Selectable mutant phenotypes of the extremely thermophilic archaebacterium Sulfolobus acidocaldarius.

As a first step toward developing the genetic potential of extremely thermophilic archaebacteria, mutant strains of Sulfolobus acidocaldarius were selected by plating cells directly on solid medium containing one of several growth inhibitors. Three spontaneous resistance phenotypes were observed (5-fluorouracil resistance, novobiocin resistance, and L-ethionine resistance), each at a different average frequency. Characterization of representative strains showed each of the three mutant phenotypes to provide a potentially useful genetic marker.     

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 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.     

Positive selection for uracil auxotrophs of the sulfur-dependent thermophilic archaebacterium Sulfolobus acidocaldarius by use of 5-fluoroorotic acid.

Uracil auxotrophs of Sulfolobus acidocaldarius were positively selected by using 5-fluoroorotic acid. The wild-type strain was unable to grow in medium containing 5-fluoroorotic acid, whereas the mutants grew normally. Positive selection could be done for the auxotrophs. Mutants deficient in orotidine-5'-monophosphate pyrophosphorylase activity were isolated.     

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 a subunit Mr of 80,000 was estimated by SDS-polyacrylamide gel electrophoresis. The temperature optimum of the enzyme was at 75 degrees C and the pH optimum was found to be 6.5. The aminopeptidase was highly active against the chromogenic substrates L-Leu-p-NA and L-Ala-p-NA. The enzyme was inhibited by EDTA, but the activity could be partially restored by removal of the EDTA and incubation with Co2+ or Mn2+. 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.     

Purification and properties of a thermophilic and thermostable DNA polymerase from the archaebacterium Sulfolobus solfataricus

A DNA-dependent DNA polymerase was obtained in homogenous form from the thermoacidophilic archaebacterium Sulfolobus solfataricus. The enzyme, purified 706-fold, has a molecular mass of about 110000 daltons as determined by gel filtration and by glycerol gradient centrifugation. It requires Mg++ for its activity and has a pH optimum of 7.7. The activity is sharply dependent on the ionic strength. The enzyme is thermostable; its properties and activity requirements were characterized. The features of this enzyme are compared to those of other DNA polymerases isolated either from prokaryotes or eukaryotes.     

Structure of solfapterin (erythro-neopterin-3''-D-2-deoxy-2-aminoglucopyranoside) isolated from the thermophilic archaebacterium Sulfolobus solfataricus.

The structure of the major fluorescent pterin present in thermophilic archaebacterium Sulfolobus solfataricus has been assigned, by analysis of the intact molecule and its hydrolytic and periodate cleavage products, as erythro-neopterin-3'-D-2-deoxy-2-aminoglucopyranoside. The trivial name solfapterin is proposed for this compound.     

Thermostable beta-galactosidase from the archaebacterium Sulfolobus solfataricus. Purification and properties

A thermophilic and thermostable beta-galactosidase activity was purified to homogeneity from crude extracts of the archaebacterium Sulfolobus solfataricus, by a procedure including ion-exchange and affinity chromatography. The homogeneous enzyme had a specific activity of 116.4 units/mg at 75 degrees C with o-nitrophenyl beta-galactopyranoside as substrate. Molecular mass studies demonstrated that the S. solfataricus beta-galactosidase was a tetramer of 240 +/- 8 kDa composed of similar or identical subunits. Comparison of the amino acid composition of beta-galactosidase from S. solfataricus with that from Escherichia coli revealed a lower cysteine content and a lower Arg/Lys ratio in the thermophilic enzyme. A rabbit serum, raised against the homogeneous enzyme did not cross-react with beta-galactosidase from E. coli. The enzyme, characterized for its reaction requirements and kinetic properties, showed a thermostability and thermophilicity notably greater than those reported for beta-galactosidases from other mesophilic and thermophilic sources.     

Purification and characterization of DNA polymerase from the archaebacterium Sulfolobus acidocaldarius.

DNA polymerase has been purified about 25,000-fold from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius. On SDS-PAGE the enzyme was observed to have a molecular weight of 100 kDa and to be about 90% pure. The native molecular weight was 108 kDa indicating that the enzyme is composed of a single polypeptide. Activity gel analysis showed an active polypeptide of about 100 kDa. Under conditions promoting proteolysis this polypeptide was degraded to a slightly smaller form of 98 kDa. The enzyme has been characterized in respect to optimal assay conditions, template specificity, sensitivity to inhibitors and associated nuclease activities. The high temperature optimum of 65 degrees C should be emphasized. No substantial similarities have been found with other prokaryotic and eukaryotic DNA polymerases, although the enzyme bears certain resemblances to prokaryotic non-replicative polymerases.     

