Structure function relationships in the ribosomal stalk proteins of archaebacteria.

The ribosomal L12 protein gene of Sulfolobus solfataricus (SsoL12) has been subcloned and overexpressed in Escherichia coli. Five protein L12 mutants were designed: two NH2-terminal and two COOH-terminal truncated mutants and one mutant lacking the highly charged part of the COOH-terminal region. The mutant protein genes were overexpressed in E. coli and the products purified. The amino acid composition was verified and the NH2 terminally truncated mutants were subjected to Edman degradation. The SsoL12 protein was selectively removed from entire S. solfataricus ribosomes by an ethanol wash. The remaining ribosomal core particles showed a substantial decrease in the in vitro translational activity. S. solfataricus L12 protein overexpressed in E. coli (SsoL12e) was incorporated into these ribosomal cores and restored their translational activity. Mutants lacking any part of the COOH-terminal region could be incorporated into these cores, as proven by two-dimensional polyacrylamide gels of the reconstituted particles. Mutant SsoL12 MC2 (residue 1-70) was sufficient for dimerization and incorporation into ribosomes. In contrast to the COOH terminally truncated mutants, L12 proteins lacking the 12 highly conserved NH2-terminal residues or the entire NH2-terminal region (44 amino acids) are unable to bind to ribosomes, suggesting that the SsoL12 protein binds with its NH2-terminal portion to the ribosome. None of the mutants could significantly increase the translational activity of the core particles suggesting that every deleted part of the protein was needed directly or indirectly for translational activity. Our results suggest that the COOH terminally truncated mutants were bound to ribosomes but not functional for translation. Cores preincubated with these COOH terminally truncated mutants regained activity when a second incubation with the entire overexpressed SsoL12e protein followed. This finding suggests that archaebacterial L12 proteins are freely exchanged on the ribosome.     

Identification of a system required for the functional surface localization of sugar binding proteins with class III signal peptides in Sulfolobus solfataricus

The hyperthermophilic archaeon Sulfolobus solfataricus contains an unusual large number of sugar binding proteins that are synthesized as precursors with a class III signal peptide. Such signal peptides are commonly used to direct archaeal flagellin subunits or bacterial (pseudo)pilins into extracellular macromolecular surface appendages. Likewise, S. solfataricus binding proteins have been suggested to assemble in higher ordered surface structures as well, tentatively termed the bindosome. Here we show that S. solfataricus contains a specific system that is needed for the functional surface localization of sugar binding proteins. This system, encoded by the bas (bindosome assembly system) operon, is composed of five proteins: basABC, three homologues of so-called bacterial (pseudo)pilins; BasE, a cytoplasmic ATPase; and BasF, an integral membrane protein. Deletion of either the three (pseudo)pilin genes or the basEF genes resulted in a severe defect of the cells to grow on substrates which are transported by sugar binding proteins containing class III signal peptides, while growth on glucose and maltose was restored when the corresponding genes were reintroduced in these cells. Concomitantly, DeltabasABC and DeltabasEF cells were severely impaired in glucose uptake even though the sugar binding proteins were normally secreted across the cytoplasmic membrane. These data underline the hypothesis that the bas operon is involved in the functional localization of sugar binding proteins at the cell surface of S. solfataricus. In contrast to surface structure assembly systems of Gram-negative bacteria, the bas operon seems to resemble an ancestral simplified form of these machineries.     

Archaeal homolog of bacterial type IV prepilin signal peptidases with broad substrate specificity

A large number of secretory proteins in the thermoacidophile Sulfolobus solfataricus are synthesized as a precursor with an unusual leader peptide that resembles bacterial type IV prepilin signal sequences. This set of proteins includes the flagellin subunit but also various solute binding proteins. Here we describe the identification of the S. solfataricus homolog of bacterial type IV prepilin peptidases, termed PibD. PibD is an integral membrane protein that is phylogenetically related to the bacterial enzymes. When heterologously expressed in Escherichia coli, PibD is capable of processing both the flagellin and glucose-binding protein (GlcS) precursors. Site-directed mutagenesis of the GlcS signal peptide shows that the substrate specificity of PibD is consistent with the variations found in proteins with type IV prepilin-like signal sequences of S. solfataricus. We conclude that PibD is responsible for the processing of these secretory proteins in S. solfataricus.     

