Thermoacidophilic Archaebacterium Research Articles

Unusual Thermal Stability of Liposomes Made from Bipolar Tetraether Lipids

The thermal stability of liposomes made from polar lipids of Sulfolobus acidocaldarius, a thermoacidophilic archaebacterium, was studied. While two analogous nonarchaebacterial liposomes examined were unstable above 50°C, liposomes made of the native tetraether lipids showed stability up to at least 80°C. Incubation of the archaebacterial liposomes at 100°C, under slight pressure, caused an approximate 15 % leakage after 60 minutes. Boiling, however, destroys the integrity of the lipid membranes. The tetraether lipid vesicles are less permeable to solute molecules, such as carboxyfluorescein, than the other lipid vesicles at any temperature. The permeability is also less temperature-sensitive than that for other lipids.

Pyrite Oxidation bySulfolobus acidocaldarius

Two strains of Sulfolobus acidocaldarius, a thermoacidophilic archaebacterium, were examined for their pyrite-oxidizing ability. S. acidocaldarius ATCC 33909 was shown to possess iron-oxidizing activity by ferrous sulfate oxidizing experiments, but S. acidocaldarius No. 7 did not have it. Pyrite-oxidizing rate of S. acidocaldarius ATCC 33909 was 1.6-fold higher than that of strain 7 though they had a similar level of sulfur-oxidizing ability. These results show that the iron-oxidizing activity accelerates pyrite oxidation.

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.

Cloning and sequencing of the gene encoding thermostable elongation factor 2 in Sulfolobus solfataricus

The gene ( aEF-2) coding for the translation elongation factor 2 (aEF-2) in the thermoacidophilic archaebacterium, Sulfolobus solfataricus, has been cloned and sequenced. The deduced primary structure of aEF-2 is composed of 735 amino acids (aa), excluding the Met start residue. There are no Cys residues and the calculated M r is 81699. In the coding region of aEF-2, the high A + T content greatly influences the codon usage. From the alignment of the primary structure of aEF-2 with that of the analogous factors from the three kingdoms, aa identities were derived. The greatest identity (82%) was found with EF-2 from Sulfolobus acidocaldarius; lower values were observed with other archaebacterial EF-2 (45–47%), eukaryotic EF-2 (38–40%) and with the functional eubacterial analogue EF-G (28–31%). aEF-2 possesses the consensus sequences required for a GTP-binding protein and the four regions which are supposed to be involved in the functional regulation of EF-2/EF-G. These data should have phylogenetic implications.

Structural features responsible for kinetic thermal stability of a carboxypeptidase from the archaebacterium Sulfolobus solfataricus

Investigations were performed on the structural features responsible for kinetic thermal stability of a thermostable carboxypeptidase from the thermoacidophilic archaebacterium Sulfolobus solfataricus which had been purified previously and identified as a zinc metalloprotease [Colombo, D'Auria, Fusi, Zecca, Raia and Tortora (1992) Eur. J. Biochem. 206, 349-357]. Removal of Zn2+ by dialysis led to reversible activity loss, which was promptly restored by addition of 80 microM ZnCl2 to the assay mixture. For the first-order irreversible thermal inactivation the metal-depleted enzyme showed an activation energy value of 205.6 kJ.mol-1, which is considerably lower than that of the holoenzyme (494.4 kJ.mol-1). The values of activation free energies, enthalpies and entropies also dropped with metal removal. Thermal inactivation of the apoenzyme was very quick at 80 degrees C, whereas the holoenzyme was stable at the same temperature. These findings suggest a major stabilizing role for the bivalent cation. Chaotropic salts strongly destabilized the holoenzyme, showing that hydrophobic interactions are involved in maintaining the native conformation of the enzyme. However, the inactivation rate was also increased by sodium sulphate, acetate and chloride, which are not chaotropes, indicating that one or more salt bridges concur in stabilizing the active enzyme. Furthermore, at the extremes of the pH-stability curve, NaCl did not affect the inactivation rate, confirming the stabilizing role of intramolecular ionic bonds, as a pH-dependent decrease in stability is likely to occur from breaking of salt bridges involved in maintaining the native conformation of the protein.

