarbonylated amino acids. An esterase activity of the carboxypeptidase was also demonstrated on different esterified amino acids and dipeptides blocked at the N-terminus. The enzyme displayed broad pH optima ranging over 5.5-7.0, or 5.5-9.0, when using an acidic or a basic benzyloxycarbonylated amino acid, respectively. With regard to thermostability, it was proven to be completely stable on incubation for 15 min at 85 degrees C. Furthermore, thanks to its relatively low activation energy, i.e. 31.0 kJ/mol, it was still significantly active at room temperature. At 40 degrees C, the enzyme could withstand 0.1% SDS and different organic solvents: particularly ethanol up to 99%. Amino acid and N-terminal sequence analyses did not evidence any similarity to carboxypeptidases A nor thermolysin. A weak similarity was only found with bovine carboxypeptidase B.

Effect of temperature on the propylamine transferase from Sulfolobus solfataricus, an extreme thermophilic archaebacterium

The thermal stability of propylamine transferase from Sulfolobus solfataricus, an extreme thermophilic archaebacterium, has been characterized thermodynamically by a Van't Hoff analysis. Conformational transitions induced by guanidine hydrochloride, as well as by temperature, have been linked together in a scheme involving six equilibria, which arise from both dissociation and unfolding. The mechanism by which the protein achieves thermal stabilization is quite unusual. It is driven by a conformational equilibrium between two forms of different stability. The stability of each form towards denaturation is characterized by a specific temperature dependence. The low-temperature form, indicated as 'form A', is stable over 12-89 degrees C. Its stability maximum is 36.8 kJ/mol at 50 degrees C. 'Form B', which is populated at higher temperature, spans the interval 28-146 degrees C. Its stability maximum is 71.6 kJ/mol at 87 degrees C. A possible explanation for the mechanism underlying this behaviour is discussed assuming that two major terms contribute to stability, i.e. hydrophobic interactions arising from burying of the accessible surface residues as well as conformational entropy. The thermal stabilization of the enzyme seems to depend on effects related to both an overall increase of flexibility and a concomitant decrease of the area buried upon folding. In this regard proteins from extreme thermophilic organisms appear to be a useful model to shed new light on the general problem of protein stability.

Effect of temperature on the propylamine transferase from Sulfolobus solfataricus, an extreme thermophilic archaebacterium. 1. Conformational behavior of the oligomeric enzyme in solution.

The effect of temperature on the molecular structure of propylamine transferase from Sulfolobus solfataricus has been investigated. Sulfolobus solfataricus is an extreme thermophilic archaebacterium with an optimum living condition at 90 degrees C. The enzyme is an oligomeric (trimer) protein of molecular mass 112 kDa. The frictional ratio for the native protein suggests an irregularly shaped compact globular structure. The protein matrix is well organized as suggested by far ultraviolet circular dichroism at 25 degrees C (18% alpha helix, 43% beta structure, 19% beta bends and 20% unordered: root mean square = 7). Structural effects of temperature were investigated over 25-85 degrees C. The protein retains its quaternary structure in this temperature range. A highly reversible subtle conformational transition was detected by numerous structure-dependent techniques over 40-50 degrees C, with a midpoint centered at 45 degrees C. Functional data also support this view. In fact, two enzyme forms, characterized by different catalytic properties, are present in solution. The Arrhenius plot suggests the occurrence of two different activation-energy-dependent processes, one at a temperature higher and one at a temperature lower than 45 degrees C. The transition has been considered as a molecular switch between two protein populations at equilibrium with different functional and structural properties, temperature modulated. A physiological role for the molecular switch has also been postulated. The protein also shows some subtle and reversible spectroscopic changes around 75 degrees C. The molecular basis of the thermophilic nature of this enzyme seems to reside in its capability to dynamically couple catalytic and structural events to the thermal properties of the ambient medium.

Functional reconstitution of membrane proteins in monolayer liposomes from bipolar lipids of Sulfolobus acidocaldarius.