An archaebacterial homolog of pelota, a meiotic cell division protein in eukaryotes

Abstract An open reading frame ( pelA ) specifying a homolog of pelota and DOM34, proteins required for meiotic cell division in Drosophila melanogaster and Saccharomyces cerevisiae , respectively, has been cloned, sequenced and identified from the archaebacterium Sulfolobus solfataricus . The S. solfataricus PelA protein is about 20% identical with pelota, DOM34 and the hypothetical protein R74.6 of Caenorhabditis elegans . The presence of a pelota homolog in archaebacteria implies that the meiotic functions of the eukaryotic protein were co-opted from, or added to, other functions existing before the emergence of eukaryotes. The nuclear localization signal and negatively charged carboxy-terminus characteristic of eukaryotic pelota-like proteins are absent from the S. solfataricus homolog, and hence may be indicative of the acquired eukaryotic function(s).     

Production of recombinant and tagged proteins in the hyperthermophilic archaeon Sulfolobus solfataricus

Many systems are available for the production of recombinant proteins in bacterial and eukaryotic model organisms, which allow us to study proteins in their native hosts and to identify protein-protein interaction partners. In contrast, only a few transformation systems have been developed for archaea, and no system for high-level gene expression existed for hyperthermophilic organisms. Recently, a virus-based shuttle vector with a reporter gene was developed for the crenarchaeote Sulfolobus solfataricus, a model organism of hyperthermophilic archaea that grows optimally at 80 degrees C (M. Jonuscheit, E. Martusewitsch, K. M. Stedman, and C. Schleper, Mol. Microbiol. 48:1241-1252, 2003). Here we have refined this system for high-level gene expression in S. solfataricus with the help of two different promoters, the heat-inducible promoter of the major chaperonin, thermophilic factor 55, and the arabinose-inducible promoter of the arabinose-binding protein AraS. Functional expression of heterologous and homologous genes was demonstrated, including production of the cytoplasmic sulfur oxygenase reductase from Acidianus ambivalens, an Fe-S protein of the ABC class from S. solfataricus, and two membrane-associated ATPases potentially involved in the secretion of proteins. Single-step purification of the proteins was obtained via fused His or Strep tags. To our knowledge, these are the first examples of the application of an expression vector system to produce large amounts of recombinant and also tagged proteins in a hyperthermophilic archaeon.     

Coordination of multiple enzyme activities by a single PCNA in archaeal Okazaki fragment maturation

Chromosomal DNA replication requires one daughter strand-the lagging strand-to be synthesised as a series of discontinuous, RNA-primed Okazaki fragments, which must subsequently be matured into a single covalent DNA strand. Here, we describe the reconstitution of Okazaki fragment maturation in vitro using proteins derived from the archaeon Sulfolobus solfataricus. Six proteins are necessary and sufficient for coupled DNA synthesis, RNA primer removal and DNA ligation. PolB1, Fen1 and Lig1 provide the required catalytic activities, with coordination of their activities dependent upon the DNA sliding clamp, proliferating cell nuclear antigen (PCNA). S. solfataricus PCNA is a heterotrimer, with each subunit having a distinct specificity for binding PolB1, Fen1 or Lig1. Our data demonstrate that the most efficient coupling of activities occurs when a single PCNA ring organises PolB1, Fen1 and Lig1 into a complex.     

