Small Abundant DNA Binding Proteins from the Thermoacidophilic Archaeon Sulfolobus shibatae Constrain Negative DNA Supercoils

Major DNA binding proteins, designated Ssh7, were purified from the thermoacidophilic archaeon Sulfolobus shibatae. The Ssh7 proteins have an apparent molecular mass of 6.5 kDa and are similar to the 7-kDa DNA binding proteins from Sulfolobus acidocaldarius and Sulfolobus solfataricus in N-terminal amino acid sequence. The proteins constitute about 4.8% of the cellular protein. Upon binding to DNA, the Ssh7 proteins constrain negative supercoils. At the tested Ssh7/DNA mass ratios (0 to 1.65), one negative supercoil was taken up by approximately 20 Ssh7 molecules. Our results, together with the observation that the viral DNA isolated from S. shibatae is relaxed, suggest that regions of free DNA in the S. shibatae genome, if present, are highly positively supercoiled.     

Enzymatic characterization and substrate specificity of thermostable beta-glycosidase from hyperthermophilic archaea, Sulfolobus shibatae, expressed in E. coli

Enzymatic properties and substrate specificity of recombinant beta-glycosidases from a hyperthermophilic archaeon, Sulfolobus shibatae (rSSG), were analyzed. rSSG showed its optimum temperature and pH at 95 degrees C and pH 5.0, respectively. Thermal inactivation of rSSG showed that its half-life of enzymatic activity at 75 degrees C was 15 h whereas it drastically decreased to 3.9 min at 95 degrees C. The addition of 10 mM of MnCl2 enhanced the hydrolysis activity of rSSG up to 23% whereas most metal ions did not show any considerable effect. Dithiothreitol (DTT) and 2-mercaptoethanol exhibited significant influence on the increase of the hydrolysis activity of rSSG. rSSG apparently preferred laminaribiose (beta1-->3Glc), followed by sophorose (beta1-->2Glc), gentiobiose (beta1-->6Glc), and cellobiose (beta1--4Glc). Various intermolecular transfer products were formed by rSSG in the lactose reaction, indicating that rSSG prefers lactose as a good acceptor as well as a donor. The strong intermolecular transglycosylation activity of rSSG can be applied in making functional oligosaccharides.     

Purification and characterization of an intracellular chymotrypsin-like serine protease from Thermoplasma volcanium

An intracellular serine protease produced by Thermoplasma (Tp.) volcanium was purified using a combination of ammonium sulfate fractionation, ion exchange, and alpha-casein agarose affinity chromatography. This enzyme exhibited the highest activity and stability at pH 7.0, and at 50 degrees C. The purifed enzyme hydrolyzed synthetic peptides preferentially at the carboxy terminus of phenylalanine or leucine and was almost completely inhibited by PMSF, TPCK, and chymostatin, similarly to a chymotrypsin-like serine protease. Kinetic analysis of the Tp. volcanium protease reaction performed using N-succinyl-L-phenylalanine-p-nitroanilide as substrate revealed a Km value of 2.2 mM and a Vmax value of 0.045 micromol(-1) ml(-1) min(-1). Peptide hydrolyzing activity was enhanced by >2-fold in the presence of Ca2+ and Mg2+ at 2-12 mM concentration. The serine protease is a monomer with a molecular weight of 42 kDa as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and zymogram activity staining.     

Cloning and characterization of glycogen-debranching enzyme from hyperthermophilic archaeon Sulfolobus shibatae

