Viruses of the extremely thermophilic archaeon Sulfolobus

Viruses of Sulfolobus are highly unusual in their morphology, and genome structure and sequence. Certain characteristics of the replication strategies of these viruses and the virus-host interactions suggest relationships with eukaryal and bacterial viruses, and the primeval existence of common ancestors. Moreover, studying these viruses led to the discovery of archaeal promoters and has provided tools for the development of the molecular genetics of these organisms. The Sulfolobus viruses contain unique regulatory features and structures that undoubtedly hold surprises for researchers in the future.     

Genetic elements in the extremely thermophilic archaeon Sulfolobus

This minireview summarizes what is known about genetic elements in the archaeal crenarchaeotal genus Sulfolobus, including recent work on viruses, cryptic plasmids, a novel type of virus satellite plasmids or satellite viruses, and conjugative plasmids (CPs), mostly from our laboratory. It does not discuss IS elements and transposons. Received: January 22, 1998 / Accepted: February 16, 1998     

Genomic comparison of archaeal conjugative plasmids from Sulfolobus

All of the known self-transmissable plasmids of the Archaea have been found in the genus Sulfolobus. To gain more insight into archaeal conjugative processes, four newly isolated self-transmissable plasmids, pKEF9, pHVE14, pARN3 and pARN4, were sequenced and subjected to a comparative sequence analysis with two earlier sequenced plasmids, pNOB8 and pING1. The analyses revealed three conserved and functionally distinct sections in the genomes. Section A is considered to encode the main components of the conjugative apparatus, where two genes show low but significant sequence similarity to sections of genes encoding bacterial conjugative proteins. A putative origin of replication is located in section B, which is highly conserved in sequence and contains several perfect and imperfect direct and inverted repeats. Further downstream, in section C, an operon encoding six to nine smaller proteins is implicated in the initiation and regulation of replication. Each plasmid carries an integrase gene of the type that does not partition on integration, and there is strong evidence for their integration into host chromosomes, where they may facilitate intercellular exchange of chromosomal genes. Two plasmids contain hexameric short regularly spaced repeats (SRSR), which have been implicated in plasmid maintenance, and each plasmid carries multiple recombination motifs, concentrated in the variable regions, which likely provide sites for genomic rearrangements.     

Folding and association of an extremely stable dimeric protein from Sulfolobus islandicus

ORF56 is a plasmid-encoded protein from Sulfolobus islandicus, which probably controls the copy number of the pRN1 plasmid by binding to its own promotor. The protein showed an extremely high stability in denaturant, heat, and pH-induced unfolding transitions, which can be well described by a two-state reaction between native dimers and unfolded monomers. The homodimeric character of native ORF56 was confirmed by analytical ultracentrifugation. Far-UV circular dichroism and fluorescence spectroscopy gave superimposable denaturant-induced unfolding transitions and the midpoints of both heat as well as denaturant-induced unfolding depend on the protein concentration supporting the two-state model. This model was confirmed by GdmSCN-induced unfolding monitored by heteronuclear 2D NMR spectroscopy. Chemical denaturation was accomplished by GdmCl and GdmSCN, revealing a Gibbs free energy of stabilization of -85.1 kJ/mol at 25 degrees C. Thermal unfolding was possible only above 1 M GdmCl, which shifted the melting temperature (t(m)) below the boiling point of water. Linear extrapolation of t(m) to 0 M GdmCl yielded a t(m) of 107.5 degrees C (5 microM monomer concentration). Additionally, ORF56 remains natively structured over a remarkable pH range from pH 2 to pH 12. Folding kinetics were followed by far-UV CD and fluorescence after either stopped-flow or manual mixing. All kinetic traces showed only a single phase and the two probes revealed coincident folding rates (k(f), k(u)), indicating the absence of intermediates. Apparent first-order refolding rates depend linearly on the protein concentration, whereas the unfolding rates do not. Both lnk(f) and lnk(u) depend linearly on the GdmCl concentration. Together, folding and association of homodimeric ORF56 are concurrent events. In the absence of denaturant ORF56 refolds fast (7.0 x 10(7)M(-1)s(-1)) and unfolds extremely slowly (5.7 year(-1)). Therefore, high stability is coupled to a slow unfolding rate, which is often observed for proteins of extremophilic organisms.     