Membranes of Sulfolobus acidocaldarius, an extreme thermophilic archaebacterium, are composed of unusual bipolar lipids. They consist of macrocyclic tetraethers with two polar heads linked by two hydrophobic C40 phytanyl chains which are thought to be arranged as a monolayer in the cytoplasmic membrane. Fractionation of a total lipid-extract from S. acidocaldarius yielded a lipid fraction which forms closed and stable unilamellar liposomes in aqueous media. Beef heart cytochrome c-oxidase could be functionally reconstituted in these liposomes. In the presence of reduced cytochrome c, a protonmotive force (delta p) across the liposomal membrane was generated of up to -92 mV. Upon fusion of these proteoliposomes with membrane vesicles of Lactococcus lactis, the delta p generated by cytochrome c-oxidase activity was capable to drive uphill transport of leucine. Electron microscopic analysis indicated that the tetraether lipids form a single monolayer liposome. The results demonstrate that tetraether lipids of archaebacteria can form a suitable matrix for the function of exogenous membrane proteins originating from a regular lipid bilayer.

A small basic ribosomal protein from the extreme thermophilic archaebacterium Sulfolobus solfataricus that has no equivalent in Escherichia coli

The structure of the gene for a small, very basic ribosomal protein in Sulfolobus solfataricus has been determined and the structure of the protein coded by this gene has been confirmed by partial amino acid sequencing. The protein shows no sequence similarity to any of the ribosomal proteins from eubacteria ( Escherichia coli) or to those that have been reported from eukaryotes.

Purification and properties of an extreme thermostable glutamate dehydrogenase from the archaebacterium Sulfolobus solfataricus

Glutamate dehydrogenase (l-glutamate: NAD(P)+ oxidoreductase, deaminating, EC 1.4.1.3.) of the extreme thermophilic archaebacterium Sulfolobus solfataricus was purified to homogeneity by (NH4)2SO4 fractionation, anion-exchange chromatography and affinity chromatography on 5′-AMP-Sepharose. The purified native enzyme had a Mr of about 270 000 and was shown to be a hexamer of subunit Mr of 44 000. It was active from 30 to 95°C, with a maximum activity at 85°C. No significant loss of enzyme activity could be detected, either after incubation of the purified enzyme at 90°C for 60 min, or in the presence of 4 M urea or 0.1% SDS. The enzyme was catalytically active with both NADH and NADPH as coenzyme and was specific for 2-oxoglutarate and l-glutamate as substrates. With respect to coenzyme utilization the Sulfolobus solfataricus glutamate dehydrogenase resembled more closely the equivalent enzymes from eukaryotic organisms that those from eubacteria.

Isolation and characterization of an intracellular aminopeptidase from the extreme thermophilic archaebacterium Sulfolobus solfataricus

An intracellular aminopeptidase (EC 3.4.11.-) was purified from the extreme thermophilic archaebacterium, Sulfolobus solfataricus. The molecular weight of the native enzyme was about 320 000, as calculated by gel-filtration studies, and subunit M r of 80 000 was estimated by SDS-polyacrylamide gel electrophoresis. The temperature optimum of the enzyme was at 75°C and the pH optimum was found to be 6.5. Th e aminopeptidase was highly active against the chromogenic substrates l-Leu-p-DNA and l-Ala-p-DNA. The enzyme was inhibited by EDTA, but the activity could be partially restored by removal of the EDTA and incubation with Co 2+ or Mn 2+. Bestatin, a typical inhibitor of aminopeptidase, fully inhibited the enzyme activity, but inhibitors of serine proteinases had no effect. Beside a high thermostability, the enzyme showed a remarkable stability against 6 M urea, organic solvents and acetonitrile.

A novel archaebacterial NAD+-dependent alcohol dehydrogenase. Purification and properties.

An NAD+-dependent alcohol dehydrogenase (alcohol: NAD+ oxidoreductase, EC 1.1.1.1) was detected in cellular extracts of the extreme thermophilic archaebacterium Sulfolobus solfataricus. The enzyme was purified to homogeneity and shown to be a dimer with a native molecular mass of 71 kDa by sucrose gradient centrifugation and SDS electrophoresis. The enzyme has a broad substrate specificity that includes linear and branched primary alcohols, linear and cyclic secondary alcohols, linear and cyclic ketones and anisaldehyde. The enzyme has an extraordinary thermophilicity and a remarkable thermostability, and appears to have some properties and a structure different from those previously described for thermophilic alcohol dehydrogenases.