Identification and characterization of sulfolobus solfataricus P2 proteome using multidimensional liquid phase protein separations

We have identified and characterized the proteome of Sulfolobus solfataricus P2 using multidimensional liquid phase protein separations. Multidimensional liquid phase chromatography was performed using ion exchange chromatography in the first dimension, followed by reverse-phase chromatography using 500 microm i.d. poly(styrene-divinylbenzene) monoliths in the second dimension to separate soluble protein lysates from S. solfataricus. The 2DLC protein separations from S. solfataricus protein lysates enabled the generation of a 2D liquid phase map analogous to the traditional 2DE map. Following separation of the proteins in the second dimension, fractions were collected, digested in solution using trypsin and analyzed using mass spectrometry. These approaches offer significant reductions in labor intensity and the overall time taken to analyze the proteome in comparison to 2DE, taking advantage of automation and fraction collection associated with this approach. Furthermore, following proteomic analysis using 2DLC, the data obtained was compared to previous 2DE and shotgun proteomic studies of a soluble protein lysate from S. solfataricus. In comparison to 2DE, the results show an overall increase in proteome coverage. Moreover, 2DLC showed increased coverage of a number of protein subsets including acidic, basic, low abundance and small molecular weight proteins in comparison to 2DE. In comparison to shotgun studies, an increase in proteome coverage was also observed. Furthermore, 187 unique proteins were identified using 2DLC, demonstrating this methodology as an alternative approach for proteomic studies or in combination with 2DE and shotgun workflows for global proteomics.     

An archaeal Holliday junction resolving enzyme from Sulfolobus solfataricus exhibits unique properties

The rearrangement and repair of DNA by homologous recombination often involves the creation of Holliday junctions, which must be cleaved by junction-specific endonucleases to yield recombinant duplex DNA products. Holliday junction resolving enzymes are a ubiquitous class of proteins with diverse structural and mechanistic characteristics. We have characterised an endonuclease (Hje) from the thermophilic crenarchaeote Sulfolobus solfataricus that exhibits a high degree of specificity for Holliday junctions via an apparently novel mechanism. Hje resolves four-way DNA junctions by the introduction of paired nicks in a reaction that is independent of the local nucleotide sequence, but is restricted solely to strands that are continuous in the stacked-X form of the junction. Three-way DNA junctions are cleaved only when the presence of a bulge in one strand allows the junction to stack in an analogous manner to four-way junctions. These properties differentiate Hje from all other known junction resolving enzymes.     

Relative quantification of proteins across the species boundary through the use of shared peptides

We show that shared peptides of proteins that are encoded in different species are suitable for cross-species relative protein quantification. A 14N-containing proteome from the thermoacidophilic archaeon Sulfolobus tokodaii was mixed with a 15N-labeled proteome from Sulfolobus solfataricus. Using three shared peptides per protein, the relative abundance of six orthologous proteins was calculated. Observed standard deviations were approximately 10%, indicating that the trypsin accessibility to cleavage sites was not altered in the orthologs. The abundance ratios of the and subunits of the Thermosome were 0.64 and 1.24 in Sulfolobus tokodaii compared to Sulfolobus solfataricus, suggesting a different stoichiometry of the complex in both species. In addition, an in silico study was performed on the occurrence of shared peptides. Inter- and intra-species peptide redundancy was investigated in the model organisms Homo sapiens, Mus musculus, Escherichia coli K12, Escherichia coli O157:H7, S. solfataricus, and S. tokodaii. M. musculus and H. sapiens share 30-50% of all peptides (6-15 residues). Moreover, approximately one-third of all proteins shared > or = 40% of their peptides with at least one other protein in the related species, thus offering strong potential for cross-species relative protein quantification. Conversely, approximately 40% of all peptides (6-15 residues) encoded in H. sapiens are encoded multiple times and therefore complicate identification and quantification.     

Identification and characterisation of a novel acylpeptide hydrolase from Sulfolobus solfataricus: structural and functional insights

