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.     

A plasmid in the archaebacterium Sulfolobus acidocaldarius

A plasmid of mol. wt. ~9 × 10⁶ has been isolated from the archaebacterium Sulfolobus acidocaldarius strain B12. Plasmid production is induced by u.v. radiation. A copy of the plasmid is probably carried by the chromosome, integrated at a specific site. The entire plasmid, and also restriction fragments of it, has been cloned into Escherichia coli plasmid vectors, and the cleavage sites on the plasmid DNA of three restriction endonucleases have been mapped.     

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.     

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.     

Glycogen-bound polyphosphate kinase from the archaebacterium Sulfolobus acidocaldarius.

Glycogen-bound polyphosphate kinase has been isolated from a crude extract of Sulfolobus acidocaldarius by isopycnic centrifugation in CsCl. Divalent cations (Mn2+ greater than Mg2+) stimulated the reaction. The enzyme does not require the presence of histones for its activity; it is inhibited strongly by phosphate and slightly by fluoride. The protein from the glycogen complex migrated in a sodium dodecyl sulfate-polyacrylamide gel as a 57-kilodalton protein band; after isoelectric focusing it separated into several spots in the pH range of 5.6 to 6.7.     

ATP-dependent DNA topoisomerase from the archaebacterium Sulfolobus acidocaldarius. Relaxation of supercoiled DNA at high temperature

A topoisomerase, able to relax negatively supercoiled DNA, has been isolated from the archaebacterium Sulfolobus acidocaldarius. Relaxation was fully efficient in vitro between 70 degrees C and 80 degrees C and was dependent on the presence of ATP and magnesium ions. The enzyme did not exhibit gyrase-like activity and was poorly sensitive to gyrase inhibitors. These properties are reminiscent of eukaryotic type II topoisomerases. However, the enzyme was unable to relax positively supercoiled DNA. This thermophilic enzyme may be used in a variety of ways to study the structure and stability of DNA at high temperature.     

Thermostable DNA polymerase from the archaebacterium Sulfolobus acidocaldarius. Purification, characterization and immunological properties

We have purified to near homogeneity a DNA polymerase from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius. Sodium dodecyl sulfate gel electrophoresis of the purified enzyme revealed a polypeptide of 100 kDa. On the basis of a Stokes radius of 4.2 nm and a sedimentation coefficient of 6 S, the purified enzyme has an estimated molecular mass of 109 kDa. These results are consistent with the enzyme being a monomer of 100 kDa. In addition a polyclonal antiserum, obtained by injection of the electroeluted 100-kDa polypeptide into a rabbit, specifically neutralized the DNA-polymerase activity. The enzyme is sensitive to both N-ethylmaleimide and 2',3'-dideoxyribosylthymine triphosphate and resistant to aphidicolin. The purified DNA polymerase has neither exonuclease nor primase activities. In our in vitro conditions, the enzyme is thermostable up to 80 degrees C and is active between 55 degrees C and 85 degrees C in the presence of activated calf-thymus DNA.     

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.     

Electron transport and energy conservation in the archaebacterium Sulfolobus acidocaldarius

Abstract 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.     

A restriction endonuclease SuaI from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius

A type II restriction endonuclease (SuaI) has been isolated from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius. 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 degrees C and is highly thermostable.     

Energy metabolism of a thermoacidophilic archaebacterium,Sulfolobus acidocaldarius

To elucidate the phylogenic status of the archaebacterium and mechanisms of acidophily, membrane bound ATPase, cytochromes and NADH dehydrogenase of a thermoacidophilic archaebacterium,Sulfolobus acidocaldarius, were studied. Typea cytochrome was found in the membrane. The organism was sensitive to cyanide and azide, and though cytochromec is lacking in this organism, these respiratory poisons inhibited a terminal oxidase, when assayed with cytochromec from other sources. NADH dehydrogenase was highly purified from the crude extract of the cells. The enzyme was able to transfer electrons from NADH to caldariellaquinone, a unique benzothiophenequinone in the genusSulfolobus. Thus, the enzyme is a possible member of the respiratory chain. Membrane fraction contained two types of ATPase, one was active at neutral pH and slightly activated by sulfate; the other was an acid apyrase and inhibited by sulfate. Typical characteristics of F₀F₁ATPase could not be found in these enzymes. These results suggest that (1) the thermoacidophilic archaebacteria are phylogenically distant from both eubacteria and eukaryotes, (2) the archaebacterial thermoacidophiles can be classified in a different subgroup from methanogens and extreme halophiles, and (3) in spite of the aerobic nature of the organism, the energy yielding mechanisms appear quite unique, when compared to those of other aerobes and mitochondria.     

