Molecular biology of fuselloviruses and their satellites.

Fuselloviruses, also known as Sulfolobus Spindle-shaped viruses (SSVs), are "lemon"- or "spindle"-shaped double-stranded DNA viruses. Among them, SSV1, SSV2 and the satellite viruses pSSVx and pSSVi have been investigated at the structural, genetic, transcriptomic, proteomic and biochemical levels, thus becoming models for dissecting DNA replication/gene expression in Archaea. Important progress has been made including elucidation of temporal genome expression during virus infection and induction of replication, SSV1 lysogeny maintenance as well as differentially expression of pSSVx replicase. Future researches focusing on these model systems would yield insightful knowledge of life cycle and DNA replication of fuselloviruses.

Cellulose degradation by Sulfolobus solfataricus requires a cell-anchored endo-ß-1-4-glucanase.

A sequence encoding a putative extracellular endoglucanase (sso1354) was identified in the complete genome sequence of Sulfolobus solfataricus. The encoded protein shares signature motifs with members of glycoside hydrolases family 12. After an unsuccessful first attempt at cloning the full-length coding sequences in Escherichia coli, an active but unstable recombinant enzyme lacking a 27-residue N-terminal sequence was generated. This 27-amino-acid sequence shows significant similarity with corresponding regions in the sugar binding proteins AraS, GlcS, and TreS of S. solfataricus that are responsible for anchoring them to the plasma membrane. A strategy based on an effective vector/host genetic system for Sulfolobus and on expression control by the promoter of the S. solfataricus gene which encodes the glucose binding protein allowed production of the enzyme in sufficient quantities for study. In fact, the enzyme expressed in S. solfataricus was stable and highly thermoresistant and showed optimal activity at low pH and high temperature. The protein was detected mainly in the plasma membrane fraction, confirming the structural similarity to the sugar binding proteins. The results of the protein expression in the two different hosts showed that the SSO1354 enzyme is endowed with an endo-ß-1-4-glucanase activity and specifically hydrolyzes cellulose. Moreover, it also shows significant but distinguishable specificity toward several other sugar polymers, such as lichenan, xylan, debranched arabinan, pachyman, and curdlan.

Biochemical properties of a novel and highly thermostable bacterial α-carbonic anhydrase from Sulfurihydrogenibium yellowstonense YO3AOP1.

A new carbonic anhydrase (CA, EC 4.2.1.1) from the thermophilic bacterium Sulfurihydrogenibium yellowstonense YO3AOP1 was identified and characterized. The bacterial carbonic anhydrase gene was expressed in Escherichia coli yielding an active enzyme, which was purified in large amounts. The recombinant protein (SspCA) was found to belong to the α-CA class and displays esterase activity. The kinetic parameters were determined by using CO(2) and p-nitrophenylacetate (p-NpA) as substrates. The bacterial enzyme presented specific activity comparable to that of bovine carbonic anhydrase (bCA II) but it showed biochemical properties never observed for the mammalian enzyme. The thermophilic enzyme, in fact, was endowed with high thermostability and with unaltered residual activity after prolonged exposure to heat up to 100°C. SspCA and the bovine carbonic anhydrase (bCA II) were immobilized within a polyurethane (PU) foam. The immobilized bacterial enzyme was found to be active and stable at 100°C up to 50 h.

Transcription termination in the plasmid/virus hybrid pSSVx from Sulfolobus islandicus.

The pSSVx from Sulfolobus islandicus, strain REY15/4, is a hybrid between a plasmid and a fusellovirus. A systematic study previously performed revealed the presence of nine major transcripts, the expression of which was differentially and temporally regulated over the growth cycle of S. islandicus. In this study, two new transcripts were identified. Then, 3' termini of all the RNAs were mapped using adaptor RT-PCR and RNase protection assays, and termination/arrest positions were identified for each transcript. The majority of the identified ending positions were located in the close vicinity of a T-rich sequence and this was consistent with termination signals identifiable for most of archaeal genes. Furthermore, termination also occurred at locations where a T-track sequence was absent but a stem-loop structure could be formed. We propose that an alternative mechanism based on secondary RNA structures and counter-transcripts might be responsible for the transcription termination at these T-track-minus loci in the closely spaced pSSVx genes.