Glutamate dehydrogenase from the thermoacidophilic archaebacterium Sulfolobussolfataricus: studies on thermal and guanidine-dependent inactivation

The hexameric NAD(P)-dependent glutamate dehydrogenase isolated from the thermoacidophilic archaebacterium Sulfolobus solfataricus shows a remarkable thermal stability which is strictly dependent on protein concentration (half-life at 95°C is 0.25 h and 0.5 h at 0.4 and 0.8 mg/ml, respectively). Temperature-dependent inactivation of the enzyme is apparently irreversible; this process is accompanied by a progressive increase increase hydrophobic surface area which leads to protein precipitation. 3 M GdnHCl increases the half-life of the enzyme at 90°C and 0.2 mg/ml 6-fold. The hexamer is the only soluble molecular species revealed by glutaraldehyde fixation after thermal inactivation. Lyotropic salts strongly affect the enzyme thermal stability: the half-life at 90°C and 0.2 mg/ml protein concentration increases more tha 6-fold in the presence of 0.4 M Na 2SO 4 and decreases 4-fold in the presence of 0.4 M NaSCN. The maximum protein thermal stability is observed around the isoelectric pH, between pH 5.2 and pH 6.8. Guanidine-dependent inactivation of the enzyme at 20°C is irreversible above 1.5 M GdnHCl. The decline in percentage of reactivation closely parallels the structural changes detected by fluorescence and the loss of hexameric structure accompanied by the dissociation to monomers, as indicated by glutaraldehyde fixation.

An Ecto-Enzyme from Sulfolobus acidocaldarius Strain 7 Which Catalyzes Hydrolysis of Inorganic Pyrophosphate, ATP, and ADP: Purification and Characterization

Membranes of Sulfolobus acidocaldarius, a thermoacidophilic archaebacterium, show novel enzymatic activities to hydrolyze PPi, ATP, and ADP at an optimal pH of 3, equal to the growth optimum. The activity increased by about 2-fold on addition of PPi and/or Pi to the growth medium, when yeast extract and casamino acids were removed. The enzyme which hydrolyzes PPi at pH 3 was solubilized and purified by successive chromatographies. The final preparation showed a 26 kDa single band on SDS-PAGE, and a molecular mass of 35 kDa on gel permeation chromatography. The Km and Vmax for PPi were 0.16 mM and 33 mumol Pi released/min/mg at 55 degrees C. ATP and ADP were also good substrates. Divalent cations were not essential for activity. Substrate inhibition at more than 5 mM PPi, ATP or ADP was observed. AMP, glucose-6-phosphate, and p-nitrophenyl phosphate were not hydrolyzed at all. The activity was 4-fold stimulated by addition of the lipid fraction extracted from the organism.

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.

Resistance of archaebacterial aEF-1α· GDP against denaturation by heat and urea

The elongation factor aEF-1α, isolated as aEF-1α· GDP from the thermoacidophilic archaebacterium Sulfolobus solfataricus, exchanges GDP for [ 3H]GDP at a rate which reaches a maximum at 95°C. The rate constants at different temperatures of the heat inactivation of aEF-1α· GDP are considerably lower compared to those referred to Escherichia coli EF-Tu· GDP. The T m values determined for both aEF-1α· GDP and EF-Tu· GDP are 97 and 53°C, respectively. The addition of GDP during the heat treatment protects significantly EF-Tu· GDP but only slightly aEF-1α· GDP. The ability of aEF-1α· GDP to exchange GDP for [ 3H]GDP is impaired at 70°C by urea at concentrations which are greater compared to those required to inactivate E. coli EF-Tu· GDP at 45°C; apparently both factors are hot protected by GDP against inactivation by urea.

Thermostable NAD(+)-dependent alcohol dehydrogenase from Sulfolobus solfataricus: gene and protein sequence determination and relationship to other alcohol dehydrogenases.