Recombination shapes the natural population structure of the hyperthermophilic archaeon Sulfolobus islandicus

Although microorganisms make up the preponderance of the biodiversity on Earth, the ecological and evolutionary factors that structure microbial populations are not well understood. We investigated the genetic structure of a thermoacidophilic crenarchaeal species, Sulfolobus islandicus, using multilocus sequence analysis of six variable protein-coding loci on a set of 60 isolates from the Mutnovsky region of Kamchatka, Russia. We demonstrate significant incongruence among gene genealogies and a lack of association between alleles consistent with recombination rates greater than the rate of mutation. The observation of high relative rates of recombination suggests that the structure of this natural population does not fit the periodic selection model often used to describe populations of asexual microorganisms. We propose instead that frequent recombination among closely related individuals prevents periodic selection from purging diversity and provides a fundamental cohesive mechanism within this and perhaps other archaeal species.     

Genetic profile of pNOB8 from Sulfolobus: the first conjugative plasmid from an archaeon

The complete nucleotide sequence of the archaeal conjugative plasmid, pNOB8, from the Sulfolobus isolate NOB8-H2, was determined. The plasmid is 41 229 bp in size and contains about 50 ORFs. Several direct sequence repeats are present, the largest of which is a perfect 85-bp repeat and a site of intraplasmid recombination in foreign Sulfolobus hosts. This recombination event produces a major deletion variant, pNOB8-33, which is not stably maintained. Less than 20% of the ORFs could be assigned putative functions after extensive database searches. Tandem ORFs 315 and 470, within the deleted 8-kb region, show significant sequence similarity to the protein superfamilies of ParA (whole protein) and ParB (N-terminal half), respectively, that are important for plasmid and chromosome partitioning in bacteria. A putative cis-acting element is also present that exhibits six 24-mer repeats containing palindromic sequences which are separated by 39 or 42 bp. By analogy with bacterial systems, this element may confer plasmid incompatibility and define a group of incompatible plasmids in Archaea. Although several ORFs can form putative trans-membrane or membrane-binding segments, only two ORFs show significant sequence similarity to bacterial conjugative proteins. ORF630b aligns with the TrbE protein superfamily, which contributes to mating pair formation in Bacteria, while ORF1025 aligns with the TraG protein superfamily. We infer that the conjugative mechanism for Sulfolobus differs considerably from known bacterial mechanisms. Finally, two transposases were detected; ORF413 is flanked by an imperfect 32-bp inverted repeat with a 5-bp direct repeat at the ends, and ORF406 is very similar in sequence to an insertion element identified in the Sulfolobus solfataricus P2 genome. Received: March 10, 1998 / Accepted: May 2, 1998     

Characterization of the maltooligosyl trehalose synthase from the thermophilic archaeon Sulfolobus acidocaldarius

We report the molecular characterization and the detailed study of the recombinant maltooligosyl trehalose synthase mechanism from the thermoacidophilic archaeon Sulfolobus acidocaldarius. The mts gene encoding a maltooligosyl trehalose synthase was overexpressed in Escherichia coli using the T7-expression system. The purified recombinant enzyme exhibited optimum activity at 75 degrees C and pH 5 with citrate-phosphate buffer and retained 60% of residual activity after 72 h of incubation at 80 degrees C. The recombinant enzyme was active on maltooligosaccharides such as maltotriose, maltotetraose, maltopentaose and maltoheptaose. Investigation of the enzyme action on maltooligosaccharides has brought much insight into the reaction mechanism. Results obtained from thin-layer chromatography suggested a possible mechanism of action for maltooligosyl trehalose synthase: the enzyme, after converting the alpha-1,4-glucosidic linkage to an alpha-1,1-glucosidic linkage at the reducing end of maltooligosaccharide glc(n) is able to release glucose and maltooligosaccharide glc(n-1) residues. And then, the intramolecular transglycosylation and the hydrolytic reaction continue, with the maltooligosaccharide glc(n-1) until the initial maltooligosaccharide is reduced to maltose. An hypothetical mechanism of maltooligosyl trehalose synthase acting on maltooligosaccharide is proposed.     

A multicopy plasmid of the extremely thermophilic archaeon Sulfolobus effects its transfer to recipients by mating.