A new archaeal beta-glycosidase from Sulfolobus solfataricus: seeding a novel retaining beta-glycan-specific glycoside hydrolase family along with the human non-lysosomal glucosylceramidase GBA2.

Carbohydrate active enzymes (CAZymes) are a large class of enzymes, which build and breakdown the complex carbohydrates of the cell. On the basis of their amino acid sequences they are classified in families and clans that show conserved catalytic mechanism, structure, and active site residues, but may vary in substrate specificity. We report here the identification and the detailed molecular characterization of a novel glycoside hydrolase encoded from the gene sso1353 of the hyperthermophilic archaeon Sulfolobus solfataricus. This enzyme hydrolyzes aryl beta-gluco- and beta-xylosides and the observation of transxylosylation reactions products demonstrates that SSO1353 operates via a retaining reaction mechanism. The catalytic nucleophile (Glu-335) was identified through trapping of the 2-deoxy-2-fluoroglucosyl enzyme intermediate and subsequent peptide mapping, while the general acid/base was identified as Asp-462 through detailed mechanistic analysis of a mutant at that position, including azide rescue experiments. SSO1353 has detectable homologs of unknown specificity among Archaea, Bacteria, and Eukarya and shows distant similarity to the non-lysosomal bile acid beta-glucosidase GBA2 also known as glucocerebrosidase. On the basis of our findings we propose that SSO1353 and its homologs are classified in a new CAZy family, named GH116, which so far includes beta-glucosidases (EC 3.2.1.21), beta-xylosidases (EC 3.2.1.37), and glucocerebrosidases (EC 3.2.1.45) as known enzyme activities.

Identification of a cell-bound extracellular protease overproduced by Sulfolobus solfataricus in peptide-rich media.

A new protease, named SsMTP was identified from the archeon Sulfolobus solfataricus. The enzyme is associated to the cell-membrane and over-produced in response to the peptide-enriched media. SsMTP has a molecular mass of 120 kDa showing optimal activity at pH 2.0 in the temperature range 70 - 90 degrees C, and a half-life of 20 days at 80 degrees C. Primary structure analysis revealed that SsMTP represents a novel type of multi-domain thermopsin-like protease containing the catalytic domain followed by two distinct domains, PKD and Y_Y_Y, which are usually involved in a range of protein-protein interactions among the extracellular proteins.

Outside the unusual cell wall of the hyperthermophilic archaeon Aeropyrum pernix K1.

In contrast to the extensively studied eukaryal and bacterial protein secretion systems, comparatively less is known about how and which proteins cross the archaeal cell membrane. To identify secreted proteins of the hyperthermophilic archaeon Aeropyrum pernix K1 we used a proteomics approach to analyze the extracellular and cell surface protein fractions. The experimentally obtained data comprising 107 proteins were compared with the in silico predicted secretome. Because of the lack of signal peptide and cellular localization prediction tools specific for archaeal species, programs trained on eukaryotic and/or Gram-positive and Gram-negative bacterial signal peptide data sets were used. PSortB Gram-negative and Gram-positive analysis predicted 21 (1.2% of total ORFs) and 24 (1.4% of total ORFs) secreted proteins, respectively, from the entire A. pernix K1 proteome, 12 of which were experimentally identified in this work. Six additional proteins were predicted to follow non-classical secretion mechanisms using SecP algorithms. According to at least one of the two PSortB predictions, 48 proteins identified in the two fractions possess an unknown localization site. In addition, more than half of the proteins do not contain signal peptides recognized by current prediction programs. This suggests that known mechanisms only partly describe archaeal protein secretion. The most striking characteristic of the secretome was the high number of transport-related proteins identified from the ATP-binding cassette (ABC), tripartite ATP-independent periplasmic, ATPase, small conductance mechanosensitive ion channel (MscS), and dicarboxylate amino acid-cation symporter transporter families. In particular, identification of 21 solute-binding receptors of the ABC superfamily of the 24 predicted in silico confirms that ABC-mediated transport represents the most frequent strategy adopted by A. pernix for solute translocation across the cell membrane.