Purification and characterization of propylamine transferase from Sulfolobus solfataricus, an extreme thermophilic archaebacterium.

The enzyme propylamine transferase, catalyzing the transfer of the propylamine moiety from S-adenosyl(5')-3-methylthiopropylamine to several amine acceptors, has been purified 643-fold in 20% yield from Sulfolobus solfataricus, an extreme thermophilic archaebacterium optimally growing at 87 degrees C. The purified enzyme (specific activity 2.05 units/mg protein), is homogeneous by criteria of gel electrophoresis, gel filtration, isoelectric focusing and ultracentrifugation analysis. The molecular mass of the native enzyme was estimated to be about 110 kDa by gel permeation and ultracentrifugation analysis. The protein migrates on SDS/polyacrylamide gel electrophoresis as a single band of 35 kDa, suggesting that the enzyme is a trimer composed by identical subunits. An optimum pH of 7.5 and an acidic isoelectric point of 5.3 have been calculated. The optimum temperature was 90 degrees C and no loss of activity is observable even after exposure of the purified enzyme to 100 degrees C for 1 h. No reducing agents are required for enzymatic activity. Substrate specificity towards the amine acceptors is rather broad in that 1,3-diaminopropane (Km = 1675 microM), putrescine (Km = 3850 microM), sym-norspermidine (Km = 954 microM) and spermidine (Km = 1539 microM) are recognized as substrates. Conversely S-adenosyl(5')-3-methylthiopropylamine is the only propylamine donor (Km = 7.9 microM) and the deamination of the sulfonium compound prevents the recognition by the enzyme. The reaction is irreversible and initial-rate kinetic studies indicate that the propylamine transfer is operated through a sequential mechanism. 5'-Methylthioadenosine, a product of the reaction, acts as a powerful competitive inhibitor with a Ki of 3.7 microM. Enzyme-substrate binding sites have been investigated with the aid of several substrate analogs and products. Among the compounds assayed, 5'-methylthiotubercidin, S-adenosyl(5')-3-thiopropylamine and S-adenosyl-3-thio-1,8-diaminooctane are the most active inhibitors.

Phase transitions of bipolar lipids of thermophilic archaebacteria

The plasma membrane of Sulfolobus solfataricus, an extreme thermophilic archaebacterium, is characterized by unusual bipolar lipids arranged in a single monolayer. They are based on two C 40 ω-ω′ biphytanyl residues, with up to four cyclopentane rings per chain, linked to either two glycerols (symmetric lipid) or to one glycerol and one branched-chain nonitol (asymmetric lipid). In this work we present a comparative calorimetric study of the symmetric and asymmetric lipid at various degrees of hydration. The results are compared with X-ray diffraction data. The thermotropic properties of the asymmetric lipid are very complex ones, showing two transitions in the dry state and at least two in the hydrated one. The low enthalpies displayed by the higher-temperature transitions indicate lipid polymorphism associated with temperature-dependent hydrogen bonds between the nonitol polar heads, while the lower-temperature transitions are mainly associated with structural changes of the hydrophobic core. The symmetric lipid exhibits a much simpler behaviour, being arranged in a lamellar structure with a single melting point. It shows, however, phase separation between hydrated and non-hydrated lipid domains, which allows some general considerations to be made on the non-random way water is absorbed and released in the interlamellar spaces.

A spin label ESR and saturation transfer-ESR study of archaebacteria bipolar lipids