Metabolism of Pentose Sugars in the Hyperthermophilic Archaea Sulfolobus solfataricus and Sulfolobus acidocaldarius

We have previously shown that the hyperthermophilic archaeon, Sulfolobus solfataricus, catabolizes d-glucose and d-galactose to pyruvate and glyceraldehyde via a non-phosphorylative version of the Entner-Doudoroff pathway. At each step, one enzyme is active with both C6 epimers, leading to a metabolically promiscuous pathway. On further investigation, the catalytic promiscuity of the first enzyme in this pathway, glucose dehydrogenase, has been shown to extend to the C5 sugars, d-xylose and l-arabinose. In the current paper we establish that this promiscuity for C6 and C5 metabolites is also exhibited by the third enzyme in the pathway, 2-keto-3-deoxygluconate aldolase, but that the second step requires a specific C5-dehydratase, the gluconate dehydratase being active only with C6 metabolites. The products of this pathway for the catabolism of d-xylose and l-arabinose are pyruvate and glycolaldehyde, pyruvate entering the citric acid cycle after oxidative decarboxylation to acetyl-coenzyme A. We have identified and characterized the enzymes, both native and recombinant, that catalyze the conversion of glycolaldehyde to glycolate and then to glyoxylate, which can enter the citric acid cycle via the action of malate synthase. Evidence is also presented that similar enzymes for this pentose sugar pathway are present in Sulfolobus acidocaldarius, and metabolic tracer studies in this archaeon demonstrate its in vivo operation in parallel with a route involving no aldol cleavage of the 2-keto-3-deoxy-pentanoates but direct conversion to the citric acid cycle C5-metabolite, 2-oxoglutarate.     

Particle weights and protein composition of the ribosomal subunits of the extremely thermoacidophilic archaebacterium Caldariella acidophila.

1. The ribosomal subunits of one thermoacidophilic archaebacterium (Caldariella acidophila) and of two reference eubacterial species (Bacillus acidocaldarius, Escherichia coli) were compared with respect to ribosome mass and protein composition by (i) equilibrium-density sedimentation of the particles in CsCl and (ii) gel-electrophoretic estimations of the molecular weights of the protein and the rRNA. 2. By either procedure, it is estimated that synthetically active archaebacterial 30S subunits (52% protein by wt.) are appreciably richer in protein than the corresponding eubacterial particles (31% protein by wt.) 3. The greater protein content of the archaebacterial 30S subunits is accounted for by both a larger number and a greater average molecular weight of the subunit proteins; specifically, C. acidophila 30S subunits yield 28 proteins whose combined mass is 0.6 X 10(6) Da, compared with 20 proteins totalling 0.35 X 10(6) Da mass for eubacterial 30S subunits. 4. No differences in protein number are detected among the large subunits, but C. acidophila 50S subunits exhibit a greater number-average molecular weight of their protein components than do eubacterial 50S particles. 5. Particle weights estimated by either buoyant-density data, or molecular weights of rRNA plus protein, agree to within less than 2%. By either procedure C. acidophila 30S subunits 1.15 X 10(6) Da mass) are estimated to be about 300 000 Da heavier than their eubacterial counterparts (0.87 X 10(6) Da mass); a smaller difference. 0.15 X 10(6) Da, exists between the archaebacterial and the eubacterial 50S subunits (respectively 1.8 X 10(6) and 1.65 X 10(6) Da). It is concluded that the heavier-than-eubacterial mass of the C. acidophila ribosomes resides principally in their smaller subunits.     

The structure of the gene for ribosomal protein L5 in the archaebacterium Sulfolobus acidocaldarius.

The gene for the ribosomal protein L5 from the archaebacterium Sulfolobus acidocaldarius has been isolated and sequenced. The gene codes for a basic protein of molecular weight 29 165 Da. This protein shows substantial similarity to the equivalent protein from other archaebacteria as well as from yeast, and considerably less similarity to the equivalent eubacterial protein. These results support the concept of the archaebacteria as a monophyletic kingdom more closely related to eukaryotes than to eubacteria.     