A plasmid of 45 kb, designated pNOB8, was found in high copy number in a new heterotrophic Sulfolobus isolate, NOB8H2, from Japan. Dissemination of the plasmid occurred in six cultures of nine different Sulfolobus strains when small amounts of the donor were added. These mixed cultures exhibited a high average copy number of the plasmid, between 20 and 40 per chromosome, and showed a marked growth retardation. Horizontal transfer of pNOB8 was proved by isolating transcipients from mating mixtures via single colonies. In these isolates, the copy number of the plasmid appeared to be subject to a control mechanism. Cell-free filtrates of donor cultures did not transmit the plasmid, and plating of the donor on lawns of recipients did not result in plaque formation, suggesting that the transfer was not mediated by a virus. Rapid formation of cell-to-cell contacts between differently stained donor and recipient partners was demonstrated after the two strains were mixed. Electron microscopic analysis of mating mixtures revealed many cell aggregates made up of 2 to 30 cells and intercellular cytoplasmic bridges connecting two or more cells. Cells that had been transformed with purified plasmid DNA as well as transcipients isolated from mating mixtures were shown to serve as donors for further transmission of pNOB8. The plasmid undergoes extensive genetic variations, since deletions and insertions were frequently observed in plasmid preparations from the donor strain and from mating mixtures.     

Biochemical and structural studies of a l-haloacid dehalogenase from the thermophilic archaeon Sulfolobus tokodaii

Haloacid dehalogenases have potential applications in the pharmaceutical and fine chemical industry as well as in the remediation of contaminated land. The l-2-haloacid dehalogenase from the thermophilic archaeon Sulfolobus tokodaii has been cloned and over-expressed in Escherichia coli and successfully purified to homogeneity. Here we report the structure of the recombinant dehalogenase solved by molecular replacement in two different crystal forms. The enzyme is a homodimer with each monomer being composed of a core-domain of a β-sheet bundle surrounded by α-helices and an α-helical sub-domain. This fold is similar to previously solved mesophilic l-haloacid dehalogenase structures. The monoclinic crystal form contains a putative inhibitor l-lactate in the active site. The enzyme displays haloacid dehalogenase activity towards carboxylic acids with the halide attached at the C2 position with the highest activity towards chloropropionic acid. The enzyme is thermostable with maximum activity at 60°C and a half-life of over 1 h at 70°C. The enzyme is relatively stable to solvents with 25% activity lost when incubated for 1 h in 20% v/v DMSO.     

Crystal structure of a thermophilic cytochrome P450 from the archaeon Sulfolobus solfataricus

The structure of the first P450 identified in Archaea, CYP119 from Sulfolobus solfataricus, has been solved in two different crystal forms that differ by the ligand (imidazole or 4-phenylimidazole) coordinated to the heme iron. A comparison of the two structures reveals an unprecedented rearrangement of the active site to adapt to the different size and shape of ligands bound to the heme iron. These changes involve unraveling of the F helix C-terminal segment to extend a loop structure connecting the F and G helices, allowing the longer loop to dip down into the active site and interact with the smaller imidazole ligand. A comparison of CYP119 with P450cam and P450eryF indicates an extensive clustering of aromatic residues may provide the structural basis for the enhanced thermal stability of CYP119. An additional feature of the 4-phenylimidazole-bound structure is a zinc ion tetrahedrally bound by symmetry-related His and Glu residues.     

Tailoring the substrate specificity of the beta-glycosidase from the thermophilic archaeon Sulfolobus solfataricus.

The substrate specificity of the thermophilic beta-glycosidase (lacS) from the archaeon Sulfolobus solfataricus (SSbetaG), a member of the glycohydrolase family 1, has been analysed at a molecular level using predictions from known protein sequences and structures and through site-directed mutagenesis. Three critical residues were identified and mutated to create catalysts with altered and broadened specificities for use in glycoside synthesis. The wild-type (WT) and mutated sequences were expressed as recombinant fusion proteins in Escherichia coli, with an added His(6)-tag to allow one-step chromatographic purification. Consistent with side-chain orientation towards OH-6, the single Met439-->Cys mutation enhances D-xylosidase specificity 4.7-fold and decreases D-fucosidase activity 2-fold without greatly altering its activity towards other D-glycoside substrates. Glu432-->Cys and Trp433-->Cys mutations directed towards OH-4 and -3, respectively, more dramatically impair glucose (Glc), galactose (Gal), fucose specificity than for other glycosides, resulting in two glycosidases with greatly broadened substrate specificities. These include the first examples of stereospecificity tailoring in glycosidases (e.g. WT-->W433C, k(cat)/K(M) (Gal):k(cat)/K(M) (mannose (Man))=29.4:1-->1.2:1). The robustness and high utility of these broad specificity SSbetaG mutants in parallel synthesis were demonstrated by the formation of libraries of beta-glycosides of Glc, Gal, xylose, Man in one-pot preparations at 50 degrees C in the presence of organic solvents, that could not be performed by SSbetaG-WT.     