A gene encoding a putative glycogen-debranching enzyme in Sulfolobus shibatae (abbreviated as SSGDE) was cloned and expressed in Escherichia coli. The recombinant enzyme was purified to homogeneity by heat treatment and Ni-NTA affinity chromatography. The recombinant SSGDE was extremely thermostable, with an optimal temperature at 85 degrees C. The enzyme had an optimum pH of 5.5 and was highly stable from pH 4.5 to 6.5. The substrate specificity of SSGDE suggested that it possesses characteristics of both amylo-1,6-glucosidase and alpha-1,4-glucanotransferase. SSGDE clearly hydrolyzed pullulan to maltotrlose, and 6-O-alpha-maltosyl-beta-cyclodextrin (G2-beta-CD) to maltose and beta-cyclodextrin. At the same time, SSGDE transferred maltooligosyl residues to the maltooligosaccharides employed, and maltosyl residues to G2-beta-CD. The enzyme preferentially hydrolyzed amylopectin, followed in a decreasing order by glycogen, pullulan, and amylose. Therefore, the present results suggest that the glycogen-debranching enzyme from S. shibatae may have industrial application for the efficient debranching and modification of starch to dextrins at a high temperature.     

Evolutionary conservation and DNA binding properties of the Ssh7 proteins from Sulfolobus shibatae

The thermoacidophilic archaeon Sulfolobus shibatae synthesizes a large amount of the 7-ku DMA binding proteins known as Ssh7. Our hybridization experiments showed that two Ssh7-encoding genes existed in the genome of S. shibatae. These two genes, designated ssh7a and ssh7b, have been cloned, sequenced and expressed in Escherichia coli. The two Ssh7 proteins differ only at three amino acid positions. In addition, the cis-regulatory sequences of the ssh7a and ssh7b genes are highly conserved. These results suggest the presence of a selective pressure to maintain not only the sequence but also the expression of the two genes. We have also found that there are two genes encoding the 7-ku protein in Sulfolobus solfataricus. Based on this and other studies, we suggest that the gene encoding the 7-ku protein underwent duplication before the separation of Sulfolobus species. Binding of native Ssh7 and recombinant (r)Ssh7 to short duplex DNA fragments was analyzed by electrophoretic mobility shift assays. Both n     

Isolation and characterization of novel neutral glycolipids from Thermoplasma acidophilum.

Several novel neutral glycolipids (GL-1a, GL-1b, GL-2a, GL-2b and GL-2c) were isolated from Thermoplasma acidophilum by high-performance liquid chromatography using phenylboronic acid-silica and preparative thin-layer chromatography. The tentative structures of these lipids were characterized by the combination of gas-liquid chromatography, the methylation procedure, and (1)H-NMR and FAB-mass spectrometries. The lipophilic portion of the neutral glycolipids was composed of a simple molecular species named caldarchaeol (dibiphytanyl-diglycerol tetraether). The sugar moieties of these glycolipids were composed of gulose and glucose which formed monosaccharide residues on one side or both sides of the core lipids. Gulose was attached to the terminal glycerol OH group of the core lipid with a beta-configuration and glucose being attached with an alpha-configuration. The proposed structure of GL-1a was gulosylcaldarchaeol and that of GL-1b was glucosylcaldarchaeol. The structures of GL-2a, GL-2b, and GL-2c were the analogs of the caldarchaeol derivatives attached by a variety of gulosyl residues or glucosyl residues on both sides of the terminal OH groups.     

The stability of the archaeal HU histone-like DNA-binding protein from Thermoplasma volcanium

The complete genome analysis of the archaeon Thermoplasma volcanium has revealed a gene assigned to encode the histone-like DNA-binding protein HU. Thermoplasma volcanium is a moderate thermophile growing around 60°C and it is adaptable to aerobic and anaerobic environment and therefore it is unique as a candidate for the origin of eukaryotic nuclei in the endosymbiosis hypothesis. The HU protein is the major component of the bacterial nuclei and therefore it is an important protein to be studied. The gene for HUTvo protein (huptvo) was cloned from the genomic DNA of T. volcanium and overexpressed in Escherichia coli. A fast and efficient purification scheme was established to produce an adequate amount of bioactive protein for biochemical and biophysical studies. Highly purified HUTvo was studied for its DNA-binding activity and thermostability. As studied by circular dichroism and high-precision differential scanning microcalorimetry, the thermal unfolding of HUTvo protein is reversible and can be well described by a two-state model with dissociation of the native dimeric state into denatured monomers. The ∆G versus T profile for HUTvo compared to the hyperthermophilic marine eubacterial counterpart from Thermotoga maritima, HUTmar, clearly shows that the archaeal protein has adopted a less efficient molecular mechanism to cope with high temperature. The molecular basis of this phenomenon is discussed. F. Orfaniotou and P. Tzamalis contributed equally to this work.     