MarR-like transcriptional regulator involved in detoxification of aromatic compounds in Sulfolobus solfataricus.

A DNA binding protein, BldR, was identified in the crenarchaeon Sulfolobus solfataricus as a protein 5- to 10-fold more abundant in cells grown in the presence of toxic aldehydes; it binds to regulatory sequences located upstream of an alcohol dehydrogenase gene (Sso2536). BldR is homologous to bacterial representatives of the MarR (multiple antibiotic resistance) family of transcriptional regulators that mediate response to multiple environmental stresses. Transcriptional analysis revealed that the bldR gene was transcribed in a bicistronic unit composed of the genes encoding the transcriptional regulator (Sso1352) and a putative multidrug transporter (Sso1351) upstream. By homology to bacterial counterparts, the bicistron was named the mar-like operon. The level of mar-like operon expression was found to be increased at least 10-fold in response to chemical stress by aromatic aldehydes. Under the same growth conditions, similar enhanced in vivo levels of Sso2536 gene transcript were also measured. The gene encoding BldR was expressed in E. coli, and the recombinant protein was purified to homogeneity. DNA binding assays demonstrated that the protein is indeed a transcription factor able to recognize site specifically both the Sso2536 and mar-like promoters at sites containing palindromic consensus sequences. Benzaldehyde, the substrate of ADH(Ss), stimulates DNA binding of BldR at both promoters. The role of BldR in the auto-activation as well as in the regulation of the Sso2536 gene, together with results of increased operon and gene expression under conditions of exposure to aromatic aldehydes, indicates a novel coordinate regulatory mechanism in cell defense against stress by aromatic compounds.

Transcriptional analysis of the genetic element pSSVx: differential and temporal regulation of gene expression reveals correlation between transcription and replication.

pSSVx from Sulfolobus islandicus strain REY15/4 is a hybrid between a plasmid and a fusellovirus. A systematic study performed by a combination of Northern blot analysis, primer extension, and reverse transcriptase PCR revealed the presence of nine major transcripts whose expression was differentially and temporally regulated over the growth cycle of S. islandicus. The map positions of the RNAs as well as the clockwise and the anticlockwise directions of their transcription were determined. Some genes were clustered and appeared to be transcribed as polycistronic messengers, among which one long transcriptional unit comprised the genes for the plasmid copy number control protein ORF60 (CopG), ORF91, and the replication protein ORF892 (RepA). We propose that a termination readthrough mechanism might be responsible for the formation of more than one RNA species from a single 5' end and therefore that the nine different RNAs corresponded to only seven different transcriptional starts. Three transcripts, ORF76 and two antisense RNAs, countertranscribed RNA1 (ctRNA1) and ctRNA2, were found to be specifically expressed during (and hence correlated to) the phase in which the pSSVx copy number is kept under stringent control, as they were completely switched off upon the onset of the induction of replication.

Gene cloning and expression in Escherichia coli of a bi-functional beta-D-xylosidase/alpha-L-arabinosidase from Sulfolobus solfataricus involved in xylan degradation.