A spin label study has been carried out on bipolar lipids extracted from Sulfolobus solfataricus, an extreme thermophilic archaebacterium growing at about 85°C and pH 3. These lipids are cyclic diisopranyl tetraether molecules, quite different from the usual fatty acid lipids. Two hydrolytic fractions of the membrane complex lipids have been studied: the symmetric lipid glycerol-dialkyl-glycerol-tetraether (GDGT) and the asymmetric lipid glyceroldialkyl-nonitol-tetraether (GDNT). The ESR spectra confirm the results previously obtained from calorimetric and X-ray diffraction experiments showing a polymorphic behaviour of these lipids and indicating the critical temperature ranges at which structural transitions occur. Moreover, the present study adds information on the dynamics of the different portions of the hydrophobic chain. ST-ESR measurements show correlation times ranging from 10-8 s up to 10-5 s, depending upon the lipid sample, the label position and the degree of hydration. At very high temperatures, i.e. the physiological temperatures of Sulfolobus solfataricus, the nonitol head groups of the asymmetric lipids form a strongly immobilized structure. Indeed, the molecular correlation times of the outermost hydrophobic portion of GDNT are higher, by a factor up to 103, than those of usual monopolar lipids. Anisotropic motional behaviour is observed even at such very high temperatures. Possible biological implications are discussed.

Effect of isoprenoid cyclization on the transition temperature of lipids in thermophilic archaebacteria

The plasma membrane of Caldariella acidophila, an extreme thermophilic archaebacterium, is characterized by unusual bipolar lipids. They are based on two C 40 ω-ω′ biphytanyl residues, with up to four cyclopentane rings per chain linked to either two glycerols (symmetric lipid) or to one glycerol and to one branched-chain nonitol (asymmetric lipid). When C. acidophila is grown at various temperatures, these lipids show a degree of cyclization of the biphytanyl components which increases as the environmental temperature increases. The rôle of cyclization in determining the temperature adaptation is studied on three lipid samples presenting four, five and six cyclopentane rings per molecule, respectively. Differential scanning calorimetry on the dry asymmetric sample as well as conductance and capacitance measurements on black films have been performed. Both sets of measurements indicate the presence of thermal transitions, three in the hydrated compounds, two in the dry system. The latter are shifted towards higher temperature values as the number of cycles increases. Calorimetric measurements show that two of these transitions are strictly related to the presence of nonitol-containing polar heads. In fact, only a single 10-fold higher transition is detected in the homologous lipid bearing two glycerol polar heads, in the dry as well as in the hydrated form. It is suggested that the two higher-temperature thermal transitions, observed on warming the sample, are induced by the breaking of hydrogen bonds between the nonitol-containing polar heads. By contrast, the lower temperature transition, present only in the hydrated compound and similar to that exhibited by the symmetrical sample, is due to a partial melting of the hydrophobic core. The large change in capacitance observed near the higher transition points by lowering temperature would thus correspond to variations in the dielectric constant due to formation of hydrogen bonds.

Monolayer black membranes from bipolar lipids of archaebacteria and their temperature-induced structural changes.

The membrane of Caldariella acidophila, an extreme thermophilic archaebacterium, is characterized by unusual bipolar complex lipids. They consist of two nonequivalent polar heads, linked by a C40 alkylic component. The molecular organization of these lipids in the plasma membrane is still a matter of study. In this paper, we present current-voltage measurements on artificial bipolar lipid membranes, indicating that molecules are indeed organized as a covalently bound bilayer, in which each molecule is completely stretched and spans its entire thickness. Furthermore, conformational transitions of these artificial membranes (which could be formed only above 70 degrees C from a lipid/squalene dispersion) are analyzed in the 80 to 15 degrees C temperature range. Abrupt variations in capacitance and valinomycin-induced conductance seem to indicate the occurrence of at least two structural changes. Measurements are also extended to different solvent systems. Results are consistent with the picture of a monolayer bipolar lipid membrane in which few solvent molecules align themselves parallel to the lipophilic chains. The amount of solvent as well as the temperature at which conformational transitions occur, depend on the solvent system in which the lipid is dispersed.

A New type of cell membrane, in thermophilic archaebacteria, based on bipolar ether lipids

The complex membrane lipids of thermophilic archaebacteria are bipolar amphipathic molecules. Indirect evidence suggests that the bipolar lipids of the membrane of these microorganisms have a monolayer organization, similar to a “unit membrane” bilayer, with molecular elements, the covalent binding of which spans its entire thickness. We report further evidence that the membrane bipolar lipids of Caldariella acidophita, an extreme thermophilic archaebacterium, can be organized as a covalently bound bilayer, in which each molecule, fully stretched, anchors the two polar heads to the inner and outer faces, respectively, of the membrane array.