Molecular cloning and biochemical characterization of the first archaeal maltogenic amylase from the hyperthermophilic archaeon Thermoplasma volcanium GSS1

Maltogenic amylases (MAases), a subclass of cyclodextrin (CD)-hydrolyzing enzymes belonging to glycoside hydrolase family 13, have been studied extensively, but their physiological roles in microbes and evolutionary relationships with other amylolytic enzymes remain unclear. Here, we report the biochemical properties of a thermostable archaeal MAase from Thermoplasma volcanium GSS1 (TpMA) for the first time. The primary structure and catalytic properties of TpMA were similar to those of MAases, such as possession of an extra domain at its N-terminal and preference for CD over starch. TpMA showed high thermostability and optimal activity at 75 degrees C and 80 degrees C for beta-CD and soluble starch, respectively. The recombinant TpMA exists as a high oligomer in a solution and the oligomeric TpMA was dissociated into dimer and monomer mixture by a high concentration of NaCl. The substrate preference and thermostability of TpMA were significantly dependent on the oligomeric state of the enzyme. However, TpMA exhibited distinguishable characteristics from those of bacterial MAases. The transglycosylation pattern of TpMA was opposite to that of bacterial MAases. TpMA formed more alpha-1,4-glycosidic linked transfer product than alpha-1,6-linked products. Like as alpha-amylases, notably, TpMA has a longer subsite structure than those of other CD-degrading enzymes. Our findings in this study suggest that TpMA, the archaeal MAase, shares characteristics of both bacterial MAases and alpha-amylases, and locates in the middle of the evolutionary process between alpha-amylases and bacterial MAases.     

A PPM-family protein phosphatase from the thermoacidophile Thermoplasma volcanium hydrolyzes protein-bound phosphotyrosine

The genomes of virtually all free-living archaeons encode one or more deduced protein-serine/threonine/tyrosine kinases belonging to the so-called eukaryotic protein kinase superfamily. However, the distribution of their cognate protein-serine/threonine/phosphatases displays a mosaic pattern. Thermoplasma volcanium is unique among the Archaea inasmuch as it is the sole archaeon whose complement of deduced phosphoprotein phosphatases includes a member of the PPM-family of protein-serine/threonine phosphatases—a family that originated in the Eucarya. A recombinant version of this protein, TvnPPM, exhibited protein-tyrosine phosphatase in addition to its predicted protein-serine/threonine phosphatase activity in vitro. TvnPPM is the fourth member of the PPM-family shown to exhibit such dual-specific capability, suggesting that the ancestral versions of this enzyme exhibited broad substrate specificity. Unlike most other archeaons, the genome of T. volcanium lacks open reading frames encoding stereotypical protein-tyrosine phosphatases. Hence, the dual-specificity of TvnPPM may account for its seemingly aberrant presence in an archaeon.     

Enzymes from Sulfolobus shibatae for the production of trehalose and glucose from starch

Enzymes that convert starch and dextrins to α,α-trehalose and glucose were found in cell homogenates of the hyperthermophilic acidophilic archaeon Sulfolobus shibatae DMS 5389. Three enzymes were purified and characterized. The first, the S. shibatae trehalosyl dextrin-forming enzyme (SsTDFE), transformed starch and dextrins to the corresponding trehalosyl derivatives with an intramolecular transglycosylation process that converted the glucosidic linkage at the reducing end from α-1,4 to α-1,1. The second, the S. shibatae trehalose-forming enzyme (SsTFE), hydrolyzed the α-1,4 linkage adjacent to the α-1,1 bond of trehalosyl dextrins, forming trehalose and lower molecular weight dextrins. These two enzymes had molecular masses of 80 kDa and 65 kDa, respectively, and showed the highest activities at pH 4.5. The apparent optimal temperature for activity was 70°C for SsTDFE and 85°C for SsTFE. The third enzyme identified was an α-glycosidase (SsαGly), which catalyzed the hydrolysis of the α-1,4 glucosidic linkages in starch and dextrins, releasing glucose in a stepwise manner from the nonreducing end of the polysaccharide chain. The enzyme had a molecular mass of 313 kDa and showed the highest activity at pH 5.5 and at 85°C. Received: October 29, 1997 / Accepted: April 29, 1998     