A novel acylpeptide hydrolase, named APEH-3(Ss), was isolated from the hypertermophilic archaeon Sulfolobus solfataricus. APEH is a member of the prolyl oligopeptidase family which catalyzes the removal of acetylated amino acid residues from the N terminus of oligopeptides. The purified enzyme shows a homotrimeric structure, unique among the associate partners of the APEH cluster and, in contrast to the archaeal APEHs which show both exo/endo peptidase activities, it appears to be a "true" aminopeptidase as exemplified by its mammalian counterparts, with which it shares a similar substrate specificity. Furthermore, a comparative study on the regulation of apeh gene expression, revealed a significant but divergent alteration in the expression pattern of apeh-3(Ss) and apeh(Ss) (the gene encoding the previously identified APEH(Ss) from S. solfataricus), which is induced in response to various stressful growth conditions. Hence, both APEH enzymes can be defined as stress-regulated proteins which play a complementary role in enabling the survival of S. solfataricus cells under different conditions. These results provide new structural and functional insights into S. solfataricus APEH, offering a possible explanation for the multiplicity of this enzyme in Archaea.     

A comparison of the DNA binding properties of histone-like proteins derived from representatives of the two kingdoms of the Archaea

Abstract The nucleoid protein composition, the enhancement of DNA electrophoretic mobility, the toroidal wrapping and the helical period of DNA complexed with nucleoid proteins from species within the archaeal kingdom Euryarchaeota was shown to contrast with the composition and properties of nucleoid proteins from Sulfolobus solfataricus , a member of the archaeal kingdom Crenarchaeota. This result was seen to support the hypothesis that archaeal histones with homology to the eukaryal hi stone consensus are a diagnostic feature of the Euryarchaeota.     

Thermoadaptation of a mesophilic hygromycin B phosphotransferase by directed evolution in hyperthermophilic Archaea: selection of a stable genetic marker for DNA transfer into Sulfolobus solfataricus

A mutated version of the hygromycin B phosphotransferase (hph(mut)) gene from Escherichia coli, isolated by directed evolution at 75 degrees C in transformants of a thermophilic strain of Sulfolobus solfataricus, was characterized with respect to its genetic stability in both the original mesophilic and the new thermophilic hosts. This gene was demonstrated to be able to express the hygromycin B resistance phenotype and to be steadily maintained and propagated also in other, more thermophilic strains of S. solfataricus, i.e., up to 82 degrees C. Furthermore, it may be transferred to S. solfataricus cells by cotransformation with pKMSD48, another extrachromosomal element derived from the virus SSV1 of Sulfolobus shibatae, without any loss of stability and without affecting the replication and infectivity of this viral DNA. The hph(mut) and the wild-type gene products were expressed at higher levels in E. coli and purified by specific affinity chromatography on immobilized hygromycin B. Comparative characterization revealed that the mutant enzyme had acquired significant thermoresistance and displayed higher thermal activity with augmented catalytic efficiency.     

Cellulose degradation by Sulfolobus solfataricus requires a cell-anchored endo-β-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-β-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.     

Genome-scale reconstruction and analysis of the metabolic network in the hyperthermophilic archaeon sulfolobus solfataricus

We describe the reconstruction of a genome-scale metabolic model of the crenarchaeon Sulfolobus solfataricus, a hyperthermoacidophilic microorganism. It grows in terrestrial volcanic hot springs with growth occurring at pH 2-4 (optimum 3.5) and a temperature of 75-80°C (optimum 80°C). The genome of Sulfolobus solfataricus P2 contains 2,992,245 bp on a single circular chromosome and encodes 2,977 proteins and a number of RNAs. The network comprises 718 metabolic and 58 transport/exchange reactions and 705 unique metabolites, based on the annotated genome and available biochemical data. Using the model in conjunction with constraint-based methods, we simulated the metabolic fluxes induced by different environmental and genetic conditions. The predictions were compared to experimental measurements and phenotypes of S. solfataricus. Furthermore, the performance of the network for 35 different carbon sources known for S. solfataricus from the literature was simulated. Comparing the growth on different carbon sources revealed that glycerol is the carbon source with the highest biomass flux per imported carbon atom (75% higher than glucose). Experimental data was also used to fit the model to phenotypic observations. In addition to the commonly known heterotrophic growth of S. solfataricus, the crenarchaeon is also able to grow autotrophically using the hydroxypropionate-hydroxybutyrate cycle for bicarbonate fixation. We integrated this pathway into our model and compared bicarbonate fixation with growth on glucose as sole carbon source. Finally, we tested the robustness of the metabolism with respect to gene deletions using the method of Minimization of Metabolic Adjustment (MOMA), which predicted that 18% of all possible single gene deletions would be lethal for the organism.     