Gene content and organization of a 281-kbp contig from the genome of the extremely thermophilic archaeon, Sulfolobus solfataricus P2.

The sequence of a 281-kbp contig from the crenarchaeote Sulfolobus solfataricus P2 was determined and analysed. Notable features in this region include 29 ribosomal protein genes, 12 tRNA genes (four of which contain archaeal-type introns), operons encoding enzymes of histidine biosynthesis, pyrimidine biosynthesis, and arginine biosynthesis, an ATPase operon, numerous genes for enzymes of lipopolysaccharide biosynthesis, and six insertion sequences. The content and organization of this contig are compared with sequences from crenarchaeotes, euryarchaeotes, bacteria, and eukaryotes.     

Cis-acting signals controlling translational initiation in the thermophilic archaeon Sulfolobus solfataricus

In this work, we have studied the in vitro translational features of a bicistronic mRNA of the extremely thermophilic Archaeon Sulfolobus solfataricus, with the aim of determining the nature of the cis-acting signals controlling the recognition of the translation initiation sites in the Archaea. We found that the most important feature for efficient initiation was the presence of a Shine-Dalgarno (SD)-like ribosome-binding motif, whose disruption entirely abolished the translation of the corresponding cistron. The influence of other features, such as the type of initiation codon, was variable and depended upon the gene and its position in the mRNA. However, the translational block caused by the disruption of the SD sequences could be removed by deleting the 5' untranslated region altogether, thereby creating a 'leaderless' mRNA. This suggests that 'leaderless' initiation operates by a default mechanism that does not require a specific mRNA-rRNA interaction and may be common to all three primary domains of life.     

Genomics and genetics of Sulfolobus islandicus LAL14/1, a model hyperthermophilic archaeon

The 2 465 177 bp genome of Sulfolobus islandicus LAL14/1, host of the model rudivirus SIRV2, was sequenced. Exhaustive comparative genomic analysis of S. islandicus LAL14/1 and the nine other completely sequenced S. islandicus strains isolated from Iceland, Russia and USA revealed a highly syntenic common core genome of approximately 2 Mb and a long hyperplastic region containing most of the strain-specific genes. In LAL14/1, the latter region is enriched in insertion sequences, CRISPR (clustered regularly interspaced short palindromic repeats), glycosyl transferase genes, toxin–antitoxin genes and MITE (miniature inverted-repeat transposable elements). The tRNA genes of LAL14/1 are preferential targets for the integration of mobile elements but clusters of atypical genes (CAG) are also integrated elsewhere in the genome. LAL14/1 carries five CRISPR loci with 10 per cent of spacers matching perfectly or imperfectly the genomes of archaeal viruses and plasmids found in the Icelandic hot springs. Strikingly, the CRISPR_2 region of LAL14/1 carries an unusually long 1.9 kb spacer interspersed between two repeat regions and displays a high similarity to pING1-like conjugative plasmids. Finally, we have developed a genetic system for S. islandicus LAL14/1 and created ΔpyrEF and ΔCRISPR_1 mutants using double cross-over and pop-in/pop-out approaches, respectively. Thus, LAL14/1 is a promising model to study virus–host interactions and the CRISPR/Cas defence mechanism in Archaea.     

Purification and characterization of a maltase from the extremely thermophilic crenarchaeote Sulfolobus solfataricus.

A soluble maltase (alpha-glucosidase) with an apparent subunit mass of 80 kDa was purified to homogeneity from Sulfolobus solfataricus. The enzyme liberates glucose from maltose and malto-oligomers. Maximal activity was observed at 105 degrees C, with half-lives of 11 h (85 degrees C), 3.0 h (95 degrees C), and 2.75 h (100 degrees C). The enzyme was generally resistant to proteolysis and denaturants including aliphatic alcohols. n-Propanol treatment at 85 degrees C increased both Km and Vmax for maltose hydrolysis.     