Type 2 isopentenyl diphosphate isomerase from a thermoacidophilic archaeon Sulfolobus shibatae.

Although isopentenyl diphosphate-dimethylallyl diphosphate isomerase is thought to be essential for archaea because they use the mevalonate pathway, its corresponding activity has not been detected in any archaea. A novel type of the enzyme, which has no sequence similarity to the known, well-studied type of enzymes, was recently reported in some bacterial strains. In this study, we describe the cloning of a gene of a homologue of the novel bacterial isomerase from a thermoacidophilic archaeon Sulfolobus shibatae. The gene was heterologously expressed in Escherichia coli, and the recombinant enzyme was purified and characterized. The thermostable archaeal enzyme is tetrameric, and requires NAD(P)H and Mg2+ for activity, similar to its bacterial homologues. Using its apoenzyme, we were able to confirm that the archaeal enzyme is strictly dependent on FMN. Moreover, we provide evidence to show that the enzyme also has NADH dehydrogenase activity although it catalyzes the isomerase reaction without consuming any detectable amount of NADH.     

Novel Medium-Chain Prenyl Diphosphate Synthase from the Thermoacidophilic Archaeon Sulfolobus solfataricus

Two open reading frames which encode the homologues of (all-E) prenyl diphosphate synthase are found in the whole-genome sequence of Sulfolobus solfataricus, a thermoacidophilic archaeon. It has been suggested that one is a geranylgeranyl diphosphate synthase gene, but the specificity and biological significance of the enzyme encoded by the other have remained unclear. Thus, we isolated the latter by the PCR method, expressed the enzyme in Escherichia coli cells, purified it, and characterized it. The archaeal enzyme, 281 amino acids long, is highly thermostable and requires Mg(2+) and Triton X-100 for full activity. It catalyzes consecutive E-type condensations of isopentenyl diphosphate with an allylic substrate such as geranylgeranyl diphosphate and yields the medium-chain product hexaprenyl diphosphate. Despite such product specificity, phylogenetic analysis revealed that the archaeal medium-chain prenyl diphosphate synthase is distantly related to the other medium- and long-chain enzymes but is closely related to eucaryal short-chain enzymes.     

Analysis of DNA cleavage by reverse gyrase from Sulfolobus shibatae B12.

Reverse gyrase is a type I-5' topoisomerase, which catalyzes a positive DNA supercoiling reaction in vitro. To ascertain how this reaction takes places, we looked at the DNA sequences recognized by reverse gyrase. We used linear DNA fragments of its preferred substrate, the viral SSV1 DNA, which has been shown to be positively supercoiled in vivo. The Sulfolobus shibatae B12 strain, an SSV1 virus host, was chosen for production of reverse gyrase. This naturally occurring system (SSV1 DNA-S. shibatae reverse gyrase) allowed us to determine which SSV1 DNA sequences are bound and cleaved by the enzyme with particularly high selectivity. We show that the presence of ATP decreases the number of cleaved complexes obtained whereas the non-hydrolyzable ATP analog adenosine 5'-[beta, gamma-imido]triphosphate increases it without changing the sequence specificity.     