The NAD(+)-dependent alcohol dehydrogenase (EC 1.1.1.1) from the thermoacidophilic archaebacterium Sulfolobus solfataricus, DSM1617 strain (SSADH), has been purified and characterized. Its gene has been isolated by screening two S. Solfataricus genomic libraries using oligonucleotide probes. The encoding sequence consists of 1041 base pairs, and it shows a high preference for codons ending in T or A. The primary structure, determined by peptide and gene analysis, consists of 347 amino acid residues, yielding a molecular weight of 37,588. A level of identity of 24-25% was found with the amino acid sequences of horse liver, yeast, and Thermoanaerobium brockii alcohol dehydrogenases. The coenzyme-binding and catalytic and structural zinc-binding residues typical of eukaryotic alcohol dehydrogenases were found in SSADH with the difference that one out of the four structural zinc-binding Cys residues is substituted by Glu. The protein contains four zinc atoms per dimer, two of which are removed by chelating agents with a concomitant loss of structural stability.

Long-term preservation of halophilic archaebacteria and thermoacidophilic archaebacteria by liquid drying

A liquid drying (L-drying) method was applied satisfactorily to the long-term preservation of extremely halophilic archaebacteria and thermoacidophilic archaebacteria that are sensitive to freezing and freeze-drying. Survival values of Halobacterium, Haloferax, Haloarcula, Halococcus, Natronobacterium, Thermoplasma, Sulfolobus and Acidianus were more than 5% after drying and more than 0.04% after being stored at 37°C for 2 weeks. The survival values of these dried specimens after long-term preservation at 5°C corresponded to those estimated from the results of the accelerated storage test. Therefore, it is considered that the accelerated storage test is useful for estimating the stability of the dried specimens of archaebacteria during preservation and that the dried specimens of even the most fragile bacterium. Thermoplasma, will remain viable for more than 15 years when they are kept at 5°C.

Molecular, functional and structural properties of an archaebacterial elongation factor 2

The elongation factor 2 (aEF-2) from the extreme thermo-acidophilic archaebacterium Sulfolobus solfataricus, is the only cytosolic target protein which is ADP-ribosylated by diphtheria toxin in presence of NAD. Once ADP-ribosylated, aEF-2 is no longer able to sustain poly(Phe) synthesis in vitro. aEF-2 displays a great thermoresistance: at the growth temperature of the archaebacterium, 87°C, its half-life is 3 h. The amino acid sequence of the N-terminal region of aEF-2 has been determined up to residue 22. In the first 15 positions such a sequence is identical to that of EF-2 from Sulfolobus acidocaldarius and very similar to that of EF-2 from other archaebacteria or eukaryotes. The same is true for the primary structure of the peptide containing the ADP-ribosylation site. The fact that the primary structure of EF-2 at the ADP-ribosylation site is highly conserved ensures either the correct recognition of the histidine residue by the enzymes involved in its modification to diphthamide, or the proper interaction with the diphtheria toxin.

A heat-stable serine proteinase from the extreme thermophilic archaebacterium Sulfolobus solfataricus

A proteinase was purified to electrophoretic homogeneity from crude extracts of the thermoacidophilic archaebacterium Sulfolobus solfataricus. Molecular mass values assessed by SDS-PAGE and gel filtration were 54 and 118 kDa, respectively, which points to a dimeric structure of the molecule. An isoelectric point of 5.6 was also determined. The enzyme behaved as a chymotrypsin-like serine proteinase, as shown by the inhibitory effects exerted by phenylmethanesulfonyl fluoride, 3,4-dichloroisocoumarin, tosylphenylalaninechloromethyl ketone and chymostatin. Consistently with the inhibition pattern, the enzyme cleaved chromogenic substrates at the carboxyl side of aromatic or bulky aliphatic amino acids; however, it effectively attacked only a small number of such substrates, thus, displaying a specificity much narrower than and clearly different from that of chymotrypsin. This was confirmed by its inability to digest a set of natural substrate proteins, as well as insulin chains A and B; only after alkylation casein was degraded to some extent. Proteinase activity was significantly stimulated by Mn2+ which acted as a mixed-type nonessential activator. The enzyme also displayed a broad pH optimum in the range 6.5-8.0. Furthermore, it was completely stable up to 90 degrees C; above this temperature it underwent first-order thermal inactivation with half-lives ranging from 342 min (92 degrees C) to 7 min (101 degrees C). At 50 degrees C it could withstand 6 M urea and, to some extent, different organic solvents; however, at 95 degrees C it was extensively inactivated by all of these compounds. None of the chemical physical properties of the enzyme, including amino-acid analysis, provided evidence of a possible relation to other well-known microbial serine proteinases.