An open reading frame encoding a putative bi-functional beta-D-xylosidase/alpha-L-arabinosidase (Sso3032) was identified on the genome sequence of Sulfolobus solfataricus P2, the predicted gene product showing high amino-acid sequence similarity to bacterial and eukaryal individual beta-D-xylosidases and alpha-L-arabinosidases as well as bi-functional enzymes such as the protein from Thermoanaerobacter ethanolicus and barley. The sequence was PCR amplified from genomic DNA of S. solfataricus P2 and heterologous gene expression obtained in Escherichia coli, under optimal conditions for overproduction. Specific assays performed at 75 degrees C revealed the presence in the transformed E. coli cell extracts of this archaeal activity involved in sugar hydrolysis and specific for both substrates. The recombinant protein was purified by thermal precipitation of the host proteins and ethanol fractionation and other properties, such as high thermal activity and thermostability could be determined. The protein showed a homo-tetrameric structure with a subunit of molecular mass of 82.0 kDa which was in perfect agreement with that deduced from the cloned gene. Northern blot analysis of the xarS gene indicates that it is specifically induced by xylan and repressed by monosaccharides like D-glucose and L-arabinose.

A spreadable, non-integrative and high copy number shuttle vector for Sulfolobus solfataricus based on the genetic element pSSVx from Sulfolobus islandicus.

The pSSVx genetic element from Sulfolobus islandicus REY15/4 is a hybrid between a plasmid and a fusellovirus, able to be maintained in non-integrative form and to spread when the helper SSV2 virus is present in the cells. In this work, the satellite virus was engineered to obtain an Escherichia coli-Sulfolobus solfataricus shuttle vector for gene transfer and expression in S.solfataricus by fusing site-specifically the pSSVx chromosome with an E.coli plasmid replicon and the ampicillin resistance gene. The pSSVx-based vector was proven functional like the parental virus, namely it was able to spread efficiently through infected S.solfataricus cells. Moreover, the hybrid plasmid stably transformed S.solfataricus and propagated with no rearrangement, recombination or integration into the host chromosome. The high copy number of the artificial genetic element was found comparable with that calculated for the wild-type pSSVx in the new host cells, with no need of genetic markers for vector maintenance in the cells and for transfomant enrichment. The newly constructed vector was also shown to be an efficient cloning vehicle for the expression of passenger genes in S.solfataricus. In fact, a derivative plasmid carrying an expression cassette of the lacS gene encoding the beta-glycosidase from S.solfataricus under the control of the Sulfolobus chaperonine (thermosome tf55) heat shock promoter was also able to drive the expression of a functional enzyme. Complementation of the beta-galactosidase deficiency in a deletion mutant strain of S.solfataricus demonstrated that lacS gene was an efficient marker for selection of single transformants on solid minimal lactose medium.

A novel thermostable sulfite oxidase from Thermus thermophilus: characterization of the enzyme, gene cloning and expression in Escherichia coli.

A novel sulfite oxidase has been identified from Thermus thermophilus AT62. Despite this enzyme showing significant amino-acid sequence homology to several bacterial and eukaryal putative and identified sulfite oxidases, the kinetic analysis, performed following the oxidation of sulfite and with ferricyanide as the electron acceptor, already pointed out major differences from representatives of bacterial and eukaryal sources. Sulfite oxidase from T. thermophilus, purified to homogeneity, is a monomeric enzyme with an apparent molecular mass of 39.1 kDa and is almost exclusively located in the periplasm fraction. The enzyme showed sulfite oxidase activity only when ferricyanide was used as electron acceptor, which is different from most of sulfite-oxidizing enzymes from several sources that use cytochrome c as co-substrate. Spectroscopic studies demonstrated that the purified sulfite oxidase has no cytochrome like domain, normally present in homologous enzymes from eukaryotic and prokaryotic sources, and for this particular feature it is similar to homologous enzyme from Arabidopsis thaliana. The identified gene was PCR amplified on T. thermophilus AT62 genome, expressed in Escherichia coli and the recombinant protein identified and characterized.

pIT3, a cryptic plasmid isolated from the hyperthermophilic crenarchaeon Sulfolobus solfataricus IT3.