Sequential polymerization of flagellin A and flagellin B into Caulobacter flagella

The mode of polymerization of two species of flagellins, flagellin A and flagellin B, in polar flagella of Caulobacter crescentus was examined. By immunological staining we found that 1 to 1.2 μm of the portion of the flagellar filament proximal to the cell was composed of flagellin B, whereas about 5 μm of the distal portion was composed of flagellin A. This result, together with the previous observation that a flagellin B-less mutant cannot form normal flagella but instead forms stubs in spite of their high level of flagellin A synthesis, indicates that flagellin B is very important for the formation of complete flagella and/or for the initiation of filament formation from the hook.     

Three conformations of an archaeal chaperonin, TF55 from Sulfolobus shibatae

Chaperonins are cylindrical, oligomeric complexes, essential for viability and required for the folding of other proteins. The GroE (group I) subfamily, found in eubacteria, mitochondria and chloroplasts, have 7-fold symmetry and provide an enclosed chamber for protein subunit folding. The central cavity is transiently closed by interaction with the co-protein, GroES. The most prominent feature specific to the group II subfamily, found in archaea and in the eukaryotic cytosol, is a long insertion in the substrate-binding region. In the archaeal complex, this forms an extended structure acting as a built-in lid, obviating the need for a GroES-like co-factor. This extension occludes a site known to bind non-native polypeptides in GroEL. The site and nature of substrate interaction are not known for the group II subfamily. The atomic structure of the thermosome, an archaeal group II chaperonin, has been determined in a fully closed form, but the entry and exit of protein substrates requires transient opening. Although an open form has been investigated by electron microscopy, conformational changes in group II chaperonins are not well characterized. Using electron cryo-microscopy and three-dimensional reconstruction, we describe three conformations of a group II chaperonin, including an asymmetric, bullet-shaped form, revealing the range of domain movements in this subfamily.     

Partial Purification and Characterization of Thermostable Acid Phosphatase from Thermoacidophilic Archaeon Sulfolobus acidocaldarius

Thermostable acid phosphatase (APase) from thermoacidophilic archaeon Sulfolobus acidocaldarius was isolated, partially purified, and characterized. The optimum pH and temperature of the enzyme for p-nitrophenylphosphate (pNPP) as a substrate were 5.0 and 70°C, respectively. The apparent K _m value was 1.9 mM. This APase showed a native molecular mass of 20 kDa on a gel filtration chromatography. Of the APase activity, 60% remained after 60 min of heat treatment at 75°C. To confirm whether the APase is active in the monomeric form, we attempted to elute the enzyme from SDS-polyacrylamide gels with Disk electrophoresis apparatus and renature the enzyme. The APase activity was recovered up to 50% in the 14- to 35-kDa range, and maximum around 25 kDa. These results suggest that this APase is monomeric protein. Received: 8 July 1999 / Accepted: 9 August 1999     

The flagellar filament structure of the extreme acidothermophile Sulfolobus shibatae B12 suggests that archaeabacterial flagella have a unique and common symmetry and design

Archaea, constituting a third domain of life between Eubacteria and Eukarya, characteristically inhabit extreme environments. They swim by rotating flagellar filaments that are phenomenologically and functionally similar to those of eubacteria. However, biochemical, genetic and structural evidence has pointed to significant differences but even greater similarity to eubacterial type IV pili. Here we determined the three-dimensional symmetry and structure of the flagellar filament of the acidothermophilic archaeabacterium Sulfolobus shibatae B12 using transmission electron microscopy (TEM) and scanning transmission electron microscopy (STEM). Processing of the cryo-negatively stained filaments included analysis of their helical symmetry and subsequent single particle reconstruction. Two filament subunit packing arrangements were identified: one has helical symmetry, similar to that of the extreme halophile Halobacterium salinarum, with ten subunits per 5.3 nm repeat and the other has helically arranged stacked disks with C₃ symmetry and 12 subunits per repeat. The two structures are related by a slight twist. The S. shibatae filament has a larger diameter compared to that of H. salinarum, at the opposite end of the archaeabacterial phylogenetic spectrum, but the basic three-start symmetry and the size and arrangement of the core domain are conserved and the filament lacks a central channel. This similarity suggests a unique and common underlying symmetry for archaeabacterial flagellar filaments.