A carboxylesterase from the hyperthermophilic archaeon Sulfolobus solfataricus: cloning of the gene, characterization of the protein

A genomic library of the hyperthermophilic archaeon Sulfolobus solfataricus strain MT4 was constructed in Escherichia coli using a cloning vector not designed for heterologous gene expression. One positive clone exhibiting acquired thermophilic acetylesterase activity was directly detected by an in situ plate assay using a colony staining procedure with the chromogenic substrate beta-naphthyl acetate. The plasmid isolated from the clone contained a 3.3 kb genomic fragment from S. solfataricus and a full-length esterase coding sequence could be identified. Expression of the active thermostable esterase in E. coli was independent of isopropyl-beta-D-thiogalactopyranoside and of the kind of vector, suggesting that the archaeal esterase gene was controlled by fortuitous bacterial-like sequences present in its own 5' flanking region, not by the bacterial lac promoter or other serendipitous vector-located sequences. The protein, partially purified by thermoprecipitation of the host proteins at high temperature and gel exclusion chromatography, showed a homo-tetrameric structure with a subunit of molecular mass of 32 kDa which was in perfect agreement with that deduced from the cloned gene. The same protein was revealed in S. solfataricus cell extracts, thus demonstrating its functional occurrence in vivo under the cell culture conditions tested. The recombinant enzyme exhibited high thermal activity and thermostability with optimal activity between pH 6.5 and 7.0. The hydrolysis of p-nitrophenyl esters of fatty acids (from C(2) to C(8)) allowed the enzyme to be classified as a short length acyl esterase.     

Characterization of a multifunctional protein disulfide oxidoreductase from Sulfolobus solfataricus

A potential role in disulfide bond formation in the intracellular proteins of thermophilic organisms has recently been ascribed to a new family of protein disulfide oxidoreductases (PDOs). We report on the characterization of SsPDO, isolated from the hyperthermophilic archaeon Sulfolobus solfataricus. SsPDO was cloned and expressed in Escherichia coli. We revealed that SsPDO is the substrate of a thioredoxin reductase in S. solfataricus (K(M) 0.3 microm) and not thioredoxins (TrxA1 and TrxA2). SsPDO/S. solfataricus thioredoxin reductase constitute a new thioredoxin system in aerobic thermophilic archaea. While redox (reductase, oxidative and isomerase) activities of SsPDO point to its central role in the biochemistry of cytoplasmic disulfide bonds, chaperone activities also on an endogenous substrate suggest a potential role in the stabilization of intracellular proteins. Northern and western analysis have been performed in order to analyze the response to the oxidative stress.     

Glucose Transport in the Extremely Thermoacidophilic Sulfolobus solfataricus Involves a High-Affinity Membrane-Integrated Binding Protein

The archaeon Sulfolobus solfataricus grows optimally at 80 degrees C and pH 2.5 to 3.5 on carbon sources such as yeast extracts, tryptone, and various sugars. Cells rapidly accumulate glucose. This transport activity involves a membrane-bound glucose-binding protein that interacts with its substrate with very high affinity (Kd of 0. 43 microM) and retains high glucose affinity at very low pH values (as low as pH 0.6). The binding protein was extracted with detergent and purified to homogeneity as a 65-kDa glycoprotein. The gene coding for the binding protein was identified in the S. solfataricus P2 genome by means of the amino-terminal amino acid sequence of the purified protein. Sequence analysis suggests that the protein is anchored to the membrane via an amino-terminal transmembrane segment. Neighboring genes encode two membrane proteins and an ATP-binding subunit that are transcribed in the reverse direction, whereas a homologous gene cluster in Pyrococcus horikoshii OT3 was found to be organized in an operon. These data indicate that S. solfataricus utilizes a binding-protein-dependent ATP-binding cassette transporter for the uptake of glucose.