Calditol tetraether lipids of the archaebacterium Sulfolobus solfataricus. Biosynthetic studies.

Lipids from the archaebacterium Sulfolobus solfataricus are based on 72-membered macrocyclic tetraethers made up from two C40 diol units differently cyclized and either two glycerol moieties or one glycerol moiety and a unique branched-chain nonitol named calditol (glycerodialkylnonitol tetraethers, GDNTs). To elucidate the biosynthesis of calditol and related tetraethers, labelled precursors, [U-14C,1(3)-3H]glycerol, [U-14C,2-3H]glycerol, D-[1-14C,6-3H]glucose, D-[6-14C,1-3H]glucose, D-[1-14C,2-3H]glucose, D-[1-14C,6-3H]fructose and D-[1-14C]galactose, were fed to S. solfataricus. Without regard to stereochemistry or phosphorylation, incorporation experiments provided evidence that the biosynthesis of calditol occurs via an aldolic condensation between dihydroxyacetone and fructose, through a 2-oxo derivative of calditol as an intermediate. The latter is in turn reduced and then alkylated to yield the GDNTs. The biogenetic origins of both glycerol and C40 isoprenoid moieties of GDNTs are also discussed.     

Structural and functional exchangeability of 5 S RNA species from the eubacterium E.coli and the thermoacidophilic archaebacterium Sulfolobus solfataricus.

The role of 5 S RNA within the large ribosomal subunit of the extremely thermophilic archaebacterium Sulfolobus solfataricus has been analysed by means of in vitro reconstitution procedures. It is shown that Sulfolobus 50 S subunits reconstituted in the absence of 5 S RNA are inactive in protein synthesis and lack 2-3 ribosomal proteins. Furthermore, it has been determined that in the course of the in vitro assembly process Sulfolobus 5 S RNA can be replaced by the correspondent RNA species of E.coli; Sulfolobus reconstituted particles containing the eubacterial 5 S molecule are stable and active in polypeptide synthesis at high temperatures.     

Indole-3-acetic acid and 2-(indol-3-ylmethyl)indol-3-yl acetic acid in the thermophilic archaebacterium Sulfolobus acidocaldarius.

Indole-3-acetic acid (IAA) and 2-(indol-3-ylmethyl)indol-3-yl acetic acid were identified in lipid extracts of Sulfolobus acidocaldarius; they occurred at concentrations of 0.57 and 0.59 mumol/g (dry weight), respectively. The amount of IAA found in these cells is more than a thousand times greater than that found in a typical extract of a plant in which IAA serves as a plant growth hormone. Neither of these compounds was detected in the other archaebacteria that were analyzed; these included Sulfolobus sulfataricus, Halobacterium salinarium, and several strains of methanogenic bacteria. This is the first report of the natural occurrence of 2-(indol-3-ylmethyl)indol-3-yl acetic acid.     

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.     

S-adenosylmethionine decarboxylase from the thermophilic archaebacterium Sulfolobus solfataricus. Purification, molecular properties and studies on the covalently bound pyruvate.

S-Adenosylmethionine decarboxylase from Sulfolobus solfataricus, a thermoacidophilic archaebacterium optimally growing at 87 degrees C, has been purified to homogeneity. The specific activity of the homogeneous enzyme is 12 nmol CO2 formed min-1 (mg protein)-1 and the overall yield 8%. The enzyme is thermophilic with an optimum at 75 degrees C, is thermostable, and does not require divalent cations or putrescine for activity. It has a molecular mass of 32 kDa, and appears to be a monomeric protein. S-Adenosylmethionine decarboxylase from S. solfataricus contains covalently linked pyruvate as prosthetic group and is inactivated in a time-dependent process by NaCNBH3, in the presence of both the substrate and the product. Incubation with decarboxylated S-adenosyl[Me-3H]methionine and NaCNBH3 resulted in the labeling of the protein at the active site.     

Functional curation of the Sulfolobus solfataricus P2 and S. acidocaldarius 98-3 complete genome sequences

The thermoacidophiles Sulfolobus solfataricus P2 and S. acidocaldarius 98-3 are considered key model organisms representing a major phylum of the Crenarchaeota. Because maintaining current, accurate genome information is indispensable for modern biology, we have updated gene function annotation using the arCOGs database, plus other available functional, structural and phylogenetic information. The goal of this initiative is continuous improvement of genome annotation with the support of the Sulfolobus research community.