The plasmid pIT3 (4,967 bp) was isolated from the hyperthermophilic archaeon Sulfolobus solfataricus, strain IT3. The completely sequenced plasmid contains six open reading frames (ORFs), the largest (ORF915) spanning more than half of the plasmid and encoding a putative protein with significant similarity to the helicase domain of viral and plasmid primase proteins, as well as to the newly described archaeal primase-polymerase domain. A small ORF, (ORF80), located upstream of this putative polymerase, encodes a putative copy number control protein. Specific transcripts corresponding to the ORF80 and ORF915, were detected by Northern blot analyses, and their transcriptional start sites were determined by primer extension. Moreover, the transfer and the maintenance of the plasmid in other Sulfolobus strains were demonstrated to be effective and stable.

Identification and characterization of a novel bacterial sulfite oxidase with no heme binding domain from Deinococcus radiodurans.

An open reading frame (draSO) encoding a putative sulfite oxidase (SO) was identified in the sequence of chromosome II of Deinococcus radiodurans; the predicted gene product showed significant amino acid sequence homology to several bacterial and eukaryotic SOs, such as the biochemically and structurally characterized enzyme from Arabidopsis thaliana. Cloning of the Deinococcus SO gene was performed by PCR amplification from the bacterial genomic DNA, and heterologous gene expression of a histidine-tagged polypeptide was obtained in a molybdopterin-overproducing strain of Escherichia coli. The recombinant protein was purified to homogeneity by nickel chelating affinity chromatography, and its main kinetic and chemical physical parameters were determined. Northern blot and enzyme activity analyses indicated that draSO gene expression is constitutive in D. radiodurans and that there is no increase upon exposure to thiosulfate and/or molybdenum(II).

Identification and autonomous replication capability of a chromosomal replication origin from the archaeon Sulfolobus solfataricus.

Here, we describe the identification of a chromosomal DNA replication origin (oriC) from the hyperthermophilic archaeon Sulfolobus solfataricus (subdomain of Crenarchaeota). By means of a cumulative GC-skew analysis of the Sulfolobus genome sequence, a candidate oriC was mapped within a 1.12-kb region located between the two divergently transcribed MCM- and cdc6-like genes. We demonstrated that plasmids containing the Sulfolobus oriC sequence and a hygromycin-resistance selectable marker were maintained in an episomal state in transformed S. solfataricus cells under selective pressure. The proposed location of the origin was confirmed by 2-D gel electrophoresis experiments. This is the first report on the functional cloning of a chromosomal oriC from an archaeon and represents an important step toward the reconstitution of an archaeal in vitro DNA replication system.

Identification of a GDP-mannose pyrophosphorylase gene from Sulfolobus solfataricus.

An open reading frame (ORF) encoding a putative GDP-mannose pyrophosphorylase (SsoGMPP) was identified on the genome sequence of Sulfolobus solfataricus P2, the predicted gene product showing high amino acid sequence homology to several archaeal, bacterial, and eukaryal GDP-mannose pyrophosphorylases such as guanidine diphosphomannose pyrophosphorylases (GMPPs) from Saccharomyces cerevisiae and Arabidopsis thaliana. The sequence was PCR amplified from genomic DNA of S. solfataricus P2 and heterologous gene expression obtained as a fusion to glutathione S-transferase in Escherichia coli, under conditions suitable to reduce the formation of inclusion bodies. Specific assays performed at 60 degrees C revealed the presence of the archaeal synthesizing GDP-mannose enzyme activity in the cell extracts of the transformed E. coli. As a positive control, the same assays were performed at the mesophilic enzyme optimum temperature on the already characterized yeast recombinant GMPP. The recombinant protein was purified to homogeneity by glutathione sepharose affinity chromatography and its thermophilic nature could be verified. The enzyme was definitively identified by demonstrating its capability to catalyze also the reverse reaction of pyrophosphorolysis and, most interestingly, its high specificity for synthesizing GDP-mannose.

A xylan-degrading strain of Sulfolobus solfataricus: isolation and characterization of the xylanase activity.