Membrane-bound ATPase of a thermoacidophilic archaebacterium, Sulfolobus acidocaldarius

The membranes of Sulfolobus, a thermoacidophilic archaebacterium showed two types of ATP hydrolyzing activity. One was that of a neutral ATPase at an optimum pH around 6.5. This enzyme was activated by 10 mM sulfate with a shift of optimum pH to 5. In these respects, the enzyme was similar to membrane-bound ATPase of Thermoplasma, another thermoacidophilic archaebacterium, reported by Searcy and Whatley [1982) Zbl. Bakt. Hyg., I. Abt. Orig. C3, 245-257). The enzyme hydrolyzed ATP and other NTPs, but not ADP or AMP. It was highly thermostable, but irreversibly inactivated in 0.1 M HCl. The other activity was that of an acidic apyrase at an optimum pH around 2.5. This enzyme was extremely stable toward high temperature and acid and inhibited by sulfate. Both of these ATP hydrolyzing enzymes were resistant to N,N'-dicyclohexylcarbodiimide (DCCD), azide, oligomycin, N'-ethylmaleimide, p-chloromercuribenzoate, orthovanadate, or ouabain. Sulfolobus ATPases differ from F1 and other transport ATPases so far described.     

Some biochemical properties of an acido-thermophilic archaebacterium,Sulfolobus acidocaldarius

To elucidate the phylogenic status of archaebacteria, some basic cellular components of an acido-thermophilic archaebacterium,Sulfolobus acidocaldarius, were studied. Poly(A) containing RNA was present in the cells, and performed the role of mRNA in a cell-free extract of reticulocyte or the archaebacteria. Poly(A) containing RNA was also found in other archaebacterial cells. The absence of cap structure was suggested in these RNAs. The cell-free protein synthesis using the archaebacterial extract was inhibited by anisomycin, a specific inhibitor for eukaryotic ribosomes. Two unique membrane-bound ATPases were detected. Based on resistance to H⁺-ATPase inhibitors, these enzymes seemed not to be F₀F₁-ATPase.     

Purification and characterization of a heat-stable esterase from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius.

A heat-stable esterase has been purified 1080-fold to electrophoretic homogeneity from Sulfolobus acidocaldarius, a thermoacidophilic archaebacterium; 20% of the starting activity is recovered. The purified enzyme shows a specific activity of 158 units/mg, based on the hydrolysis of p-nitrophenyl acetate. The esterase hydrolyses short-chain p-nitrophenyl esters, aliphatic esters and triacylglycerols. It is strongly inhibited by paraoxon and phenylmethanesulphonyl fluoride, but only weakly by eserine. From sedimentation-equilibrium data and molecular sieving in polyacrylamide gels, the Mr of the esterase is estimated to be 117000-128000. SDS/polyacrylamide-gel electrophoresis reveals a single band of protein, of Mr 32000. The purified esterase crystallizes in the presence of poly(ethylene glycol) in short rods. The enzyme is inactivated only on prolonged storage at temperature above 90 degrees C.     

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.     

SAV 1, a temperate u.v.-inducible DNA virus-like particle from the archaebacterium Sulfolobus acidocaldarius isolate B12

Sulfolobus acidocaldarius, strain B12, which harbours a double-stranded DNA species both as a plasmid and in a linear form, which is integrated at a specific site of the chromosome, produces virus-like particles upon u.v. irradiation. These particles contain the same circular DNA and a number of coat proteins and are probably surrounded by a lipid membrane. They are lemon shaped, 100 x 60 nm in size and carry tail structures at one pole. The host cell recovers and remains lysogenic after virus production. Though a large fraction of liberated particles is found attached to structures derived from the cells, neither adsorption nor infection of a number of Sulfolobus isolates has so far been observed.     

The genome of the thermoacidophilic archaebacterium Sulfolobus acidocaldarius does not contain repetitive sequences

Characteristics of genome organization in the sulfur-dependent thermoacidophilic archaebacterium Sulfolobus acidocaldarius have been studied. By means of hybridization analysis it is shown that the genome of S. acidocaldarius, unlike the genome of the extremely halophilic archaebacterium Halobacterium halobium, does not contain repetitive sequences.