Expression and characterization of the HMG-CoA reductase of the thermophilic archaeon Sulfolobus solfataricus

The thermostable class I HMG-CoA reductase of Sulfolobus solfataricus offers potential for industrial applications and for the initiation of crystallization trials of a biosynthetic HMG-CoA reductase. However, of the 15 arginine codons of the hmgA gene that encodes S. solfataricus HMG-CoA reductase, 14 (93%) are AGA or AGG, the arginine codons used least frequently by Escherichia coli. The presence of these rare codons in tandem or in the first 20 codons of a gene can complicate expression of that gene in E. coli. Problems include premature chain termination and misincorporation of lysine for arginine. We therefore sought to improve the expression and subsequent yield of S. solfataricus HMG-CoA reductase by expanding the pool size of tRNA(AGA,AGG), the tRNA that recognizes these two rare codons. Coexpression of the S. solfataricus hmgA gene with the argU gene that encodes tRNA(AGA,AGG) resulted in an over 10-fold increase in enzyme yield. This has provided significantly greater quantities of purified enzyme for potential industrial applications and for crystallographic characterization of a stable class I HMG-CoA reductase. It has, in addition, facilitated determination of kinetic parameters and of pH optima for all four catalyzed reactions, for determination of the K(i) for inhibition by the statin drug mevinolin, and for comparison of the properties of the HMG-CoA reductase of this thermophilic archaeon to those of other class I HMG-CoA reductases.     

Expression, purification, and characterization of recombinant S-adenosylhomocysteine hydrolase from the thermophilic archaeon Sulfolobus solfataricus

S-Adenosylhomocysteine hydrolase from Sulfolobus solfataricus was expressed in Escherichia coli by inserting the genomic fragment containing the gene encoding for S-adenosylhomocysteine hydrolase downstream the isopropyl-beta-d-thiogalactoside-inducible promoter of pTrc99A expression vector. An ATG positioned 25 bp upstream of the gene which is in frame with a stop codon was utilized as the initiation codon. This construct was used to transform E. coli RB791 and E. coli JM105 strains. The recombinant protein, purified by a fast and efficient two-step procedure (yield of 0.4 mg of enzyme per gram of cells), does not appear homogeneous on SDS-PAGE because of the presence of a protein contaminant corresponding to a "truncated" S-adenosylhomocysteine hydrolase subunit lacking the first 24 amino acid residues. The recombinant enzyme shows the same molecular mass, optimum temperature, and kinetic features of S-adenosylhomocysteine hydrolase isolated from S. solfataricus but it is less thermostable. To construct a vector which presents a correct distance between the ribosome-binding site and the start codon of S-adenosylhomocysteine hydrolase gene, a NcoI site was created at the translation initiation codon using site-directed mutagenesis. The expression of the homogeneous mutant S-adenosylhomocysteine hydrolase was achieved at high level (1.7 mg of mutant protein per gram of cells). The mutant S-adenosylhomocysteine hydrolase and the native one were indistinguishable in all physicochemical and kinetic properties including thermostability, indicating that the interactions involving the NH(2)-terminal sequence of the protein play a role in the thermal stability of S. solfataricus S-adenosylhomocysteine hydrolase.     

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 synthetic arabinose-inducible promoter confers high levels of recombinant protein expression in hyperthermophilic archaeon Sulfolobus islandicus

Despite major progresses in genetic studies of hyperthermophilic archaea, recombinant protein production in these organisms always suffers from low yields and a robust expression system is still in great demand. Here we report a versatile vector that confers high levels of protein expression in Sulfolobus islandicus, a hyperthermophilic crenarchaeon. Two expression vectors, pSeSD and pEXA, harboring 11 unique restriction sites were constructed. They contain coding sequences of two hexahistidine (6×His) peptide tags and those coding for two protease sites, the latter of which make it possible to remove the peptide tags from expressed recombinant proteins. While pEXA employed an araS promoter for protein expression, pSeSD utilized P(araS-SD), an araS derivative promoter carrying an engineered ribosome-binding site (RBS; a Shine-Dalgarno [SD] sequence). We found that P(araS-SD) directed high levels of target gene expression. More strikingly, N-terminal amino acid sequencing of recombinant proteins unraveled that the protein synthesized from pEXA-N-lacS lacked the designed 6×His tag and that translation initiation did not start at the ATG codon of the fusion gene. Instead, it started at multiple sites downstream of the 6×His codons. Intriguingly, inserting an RBS site upstream of the ATG codon regained the expression of the 6×His tag, as shown with pSeSD-N-lacS. These results have yielded novel insight into the archaeal translation mechanism. The crenarchaeon Sulfolobus can utilize N-terminal coding sequences of proteins to specify translation initiation in the absence of an RBS site.