Two strains (O(alpha) and X(2)) of the hyperthermophilic crenarchaeon Sulfolobus solfataricus strain MT4 were selected and isolated for their ability to grow on xylan. O(alpha) and X(2), grown on media containing oat spelt xylan and birchwood xylan as the sole nutrient source, respectively, produced the same thermostable xylanase that was demonstrated to be inducible in xylan cultures. In an oat spelt medium, S. solfataricus O(alpha) underwent interesting morphological changes in the cell envelope, exhibiting mobile appendages not present in the typical coccal shape. The enzyme was prevalently membrane associated and showed a molecular mass of approximately 57.0 kDa. It was also highly thermostable, with a half-life of 47 min at 100 degrees C, and exhibited an optimal temperature and pH of 90 degrees C and 7.0, respectively. Xylo-oligosaccharides were the enzymatic products of xylan hydrolysis, and the smallest degradation product was xylobiose, thus indicating that the enzyme was an endoxylanase. The enzyme was able to bind weakly to crystalline cellulose (Avicel) and more strongly to insoluble xylan in a substrate amount-and temperature-dependent manner.

Transcriptional regulation of the gene encoding an alcohol dehydrogenase in the archaeon Sulfolobus solfataricus involves multiple factors and control elements.

A transcriptionally active region has been identified in the 5' flanking region of the alcohol dehydrogenase gene of the crenarchaeon Sulfolobus solfataricus through the evaluation of the activity of putative transcriptional regulators and the role of the region upstream of the gene under specific metabolic circumstances. Electrophoretic mobility shift assays with crude extracts revealed protein complexes that most likely contain TATA box-associated factors. When the TATA element was deleted from the region, binding sites for both DNA binding proteins, such as the small chromatin structure-modeling Sso7d and Sso10b (Alba), and transcription factors, such as the repressor Lrs14, were revealed. To understand the molecular mechanisms underlying the substrate-induced expression of the adh gene, the promoter was analyzed for the presence of cis-acting elements recognized by specific transcription factors upon exposure of the cell to benzaldehyde. Progressive dissection of the identified promoter region restricted the analysis to a minimal responsive element (PAL) located immediately upstream of the transcription factor B-responsive element-TATA element, resembling typical bacterial regulatory sequences. A benzaldehyde-activated transcription factor (Bald) that specifically binds to the PAL cis-acting element was also identified. This protein was purified from heparin-fractionated extracts of benzaldehyde-induced cells and was shown to have a molecular mass of approximately 16 kDa. The correlation between S. solfataricus adh gene activation and benzaldehyde-inducible occupation of a specific DNA sequence in its promoter suggests that a molecular signaling mechanism is responsible for the switch of the aromatic aldehyde metabolism as a response to environmental changes.

Characterization and functional complementation of a nonlethal deletion in the chromosome of a beta-glycosidase mutant of Sulfolobus solfataricus.

LacS(-) mutants of Sulfolobus solfataricus defective in beta-glycosidase activity were isolated in order to explore genomic instability and exploit novel strategies for transformation and complementation. One of the mutants showed a stable phenotype with no reversion; analysis of its chromosome revealed the total absence of the beta-glycosidase gene (lacS). Fine mapping performed in comparison to the genomic sequence of S. solfataricus P2 indicated an extended deletion of approximately 13 kb. The sequence analysis also revealed that this chromosomal rearrangement was a nonconservative transposition event driven by the mobile insertion sequence element ISC1058. In order to complement the LacS(-) phenotype, an expression vector was constructed by inserting the lacS coding sequence with its 5' and 3' flanking regions into the pEXSs plasmid. Since no transformant could be recovered by selection on lactose as the sole nutrient, another plasmid construct containing a larger genomic fragment was tested for complementation; this region also comprised the lacTr (lactose transporter) gene encoding a putative membrane protein homologous to the major facilitator superfamily. Cells transformed with both genes were able to form colonies on lactose plates and to be stained with the beta-glycosidase chromogenic substrate X-Gal (5-bromo-4-chloro-3-indoyl-beta-D-galactopyranoside).