Purification and properties of pyruvate kinase from Thermoplasma acidophilum

Thermoplasma acidophilum is a thermoacidophilic archaebacterium occupying a paradoxical place in phylogenetic trees (phenotypically it is a thermoacidophile but phylogenetically it classifies with the methanogens). To better understand its phylogeny, the pyruvate kinase from this organism is being investigated as a molecular marker. The enzyme has been purified and has a native M r of 250 000. It consists of four, apparently identical subunits each of M r 60 000. No remarkable kinetic differences have been found between this thermophilic enzyme and its mesophilic counterparts other than its greater thermostability. Its amino acid composition has been determined and some partial sequencing has been done.

Purification and properties of pyruvate kinase fromThermoplasma acidophilum

Thermoplasma acidophilum is a thermoacidophilic archaebacterium occupying a paradoxical place in phylogenetic trees (phenotypically it is a thermoacidophile but phylogenetically it classifies with the methanogens). To better understand its phylogeny, the pyruvate kinase from this organism is being investigated as a molecular marker. The enzyme has been purified and has a native Mr of 250 000. It consists of four, apparently identical subunits each of Mr 60 000. No remarkable kinetic differences have been found between this thermophilic enzyme and its mesophilic counterparts other than its greater thermostability. Its amino acid composition has been determined and some partial sequencing has been done.

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.

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.

An extremely stable inorganic pyrophosphatase purified from the cytosol of a thermoacidophilic archaebacterium, Sulfolobus acidocaldarius strain 7.

A highly active inorganic pyrophosphatase was purified to electrophoretical homogeneity from the cytosol of Sulfolobus acidocaldarius strain 7, an extremely thermoacidophilic archaebacterium. The enzyme has an apparent molecular mass of 80 kDa as estimated by gel permeation chromatography, and showed a 21-kDa polypeptide on SDS-PAGE, suggesting that the archaebacterial enzyme is similar to most of the eubacterial pyrophosphatases rather than eukaryotic ones. The pI = 5.1. The enzyme showed relatively high content of Pro and low content of Ser plus Thr. The optimal pH was 6.5 (at 56 degrees C). From the Arrhenius plot an activation energy of 11.2 kcal/mol was obtained between 37-95 degrees C. The specific activity was 617 mumol Pi release min-1 mg-1 at 56 degrees C. The S. acidocaldarius pyrophosphatase was extremely stable. Complete activity remained after incubation at 100 degrees C for 10 min. No dissociation into subunit or unfolding of polypeptide chain occurred in the presence of 8 M urea. Experiments using guanidine-HCl suggested that the transition between a native tetrameric state and an unfolded state is completely reversible, and essentially independent of any additional factors such as divalent metal cation or dithiothreitol.

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.

Expression of the thermostable beta-galactosidase gene from the archaebacterium Sulfolobus solfataricus in Saccharomyces cerevisiae and characterization of a new inducible promoter for heterologous expression

The lacS gene from the extremely thermoacidophilic archaebacterium Sulfolobus solfataricus encodes an enzyme with beta-galactosidase activity that, like other enzymes from this organism, is exceptionally thermophilic (optimal activity above 90 degrees C), thermostable, and resistant to common protein denaturants and proteases. Expression of the gene in mesophilic hosts is needed to uncover the molecular nature of these features. We have obtained expression of beta-galactosidase in Saccharomyces cerevisiae under the control of the galactose-inducible upstream activating sequence of the yeast genes GAL1 and GAL10. The expressed enzyme is identical in molecular mass, thermostability, and thermophilicity to the native enzyme, showing that these features are intrinsic to the primary structure of the enzyme. We also present a new promoter for the expression of thermostable proteins in S. cerevisiae. This promoter contains a sequence isolated from the nematode Caenorhabditis elegans that works as a strong, heat-inducible upstream activating sequence in S. cerevisiae. Transcription of the lacS gene under the control of this sequence is rapidly and efficiently induced by heat shock. The availability of a plate assay for monitoring beta-galactosidase activity in S. cerevisiae may allow screening for mutants affecting the efficiency and activity of the enzyme.